Joanne Sarah

Sonia

Fabienne

Julie Lisou

Nor' Guillaume Kévin

Cyrielle, Morgane, Greg, Camille, Wiki, Valentin, Léo, Loïc, Agnès, Sarah, Jérém', Ellen, Hélène, MJ, Flo, Paulinette, Lise, Bibine, Bérénice, Férouze, Muriel Nathalie Sandra

Aurél'

Diane Poulpi

Anneke Jana Sam

Pauline Marangoni

Cyril Charles

Youngwook Ahn

Kerstin Seidel

Andrew Jheon

Sarah Alto

Robb Krumlauf

Laurent Viriot
email: laurent.viriot@ens-lyon.fr

Ophir Klein
email: ophir.klein@ucsf.edu

UNRAVELING DEVELOPMENT AND AGEING DYNAMICS OF THE RODENT DENTITION

Keywords: Rodent dentition, ERK-MAPK pathway, comparative dental anatomy, incisor stem cell niches, ageing, gene expression profile evolution, diversity & ageing B MAPK signalling pathway, dentition evolution, dental formula, Implications FGF signaling pathway, tooth shape, enamel mineralization, syngnathia

I value the time I spent with Guillaume, Mélisandre, Juli, and Julie. We all came here for various reasons, but at least we got to create friendships that will hopefully last longer than our respective PhDs… Cyrielle & Domi (and of cours Kévin & Jeff'), please know that you're missed every single day, and that I cannot wait to be on the same continent than you! One last thing: when I grow up, I want to be just like you  Delphine, thank you for being an active member of my PhD follow up committee. You have been incredibly supportive, and

As it is the tradition, I would like to start by thanking my two supervisors... Laurent, you made me enter the research world working on a biological question I did not even know I could be interested in. And here I am, about six years after you first talked to me about the importance of the fossil record in evolutionary biology. And then Cyril, we met in San Francisco where you let the chatty French intern crush on your coach. If I recall well, Laurent told me that it would be difficult to make you talk. I guess I am contagious. Thank you both for supervising me during this PhD, I really appreciated the independence you let me have, and I value the advices you gave me on the road.

I am also very grateful to the members of my jury, for having agreed to be part of this adventure. Ophir, thank you for coming such a long way just for my defense, it means a lot to me. Fred, thank you for having a nice word to say every single time, and for asking me questions that make me re-think entire parts of my projects in the blink of an eye. Yann, you monitored closely this PhD project, thank you for your advices during my PhD committee meetings, and thank you for being part of this jury to complete the circle! Ariane, I am very grateful that you agreed on coming, and I am looking forward to hearing your questions and remarks.

I am honored to have been part of the IGFL. During all the internships I performed at the old LR4, and in our shiny and new building, I have met and worked with amazing people.

Starting with the team Evo-Devo of the Vertebrate Dentition. Béatrice, I am so sad that you will not be at my defense. It has been a pleasure sharing your office, talking about dance, cooking, theater, music, and occasionally about science! Johan, you are the PhD student in charge now, thanks for supporting me even in my poor sport choices  Ana Rosa, this has been a real pleasure having you around, I will miss your accent for sure. Florence, you are officially my qPCR role-model. Thank you for being so kind with me. And of course, thanks to those who have already left the team, but are still present in our minds: Antoine, for the insightful discussions we have had, Helder, especially for the pep talk before my PhD funding examination, Manu, for the amazing ride to La Londe in that van, Maïtena, for your contagious laugh… I shall not forget Henry and Marie-Lise: your kindness is beyond words, and I really enjoyed working next to you. We all make a hell of a team, not quite conventional I have to admit, but who cares? Tab. 3: Genes of interest to understand the molecular impact of ageing on the stem cell niches, and the setting of the aplastic groove Suppl. Tab. 2: List of the differentially expressed genes between two age conditions from the growth region transcriptomic analysis Suppl. Tab. 3: List of the genes differentially expressed between two age conditions from the entire incisor transcriptomic analysis Suppl. Tab. 4: List of the genes differentially expressed in the presence or the absence of aplastic groove

INTRODUCTION & OBJECTIVES

ince the very first steps of vertebrate paleontology [START_REF] Cuvier | Recherches sur les ossements fossiles de quadrupèdes[END_REF], the comparative anatomy of teeth and dentitions occupies a very important position in mammal paleobiology.

This status stands for several reasons: firstly because teeth are made of the hardest mineralized tissues of the body and as a consequence often become selectively preserved in comparison with other parts of the skeleton during fossilization; secondly because teethand especially postcanine teeth (premolars and molars)provide appropriate characters to study mammalian diet, taxonomy and evolution [START_REF] Gregory | Polyisomerism and Anisomerism in Cranial and Dental Evolution among Vertebrates[END_REF][START_REF] Mckenna | Toward a phylogenetic classification of the Mammalia[END_REF]. Given the morphological diversity among extant species and the available fossil record, biologists and paleobiologists have started to address the question of the mechanisms underlying the setting up of the mammalian dental diversity.

To understand the basis of mammal dental evolution, it is necessary to consider mammals in the larger framework of the synapsid evolution, thus supplementing the living species with many extinct taxa with no current descendants. The earliest members of the lineage leading to mammals lived about 300 million years (Ma) ago [START_REF] Pough | Vertebrate life[END_REF].

They displayed a dentition made up of numerous simple, conical and pointed teeth (haplodonty), which were almost identical along the tooth row (homodonty), and which were continuously replaced throughout their life (polyphyodonty) [START_REF] Ungar | Mammal teeth: origin, evolution, and diversity[END_REF]. The earliest mammals sensu lato, known at about 200 Ma, had a dentition made of teeth with various shapes (heterodonty) including tricuspid postcanine teeth (plexodonty) that were replaced S once during their lifetime (diphyodonty) [START_REF] Luo | Evolution of dental replacement in mammals[END_REF]. Further evolutionary trends of the dentition from these earliest mammals sensu lato to the placental mammals (eutherians) is characterized by a reduction of the dental formula coupled with a specialization of postcanine teeth leading to the acquisition of the mammalian mastication [START_REF] Weijs | Evolutionary Approach of Masticatory Motor Patterns in Mammals[END_REF]. Basal eutherians had a fixed maximal number of teeth (FIG. 1A) and this number continuously decreased over the evolution of most modern eutherian groups such as euarchontoglires (FIG. 1B). Along with this decrease, a higher specialization of the postcanine teeth (especially the molars) was achieved.

Mammals thus progressively acquired a limited dentition replacement, a sectorization of the dentition into three dental types (incisor, canine and postcanine), as well as an increased complexity of their postcanine crown pattern. Because the various steps of these evolutionary processes are only documented by fossils, the evolution of the mammalian dentition has first mainly interested paleontologists, and for the past two decades this interest has expanded to the evo-devo community.

Evo-devo is a dual discipline that compares individual development with phenotypic changes during evolution [START_REF] Müller | Evo-devo: extending the evolutionary synthesis[END_REF]. In order to understand how evolution acted on the biological properties of extinct and extant species, investigations aim at understanding how developmental processes are set up and modulated in extant species. Established correlations allow us to infer that similar processes were acting in the course of evolution [START_REF] Carroll | Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution[END_REF].

Besides being a major tool to study mammalian evolution, teeth are also a well-studied example of ectodermal organs arising from the interaction of an epithelial layer with the underlying mesenchyme, similarly to many exocrine glands, feathers, scales, nails, and hair [START_REF] Pispa | Mechanisms of ectodermal organogenesis[END_REF]. Historically, the evo-devo community first asserted itself by comparing various body plans and analyzing the importance of toolkit genes in setting them [START_REF] Duboule | The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes[END_REF]. Dental evo-devo rapidly grew as a major focus of the discipline. As the field developed, a new interest arose on the genetic and epigenetic determination of odontogenesis (Maas and Bei, 1997;[START_REF] Peters | Teeth: where and how to make them[END_REF][START_REF] Jernvall | Reiterative signaling and patterning during mammalian tooth morphogenesis[END_REF]Thesleff, 2003;Tucker and Sharpe, 2004;[START_REF] Catón | Current knowledge of tooth development: patterning and mineralization of the murine dentition[END_REF][START_REF] Jheon | Expression of MicroRNAs in the Stem Cell Niche of the Adult Mouse Incisor[END_REF], and computational tools helped to better understand the evolution of dentition features (Salazar-Ciudad and [START_REF] Salazar-Ciudad | A computational model of teeth and the developmental origins of morphological variation[END_REF]. Since there are about 5,500 species of mammals [START_REF] Wilson | Mammal species of the world: a taxonomic and geographic reference[END_REF], researchers have to focus on a few species and use them as proxies to collect data (Minelli and Baedke, 2014). A classic extrapolation has been to work on rodents. Indeed, the order Rodentia is a rather large order encompassing about 40% of all mammal species diversity [START_REF] Wilson | Mammal species of the world: a taxonomic and geographic reference[END_REF]. This orderand especially murine rodentsshows a reduction of the maximal dental formula compared to the euarchontoglires (FIG. 1C). Rodents gained the status of well-established mammal representatives within the "tooth community" because of the very interesting dental diversity they display, but also because one of their members, the mouse (Mus musculus) has been used as a model for mammalian development for decades (Hedrich, 2004).

Mice are extremely derived in terms of dentition properties. They display only one highly specialized continuously growing incisor, as well as three complex molars that are not replaced per jaw quadrant (FIG. 2). If rare mammalian species display continuous dental replacement (see annex 1: Rodrigues et al., 2011), monophyodonty marks the final limitation in tooth renewal, and is observed in many taxa including muroid rodents, bats or shrews [START_REF] Jernvall | Tooth shape formation and tooth renewal: evolving with the same signals[END_REF]. Mouse dentition recapitulates the evolutionary trends that are characteristic of mammalian dental evolution, and as a consequence, the understanding of this evolutionary dynamics is of prime interest.

The present work aims at addressing two levels of mouse dentition dynamics: (1) the evolutionary dynamics of tooth number determination and shape setting in the postcanine area, and (2) the lifespan dynamics of the continuously growing incisors. Using a classic evodevo approach on postcanine tooth number and shape, I first tackle the evolutionary potential of the well-documented FGF-activated ERK-MAPK pathway. This is an example of huge signaling pathway modulating most of the cellular processes, also involved at various stages of tooth development (Laugel-Haushalter et al., 2014). By scrutinizing the dentition of mice bearing mutation for various genes expressed in the same signaling pathway, I address the effects of genes acting at different steps of the pathway. From the mineralized tooth phenotype, I draw conclusions regarding the plausible evolutionary relevance of these genes.

In a second part, I focus on the mouse continuously growing incisors. By looking at the diversity they display in terms of ornamentation, color and shape, I highlight their importance in the evo-devo field. Switching focus to the modification of their phenotype during the animal lifetime, I address the question of stem cell ageing in combining traditional in vivo monitoring and histological studies with cutting-edge transcriptomics to provide a further insight into the use of the mouse model. Tooth development (odontogenesis) is ruled by specific time-space interactions between the oral epithelium of the first branchial arch and the underlying mesenchyme deriving from the neural crests [START_REF] Peters | Teeth: where and how to make them[END_REF]. Odontogenesis begins with a succession of histomorphogenetical transformations named from the shape of the dental epithelium (FIG.

A.1).

Mouse gestation lasts around 20 days, and tooth development is initiated around Embryonic day 9.5 (Tucker and Sharpe, 2004). The secretion of growth factors combined with the activity of transcription factors in both upper and lower cheek teeth presumptive areas induces cell proliferation in both odontogenic epithelium and underlying mesenchyme (Thesleff, 2003). The first histo-morphogenetical sign of tooth development is the thickening of the dental epithelium until it reaches the placode stage. Then the placode grows toward the mesenchyme to form a bud (E13). At that stage, two distinct populations of epithelial cells can be observed: (1) a well-organized basal epithelium which remains at the interface with the odontogenic mesenchyme, and (2) a loosely arrange group of cells located towards the mouth cavity called stellate reticulum. Later on, the basal surface of the bud invaginates at its lingual and vestibular ends (cervical loops) as the underlying dental mesenchyme condenses. At the cap stage, the primary enamel knot (pEK, dark blue in FIG.

A.1) forms in the basal dental O epithelium [START_REF] Jernvall | Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene[END_REF]Jernvall et al., 1998;Vaahtokari et al., 1996a;[START_REF] Vaahtokari | Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4[END_REF]. The pEK is a dense non-proliferative group of cells secreting many molecules including Sonic Hedgehog (SHH), Fibroblast Growth Factors (FGFs), Bone Morphogenetic Proteins (BMPs) and WNTs [START_REF] Thesleff | Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation[END_REF]. The combination of these molecules plays a major role in inducing the proliferation of surrounding cells, and thus in initiating the presumptive crown shape [START_REF] Coin | Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area[END_REF]. From that stage on, odontogenesis is ruled by this epithelial signaling center.

Then, the cervical loops continue to expand into the mesenchyme, and the cap takes a bell shape. As the cytodifferentiation goes further, secondary enamel knots (sEKs, light blue in FIG.

A.1) are formed at the top of the presumptive cusps (Tucker and Sharpe, 2004). At the late bell stage, the dimensions of the presumptive crown are set and the mineralization of the secreted dentin and enamel matrixes starts (Tucker and Sharpe, 1999). These matrixes are respectively secreted by odontoblasts and ameloblasts, two cell types which constitute the epithelio-mesenchymal interface (Arana-Chavez and Massa, 2004;[START_REF] Goldberg | The dentino-enamel junction revisited[END_REF].

Ameloblasts derive from the basal epithelial layer, while odontoblasts derive the mesenchymal dental papilla [START_REF] Thesleff | Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation[END_REF]. By the end of this sequence, the crown formation is achieved. After the eruption (starting at about postnatal day 10 for the first molar) is achieved, the mouse possesses one incisor separated from three molars by a toothless gap called diastema in each dental quadrant [START_REF] Nanci | Ten Cate's Oral Histology-Pageburst on VitalSource: Development, Structure, and Function[END_REF]. Incisor development follows the same overall developmental series, the specificities of which will be discussed below.

This sequence of morphological changes is driven by precise molecular networks acting together to tightly regulate its successive steps [START_REF] Bei | Molecular genetics of tooth development[END_REF]. One example of important network is the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) signaling pathway that can be triggered by receptor tyrosine kinase (RTK) activation.

This cascade has the specificity of encompassing two signaling pathways necessary for proper tooth development: FGF signaling pathway (Li et al., 2014), and the ERK signaling pathway (Corson et al., 2003). Numerous RTK-activated ERK-MAPK members are expressed in dental tissues. They have been demonstrated to act during tooth development (TAB. A.1).

In E gene are responsible for the development of this syndrome in human [START_REF] Trivier | Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome[END_REF][START_REF] Jacquot | Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations[END_REF][START_REF] Delaunoy | Mutations in the X-linked RSK2 gene (RPS6KA3) in patients with Coffin-Lowry syndrome[END_REF]. The disorder has an estimated prevalence of 1 in 40,000 to 50,000 people, with females displaying a higher feature variability than males (Hanauer and Young, 2002). Clinically, some defects resulting from this syndrome are detectable at birth, while others will progressively be established until the patient reaches the age of 2 [START_REF] Hunter | Coffin-Lowry syndrome: A 20-year follow-up and review of long-term outcomes[END_REF]. Growth defects, skeletal and craniofacial dismorphia, as well as mental retardation make up the wide range of features associated with the syndrome (Hanauer and Young, 2002;[START_REF] Herrera-Soto | The musculoskeletal manifestations of the Coffin-Lowry syndrome[END_REF].

M E M E M E M E M E M E M E M E M E M E M E M Egf + - Egr1 - - - - -+ + + + + + + Fgf1 + + + + + + + + + + * Fgf2 + + + -+ + + - Fgf3 - - - - + + + + -+ - - Fgf4 - - - - + -+ -+ - - - - - - - + -+ - - - - - Fgf7 - - - - - - - - - - - - -+ Fgf8 + - + - - - - - - - - -+ - + - - - - - - - - - Fgf9 + - + - + -+ + + -+ -+ - + - + -+ + + -+ - Fgf10 + + - + -+ - - -+ - - Fgf15 - - + - - -+ - -+ - - Fgf16 - - - - - -+ + + -+ - Fgf17 + - - - - -+ + - - - - Fgf18 - - - + -+ -+ - - - - Fgf20 - - + - + -+ -+ - - - Fgfr1IIIb - - - - - -+ -+ + + + + + Fgfr1IIIc -+ - + -+ + + + + + + + + Fgfr2IIIb + - + - + - - - - - - - + - Fgfr2IIIc - - - - -+ + + -+ - - + - Fgfr3IIIb -+ Fgfr3IIIc - - - - - - - - - - - - -+ Fgfr4 - - - - - - - - - - - - ° Tgfb1 - - + + + - -+ + + Tgfb2 - - -+ Tgfb3 - -+ -+ -+ + Tgfbr2 -+ -+ Rasa3 + + Hras1 + - Rsk1 + + + -+ -+ - + -+ -+ + + + Rsk2 - + -+ -+ -+ -+ -+ -+ -+ -+ Rsk3 + + -+ -+ -+ + - -+ + -+ - Rsk4 + -+ -+ -+ - + + + + + + + + Spry1 - -+ + + + Spry2 + - + - Spry4 -+ -+ I take

A.1 RSK2 IS A MODULATOR OF TOOTH DEVELOPMENT

Our collaborators analyzed the skull phenotype of the two Rsk mutant mice, and they designed a microarray experiment to address molecular changes in the regulatory network. I aimed at documenting adult molar phenotype in Rsk2 -/Y as well as triple Rsk1,2,3 KO mutants in order to complete the description of Rsk2 gene role in craniofacial development.

A.1.1 Material and methods

A first cohort of 6 Rsk2 -/Y male mice and 6 WT littermates has been studied. They have been bred at the IGBMC mouse breeding facility (Illkirch, France). A second cohort including 7 Rsk1,2,3 -/-compound mutants was also provided to us. Molar rows were imaged using highresolution X-ray microtomography with the Nanotom S (GE) equipment available at the UMS3444 (Lyon). A cubic voxel of 3µm was used. 3D-reconstructions from the acquired 2,000 projections were obtained using datos|x software and algorithm with a beam hardening correction. Pictures of the reconstructed molar row were taken in occlusal view to observe dental features of each specimen. Measures of the tooth length (mesio-distal distance) and width (vestibule-lingual distance) have been made on occlusal-oriented pictures of the molar rows. Statistical significance of the measures has been verified using the Student t-test for 2 group comparisons, with a p-value threshold of 0.05.

A.1.2 Results

Despite the absence of abnormal phenotype in incisors, defects are seen in the molar rows. The most striking abnormality observed in both Rsk2 -/Y and Rsk1,2,3 null mutant mice is the presence of a ST in both upper and lower jaws (arrowheads,FIG. A.3). This extra tooth is erupted in alignment with the molar row, in a mesial position. The upper ST occurs in 65%

of Rsk2 -/Y mice and in 83% of the compound mutant mice. The lower ST occurs in 31% of the simple mutant mice and in 11% of the Rsk1,2,3 null mice. Overall, the shape of the ST ranges from a monocuspid tooth to complex molar-like tooth. The inactivation of Rsk2 alone is sufficient to generate the abnormal ST-displaying phenotype. It is also sufficient to alter tooth proportions (FIG A.4). In compound mutants, the M 3 is absent in 39% of the specimens (e.g.

in FIG.

A.3). The absence of any tooth socket calls for agenesis, which is a missing tooth due to a developmental failure.

A.1.3 Discussion

Coffin-Lowry syndrome orodental features do not relate to Rsk-KO mutant phenotype

Coffin-Lowry syndrome is characterized by psychomotor and growth retardation, digit abnormalities, progressive skeletal malformations, as well as craniofacial dysmorphism (Temtamy et al., 1974;Temtamy et al., 1975;Hanauer and Young, 2002). Focusing on those craniofacial dysmorphism, cases report a high narrow palate, a midline lingual furrow, malocclusion, peg-shaped incisors, and hypodontia [START_REF] Young | The Coffin-Lowry syndrome[END_REF]. Due to rare availability of , meaning that modern mice lost 2 incisors, 1 canine and 4 premolars along their evolutionary history. In our mutants, the ST occurs in a position corresponding to a 4 th premolar, at the back of the diastema. A rudimentary tooth primordium was reported to develop in that same distal part of the diastema though it is aborted by the cap stage (E14) [START_REF] Peterka | Position and growth of upper and lower tooth primordia in prenatal mouse--3D study[END_REF]Viriot et al., 2000;[START_REF] Peterková | The developing mouse dentition: a new tool for apoptosis study[END_REF]. The ST found in Rsk2 -/Y and Rsk1,2,3 null mutants is likely to be erupting from the completed development of this rudimentary primordium. This finding recalls another mutant studied in the light of human craniofacial syndrome that finally revealed its evolutionary potential: the Tabby mice.

Heterozygous Tabby mice display ST in their upper molar rows, while K14-Eda mice display ST in their lower molar rows [START_REF] Pispa | Cusp patterning defect in Tabby mouse teeth and its partial rescue by FGF[END_REF]Kangas et al., 2004;Charles et al., 2009a).

But these mice also display a lot of cusp defects, which do not seem to occur in Rsk-knockout mutants. However, the specimens in our sample were not collected at the same age. Due to the different wear state of the molar rows, it is thus difficult to rule out the existence of such cusp defects.

A.1.4 Conclusions

Rsk2 -/Y and Rsk1,2,3 null mice were screened to test whether their craniofacial phenotypes match the clinical picture of the Coffin-Lowry syndrome. Only two of the displayed phenotypes mimic what is observed in human patients. The occurrence of tooth agenesis has indeed been addressed, but the mice are also displaying variable skull phenotype ranging from WT-like to severe dysmorphy associated with nasal bone deviation. Thus, these mutant mice cannot be considered as the best models to address the Coffin-Lowry syndrome.

Interestingly, the occurrence of ST mesially to the molar rows reveals the evolutionary potential of Rsk2. The small size of the sample and the variation in the mouse age prevent us from scrutinizing the exact extent of dental abnormalities and the precise evolutionary relevance.

A.2 UNRAVELING THE EVOLUTIONARY POTENTIAL OF THE ERK-MAPK PATHWAY

The occurrence of ST in mesial position to the molar row is a feature that is displayed in several mutant mice already published. Mice carrying mutant for Gas1, Shh, Wnt1, Wise, Lrp4, Eda or Edar, Spry2 and Spry4 areamong other dental characterscharacterized by the similar presence of a ST (respectively Ahn et al., 2010;Charles et al., 2009a;Kangas et al., 2004;Ohazama et al., 2009;Klein et al., 2006;Lagronova-Churava et al., 2013). Among this list, Spry2 and Spry4 mutant drew our attention, because of the molecular role of Sprouty proteins. Sprouty proteins are indeed general Receptor Tyrosine Kinase (RTK) inhibitors (Reich et al., 1999), and as so, they can inhibit FGFR-mediated activation of the ERK-MAPK cascade.

Taking advantage of the availability of Spry1 -/-, Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice, we conduct a comparative analysis to address the relative role of these components in the building of the mouse postcanine dentition. Eventually, this study helps us perfecting the analysis of Rsk2 mutant dental phenotype, and addressing the role of the entire pathway in the course of muroid rodent evolution. This study is currently submitted to Scientific Reports.

Phenotypic and evolutionary implications of modulating the ERK-MAPK cascade using the dentition as a model

Pauline Marangoni 1* , Cyril Charles 1* , Paul Tafforeau 2 , Virginie Laugel-Haushalter 3 , Adriane Joo 4 , Agnès Bloch-Zupan 3,5,6 , Ophir D. Klein 4,7 , and Laurent Viriot

INTRODUCTION

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a central regulator of tooth development. This cascade is typically initiated by the binding of a growth factor to a receptor tyrosine kinase (RTK), which triggers the phosphorylation of successive kinases and culminates in activation of effector kinases and the transcription of target genes (Sebolt-Leopold and Herrera, 2004). The MAPK signaling pathway has been intensively studied by cancer biologists because of its effects on regulation of cell proliferation and survival (Downward, 2003;Sebolt-Leopold and Herrera, 2004), but this pathway is also important throughout mouse embryogenesis (Massague, 2003). The pathway has been investigated in numerous embryonic processes, including development of the central nervous system and mesodermal derivatives (Campos et al., 2004), skeletal development (Ge et al., 2007), and tooth development (Thesleff and Mikkola, 2002;Klein et al., 2006;Tompkins, 2006;Xu et al., 2008;Laugel-Haushalter et al., 2014).

Tooth development is a well-documented example of ectodermal organ development. It is a tightly regulated process arising from the crosstalk between dental epithelium and its underlying mesenchyme (Tucker and Sharpe, 2004). The signaling networks responsible for properly building the dentition have been heavily investigated, and numerous members of the ERK-MAPK signaling pathway are known to play a role in tooth development. Early studies examined the fibroblast growth factors (FGFs) that are FGFR ligands, and thus trigger the ERK-MAPK phosphorylation cascade (Neubüser et al., 1997;Thesleff, 2003). Investigations then moved to further steps of the cascade in order to determine which components were involved in tooth development (Goodwin et al., 2013;Laugel-Haushalter et al., 2014). An exciting current challenge is to understand the complexity of feedback regulation in this signaling pathway that can be time-and/or tissue-specific. In the present study, we compare the phenotype of molar teeth in mice carrying mutations in Sprouty1, Sprouty2, Sprouty4, and Rsk2 genes, which are involved at various levels in the MAPK cascade.

The Sprouty (Spry) family of genes encodes general RTK inhibitors (Hacohen et al., 1998;Reich et al., 1999). After stimulation by growth factors, the Sprouty proteins are thought to translocate to the plasma membrane where their phosphorylation prevents the formation of an FGFR adaptor complex, thus having a negative effect on the activation of the rest of the cascade (Hanafusa et al., 2002). Spry1 is expressed in both the epithelium and the mesenchyme, with exception of a cluster of non-proliferating epithelial cells that serve as a signaling center called the enamel knot. Spry2 is expressed only in the epithelium adjacent to the dental mesenchyme, including the enamel knot, and Spry4 is expressed in the dental mesenchyme (Zhang et al., 2001;Klein et al., 2006). Whereas the morphogenesis of molar teeth in Spry1 -/-mice has not yet been examined, Spry2 -/-and Spry4 -/-mice are known to have abnormal dentition, which sometimes includes ST located immediately in front of the first lower molar (Klein et al., 2006). These supernumerary teeth, which occur at differing frequencies depending on the genetic background (Klein et al., 2006;Ohazama et al., 2009;Lagronova-Churava et al., 2013) are believed to derive from evolutionary vestigial tooth buds that normally undergo apoptosis in wild-type embryos (Peterková et al., 1998;Viriot et al., 2000;Viriot et al., 2002). Lagronova-Churava and colleagues (2013) showed that although all Spry2 -/-and Spry4 -/-embryos present a revitalization of tooth rudiments at ED13.5, only 2% of Spry4 -/-and 27% of Spry2 -/-specimens had a lower ST. However, the role of Spry1, Spry2

and Spry4 in the development of upper molars is not known, and the adult molar morphology has not been scrutinized in these mutants.

RSKs (90kDa ribosomal S6 kinases) are effector kinases belonging to the eponymous family of highly conserved serine/threonine kinases (Frödin and Gammeltoft, 1999;Romeo et al., 2012). Out of the four isoforms found in vertebrates, Rsk2 has been recently demonstrated to be involved in craniofacial development. Rsk2 -/Y mice display a deformation of the nasal bone, as well as diastemal ST which affect the mesial part of both upper and lower first molars (Laugel-Haushalter et al., 2014). Mutations in the RSK2 gene have been associated

with Coffin-Lowry syndrome (OMIM #303600), a condition characterized by mental and growth retardation along with craniofacial and other skeletal abnormalities (Temtamy et al., 1974;Temtamy et al., 1975;Hanauer and Young, 2002).

Phenotypic convergence across the ERK-MAPK signaling pathway remains poorly documented. By studying the dental phenotype resulting from mutations in genes located upstream (Sprouty) and downstream (Rsk2) of the ERK-MAPK cascade, we address the question of whether an ERK-MAPK signature phenotype exists. To answer this question, (1)

we have characterized the molar phenotype in several mutant populations in order to evaluate the distribution of the various changes affecting both upper and lower molar rows; (2) precisely quantified the occurrence of supernumerary teeth (ST) as well as their impact on the other teeth of the row, and finally; (3) addressed the evolutionary role of the ERK-MAPK cascade by comparing specific dental traits of mutants with dental traits of other extant or extinct rodents.

MATERIAL AND METHODS

Sprouty mutant mice

All the studied Sprouty mutant mice were generated in backgrounds resulting from crossing between several lineages. The three mutants and the wild type mice were generated by inbreeding at UCSF. The sample set was composed of homozygous mice as indicated: 25

Spry1 -/-, 50 Spry2 -/-, 50 Spry4 -/-, and 60 WT individuals (littermates of the various mutants).

For each specimen, left and right, upper and lower tooth rows were studied independently.

The age of the specimens ranged from 1 month to 2.5 months. Animal experimentation was carried out in compliance with the policies and procedures established by the UCSF Institutional Animal Care and Use Committee.

Rsk2 -/Y mice

The Rsk2 mutant mouse line was generated as previously described (Yang et al., 2004).

Since Rsk2 is located on the X chromosome, analyses were performed on Rsk2

Observation and imaging of dental rows

All the heads were prepared in order to remove all non-mineralized tissues to allow good observation and measurement of the dental rows. They were examined and photographed using a Leica stereomicroscope. The measurements were obtained by following the outline of each tooth from the photos of occlusal view of the row. Thus, the length, width, and area of the tooth were produced by Leica software. Some Sprouty mutant dental rows were imaged using X-ray-synchrotron Radiation Facility (ESRF, Grenoble, France), beamline ID 19 and BM5, with a monochromatical beam at energy of 25 keV. X-ray synchrotron microtomography has been demonstrated to bring high-quality results for accurate imaging of small teeth (Tafforeau et al. 2006). A cubic voxel of 7.46µm was used. Some Rsk2 -/Y mutant dental rows were imaged using the X-ray cone-beam computed microtomography with a Nanotom machine (GE) at an energy of 100keV with a cubic voxel of 3µm. All 3D renderings were performed using VGStudiomax software.

Statistics

Student's t-tests were used to verify the significance of differences in tooth size between the mutant mice but also between the mutant and the WT mice. A threshold value of 0.05 (pvalue) was used to assess the significance of the observed differences.

RESULTS

The mouse (Mus musculus) is a muroid rodent. Like all the members of this superfamily, mice have a simplified dentition composed only of incisors and molars separated by a long toothless gap called diastema. Three lower (M 1 M 2 M 3 ) and three upper (M 1 M 2 M 3 ) molar teeth are present in each mouth quadrant. The crowns of molar teeth bear a relatively stable number of cusps that are: 8 for M 1 , 6 for M 2 , 4 for M 3 , 7 for M 1 , 5 for M 2 , and 4 for M 3 .

Crowns of upper molars are made of rows of 3 cusps arranged in linguo-vestibular chevrons pointing mesially (except the third one, which is incomplete), whereas crowns of lower molars are made of rows of 2 cusps linked by linguo-vestibular and rather straight crests called lophs (Fig. 1, first column). The two mesial lophs of the M 1 are also linked together by a mesio-distal connection.

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Diversity of the dental phenotypes in Sprouty mutants

We examined the arrangement and shape of the postcanine dentition in four populations comprising 25 Spry1 -/-, 50 Spry2 -/-, and 50 Spry4 -/-mice. Area measurements showed that the occlusal surface of molars in Spry1 -/-and Spry4 -/-mice is larger than in the WT mice, whereas the molar occlusal surface in Spry2 -/-mice is smaller than in the WT mice (t test, p value<0.05, Fig. 2).

The molar teeth of Spry1 -/-and WT mice are globally similar in shape, and 51% of the Spry1 -/-dental rows display a WT-like phenotype. The defects are numbered as character# (c#) from the mesial to the distal part of the row. The main defects of the Spry1 -/-postcanine dentition are: (c8) the occurrence of a supplementary distal cusp on the M 1 (53%); (c9) the disconnection of the mesio-lingual cusp from the first chevron of the M 2 (40%); and (c12) the absence of the mesio-vestibular cusp of the M 1 (8%) (Fig. 1, Fig. 3B, Suppl. Fig. 1A). The postcanine dentition in Spry2 -/-mice had stronger differences compared to WT samples, and only 21% of Spry2 -/-dental rows still display a WT-like phenotype. The main changes of the Spry2 -/-postcanine dentition are: (c7) the connection between the two lingual cusps of the M 1 (38%); (c8) the occurrence of a supplementary distal cusp on the M 1 (46%); (c10) the connection between the two lingual cusps of the M 2 (36%); (c11) the occurrence of a lower ST (27%); and (c13-14) an abnormal shape, number, and/or interconnection of the mesial cusps of the M 1 (29%) (Fig. 1, Fig. 3C). Spry4 -/-molar tooth phenotype is the most variable among the 3 Sprouty mutants. These teeth ranged from a WT-like phenotype (24%) to relatively severe anomalies, especially in the M 1 . The main defects of the Spry4 -/-postcanine dentition are: (c1) the occurrence of an upper ST (17%); (c2-3-4) the presence of lingual cusp disconnection, straight mesial cusp and absence of the vestibular cusp affecting the first chevron of the M 1 (66% combined); (c5) the occurrence of a supplementary lingual cusp between the first and the second chevron (14%); (c6) the disconnection of the lingual cusp from the second chevron of the M 1 (20%); (c8) the occurrence of a supplementary distal cusp on the M 1 (36%); (c9) the disconnection of the mesio-lingual cusp from the first chevron of the M 2 (64%); (c11) the occurrence of a lower ST (3%); (c12-14) an abnormal number and/or interconnection of the mesial cusps of the M 1 (16%) (Fig. 1, Fig. 3D).

Except for three characters, the dentition of Spry1 -/-mice thus resembles that of WT mice, whereas Spry2 -/-and Spry4 -/-mice show extra cusps and crest disconnections, as well as severe reductions and defects in the mesial parts of the M 1 (Spry4 -/-only) and in the M 1 (rarely in Spry4 -/-, frequently in Spry2 -/-). Spry4 -/-mutants develop ST in both upper and lower jaws, whereas Spry2 -/-only display lower ST. Lower ST have been observed in Spry2 -/-and Spry4 -/-molar rows, whereas upper ST only occurred in Spry4 -/-molar rows. Spry1 -/-mutants thus never develop any ST, and only Spry4 -/-mutants display ST in both upper and lower tooth rows. These findings suggest a potential relationship between the occurrence of ST and abnormal arrangement of the mesial parts of the first molars.

Similarities and differences of Rsk2 dental phenotype as compared to Sprouty dentitions

We next examined a cohort of 45 Rsk2 -/Y mice. The postcanine occlusal surface area in Rsk2 -/Y mice is smaller than in WT mice (M 1 and M 1,2 being significantly smaller, p-value<0.05, Fig. 2). Although many Rsk2 -/Y specimens display relatively severe dental defects, 45% of the examined dental rows display a WT-like phenotype. The main defects of the Rsk2 -/Y postcanine dentition are: (c1) the occurrence of a upper ST (14%); (c2-3-4) the presence of many defects on the first chevron of the M 1 (71%); (c11) the occurrence of a lower ST (14%); (c12+14+16) an abnormal number and/or interconnection of the mesial cusps of the M 1 (19%); (c17) abnormal mesio-distal connections between the second and the third lophs of the M 2 (9%) (Fig. 1, Fig. 3E). The frequency of ST occurrence is lower than what has been previously reported (Laugel-Haushalter et al., 2014), and this may be explained by the examination of a larger sample or by shifts in the genetic background over time.

The comparison of the postcanine dental phenotypes between Rsk2 -/Y and the 3 Sprouty mutant mice shows that Rsk2 -/Y , Spry2 -/-and Spry4 -/-mice all develop ST, at varying frequencies. Spry2 -/-mice never have an upper ST, but they frequently have a lower ST (27%). Spry4 -/-mice frequently have an upper ST (17%), but they rarely have a lower ST (3%). Rsk2 -/Y mice develop both upper and lower ST relatively frequently (14%). In addition to developing lower and upper ST, Rsk2 -/Y and Spry4 -/-postcanine dentitions share many similar defects affecting the mesial parts of both M 1 and M 1 . The occurrence of defects on mesial parts of the M 1 is much more frequent than the occurrence of an upper ST, but the defects in the mesial parts of the M 1 and M 1 are also associated with a shortening in length of both teeth (Fig. 2). Finally, Rsk2 -/Y mutants do not develop the supplementary distal cusp of the M 1 common to all Sprouty mutants, nor do they share with Spry1 -/-and Spry4 -/-mutants the trend towards having larger teeth than the WT mice.

The presence of ST impacts both the shape and size of the other teeth in the row

The size and shape of ST range from small rounded monocuspid teeth to large complex multicuspid teeth that can comprise up to five cusps (Fig. 4). In complex upper ST, a large central cusp is always present surrounded by a variable number of cusps linked by an almost circular crest. The most complex upper ST tend to have a mesial chevron pointing mesially (especially in Spry4 -/-) whereas the lower ST mainly have a bicuspid shape (especially in Spry2 -/-). These features show that ST have a clear murine shape identity.

The larger the ST is, the more the mesial parts of the neighbouring M 1 and M 1 are impacted. The occurrence of a ST leads to compression and flattening of the mesial part of the following tooth. This is particularly visible on M 1 of Spry4 -/-, and on both M 1 and M 1 of Rsk2 - /Y mutants. As a consequence, the overall occlusal area of the molar row is smaller in mutants displaying a ST, with the exception of the Spry4 -/-upper row in which the area of the M 3 seems to increase in compensation for the decrease of M 1 area (Fig. 2).

In the most severely impacted phenotypes, the mesial contour of the M 1 becomes rounded, whereas it is triangular in WT mice (e.g. Fig. 3E without ST). In addition, the two mesial-most cusps of the first chevron become extremely reduced into a simple crest (53% Spry4 -/-and in Rsk2 -/Y ), and the mesio-vestibular cusp may completely disappear (20% Spry4 -/- and 4% in Rsk2 -/Y ). Interestingly, the mesial shrinkage of the M 1 is associated with a change in the tilt angle of both cusps and roots of the first chevron (Suppl Fig. 1B). In WT mice, the slope of the M 1 mesial root is in continuity with the tilt of 50° that makes the central cusp of the first chevron with the dental neck. In mutants having a ST, as well as in some mutants that do not display any ST, the tilt between the root axis and the tooth neck tends to be more vertical (about 70°), as does the slope of the first chevron central cusp (about 60°). The same type of defects can be seen when M 1 is preceded by a ST in the three mutants: the first loph of the M 1 is shortened and cusps appear as crushed by the presence of the ST. Yellow angle is 50° in WT mice, and 60° in mutant mice. The root orientation itself is also modified, as depicted in orange.

DISCUSSION

Similar yet distinct dental phenotypes in Sprouty and Rsk2 mutants

Morphological comparisons show that the occurrence of a supplementary cusp at the distal extremity of the M 1 can be considered as a phenotypic signature of the Sprouty mutant dentitions, and Rsk2 mutants never develop this supplementary cusp. These comparisons also highlight that Spry4 -/-and Rsk2 -/Y mutants share many phenotypic features, such as the occurrence of upper and lower ST as well as abnormal arrangements in the mesial parts of the first molars. Although these mesial abnormalities in these teeth appear to be caused by the occurrence of ST, a question remains concerning the higher frequency of modifications of the M 1 first chevron in Spry4 -/-and Rsk2 -/Y mutants (66-71%) when compared to the frequency of occurrence of ST (14-17%). It has already been shown in Eda Ta mice that a supplementary dental germ can develop mesially to the M 1 until advanced stages without necessarily giving rise to a mineralized tooth (Charles et al., 2009a). In these cases, the development of a supplementary dental germ may be sufficient to cause disorders in the development of the mesial part of the following tooth. We can thus infer that about 70% of the specimens develop a supplementary upper tooth germ that will impact on the phenotype of the M 1 .

Roles of Sprouty and Rsk2 genes within the ERK-MAPK cascade

Sprouty genes are thought to encode proteins that inhibit signaling through the FGFR pathway (Hacohen et al., 1998;Hanafusa et al., 2002;Kim and Bar-Sagi, 2004). Examination of the dental phenotypes in mutant mice led to the discovery of shared features. All three Sprouty mutants share the presence of a supernumerary distal cusp, arguing in favor of shared functions. But distinct characters were also observed in each mutant, pointing to some specific functions of these genes in tooth morphology. The phenotypic diversity in the Sprouty mutants may be ascribed to differences in spatial and temporal expression patterns of these genes. Our data complement previous findings concerning the generation of similar phenotypes with the loss of function of Spry2 and Spry4 genes. We have previously reported that the Spry4 -/-phenotype penetrance was lower than the one in Spry2 null mutants (Klein et al., 2006). Our results show that the differences between the two proportions are even larger than previously reported. This trend is reversed for the upper rows, for which we did not observe ST in Spry2 -/-mice, whereas these were found in 17% in Spry4 -/-mice. The proportion of ST in mutant dental rows was comparable to what Lagronova-Churava et al.

(2013) reported, but lower than our first publication on the topic (Klein et al. 2006). Such a change in penetrance could be explained by a shift in the genetic background over generations. Finally, neither of our previous reports (Klein et al., 2006;Lagronova-Churava et al., 2013) studied the effect of loss of function of Spry1. Our work shows that Spry1 -/-mice also display modifications in the shape of the M 1 and M 2 , as well as in overall size of the dental row.

RSK2 is an effector kinase that functions downstream of the MAPK cascade, whereas Sprouty genes modulate the cascade through their inhibitory effect on the pathway. The possible negative feedback regulation from Rsk2 on the adaptor protein SOS, which recruits Ras (Frödin and Gammeltoft, 1999), may lead to similarities in the phenotypes of Sprouty and Rsk2 mutants. Both Spry4 and Rsk2 are expressed during odontogenesis in the dental papilla (Zhang et al., 2001;Corson et al., 2003;Laugel-Haushalter et al., 2014), which may explain the high degree of similarity in the phenotypes associated with their loss of function. Our study thus provides an example of molecular and phenotypic convergence of various steps in the same signaling pathway.

Mutations in Sprouty genes and Rsk2 lead to more teeth and more cusps, which is the opposite trend of mutations in the Fgf genes which lead to phenotypes with fewer cusps (e.g.

Fgf3, Wang et al., 2007;Charles et al., 2009b). This contrast in phenotypic impacts of the mutations between Rsk2 and Fgf3 is concordant with the current knowledge of regulatory feedbacks acting on the whole pathway.

Molecular basis of ST development

In addition to Sprouty and Rsk2 null mice, other mutants have been described with ST mesially to the first molars. Some of these mutations are in genes encoding proteins that interact with the ERK-MAPK pathway. The balance between SHH and Wnt signals has been demonstrated to be crucial for the development of the diastemal rudimentary tooth germ. The link between the two pathways involves regulation of expression of Fgf pathway members by the Wnt signalling pathway. Mice mutant for Gas1, Shh, Wnt1, Wise, Lrp4, Apc, Ctnnb1, Eda

or Edar genes are characterized by a disruption of this important molecular equilibrium and display ST (respectively Ahn et al., 2010;Charles et al., 2009a;Kangas et al., 2004;Ohazama et al., 2009;Wang et al., 2009). Apart from the Apc and Ctnnb1 null mutants, the The Apc and Ctnnb1 null mutants display many more ST located in the vicinity of the incisor region (Wang et al., 2009). Authors hypothesized that incisor stem cells might be migrating, generating odontogenesis-competent foci. The difference between one ST being displayed in a mesial position (compared to the molar row), and multiple ST being displayed in a disordered manner is likely to be caused by the direct interaction with Ras and ERK.

Indeed, such interactions have been demonstrated in the context of multiple cancer models in the mouse (Luo et al., 2009;Moon et al., 2014).

Potential roles of Sprouty and Rsk2 gene in the evolution of rodent dentition

The most striking phenotypic trait of Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutants is the occurrence of ST located in front of the first molars at both the lower and the upper jaws. It has long been known that the rodent dentition has evolved towards a reduction in the number of teeth over the course of evolution in parallel with a specialization of the molar teeth [START_REF] Hershkovitz | Evolution of Neotropical Cricetine Rodents (Muridae): With Special Reference to the Phyllotine Group[END_REF]. Basal rodents had lower and upper premolars and it is only since the rise of muroid rodents that premolars have been completely lost in the lineage leading up to the murine rodents. It has however been demonstrated that rudimentary dental germs are maintained in both the upper and the lower diastema during mouse early dental development (Peterková et al., 1998;Viriot et al., 2000). Because these rudimentary germs are located in the vicinity of the first molar germs, they have been considered to be rudiments of premolar germs lost over evolution (Viriot et al., 2002). These rudimentary germs rapidly abort by apoptosis and do not become autonomous mineralized teeth. It has been demonstrated that they can contribute to the development of the M 1 by merging with the M 1 germ (Viriot et al., 2002). Although it has not yet been formally demonstrated, it is likely that the same mechanism occurs with the upper rudimentary germ immediately adjacent to the M 1 (Peterková et al., 1996) for participation in the development of the first chevron.

Here we deliver new insights into the role of the MAPK signaling pathway members beyond what has been previously reported (Klein et al., 2006;Lagronova-Churava et al., 2013;Laugel-Haushalter et al., 2014). The loss of function of Spry2, Spry4 or Rsk2 allows the continuation of the development of some rudimentary premolar germs until the formation of autonomous mineralized teeth. The segmentation of the postcanine dental row is thus impacted because it comprises four teeth instead of three and the mesial parts of the first molars are accordingly reduced (Fig. 3). We can thus assign the supplementary teeth located immediately in front of the first molars to deciduous fourth premolar teeth (dP 4 and dP 4 ) that will not be replaced. Teeth located at the fourth premolar position are known in the earliest muroid rodents (Li et al., 2012) as well as in some extant dipodoids such as Sicista and Zapus (Pucek et al., 1982;Rodrigues et al., 2011), the sister group of muroids. Fourth premolars are present in the squirrel-and in the guinea pig-related clades, which arose earlier in the history of rodents than the mouse-related clade (Fabre et al., 2012). The loss of function of Spry2, Spry4 or Rsk2 involves developmental mechanisms that revitalize autonomous premolar teeth and that simplify accordingly the mesial parts of the first molars. The dental phenotype in Spry2 -/-, Spry4 -/-, and Rsk2 -/Y mutants thus represents a reversal in the evolutionary trends of the dental phenotype through the transition from pre-muroid to muroid rodents (Fig. 5A), which is documented within the fossil record (Tong, 1992;Li et al., 2012;) to have probably happened between 50 and 45 million years ago (Ma).

As discussed above, many other mouse mutants display ST mesially to first molars, but both the phenotypical and the developmental aspects of the postcanine row segmentation has solely been scrutinized in mutants of the EDA pathway (Charles et al., 2009a). From these studies, it is clear that dental phenotypes in Eda Ta (Tabby) and Edar dl-J (Downless) mice radically differ from those of Spry2 -/-, Spry4 -/-, and Rsk2 -/Y mice. Tabby and Downless mice have postcanine teeth with simplified occlusal patterns characterized by the absence of many cusps, as well as by abnormal arrangements of the lophs and the chevrons respectively in the lower and upper molars (Charles et al., 2009a). In contrast, Spry2, Spry4, or Rsk2 losses of function provoke rearrangements of the M 1 and M 1 mesial parts, but minimally impact the rest of the dental rows. From these arguments, we propose that Spry2, Spry4 or Rsk2 are candidate genes for a major role in the evolutionary trend towards reduction of the dental formula in muroid rodents 45 Ma.

Interestingly, a character that stands out from the mutant mice is the occurrence of a supplementary distal cusp, named the posterocone, on the M 1 of the three Sprouty mutants (arrowhead in Fig. 5B). The posterocone is rarely present in extant species of murine rodents, whereas this cusp or the equivalent crest located at the same location, the posteroloph, were almost always present in the basal fossil murine genera such as Potwarmus, Antemus, and Progonomys. However, neither the modern mouse (Mus musculus) nor its oldest ancestor (Mus auctor) display an individualized posterocone (Fig. 5B). In the lineage leading to the modern mouse, the posterocone has been lost during the transition from Progonomys debruijni to Mus auctor, and this transition is documented by the fossil record in the Siwaliks of Pakistan to have occurred between 9.2 and 6.5 Ma (dating from Kimura et al., 2013). Apart from the presence of a posterocone, Spry1 -/-, Spry2 -/-, and Spry4 -/-mice share other dental features with these basal murine genera such as the trend of the lingual cusps of the M 1 to be least connected to the central cusps. All of these similarities indicate that Spry1, Spry2, or Spry4 could have played a major role during the transition from Progonomys to Mus, which constitutes a major transition in the history of murine rodents.

CONCLUDING REMARKS

Spry1, Spry2, Spry4, and Rsk2, which encode components of the ERK-MAPK signalling pathway are identified here as putative major actors in the evolution of the dentition chevrons. Charles et al (2009) reported that Fgf3 -/-mice have M 1 with lingual cusps that are completely disconnected from the central cusps. Here we report that Sprouty mice, especially Spry2 -/-and Spry4 -/-mice, often have M 1 with disconnected lingual cusps. As Sprouty and Fgf genes are known to play antagonist roles during tooth morphogenesis, it is interesting to note that the loss of function of the two antagonist results in an equivalent impact on the lingual cusps of the M 1 .

A.3 MODULATIONS OF TOOTH DEVELOPMENT IN

K14-SPRY4 MICE

Work on direct knock-out mutants for FGF signaling pathway has provided strong evidence of their importance during tooth development, but so has the work on the FGFR inhibitors. Sprouty genes have been established for the past 10 years as major modulators of tooth development. Li and colleagues (2014) recently reviewed the implication of the pathway during odontogenesis. FGFs and their receptors are necessary to initiate the right type of tooth at the right time and place (Tucker and Sharpe, 1999). They regulate the invagination of the dental epithelium, and are needed for the dental papilla to be condensed (Kettunen et al., 2000;Wang et al., 2004). Specific expression patterns in the pEK and sEKs of some Fgf genes are indispensable for correct tooth shape and cusp formation (Tucker and Sharpe, 2004), as well as to set proper ameloblast and odontoblast differentiation (Ruch et al., 1995;Wang et al., 2004). In adult mice, they play a crucial role in the maintenance of the incisor stem cell niche (Klein et al., 2008;Chang et al., 2013).

In the following article, I aim at addressing the global role of FGF signaling pathway in the dental epithelium. When tooth development is initiated, the competence to form a tooth germ lies in the mesenchyme, which induces the thickening of dental epithelium. After that first step, the odontogenic competence is shifted back to the epithelium (Maas and Bei, 1997).

I take advantage of the existence of a transgenic mouse line to polish our understanding of the role of Fgf genes in shaping dental epithelium. Mice carrying the Tg(KRT14-Spry4)#Krum transgene (http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=alleleDetail&id=MGI:5466567)

were designed to overexpress the Spry4 gene in the oral epithelium leading thus to a downregulation of FGF signaling pathway in this region. By linking erupted tooth morphology and histology of the developing tooth germ, I review the importance of Fgf genes in correctly shaping dental epithelium.

INTRODUCTION

FGF signaling pathway is a major actor of tooth development. For the past decade, the role of Fgf genes in setting the very specific developmental sequence of tooth development has been investigated (Li et al., 2014). Given the number of available mouse mutants for this specific signaling pathway, research has also been able to hypothesize on their evolutionary implications in setting the complex mouse molar cusp pattern (Kavanagh et al., 2007;Charles et al., 2009;Harjunmaa et al., 2014). Most of our knowledge on FGF signaling pathway and tooth evo-devo has been provided by loss of function mice that were simple or double mutants for Fgf genes or for Sprouty (Spry) genes. The latter are very important when looking at the tight time-and tissue-specific regulation of tooth development.

Indeed, Spry genes were first identified as inhibitors of FGF receptors (FGFR) in Drosophila (Hacohen et al., 1998), and their inhibitory effects were extended to the mouse (Mason et al., 2006). Four Spry orthologs are found in Mus musculus (de Maximy et al., 1999). Spry1, Spry2 and Spry4 are expressed during tooth development (Klein et al., 2006).

Their expression is induced by the stimulation of growth factors, and they inhibit in FGFRmediated activation of the ERK-MAPK signaling pathway (Hanafusa et al., 2002). In terms of their implication during tooth development, Spry2 and 4 genes are known to prevent the development of diastemal buds and to restrict the ameloblast differentiation in the lingual side of the incisor (Klein et al., 2006;Klein et al., 2008;Lagronova-Churava et al., 2013, Marangoni, Charles et al. in prep.). In addition, along with Spry1, all three are important for setting the right murine cusp pattern (ibidem).

Here we focus on a transgenic mouse line carrying a transgene that has newly been implemented in the JAX allele databasethe Tg(KRT14-Spry4)#Krum transgenein which the expression of Spry4 mouse cDNA is driven under the control of the keratin-14 human promoter (http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=alleleDetail&id=MGI:5466567).

This line will be referred to as K14-Spry4 line in the following article. It was designed to mimic a global down-regulation of epithelial FGF signaling pathway. Although in the course of tooth development Spry4 gene is normally expressed in the dental mesenchyme (Klein et al., 2006), the transgene is expressed in the whole oral epithelium which includes dental epithelium. The erupted molar morphology in the transgenic specimens displays numerous signs of enamel layer irregularities. Histologic analyses of the developing tooth germs highlights a developmental delay that affects the formation of the tooth primary signaling center: the primary enamel knot (pEK). Preliminary gene expression pattern study reveals a shrinking of the lower tooth germ pEK that make Shh expression hardly detectable. Finally, the defects are not only seen in the developing tooth region, but a rare fusion of the upper and lower jaws also occurs in that genetic background.

MATERIAL AND METHODS

Transgenic mice

K14-Spry4 mice were designed and produced in the Stowers Institute for Medical

Research, Kansas City (USA). The mouse Spry4 cDNA (904b) was inserted downstream of a human KRT14 promoter (2.2kb) preliminary amplified from a BAC clone (RP11-434D2) using the following primers: 5'-AAGATCTAGG-TGCGTGGGGTTGGGATG-3' and 5'-GAAGCTTGAGCGAGCAG-TTGGCTGAGTG-3'. The gene was then subcloned into the pCMS-EGFP vector (Clontech) replacing the CMV promoter. After linearization and gelpurification, the 3772 bp-long pKRT14-Spry4 construct was injected into a CBA/J x C57BL/10J one-cell zygote.

The line was maintained since 2008 by breeding transgenic males with CBA/J x C57BL/10J females. The transgenic offspring displays a less furnished fur and abnormal and/or shorter tail. Embryos for the present study were generated by breeding transgenic males with C57BL/6J females. In those embryos, the transgene displayed a mosaic expression pattern detectable from the embryonic day 14 (E14). Mice were housed at the UCSF.

Characterization of erupted dentition

A sample of 25 transgenic adults, and 15 WT littermates was generated in the LARC (UCSF, USA) by breeding C57BL/6J females with K14-Spry4 males. At five weeks, animals were sacrificed by CO 2 asphyxia and cervical dislocation. Their heads were placed in a

Dermestes maculatus colony to clean the skulls, which were then imaged using X-ray microtomography (cubic voxel size of 3µm). Crown surface was measured on the occlusaloriented pictures of the scanned volumes by drawing the outline of the molars with Canvas.

Histological analyses

Noon of the day on which the vaginal plug was detected was considered as the E0.5.

Whole litters (125 embryos in total) were collected every 12 hours from E13 to E18.5.

Littermates were genotyped using the following primers: 5'-CTGGGCAGGTAAGTATCAAGG-3' and 5'-TGGTCAATGGGTAAGATGGTG-3'. PCR was performed according to: 2 min at 94°C, 25 cycles of 30 s at 94°C -30 s at 54.8°C -1 min at 72°, and 5 min at 72°C. K14-Spry4 transgenic embryos display a 354bp fragment specific to the construct. Embryos were collected in 1X PBS and fixed overnight in 4% PFA.

After dehydration in graded ethanol, embryos were processed in paraffin and serially sectioned (7 µm thick) using a Leica Autocut 2055 microtome. Masson's trichrome was used to conduct the histological analyses of the sectioned dental germs (hemalum, 8 min; fushine, 2 min; aniline blue,1 min). Pictures were taken with an Olympus microscope equipped with a CCD camera and Cell F™.

Gene expression patterning

DIG-labeled probes for Sonic hedgehog gene (Shh) were generated from a plasmid (Molecular Zoology team, IGFL). Plasmid linearization was carried out with HindIII restriction enzymes (New England Biolabs) at a concentration of 5 units for 1µg DNA. The linearized DNA fragments were transcribed in vitro using T3 RNA polymerase (Invitrogen).

and labeled with a digoxigenin labeling mix (Roche). In situ hybridizations were performed on E14.5, and E15.5 beforehand sectioned WT and transgenic embryos according to standard protocols. Samples were deparaffinized with xylene, rehydrated in graded ethanol and treated with 10 µg/mL proteinase K (Roche) before being hybridized with probe overnight at 70°C.

Staining was repeated on serial sections. Pictures were taken with a Leica microscope equipped with a CCD camera and Cell F™.

RESULTS

Fgf gene down-regulation leads to strong enamel irregularities, mild cusp defects, and smaller teeth

We investigated the arrangement and shape of the molar rows in a population of 25 transgenic mice and compared it to what 15 of their WT littermates display. The mice were collected at 5-week-old to study fully erupted but still only slightly worn molars.

Analysis of molar tooth phenotype shows that the transgenic molar rows display a great diversity of cusp defects, but also defects in the tooth neck. The latter marks the anatomic separation between the crown covered with enamel and the roots covered with cementum.

The most prevalent defects in the upper molar rows are (1) the presence of indentations of the tooth neck (62%), which are especially visible on the mesial side of the M 1 (35%), and (2) the presence of pits and irregularities on the crown enamel surface (50%) (Fig. 1A,B). On the lower molar rows, the presence of indentations of the tooth neck is visible in the entire transgenic population, the vestibular side being often the most severely affected (vestibular and lingual views, Fig. 1B,C). Moreover, in 50% of the transgenic cohort, the distal-most part of both M 1 and M 2 is reduced or absent, while the mesio-vestibular cusp of the M 1 is reduced in 30% of the transgenic specimens (occlusal views, Fig. 1B,C).

A variable series of cusp defect affects both M 1 and M 1 transgenic molars to a lesser extent (Fig. 2). Indeed, M 1 occasionally display (1) an ectopic connection linking the lingual cusps of both 1 st and 2 nd chevrons (4%), (2) a disconnection of the lingual-most cusp of the 1 st chevron (2%), (3) an ectopic crest linking the vestibular cusps of both 1 st and 2 nd chevrons (2%), and (4) a splitted and individualized 1 st chevron central cusp (2%) (Fig. 2B). M 1 occasionally display ectopic connection of the distal-most part of the tooth (4%), a bigger mesio-ligual cusp (4%), a splitted mesio-lingual cusp (2%), and cingular cusps (2%) (Fig. 2D). Tooth surface measurements confirmed that both the upper and lower transgenic molars are smaller than the WT molars (Fig. 3). The range of tooth surface displayed in the transgenic cohort is wider than the WT one, once again illustrating the variability of the phenotypes obtained in the transgenic mice.

FGFs are essential to ensure the pEK formation and the right dental epithelium shape

In the tooth developmental sequence, the formation of a group of non-dividing cells called the primary enamel knot (pEK) at the cap stage is necessary to drive the next steps [START_REF] Jernvall | Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene[END_REF]. The pEK is a cluster of cells specifically expressing some genes among which is Fgf4. The secreted FGF4 protein will direct further invagination of the epithelium, thus playing a role in the crown patterning (Jernvall et al., 1998;Vaahtokari et al., 1996). We firstly focused on the cap stage which starts at about E14.0 in WT mice, and on the immediate surrounding stages (Fig. 4). At the bud stage, the transgenic upper and lower first molar buds look similar to the WT ones (E13.0 and E13.5 in Fig. 5). At E14.0, observations reveal a developmental delay with the absence of invagination of the transgenic cervical loops (black arrowheads in the WT). The delay is confirmed at E14.5 with the absence of a fully formed pEK in about 50% the transgenic mice. Sections also reveal that the developmental delay appears caught up by E15.5, the transgenic molar germs remaining only smaller. Focusing on late bell stage (E16.5 onwards), we tried to find an histological explanation to the irregular surface of the crown enamel and to the indentations. Overall, the transgenic dental germs, and especially the lower ones, display a rather irregular shape of their inner enamel epithelium in the sectioned embryos, with abnormal cell morphology being mostly visible on the cervical loops (dotted line, Fig. 5).

Our analysis of the late bell tooth germ histology was confronted to the relative difficulty of harvesting transgenic embryos. Given the crossbreeding realized, we expected to get an approximate transgenic vs. WT embryos ratio of 0.5 per litter. However, we noticed during our embryo collection a decrease in the transgenic embryo proportion (Mann-Whitney Wilcoxon sum rank test, p-value<0.05). This decrease starts to occur at E16.5, while the size of the litters stays in the range of what is described in WT laboratory mice (Tab.1).

FGF signaling pathway down-regulation impacts the expression Shh gene

As pEK is a major signaling center that sends morphogenesis signals to perfect tooth formation, we focused on this structure to tackle the question of possible deregulation of the molecular networks involved. Some genes are already known to be expressed in these rounded structures. The list includes Fgf3, 4 and 9, some Bmps, Edar, a couple of Wnts and Shh (Jernvall et al., 1998;Kettunen and Thesleff, 1998;Yamashiro et al., 2003). Our attention was drawn to Shh because it is a member of the eponymous SHH pathway, because its expression is restricted to EKs only, and because in vitro addition of exogenous SHH resulted in abnormal epithelial invagination (Hardcastle et al., 1998). Investigating the expression pattern of the candidate gene Shh, we observed at E14.5 and E15.5 that Shh expression was located in a smaller area in the lower dental germs, making it hardly detectable in the lower tooth germs (Fig. 6).

Tab. 1: Evolution of both the proportion of transgenic embryos in the collected litters and the mean number of embryos per stage. The proportion of transgenic embryos drops starting E16.5

(Mann-Whitney Wilcoxon sum rank test, p-value<0.05), while the number of embryos collected in every litter remains consistent (no statistically significant difference detected, p-value=0.1).

FGF signaling pathway down-regulation can lead to a defect in maintaining the upper and lower jaws separated

Along with this, a rare but severe defect affects 8% of the transgenic cohort. It consists in the failure to keep the upper and lower jaws properly separated (Fig. 8). This failure results in a fusion of the upper and lower jaws. The fusion ranges from a simple juxtaposition and sealing of the upper and lower dental epithelium in the region where teeth develop (Fig. 7A), to a severe sealing associated with cell proliferation between the two initial layers (Fig. 7B).

DISCUSSION

Fgf gene importance in setting tooth shape and proportions is confirmed

The global down-regulation of FGF signaling pathway in mice carrying the Tg(KRT14-Spry4)#Krum transgene causes a reduction in the tooth surface. This result is highly similar to what is observed in other Fgf mutant mice (namely in Fgf3 -/-mice, Charles et al., 2009).

However, the transgenic mice also display strong irregularities of the enamel layer in both upper and lower molars, as well as a reduction of the distal-most parts of the M 1 and M 2 ,.

Ameloblasts are responsible for enamel secretion, and they are specifically polarized cells (Zeichner-David et al., 1995). Abnormalities observed at the histological scale in the morphology of the inner enamel epithelium and the cervical loop cells could result in an abnormal enamel secretion and mineralization. Because the mineralization defects are the most prevalent defects overall, the role of Fgf genes in that process can be addressed in in vivo models, which will help specifying the conclusions obtained from cultured tooth germs (Tsuboi et al., 2003).

The reduction or absence of the distal-most parts of the M 1 and M 2 raises the interesting question of the sequence of cusp addition. The molar developmental sequence progresses from the mesial to the distal part of the presumptive row (Viriot et al., 2002), but the sequence of cusp formation has not been characterized yet. In that context, it is difficult to link the delayed formation of the pEK to this reduction, which both occur in 50% of the transgenic cohort (embryo or adults).

The K14-Spry4 line displays a great phenotypic variability

The display of highly variable cusp defects in both M 1 and M 1 , as well as much more frequent defects, is interesting. This might be imputable to transgene expression variations, resulting in gene dosage changes. When a gene is translocated or a transgene inserted into a locus, it is not rare to witness cis inactivation when the final locus is in or next heterochromatin. This effect is called variegation, and was described first in Drosophila melanogaster (Muller, 1930). It is also well-known in plants, in which it is responsible for the differential color sometimes displayed in the leaves (Chen et al., 2000). This same phenomenon could be the cause of the mosaicism in the transgene expression observed, resulting in highly variable phenotype on top of the signature phenotypes of the transgenic line.

More generally, given the increase in transgenic embryo lethality, the adult morphologies we observe are likely to be only a subset of the tooth defects that could be developed. We should not forget that the transgene ectopically expresses a general RTK inhibitor in a developmental context where teeth are not the only organ forming. Tooth development is not the only process affected by the transgene expression, as is evidenced by the presence of an abnormal hair phenotype on these mice. Spry genes are known to antagonize Endothelial Growth Factor (EGF) signaling, and given the role of Vascular EGFs (VEGFs) in patterning the cardiovascular system, we can hypothesize that the embryonic lethality would be associated with defects in this developmental process (Ferrara, 2005).

Fgf genes are major actor of the tooth development regulation

Preliminary results on the expression pattern of Fgf gene interactors in the pEK reveal that Shh gene expression is hardly detectable in lower transgenic germs, the size of pEK being apparently smaller. According to the established model of signaling in the molar pEK, epithelial FGFs stimulate SHH signaling pathway thanks to a dialog with mesenchymal FGFs (Klein 2006). The absence of modulation of Shh gene expression in the upper transgenic molar germ suggests the existence of a compensatory regulation specific to the upper molar germ.

The upper and lower jaws, and their respective derivatives, do not share the same embryonic origin (Tucker and Sharpe 2004). Neither are they formed under the same molecular control (Tucker andSharpe 1999, Cobourne andSharpe 2003). This so-called 'odontogenic homeobox code' could explain the differences in the phenotypes observed between upper and lower transgenic molars. Given that the majority of the studies investigating tooth development molecular regulation have been conducted in KO mutants, we expect from future analyses in this transgenic mouse line to perfect our understanding of those upper vs. lower tooth development differences.

FGF signaling pathway down-regulation leads to congenital defects

The epithelial fusion is certainly rare in the transgenic mouse line studied here, but it has a promising implication in better understanding the development of a craniofacial syndrome. Equivalent cases of partial closing of the oral cavity (syngnathia) have been reported in fewer than sixty cases in human patients (e.g. Dawson et al., 1997;Fallahi et al., 2010). A recent study elegantly demonstrated that Fgf8 was implicated in the setting of the fusion in a dosage-dependent manner (Inman et al., 2013). Fgf8 is expressed in the early tooth development, as well as in the 1 st pharyngeal arch (Maas and Bei, 1997). The defects we see in the tooth developmental sequence occur from E14.0 on, but we cannot rule out that the development of the syngnathia in our transgenic mice actually starts earlier, which might indicate that Fgf8 down-regulation is involved here.

Syngnathia also occurs in other mutant mice, especially in the Jag2 -/-mouse embryos that display a similar fusion (Mitsiadis et al., 2005). JAG2 is one of NOTCH ligand. NOTCH signaling pathway is involved in what is called lateral inhibition which is one of the mechanisms controlling cell differentiation (Kopan and Ilagan, 2009). Jag2 null mice die at birth and display abnormal morphology of dental germs as well as fusion between the palatal and mandibular shelves. The penetrance of the fusion in Jag2 null mutants seems to be more important and the histology of the defect looks similar in every reported case. However, it has not been used yet to address the human pathology, and our results point towards the existence of convergent molecular mechanisms leading to this defect.

The upper and lower jaws do not originate in the same embryonic annex. The upper jaw is formed with cells from the fronto-nasal process and from the first branchial arch, whereas the lower jaw is formed with 1 st branchial arch cells (Cobourne and Sharpe, 2003). The observed sealing and fusion of the epithelia is due to a defect in maintaining the jaws separated. A change in the cellular junction patterning is likely to cause the sealing of the two epithelial layers. FGFs are known to interact with the actin cytoskeleton (Chihara et al., 2003;Wheelock and Johnson, 2003). This interaction is mediated by the regulation of the Rac kinase activity. The more phosphorylated the actin is, the less stable it is, preventing it from polymerizing. When FGF signaling is reduced, so is Rac activity, leading to the stabilization of actin. It might result in the polymerization of actin filaments that is likely to increase the cellular junctions interacting with actin cytoskeleton.

CONCLUDING REMARKS

In summary, the K14-Spry4 mouse line has put the focus on the importance of Fgf genes to form a smooth and regular enamel layer. Epithelial Fgf genes were found essential to develop the pEK in the correct time frame. This project highlighted how specific the regulation of odontogenesis, especially when unravelling the molecular networks affected by the transgene expression. The modifications seen at embryonic stages will generate modifications of the mineralized molar row, but given the embryonic lethality, and the transgene variegation effects, the exact causality of the defects on the mineralized teeth are not completely clear. The implication of this family of genes in syngnathia reinforces the beneficial association often existing between developmental biology and clinical work.

A.4 Conclusions

Evo-devo stands for evolutionary developmental biology. Dental evo-devo takes advantage of the well-documented tooth germ developmental sequences, and uses the molecular assumptions built by developmental biologists to test evolutionary hypotheses. The use of molar shape and postcanine tooth number to address evolutionary transitions is wellaccepted in the community (Harjunmaa et al., 2014).

In this part of my PhD work, I aimed at further expanding our knowledge of the ERK-MAPK signaling pathway involvement in tooth evolution and development. After confirming the role of Rsk2 in craniofacial development, I focused on its postcanine dentition, and compared it with the one of Spry1 -/-, Spry2 -/-, and Spry4 -/-mutant mice. The high interest of this comparison relies on the fact that they are both members of the pathway, but that they act at various steps.

Similarities are observed between the dentitions, with several features being commonly displayed in Sprouty and Rsk2 mutant mice. Notably, the occurrence of a ST in the equivalent of a 4 th premolar position in Spry2 -/-, Spry4 -/-and Rsk2 -/Y mice, along with the Sprouty-specific display of a posteroconewhich is a dental feature that disappeared from the Mus lineage 9.2 to 6.5 Ma agoled us to hypothesize that the premolar loss might have been triggered by Rsk2 gene dosage augmentation, while the posterocone loss might be imputable to Sprouty gene dosage augmentation. Overall, these results emphasize that vestigial tooth buds located in the diastemal region can be reactivated to form ST by genetic modifications, thus contributing to our understanding of the dental formula control in the order Rodentia.

Stepping back to the actors that can activate the ERK-MAPK cascade, my work on the K14-Spry4 mice confirmed the role of epithelial Fgf genes in the enamel mineralization process. If my conclusions regarding this part are mainly developmental, they also enlighten the role that complementary models of genetically engineered mice have to play in understanding tooth evo-devo.

If mouse molars are traditionally used in the field, it does not mean that they are the only type of tooth that will help us catch a glimpse at the complex evolutionary history of teeth. Indeed, mouse incisors have sometimes also been used to address the question of tooth number control, notably by addressing the Sprouty-mediated modulation of RTK pathway (see annex 3, [START_REF] Charles | Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling[END_REF]. More globally, the work on rodent hypsodont -highcrowned teethand hypselodont teethever-growing teethhas also helped scientists raise the questions of the adaptation to differential diets and of prolonged growth [START_REF] Renvoisé | An Evo-Devo perspective on ever-growing teeth in mammals and dental stem cell maintenance[END_REF].

PART B RODENT INCISORS: EVOLUTION, DIVERSITY, & AGEING

dult stem cells are necessary for the organism to ensure tissue homeostasis. One original feature of rodents (and lagomorphs) is that their incisors are continuously growing throughout the animal's lifespan thanks to the presence of stem cell niches located in their growing zone [START_REF] Meng | Gomphos elkema (Glires, Mammalia) from the Erlian Basin: Evidence for the Early Tertiary Bumbanian Land Mammal Age in Nei-Mongol, China[END_REF]. In return, the protruding tips of the incisors continuously wear down when the animal gnaws. Because of their easy access, mouse incisors constitute a pivotal model to address the maintenance of such a dynamic balance.

The The epithelial stem cell niches are asymmetric (Harada et al., 2002a;Harada et al., 2002b;Klein et al., 2008). The labial cervical loop (laCL) is bigger, and contains a consequent population of stem cells located in the stellate reticulum (SR, between the inner and outer epithelium layers). The lingual cervical loop (liCL) however only contains a few stem cells [START_REF] Thesleff | Regulation of epithelial stem cells in tooth regeneration[END_REF][START_REF] Juuri | Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors[END_REF]. The loops and the inner and outer dental epithelium project in 3D and define a sheath in which the incisor will grow [START_REF] Harada | New perspectives on tooth development and the dental stem cell niche[END_REF]. Label-retaining and lineage tracing experiments have demonstrated that the cervical loop acts as a reservoir for epithelial stem cells which will drive the continuous growth of the incisor (Seidel et al., 2010;[START_REF] Juuri | Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors[END_REF]. This asymmetry results in the be found (Klein et al., 2008;Seidel et al., 2010). Dentin is secreted by odontoblasts, which dynamic turnover is supported by mesenchymal stem cells (MSCs). In a recent study, the neurovascular bundlelocated in the pulp cavity of the incisorwas acknowledged as the likely niche of MSCs (Zhao et al., 2014). Finally, the lingual side of mouse incisors is covered with a thin layer of cementum and is thus considered as the root analog whereas the vestibular side is considered as the crown analog [START_REF] Tummers | Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species[END_REF]Juuri et al., 2013). The presence of enamel only on vestibular sides and differences in both curvature and size generate a beveled wear pattern at the occlusal contact between upper and lower incisors.

The acquisition of features allowing this bevel-cut shape attests to complex coordinated changes.

Studies of the genetic networks report that members of the TGFß, BMP, Wnt, FGF, Hedgehog and Eda pathways as well as their modulators (Follistatin, Ectodin, Spry) are involved in incisor development [START_REF] Begue-Kirn | Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro[END_REF][START_REF] Tucker | Transformation of tooth type induced by inhibition of BMP signaling[END_REF][START_REF] Tummers | Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species[END_REF]Wang et al., 2004;Seidel et al., 2010;Suomalainen and Thesleff, 2010;Chang et al., 2013;Li et al., 2014). Stem cell maintenance has been a crucial topic in the field, and the role of Fgf signaling pathwayand especially FGF10is constantly specified (Chang et al., 2013;[START_REF] Renvoisé | An Evo-Devo perspective on ever-growing teeth in mammals and dental stem cell maintenance[END_REF].

Despite the interest of the mouse incisor model in terms of regeneration potential, which constitutes an active field in dental research, incisors are usually considered as very simple teeth, and the problem of the diversity in shape, size and color of incisors at the scale of the order Rodentia has never been properly tackled. In this part of my thesis, I intend to focus on various aspects of the diversity of rodent incisors in relation to their evolutionary history.

Beyond a relatively simple-looking circular shape, incisor displays a high degree of specialization, stability and variability at various scales, from age-related individual variations to intra-order variations.

B.1 INTRA-AND INTER-SPECIFIC VARIATION OF THE

RODENT INCISORS

Glires is a taxonomic grouping encompassing the living Rodentia and Lagomorpha as well as all their extinct relatives. The acquisition of continuously growing incisors is a synapomorphy of Glires, including the extinct orders Mimotonida and Mixodontia [START_REF] Mckenna | Classification of mammals above the species level[END_REF]. Rodents and the mixodonts together form the Mirorder Simplicidentata, socalled because simplicidentates only possess two upper and two lower enlarged continuously growing incisors, which have been assigned to the second decidual incisors (dI 2 and dI 2 ) [START_REF] Luckett | Superordinal and intraordinal affinities of rodents: developmental evidence from the dentition and placentation[END_REF]. Lagomorphs and the mimotonids together form the Mirorder Duplicidentata, so-called because duplicidentates have four upper and two lower continuously growing incisors. The upper dentition of duplicidentates includes two tiny continuously growing third permanent incisors (I 3 ), which are located immediately behind the dI 2 [START_REF] Ooë | Développement embryonnaire des incisives chez le lapin (Oryctolagus cuniculus L.). Interprétation de la formule dentaire[END_REF][START_REF] Luckett | Superordinal and intraordinal affinities of rodents: developmental evidence from the dentition and placentation[END_REF]. Within Glires, the position of Gomphos remains problematic because this genus has four upper and four lower continuously growing incisors. After being considered as a stem rodent, it has being reassigned to stem lagomorphs [START_REF] Kraatz | Gomphos ellae, a new mimotonid from the Middle Eocene of Mongolia and its implications for the origin of Lagomorpha[END_REF].

Because they protrude out their oral cavity, incisors are the most obviously visible teeth of both rodents and lagomorphs. Their vestibular surface, covered with enamel is facing the observer. Upper incisors are especially visible because they partially hide the lower ones.

In lagomorphs, upper incisors are very alike across the phylogeny with a clear vestibular groove being displayed on the vestibular surface. In rodents however, it is not rare to observe a great variability.

B.1.1 Morphometrics

Incisors are asymmetric teeth displaying a beveled wear pattern at the occlusal contact between upper and lower incisors. The absence of enamel on the lingual side of both upper and lower incisors also confers flexibility on the rodents when they gnaw. The risk of breakage is minimized, but not completely avoided, what remains an issue for the animal.

Also on the down-side of the system are the opposite cases of malocclusion. Indeed, when incisors are not properly occluding, they grow out without being worn down, and eventually inward, running through the rodent jaws. Laboratory mice are for example exposed to this issue because of the hardness of their food or trauma from fighting or improper chewing of the cages. There might also be a genetic predisposition to malocclusion, but this has only been demonstrated in rabbits [START_REF] Fox | Mandibular Prognathism in the Rabbit: Genetic studies Genetic studies[END_REF][START_REF] Petznek | Reduced body growth and excessive incisor length in insertional mutants mapping to mouse Chromosome 13[END_REF].

On their proximal end, rodent lower incisors growth region is located distally to the molar row (Harada et al., 1999). As a consequence, their curvature radius is higher than in the upper incisors, which growth region is located more mesially [START_REF] Lin | Morphological adaptations of digging apparatus and population genetics of the subterranean rodent Myospalax cansus[END_REF]. Interestingly, there is an inter-specific variability in the localization of the growth region in the upper jaws:

B.1.2 Incisor ornamentation

The five suborders Anomaluromorpha, Castorimorpha, Hystricomorpha, Myomorpha,

and Sciuromorpha together make up the order Rodentia. The myomorph "mouse-like" rodents represent about 70% of the living rodent species and almost a quarter of all living mammalian species [START_REF] Wilson | Mammal species of the world: a taxonomic and geographic reference[END_REF]. The presence of vestibular grooves on incisors is a widely spread character within the order Rodentia. The distribution of this character within the rodent phylogeny clearly shows that it has independently appeared several times (FIG. B.4A).

Vestibular grooves almost exclusively occur on upper incisors, and the unbalanced distribution constitutes an important issue for understanding the morphological function of this character.

Within the suborder Sciuromorpha, only two out of the 61 genera, namely Aeretes and Rheithrosciurus, have been documented as displaying grooves [START_REF] Nowak | Walker's Mammals of the World[END_REF] The vast majority of grooved-tooth rodent display only one groove, but some species display up to 10 thin grooves (Rheithrosciurus genus, [START_REF] Wilson | Mammal species of the world: a taxonomic and geographic reference[END_REF]. Grooves

B.1.3 Color

Tooth pigmentation is quite common in nature. Several vertebrate taxa have been used

as model to study this diversity, including shrews, salamanders and fish [START_REF] Motta | A quantitative analysis of ferric iron in butterflyfish teeth (Chaetodontidae, Perciformes) and the relationship to feeding ecology[END_REF][START_REF] Sparks | An analytical electron microscopy study of iron-rich teeth from the butterflyfish (Chaetodon ornatissimus)[END_REF][START_REF] Clemen | Competence and reactions of early-larval and late-larval dental laminae in original and not-original dental systems of (1) Ambystoma mexicanum shaw[END_REF][START_REF] Suga | Iron in the enameloid of perciform fish[END_REF][START_REF] Anderson | Early Iron Deposition in Teeth of the Streamside Salamander, Ambystoma barbouri[END_REF] Wen and Paine ( 2013) have demonstrated that the enamel layer specific pigmentation is linked to the incisor capacity to incorporate iron. The more colored the tooth is, the higher the iron enrichment is. Working on Blarina bricauda heavily pigmented teeth, Dumont and colleagues (2014) have resolved the structural and chemical organization of the pigments within the enamel layer. The pigments have thus been shown to be concentrated in a thin layer of aprismatic enamel at the surface of the incisor. In this study, pigmented teeth have also been shown to be 30% harder than less pigmented teeth, which is likely to represent an asset against very abrasive diets. Thus, even without a specific cusp pattern like in molars, incisors might also have played a role in the diversification of rodent food intake.

B.2 PHENOTYPIC DISRUPTIONS OF THE UPPER INCISORS IN AGEING MICE

Rodent incisors share very conserved and stable features across their phylogeny, as well as much more variable properties. Their ever-growing capacity has been focused on, especially to understand the importance of stem cell environment and the decision between self-renewal and differentiation. But when an organism ages, acquired and inherited factors affect its cells, and especially its stem cells (Sharpless and DePinho, 2007). Consequently, an organism is considered to be "as old as it stem cells" (Ho et al., 2005). Because of their role in continuous growth, tissue repair, and regeneration, the age-related decrease in adult stem cell competence is a crucial issue.

Working on the phenotypic aspect of rodent incisors, we have already stated that features like the enamel pigmentation vary within a species depending on the age of the observed specimen. Incisor ornamentation also varies along the mouse lifespan. In the wild, mice live less than a year. However in the absence of predation in laboratory conditions, scientists are able to breed and maintain mice for a longer time period. The current longevity record is 1819 days (almost 5 years) for a dwarf mouse which growth hormone receptor had been inactivated (Bartke and Brown-Borg, 2004), and 4 years for a laboratory WT mouse (Miller et al., 2002). In 1971, Robins and Rowlatt first reported the existence of dental abnormalities in the upper incisors of 22-to 36-month-old mice including the display of a discolored and grooved enamel surface, as well as the increase in the proportion of broken teeth. They also noticed the rare overgrowth of the mandibular incisors which occurred when they did not enter in contact with the maxillary incisors, which had been known in other rodents for decades (e.g. in the beaver: Bland Sutton, 1884; in the porcupine: Shadle et al., 1944). The defects they saw on the upper incisors remind of the variable features discussed above: the occurrence of a vestibular groove and the difference in enamel color.

Despite the physiological interest in age-related pathologies attested by the WHO

Global Forum on Innovation for Ageing Populations (WHO | WHO Global Forum on

Innovations for Ageing Populations), and more generally the scientific interest in stem cell ageing (Liu and Rando, 2011;Signer and Morrison, 2013), the case of age-related defects of the mouse upper incisors has remained poorly focused on. Indeed, because of the continuous growth, incisors constitute a relatively easy model to work on how stem cells react to face ageing. I address here this phenomenon in WT mice to perfect our understanding of dental stem cell ageing and to scrutinize the dynamics of the defect occurrence.

INTRODUCTION

The majority of organs and tissues that constitute a given organism are continuously renewed. It is not rare to see these life-long renewing organs affected by ageing. In rodents for example, neural stem cells lose part of their proliferative capacities with ageing, resulting in the decline of the number of newly formed neurons (Kuhn et al., 1996). Likewise, mouse melanocyte stem-cell maintenance decreases with ageing has been partly responsible for hair graying (Nishimura et al., 2005). The quiescent state of the muscle stem cell niche has also been shown to be disrupted by ageing (Chakkalakal et al., 2012). The existence of age-related defects like discolored and/or grooved enamel surface of the upper incisors, as well as the occasional overgrowth of the lower incisors prove that incisor stem cells are no exception to the rule (Robins and Rowlatt, 1971). Rodent incisors continuously grow throughout their lifespan, and wear continuously as the animal gnaws. The dynamic equilibrium is allowed by the presence of stem cells persisting into the mouse adulthood which are located in the incisor growth region (Fig. 1).

Epithelial stem cells are located within specific histological structures called cervical loops (Harada et al., 1999), whereas mesenchymal stem cells are scattered in the pulp in-between (Zhao et al., 2014). Both are necessary to ensure incisor proper formation.

The characterization of upper incisor age-related phenotype has only been sporadically addressed in WT mice (Robins and Rowlatt, 1971), in 224 Ra-treated mice (Humphreys et al., 1985), and in the Senescence Accelerated Mouse line (Sashima et al., 1987). Only one rodent species has been shown to display the same kind of age-related abnormalities: the sand rat Psammomys obesus (Ulmansky et al., 1984). If phenotypic descriptions were provided, the studies lack the dynamic aspect of ageing. The mouse incisors grow at a rate of 365 µm/day and the visible tissues are worn out and replaced in approximately 12 days, highlighting the necessity of a repeated monitoring to perfect the characterization (Hwang and Tonna, 1965;Li et al., 2012).

Here, we focus on the effects of incisor stem cell ageing in a WT context, in order to provide a chrono-morphological study of age-related defects occurrence. By monitoring in vivo mouse cohorts and further supplementing our histological analyses with classical tissue staining and X-ray microtomography, we specify that age-defects most frequently affect the enamel of upper incisors, and that they are set up earlier than expected. By conducting histological analyses on serial sections of the growth region, we highlight the disruption of the tissue arrangement and we show that the most frequent defect, the presence of an enamel groove, is due to the deep folding of the ameloblast layer. Finally, we verify that Psammomys obesus upper incisors are also affected by ageing, and we described new age-related defects.

MATERIAL & METHODS

Mice

We performed in vivo monitoring on two cohorts of WT mice housed in the PBES facility (ENS de Lyon, UMS3444). We started with 30 female mice from the CD1 "swiss" line (an outbred line), and with 30 female mice from the C57Bl/6N line (an inbred line). Our protocol consisted in (1) a daily monitoring of the mouse cohorts, and (2) picture taking every 2 weeks (ethics protocol #ENS-2012-015). For that matter, mice were anesthetized using

Vetflurane and pictures of their upper incisors were taken with a Leica stereomicroscope equipped with a CCD camera. What is observed on the visible enamel surface at any given week has been formed about 2 weeks before. 10 mice of each background were euthanized by cervical dislocation at the age of 6 months, 10 more of each background at the age of 12 months, and the rest of the colony (10 mice each) was euthanized at the age of 18 months.

A total of 6 CD1 "swiss" and 15 C57Bl/6N mice not included in the in vivo monitoring of age-related modifications were also collected from PBES users to increase our dataset on tissue histology. These mice were euthanized according to the protocol previously described.

Similarly, a total of 15 C57Bl/6J and 24 B6BCA (C57Bl/6J-derived mice) male mouse heads were collected from the CDTA facility (Orléans, UPS44) to increase our dataset on X-ray microtomographic phenotypic characterization. Age ranking was the following: 7 mice of 11 months, 3 mice of 12 months, 5 mice of 13 months, 10 mice of 16 months, 9 mice of 17 months, 3 mice of 18 months, and 2 mice of 24 months.

Sandrats

Specimens of the sandrat species Psammomys obesus were provided by the University of Deakin (Australia). The dataset consists in 12 samples as following: two newborns, two 1month-old specimens, one 3-month-old specimen, two 12-month-old specimens, one 14-month old specimen, three 18-month-old specimens, and one 24-month-old specimen. Heads were retrieved from ethanol, dried and phenotyped using X-ray microtomography.

Tissue histology

Freshly dissected mouse incisors were fixed overnight in 4% PFA. Then, they were decalcified for 4 weeks in a solution of 2% PFA and 12.5% EDTA or for 3 weeks in 0.5 M EDTA, washed and dehydrated in graded ethanol, and finally processed overnight in Histoclear (Leica) and paraffin using Leica tissue processor TP1020. Samples were embedded in paraffin blocs and kept at +4°C to be later sectioned (7 µm thick) using the Leica RM2255 microtome. Paraffin was removed from the slides using xylene, and samples were rehydrated using graded EtOH. Most of the samples were stained in hematoxylin (4 min), Scott's water for blue nuclei (3 min) and eosin (1 min 15 s). Slides were mounted using Cytoseal 60 (Thermo scientific). Two specimens with aplastic enamel grooves were stained with Masson's trichrome (hemalum, 8 min; fushine, 2min; aniline blue, 1min) to ensure a better discrimination between tissues.

X-ray microtomography analyses

C57Bl/6J-derived mouse heads retrieved from the CDTA facility were imaged using an X-ray cone-beam computed microtomography with a Nanotom machine (GE) at an energy of 100 keV with a cubic voxel of 5 µm. All 3D renderings were performed using VGStudiomax software. Psammomys obesus heads were similarly imaged, with a cubic voxel size of 5 to 8 µm depending on the skull size.

To obtain transverse sections of the mouse upper incisors, the 3D renderings were oriented sagittaly along the medial plan of the tooth. A VGStudiomax algorithm allowed the virtual section of the incisor around the axis that is perpendicular to the plan defined by the circle subscribed to the tooth.

RESULTS

Multiple age-related abnormalities affect the mouse upper incisors

Focusing on defects affecting the visible part of the upper incisors that is the enamel layer covering their vestibular side, we have addressed the specific in vivo chronology of occurrence of age-related defects. Five abnormalities have been observed in total: enamel dysplasias, a single enamel groove, hair growing out of the dental cavity, an aplastic groove, two aplastic grooves , all of them found in the C57Bl/6N cohort (from left to right in Fig. 2A).

However, only three of them (enamel dysplasia, a single enamel groove and hair growing out of the dental cavity) have been observed in the CD1 "swiss" cohort.

We show an earlier onset comparing to what has been published by Robins and Rowlatt (1971). Indeed, the detection of enamel dysplasia is possible from 6 months on in both CD1

"swiss" and C57Bl/6J cohorts (Fig. 2B). A week later, the first grooves start to be displayed, followed by the detection of hair growing out of the cavity. The presence of this thin air growing out of the dental cavity does not come from littermate grooming because the texture and color do not match the mouse fur. Aplastic enamel grooves start to be seen later on, followed by the occurrence of a 2 nd groove only in our C57Bl/6J cohort.

The persistence of the defects varies. Given the quick continuous growth of the incisor, enamel dysplasias are worn out relatively quickly and can be traced for more than two weeks.

The hair growing out of the dental cavity looks very thin and fragile. It could thus be easily pulled out when the mouse grooms its littermates, feeds or chews on the cage. As for the enamel groove, which are the most frequent defect seen during the entire in vivo monitoring (in 43% of both the CD1 "swiss" and the C57Bl/6N cohorts), a reversion of the phenotype can be observed in 50% of the case (Fig. 3). In the example depicted, the reversion of one large and aplastic 2 nd groove in a C57Bl/6N mouse is observed 2 weeks after it has been set up.

Age-related defects also affect other areas of the mouse upper incisors

Using X-ray computed microtomography, we further characterize the age-related abnormalities using a complementary dataset we collected. Phenotypic analysis of the 3Drenderings of the specimens coming from the CDTA facility corroborates that the proportion of defects increases with the age of the specimen (Fig. 4A). However in this static dataset, defects have not been observed in the younger class (11-month-old mice). The sequence of abnormality occurrence is similar to our in vivo findings. This analysis helps describing a new defect that is the presence of a fold in the dentin (uni-or bilateral) starting 13 months (blue, Figure 4B). It affects between 5 and 18% of the incisors between 13 and 17 months of age. In the last three age classes, dentin fold is present in 50% of the defect combination, along with enamel dysplasia and dens in dentes (yellow, Fig. 4B). The occurrence of one or more enamel grooves still stands out as being the most frequent defect displayed. Interestingly, the gradual increase in the proportion of age-related defects reach a maximum in the oldest class (24month-old mice), where no normal incisor is displayed anymore. Given rodent incisor specific growth pattern, what is seen on the protruding part of the incisor has been generated in the growth region and has progressed away from it. This is especially visible when looking at enamel grooves, because the folding is observable in the less mineralized part of the incisors that corresponds to the growth region (Suppl. Fig. 1).

Suppl. Fig. 1. Grooves are traceable to the growth region.(A) 11-month-old specimen with no defect displayed. (B) 17-month-old specimen with two grooves. Note that the grooves visible on the vestibular enamel surface affect the dentine surface as well, because the mineralized tissue visualized here is the dentin.

Psammomys obesus also displays age-related abnormalities of its upper incisors

Mice are not the only rodents starting to display abnormalities on their upper incisors as they age. Abnormalities are displayed in our Psammomys obesus samples from 12 months on (Fig. 5). From that time point, a single dentin groove is observed centrally in all the upper incisors (yellow arrowheads, Fig. 5). Three incisors from 12-month-old specimens, one from a 18-month-old-specimen, and one from the 24-month-old specimen additionally display what we identified as a growth region odontoma (black arrowhead, Fig. 5B). Along with this defect come various signs of previous disruption events in the growth system (red arrowheads, Fig. 5B).

Linear elongation of the upper incisors occurs as the mice age

Mouse incisors start to develop during embryogenesis, and start to erupt ten days after the pup is born. These teeth are fully functional in the juvenile, and since they are continuously growing, they never get replaced over the lifespan of the animal. The incisors of the adult mice are bigger than the ones from the juveniles. We were interested in that constant modification of the incisors, and wanted to test whether or not ageing would impact the phenomenon. By measuring the upper incisors mesio-distal length in our cohorts, we show that the increase is linear in both mouse lines (C57Bl/6N: y=0.00167x+0. 53455, R²=0.9642, p-val<2.2e-16;CD1 "swiss": y=0.001279x+0.542190, R²=0.9698, p-val<2.2e-16; in Fig. 6). C57Bl/6J upper incisors are significantly longer than CD1 "swiss" upper incisors . The distribution of the mesio-distal length in grooved incisors differs between the two cohorts (blue triangles, Fig. 6). These numerical values have not been included in the regression and in the standard deviation calculation. The dispersion of the values remains equivalent at each time point in the C57Bl/6N cohort. In the CD1 "swiss" cohort however, the dispersion is characterized by two phases. Up to 55 weeks (about one year), grooved incisors are significantly longer than smooth incisors (Mann- .9642, p-val<2.2e-16); CD1 "swiss": y=0.001279x+0. 542190 (R²=0.9698, p-val<2.2e-16).

Whitney Wilcoxon, p-value<0.05), but in the last six months of the monitoring, there is no significant difference anymore. This might indicate a change in the groove setting in this background.

Grooves occur in the same percentage of mice from both cohorts, but we find that in mouse where only one upper incisor displays a vestibular enamel groove, the smooth incisor shows a lower mesio-distal length when compared to the grooved incisor (Mann-Whitney Wilcoxon, p-value<0.05).

Various histological defects are displayed in the growth region

Mice that were monitored in vivo have been sacrificed at three ages (6, 12 and 18 months) in order to address modification of the normal organization of the upper incisor growth region (see also Fig. 1B). Histological analyses confirm the occurrence of tissue organization defects and re-state that the age-related abnormalities are set up in the growth region. The serial sections realized on the two upper incisors of a C57Bl/6N mouse of 18 months help comparing tissue organization in the absence and in the presence of enamel grooves (Fig. 7). The left incisor still displays the normal smooth enamel surface, but the right incisor displays both a fine groove and a marked groove (green and yellow arrowheads respectively, Fig. 7B). Exploring the series of sections reveals that a strong folding of the ameloblast layer is responsible for the phenotype seen protruding out of the mouth, folding that also impact the organization of the odontoblast layer (Fig. 7C,D).

In a 12-month-old C57Bl/6N, a joint folding of the odontoblast, dentin and ameloblast layer can be traced in successive slices in the transit-amplifying zone (yellow arrowheads Figure 8A). This mouse also displays a hyperplastic laCL when compared to a normallooking laCL of one littermate (yellow dotted line in Fig. 8A). In a 6-month-old CD1 "swiss" mouse, we have been able to observe the duplication of the ameloblast layer which seem to be caused by an erratic cell proliferation event (Fig. 8B).

DISCUSSION

An updated chrono-morphology of age-related defects is provided

In 1971, Robins and Rowlatt considered a bigger mouse colony, all animals being from a C57Bl/6Icrf genetic background. Their experimental design aimed at comparing upper incisor phenotypes of numerous mice on three separated occasions, withouth tracing the becoming of the individuals. Robins and Rowlatt concluded that age-related defects were virtually absent on the incisors of 4 to 16-month-old specimens but present in the 22 to 36month-old groups. On the contrary, our study provides an individual monitoring of ageing mice. This allows us to understand the quick dynamics of abnormality setting. Furthermore, we show a much earlier onset than what was thought, with mice starting to display enamel mineralization defects from about 6 months of age. Furthermore, our experimental design allows us to address the persistence of the defects displayed. The reversion of enamel grooves is observed in 50% of the specimens we monitored. Stem cell ageing has often been studied in the light of cancer biology because of a decline in the replicative function of various stem cell types (Signer and Morrison, 2013).

When an organism ages, DNA damages indeed accumulate and its genomic instability increases, leading to an increase in tumor formations (Finkel et al., 2007). This phenomenon is usually viewed as irreversible. Although grooves are not tumors, our results prove the existence of correction mechanisms that allow the ageing growth region and the stem cell niches to get back to a normal functioning.

The modifications observed depend on the genetic background

By opting for an in vivo comparison between an inbred mouse line (C57Bl/6N) and an outbred mouse line (CD1 "swiss"), we address whether or not the genetic background could influence the apparition of age-related defects. We find that although both lines eventually develop age-related defects, C57Bl/6N mice end up with the strongest defects (higher number of aplastic vestibular grooves). The genetic identity mirrors the degree of consanguinity obtained by crossing littermates over 20 generations (Hedrich, 2004). Thus, inbred mice can be considered as genetically identical (Charles River's C57Bl/6N model information sheet).

Outbred mice are animal in which a certain genetic diversity is maintained. CD1 "swiss" mice display about 28% of diversity in 110 tested loci (Charles River's CD1 model information sheet). Although the effects of consanguinity have never been linked with ageing pathologies or progeria-like syndromes, the disadvantages associated with human inbreeding has already been reviewed in the population genetics field (Bittles and Neel, 1994). The results we obtained here tend to prove that the same trend is displayed in the mouse, with a set of agerelated abnormalities that can be extended in consanguine specimens. Robins and Rowlatt (1971) put much emphasis on their finding of a high hair impaction in the gingival tissues of both maxillary and mandibular incisors that is caused by grooming regardless of the age of the mice. They use this finding and the reported presence of cysts in mice aged between 11 and 29 months (van Rijssel and Mühlbock, 1955) to hypothesize that the hair inserted in the gingiva could favor the development of cysts in the maxillary tissues that could disrupt the growth region, thus generating the grooves. Our histological studies did not show any inflammatory infiltration in the case of the enamel groove occurrence, but demonstrated that a folding of the ameloblast layer was the mechanical cause of the grooved phenotype. Surprisingly, we do not find hair impaction to be characteristic of our mouse colonies. However, we have been able to spot ectopic development of hair in the dental socket. The only organs normally growing out of the dental cavity of a mouse are its teeth. Nonetheless, the presence of very thin and colorless hair addresses the very interesting question of cellular identity. The stem cell niches of the incisors and of the hair follicle originate from the ectoderm, and share similarities in terms of molecular regulation of the niche maintenance and function (Naveau et al., 2014). If the histological organization of both niches differs, it does not rule out the possibility of a change in the differentiation program of the incisor epithelial stem cells. This change in cell fate would be supported by the action of major signaling pathways in either of the organs discussed here.

Hairy teeth raise the question of stem cell differentiation

Grooved incisors are reminiscent of a convergent feature of rodent dentition

The presence of vestibular grooves is the most frequent defect we found in the cohorts we monitored in vivo, as well as in the samples we collected for further characterization of the age-related defects. Interestingly, the mouse is not the only rodent dislaying more and more abnormalities as it ages. The Gerbillinae species Psammomys obesus also naturally does. In the previous study of the phenomenon on this species, Ulmansky and colleagues (1984) described the occurrence of tooth fracture, frequent overgrowth, and the detection of a gingival inflammatory process. We opted for a X-ray microtomographic approach in order to see whether or not ageing sand rats, like ageing mice, were displaying mineralization defects.

The dentin grooves detected on the lingual side of the upper incisors are highly similar to what is observed on the mouse vestibular side. It highlights that the mesenchymal stem cell niche is likely to be affected to ageing as well. However, the presence of odontoma-like masses appears specific to P. obesus. This phenotype is interesting because an increase in the occurrence frequency of this defect has been documented in ageing osteopetrotic mice (Amling et al., 2000).

Another slightly different case has been reported in wild-type animals: a specimen of Zapus princeps displays a supernumerary groove mesially to the first one that is characteristic of its species (specimen MVZ-36877, C. Charles' PhD dissertation). Interestingly, the presence of vestibular grooves is a dental features shared by 61 genera out of the 481 that constitute the Rodentia order. The absence of any phylogenetic signal in the repartition of the character highlights its independent and convergent acquisition. Peramorphic developmental heterochronies could explain the diversity of ornamentation in the Rodentia order.

Molecularly speaking, modulations of the gene expression profiles in the ageing stem cell niches of the mouse or the sand rat upper incisors might be re-activating the mechanisms leading to groove development.

CONCLUDING REMARKS

In summary, we have showed that two different types of age-related modifications affect the mouse upper incisors. The first one occurs from the day the incisor germ starts to develop, and consists in the continuous modification of the tooth size. Ageing beyond the mouse life expectancy does not modify this property, the mesio-distal length of the incisor linearly increasing over the time of our study. The second type of age-related modifications encompasses all the defects seen in the mineralized tissues: dysplasia, aplasia, vestibular enamel groove (aplastic or not), and dentin folding. Taken together, our findings will be the basis of further transcriptomic studies aiming at understanding the molecular disruption of the upper incisor stem cells caused by ageing, and we will thus be able to fully characterize from the phenotype to the genotype the impact of ageing on our model.

B.3 TRANSCRIPTOMIC DISRUPTIONS OF THE UPPER

INCISORS IN AGEING MICE

Among the abnormal phenotypes developing on the mouse upper incisors, the occurrence of vestibular grooves stands out as being the most frequent defect set up. It also stands out because previous work in the tooth evo-devo field acknowledged the development of similar phenotypes in mutant mice. Indeed, Lrp4 hypo/hypo mice, as well as Dcr K14-/-mice were described as displaying such grooves (respectively in [START_REF] Ohazama | A role for suppressed incisor cuspal morphogenesis in the evolution of mammalian heterodont dentition[END_REF][START_REF] Michon | Tooth morphogenesis and ameloblast differentiation are regulated by micro-RNAs[END_REF]. However, based on a large-scale characterization of hematopoietic stem cell ageing (Rossi et al., 2007), a candidate gene-based approach is likely to be too restrictive to get a good overview of the plausible large-scale changes in the gene expression profiles.

Taking advantage of Next-Generation Sequencing (NGS) technologies as a powerful tool for transcriptome analysis, I propose a comparative approach to unravel the existence of age-related changes in gene expression profiles, which consists in focusing on the incisor growth region solely or on the entire incisor. Results prove that the transcriptomic approach was indicated, although sampling questions are raised. I also aim at understanding how enamel grooves can be set up in a WT ageing context by comparing incisors of the same age which do not display the same phenotype.

Independent transcriptomic analyses reveal gene expression changes in ageing mouse incisor stem cell niches

Pauline Marangoni 1 , Benjamin Gillet 2 , Sandrine Hughes 2 , Florence Carrouel 1 , Cyril Charles 1 , and Laurent Viriot

INTRODUCTION

Mouse incisors are continuously growing organs made of two highly mineralized tissues: enamel and dentin (Harada et al., 2002a). These tissues are generated from differentiated cells that display a very precise origin. Enamel is secreted by ameloblasts which differentiate from epithelial stem cells located in the cervical loop structures, while dentin is secreted by odontoblast which derive from mesenchymal stem cells located in the pulp (Arana-Chavez and Massa, 2004;Seidel et al., 2010). All the stem cell containing structures are located at the proximal end of the incisors, referred to as the growth region (Fig. 1A,B).

Although the system lasts during the mouse lifetime, it is affected by ageing. It has been shown that age-related defects develop on the mouse upper incisors from 6 months on (Robins and Rowlatt, 1971;Marangoni et al. in prep, please see Chap. B.2). These defects are set up in the growth region and they become visible to the observer once they reach the protruding part of the tooth. They mostly include abnormalities of the enamel layer covering the vestibular (labial) side of the incisor. The presence of an enamel groove is the most frequent age-related abnormality observed (Fig. 1C).

The organization of the incisor growth region is very precise, and allows the special circular shape to develop. Stem cell maintenance, dental cell differentiation, enamel and dentin secretion and mineralization are tightly regulated processes which all involve the growth region (Jussila et al., 2013). But the capacity to ensure the upper incisor specific shape is obviously impacted by ageing through histological rearrangements. In order to better understand the molecular disruption of the stem cell niches resulting in those rearrangements, we performed transcriptomic analyses. We aimed at addressing the plausible age-related changes in the gene expression profiles of the mouse upper incisor growth region and at identifying candidate genes to further study dental stem cell ageing.

Because of the organization of the incisor in several areas (growth region, transitamplifying portion and differentiated zone), we chose a double approach to assess stem cell ageing, analyzing the gene expression profiles of (1) the sole growth region, which was extracted by laser capture, and (2) entire incisors with or without a vestibular groove. By combining the results, we show that both the growth region and the entire incisor transcriptomes vary depending on the age. Most of the genes differentially expressed between age conditions are down-regulated in old samples. We were able to identify a list of 20 genes From left to right are displayed a normal (smooth) incisor, one displaying enamel dysplasia (orange), one displaying a vestibular enamel groove (purple), one with a hair growing out of the tooth socket (gray), and one aplastic vestibular groove (no enamel left). Scale bars: 0.5 mm which biological function is interesting in the light of ageing, among which Fgf7, Krt7, Mmp20 and Wnt2b stand out. We also provide an extensive comparative analysis between smooth and grooved incisors of similar age. We show a strong correlation between the presence of an aplastic groove and an immune response with hundreds of genes involved in the NF-κB innate immune response being up-regulated.

MATERIALS & METHODS

Sample preparation

Two transcriptomic analyses were performed: one specifically focuses on the growth region, while the second addresses entire incisors. For the construction of the 1 st dataset, the upper incisors of 5 CD1 "swiss" mice of 6 months (CD1-6) were dissected from the mouse skull tissues in 1X PBS, soaked in 30X sucrose for 4h, and frozen in liquid nitrogen immediately after. The protocol was repeated for 5 CD1 "swiss" mice of 12 months (CD1-12), 5 CD1 "swiss" mice of 18 months (CD1-18), 5 C57Bl6/N mice of 6 months (B6-6), 5

C57Bl6/N mice of 12 months (B6-12), and 5 C57Bl6/N mice of 18 months (B6-18). The choice of the two mouse lines was dictated by the previous study we performed (Marangoni et al., in prep.), in order for us to investigate the importance of the background in the molecular disruption. Samples were stored at -80°C, and were destined for a Laser-Capture Microdissection (LCM) and a RNA-seq experiment.

Secondly, the incisors of 3 C57Bl6/N mice of 6 months (B6-6) were dissected in 1X PBS, and frozen in liquid nitrogen. Dissection was performed under a stereomicroscope to ensure that the collected upper incisors did not display any abnormality. The protocol was repeated for 3 C57Bl6/N mice of 18 months without any defects (B6-18 S for smooth), and for 3 C57Bl6/N mice of 18 months displaying an aplastic groove (B6-18 G for grooved). All samples were stored at -80°C, and destined to a RNA-seq experiment.

A summary of the entire experimental strategy is provided in the Fig. 2. All the mice used for the dissection were housed at the PBES facility (ENS de Lyon, UMS3444), and euthanized using cervical dislocation. The animal experimentation procedure was validated by the ethics committee of the PBES (protocol #ENS-2012-015).

Fig. 2. Comparison of the two experimental strategies.

On one hand incisors were dissected, and their growth region (hatched ellipse, top picture) was micro-dissected using Laser Capture Microdissection (LCM). cDNA libraries were obtained with a protocol dedicated to small amount or degraded RNA with cDNA amplification step before fragmentation. Validated banks were then sequenced using a Illumina Hiseq 2500 apparatus. On the other hand, incisors were dissected and total RNA extracted. cDNA libraries were generated using a starter/stopper system developed by Lexogen. Data were sequenced using a SOLiD 5500W apparatus.

LCM and Illumina library preparation

In order to dissect as precisely as possible the incisor stem cell niches, we designed a LCM approach, performed at the ProfileXpert platform (University of Lyon, IBISA platform).

The six groups of frozen incisors (CD1-6, CD1-12, CD1-18, B6-6, B6-12, and B6-18) were embedded using as little Tissue Tech (Fisher) as possible and sectioned using a Leica cryostat (10-12 µm thick). Slides were thawed out at room temperature, dehydrated in graded ethanol and xylene. Slides were dried in a vacuum dome for 10 min. LCM was then performed using PixCell LCM System (Arcturus Engineering) and CapSure® Macro LCM caps (Arcturus Engineering). Actual microdissection was allowed by the application of laser spots (7.5 µm size) through the cap, sealing the transfer film to the region of interest. The region was then removed from the slide by pulling the cap up.

Total RNAs were extracted directly from the caps using the RNeasy extraction kit (Qiagen). RNA quality and quantity were then assessed using a 2100 Bioanalyzer (Agilent Technologies). Due to the results of the test phases, samples of a same age and genotype were pulled together in order to make a single sample per age and background condition.

AmplifiedcDNA was prepared from 5ng of pooled total RNA using the Ovation RNA-seq system V2 and following the manufacturer instructions (NuGEN Technologies, Inc.). 500 ng of amplified cDNA were purified using AMPure XP beads (Agencourt Biosciences Corporation), and fragmented by sonication using a Covaris E210 instrument, and used to construct the Illumina libraries. DNA library quality was assessed using 2100 Bioanalyzer (Agilent Technologies) and quantified using NGS qPCR library quantification kit with EvaGreen (#QUANT-01-0000S, Solis Biodyne). The libraries were loaded in the flow cell at a 14pM concentration and sequenced in the Illumina Hiseq 2500 as single-end 50 base reads.

Library preparation and sequencing were performed at the IGBMC Microarray and Sequencing platform (member of France Génomique program, Illkirch, France).

SOLiD library preparation and sequencing

Total RNAs were extracted from the incisors of the second dataset consisting in nine mouse upper incisors (three groups: 6 months, 18 months smooth, 18 months grooved).

Samples were ground in liquid nitrogen and re-suspended in lysis buffer. Extraction was carried out using RNeasy extraction kit (Qiagen). Samples were treated independently, and RNA concentration was measured using a Qubit 2.0 Fluorometer (Lifetechnologies).

Barcoded libraries were prepared from 750 ng of total RNA using the SENSE mRNA-SEQ Library Prep Kits for SOLiD (Lexogen). Then, libraries were converted to be sequenced using SOLiD Wildfire technology using 5500 W conversion primer kit as recommended by Lifetechnologies. Quantification and quality assessment of the libraries was performed on 2200 Tapestation analyzer (Agilent Technologies). Libraries were finally diluted to 4nM, mixed in equimolar proportions and placed in two lines of the flow-cell (four samples on lane 1 and five samples on lane 2) following the Wildfire Template Amplification Protocol for ICS 2.1 provided by Lifetechnologies. Sequencing was performed on the sequencing platform of the IGFL (UMR5242, Lyon, France) using a 5500 W SOLiD sequencer upgraded with the Wildfire technology.

Analysis workflow

After the removal of over-represented sequences using FASTQC (Babrahan Bioinformatics group), Illumina reads were aligned to the USCS_mm10 reference genome using Bowtie2 (Langmead and Salzberg, 2012) available on our local web-interface Galaxy (Goecks et al., 2010). Similarly, after a first step of filtering (to eliminate possible reads from ncRNA, tRNA or ribosomal RNA), SOLiD color-coded reads were aligned in colored space to the USCS_mm10 reference genome (with Ensembl gtf file for annotation) using the LifeScope software with default parameters.

For both Illumina and SOLiD aligned dataset, Galaxy was then used to assess the number of reads per feature with HTSeq count for each sample (Anders et al., 2014, intersection nonempty mode).The differential expression analyses was performed on R software using DESeq2 package (Love et al., 2014) by taking advantage of an in-house program to analyze the output generated (F. Chatonnet). We used the following parameters to determine differentially expressed genes: only genes which had a minimal number of counts of 10 were considered, differences in their expression level were assessed with a p-value≤0.05, and finally only those that displayed a Fold Change (FC) superior or equal to 2 in their compared expression level were considered differentially expressed. Gene ontogeny was assessed using the Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GORILLA, Eden et al., 2009).

Validation of candidate gene expression changes

The validation of changes in gene expression levels detected during both RNA-seq experiments by RT-qPCR in ongoing. For the both types of samples, cDNA has been amplified from the remaining RNA samples using SuperScript II reverse transcriptase (Lifetechnologies). RT-qPCR using the iQ SYBR Green Supermix (BioRad) and a CFX96 apparatus (Biorad) will help prioritizing further studies. Transcript levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), results being expressed as normalized expression values (equal to 2 -ΔCq ). Primers were designed using NCBI Primer-BLAST software, and sequences used are listed in the Suppl. Tab.1.

Gene

Forward primer

Reverse primer

Dspp AGGCTTTCACACGCTGCTAT TTTCTGGTGAGGGGTCTGGA Fgf7 CATGCTTCCACCTCGTCTGT CAGTTCACACTCGTAGCCGT Krt6a AGTAACTGTGACTGCCCCGT GCCAAGAGCATCAAGGAAAGC Mmp20 CTGCAGACCCCAACCTACTG GCAAGGTGGTAGTTGCTCCT Podn AACGTACACGTGCTCAAGGT GTAGAGTTCCCGAAGCTGGG Sost TCTGCCTACTTGTGCACGC GACCTCTGTGGCATCATTCCT Cxcr4 CCATGGAACCGATCAGTGTG GAAGCAGGGTTCCTTGTTGG Cyp26a1 CCGACCGCTTTATAGTGCCT GGAATGAAGCTGAACCGGGA Gfi1 AGAGGGGCACACAGTTTAGC CCACATGCCCACCAGATGTT Glipr2 AGTGTGGAGAGAACCTTGCG CTGTACCACCTATCGGCCAC Grem1 CAAACACCAGCCGGAAACAG GGGCATTTCCGACCATCTGA Hand2 ACGGACACGTTCCTTACAGC TGGGTTCTTGGGCGCTTATT Hrh2 AGAGAACACAAAGCCACCGT TCCACGGTAAACGAAGGCAG Ibsp TGACAGCCGGGAGAACAATC AACCTTCCTTTCGGCAGTCC Krt7 GAACCGCTCTATCCAGAGGC GGACTCTAACTTGGCACGCT Mmp25 AGTCCCCCGAAAACGACATC AGCTCGCAGTGACAATCACA Sfrp4 CCTGGCAACATACCTGAGCA CACTCCTCTGGACGGCTTTT Spp1 GGGCAGCCATGAGTCAAGT CATCCGACTGATCGGCACTC Wisp CACCTGTGGGTTGGGCATAG CTCCAGTTGGCAGAATCGGT Wnt2b GTGTCAACGCTACCCAGACA CACTCTCGGATCCATTCCCG
Suppl. Tab. 1. List of the primers used for RT-qPCR validation of the candidate gene expression levels. Sequences were retrieved using Primer-BLAST software (NCBI).

RESULTS

Micro-dissected incisor growth region compose an heterogeneous dataset

We consider here the 6-month-old mouse cohort as the control for young specimens, and we compare their gene expression profile to the two older cohorts (12-and 18-month-old mice) Our previous analyses showed that mice were impacted by ageing regardless of their background, the most striking difference between CD1 "swiss" and C57Bl6/N being that CD1 mice did not display aplastic grooves for the duration of our monitoring (Marangoni et al., in prep.; please see Chap. B.2). For all our analyses, we have started by working on the raw data.

However, in order to minimize the number of differentially expressed genes resulting from a change in the ratio between epithelial and mesenchymal cells imputable to the sample dissection, we decided to also work on normalized data. Because Sox2 is a known marker for epithelial stem cells (Juuri et al., 2013), and because it is not a differentially expressed gene, we realized a first normalization using Sox2 count number. For the same reasons, we also normalized the count tables with Fgf10 count value. Indeed, Fgf10 is an essential actor that helps maintaining the functioning of the stem cell niche (Harada et al., 2002b), and it is expressed in the mesenchyme surrounding the cervical loops (Klein et al., 2008).

The dataset has the specificity of being generated by pulling 6 micro-dissected growth regions for each of the age cohorts. The most striking finding resulting from the analysis of this data is that the dataset displays heterogeneity that could be due to individual variability and/or to the absence of transcriptomic change along with ageing. The Principal Component Analysis (PCA) (Fig. 3A) recapitulates this observation, with no age-dependent clustering of the samples being observed. The heatmaps and dendrograms argue in favor of the sample variability, which could either come from biases in the sampling or from differences between the genetic background of the mice (Fig. 3B). However, the detection of differentially expressed genes matching our conditions (Fig. 3D, E; Tab.1) tend to show that age modifications can be found, and that this dataset might benefit from ongoing investigations about the genetic diversity between mouse line.

The transcriptome of the mouse incisor growth region varies when the animal ages

The comparison of the number of read per feature reveals that 17 genes are differentially expressed with a FC≥2 threshold when comparing 12-month-old mice with 6month-old mice, regardless of their genetic background (Fig. 3C, Tab.1). In all cases, these 17 genes appear down-regulated in the older mice (Suppl. Tab.2). One of them stands out because of its very specific expression pattern. Indeed, Fgf7 is known for being expressed in the mesenchyme directly surrounding the cervical loops. Similarly, 21 genes are differentially expressed with a FC≥2 when comparing 18-month-old mice to our 6-month-old mice (Figure 3D, Tab.1). As previously observed, most of them (18/21) are down-regulated in the older cohort (Suppl. Tab.2). Among them, two are of special interest: Mmp20 and Sost, both strongly down-regulated in the older cohort. pathway, which help regulating the development of the correct number of incisors (Fujimori et al., 2010).

In the Sox2-normalized analysis, no gene appears differentially expressed in the two comparisons (2 nd column, Fig. 3C,D). In the Fgf10-normalized analysis, no gene appears differentially expressed when comparing 12-to 6-month old mice (3 rd column, Fig. 3C,D).

However, four genes stand out as being differentially expressed with a FC≥2 in the 2 nd comparison: Bglap, Col1a1, Col1a2, and Dspp. Both collagen genes can be mutated in dentinogenesis imperfecta, as well as Dspp gene (Sreenath et al., 2003). However, none of these 4 genes were picked in the analysis of the raw data.

___________________________________________________________________________ Fig. 3. [following page] Genetic variation and differentially expressed genes depending on the specimen age. These data refer to the growth region transcriptomic analysis. Each column is organized similarly with (A) PCA plot of the variability in the specimen gene expression profile, the percentage indicate the contribution of the principal axes of variation; (B) heatmaps and dendrogram indicating the relationships between the samples based on their gene expression; (C) correlation graphs where black dots represent the filtered genes, pink dots represent the differentially expressed genes, and blue dots represent those with a FC≥2. 1 st column is the raw data, 2 nd the Sox2-normalized data, and 3 rd the Fgf10-normalized data.

Second RNA-seq confirms the existence of age-related changes in the transcription levels

A second dataset has been designed to compare more easily the gene expression profile of mice of various ages. In an effort to generate a more homogeneous dataset, we opted for an analysis on whole dissected incisors, subdivided in three cohorts: young and smooth (6-month-old mice), old and smooth (18-month-old mice), and finally old and grooved (18-month-old mice) samples. In this dataset, we have three replicates for each condition. We then performed the same analytic workflow on this dataset.

The variation between the samples was assessed by PCA. It shows that samples from the same cohort tend to plot together, regardless of the normalization applied (Fig. 4A). The segregation between the young and the old and smooth samples is much finer than the separation between smooth and grooved old specimens. The latter is strongly displayed on PC1 where 2/3 of the 'old and grooved' replicates plot completely separately from the rest of the replicates (Fig. 4A, all columns). This highlights the huge variability of the gene expression profile in the case of grooved incisors. The heatmap and dendrogram plots provided by the DESeq2 package confirm this repartition, one grooved incisor being retained within the smooth tooth group (Fig. 4B).

The comparison between 'young and smooth' and 'old and smooth' specimens indicates that 28 genes are differentially expressed with a FC≥2. The number is brought to 31 when working with the Sox2-normalized count table, and to six when working with the Fgf10-normalized count table (Fig. 4C, Tab.2). When comparing the gene lists, 24 genes are found in the raw data and the Sox2-normalized data, four in the raw data and the Fgf10normalized data, as well as in both normalized data (Suppl. Tab.3). Most of the genes highlighted here are down-regulated in the older specimens (all in raw data and Fgf10normalized data, and 5/31 in Sox2-normalized data, Suppl. Tab.3).

If the global down-regulation affects the same proportion of genes that in the study of the growth region transcriptome, none of the genes found in the entire incisor transcriptomic analysis was found differentially expressed in the first analysis. Gene ontology study using GOrilla software does not reveal enrichment in any biological process or function in the case of the two age comparisons from the 1 st RNA-seq performed. The same search on the comparison between young and old and smooth incisors shows a detectable enrichment in metabolism regulation, but the signal is lost when entering the list of genes from the two normalized datasets. (C) correlation graphs where black dots represent the filtered genes, pink dots represent the differentially expressed genes, and blue dots represent those with a FC≥2. 1 st column is the raw data, 2 nd the Sox2-normalized data, and 3 rd the Fgf10-normalized data.

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Entire incisor

Huge differences occur between grooved and smooth incisors

Regarding the defect factor, the number of genes differentially expressed with a FC≥2 is much bigger. 442 genes fulfill our condition in the raw data, 502 in the Sox2-normalized data, and 145 in the Fgf10-normalized data (Fig. 4D, Tab.2, Suppl. Tab.4). When cross-referencing those genes between the datasets, 96% of the genes are redundantly found in the raw data and the Sox2-normalized data, and of 12% when comparing the raw data and the Fgf10normalized dataset.

The biggest finding in terms of gene ontology enrichment comes from this comparison between grooved and smooth incisors. Indeed, we detect a very strong enrichment in immune system response. Going through the list of genes differentially expressed, we find about 35% of them involved in immune processes (Suppl. Tab.4). The vast majority of them are involved in the NF-κB mediated immune response, with notably many metalloproteases, chemokines, complements, and cytokines stimulated. All these genes of interest are strongly up-regulated in the grooved incisors, thus establishing a strong correlation between the presence of an aplastic groove and a large immune response.

Picking candidate genes to perfect our understanding of the ageing impact of the continuously growing incisors Documentation of the various lists of differentially expressed genes obtained in our dataset has allowed us to pick several candidate genes to address more precisely dental stem cell ageing. We picked 20 genes of high interest for further examination (Tab.3). Dspp, Fgf7, Krt6a, Mmp20, Podn and Sost are the genes of interest chosen to further address the ageing process within the growth region. Cxcr4,Cyp26a1,Gfi1,Glipr2,Grem1,Hand2,Hrh2,Ibsp,Krt7,Mmp25,Sfrp4,Spp1,Wisp,and Wnt2b are the genes of interest chosen to further address the setting up of the enamel groove.

AGE FACTOR

GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Dspp ↘ Krt6a ↘ Podn ↘ Fgf7 ↘ Mmp20 ↘ Sost ↘ DEFECT FACTOR GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Cxcr4 ↘ Hand2 ↗ Sfrp4 ↘ Cyp26a1 ↗ Hrh2 ↘ Spp1 ↘ Gfi1 ↘ Ibsp ↘ Wisp ↘ Glipr2 ↘ Krt7 ↘ Wnt2b ↘ Grem1 ↘ Mmp25 ↘
Tab. 3. Genes of interest to understand the molecular impact of ageing on the stem cell niches, and the setting of the aplastic groove. The orientation of the arrow in the UP/DOWN column indicates if there is a higher (up) or a lower (bottom) expression of the gene in the old and smooth or old and grooved groups when compared to their respective control.

GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN AU040972 ↘ 5730405O15Rik ↗ Bglap ↘ Acpp ↘ 9930013L23Rik ↘ Col1a1 ↘ Bnc2 ↘ Ankrd45 ↘ Col1a2 ↘ Celf3 ↘ Arhgap4 ↗ Dspp ↘ Col14a1 ↘ Bcan ↘ Col8a1 ↘ Cpz ↘ Cyp2e1 ↘ Creb3l1 ↘ Fgf7 ↘ Dpf3 ↘ Flt4 ↘ Emid2 ↘ Harbi1 ↘ Endod1 ↘ Itga11 ↘ Fam65c ↘ Lrrc32 ↘ Gabra4 ↘ Ltbp2 ↘ Gale ↘ Rab30 ↘ Gpr133 ↘ Smcr7 ↘ Itga11 ↘ Susd5 ↘ Ltbp2 ↘ Thsd7b ↘ Mmp20 ↘ Ncam2 ↘ Sgol2 ↗ Slc13a5 ↘ Sost ↘ Suppl.
Tab. 2. List of the differentially expressed genes between two age conditions from the growth region transcriptomic analysis. All the genes listed above are differentially expressed with a FC≥2. Genes highlighted in yellow are the genes of interest to conduct further studies on the ageing of the incisor growth region. In black are the lists obtained analyzing the raw data, in grey is the list obtained analyzing the Fgf10-normalized data.

GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Aqp3 ↘ Abpb ↘ Krt6a ↘ Calml3 ↘ Aqp3 ↘ Lcn13 ↘ Cecr6 ↗ Calml3 ↘ Ltf ↘ Chi3l1 ↘ Casp14 ↘ Lypd3 ↘ Chit1 ↘ Cd24a ↗ Obp1a ↘ Fgg ↗ Cecr6 ↗ Sprr1a ↘ Fxyd2 ↘ Chi3l1 ↘ Inmt ↘ Chit1 ↘ Krt4 ↘ Fgg ↗ Krt6a ↘ Fxyd2 ↘ Lcn11 ↘ Gm14744 ↘ Lcn13 ↘ Inmt ↘ Lcn14 ↘ Klk13 ↘ Ltf ↘ Krt4 ↘ Ly6g6c ↘ Krt6a ↘ Lypd3 ↘ Lcn11 ↘ Mup4 ↘ Lcn14 ↘ Nm1l ↗ Ltf ↘ Obp1a ↘ Lypd3 ↘ Pdzk1ip1 ↘ Mup4 ↘ Pglyrp1 ↘ Nrn1l ↗ Podn ↘ Obp1a ↘ Psapl1 ↘ Pdzk1ip1 ↘ Slc38a3 ↗ Pglyrp1 ↘ Spink5 ↘ Podn ↘ Sprr1a ↘ Psapl1 ↘ Tac2 ↗ Psors1c2 ↘ Tfap2b ↘ Slc38a3 ↘ Spink5 ↘ Tac2 ↘ Tfap2b
↘ Suppl. Tab. 3. List of the genes differentially expressed between two age conditions from the entire incisor transcriptomic analysis. All the genes listed above are differentially expressed with a FC≥2. Genes highlighted in yellow are the genes of interest to conduct further studies on the ageing of the incisor growth region. In black is the list obtained analyzing the raw data, in blue the one obtained analyzing the Sox2-normalized data, and in grey is the list obtained analyzing the Fgf10-normalized data. In bold are the genes found in at least 2 of the 3 analyses

Grooved vs. Smooth

GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN 2010001M09Rik ↗ Aqp9 ↗ Ccr7 ↗ 2210020M01Rik ↗ Arg1 ↗ Cd109 ↗ 2310046A06Rik ↗ Arhgap25 ↗ Cd177 ↗ 2610109H07Rik ↗ Arhgdib ↗ Cd2 ↗ 2610528A11Rik ↗ Arl11 ↗ Cd247 ↗ 5330426P16Rik ↗ Atf3 ↗ Cd300lb ↗ 5830416P10Rik ↗ Atp10a ↗ Cd300ld ↗ A4galt ↗ Atp2a3 ↗ Cd33 ↗ AA467197 ↗ Atp6v0d2 ↗ Cd37 ↗ AB124611 ↗ Atp8b1 ↗ Cd48 ↗ AF251705 ↗ Atp8b4 ↗ Cd68 ↗ AW112010 ↗ B2m ↗ Cd72 ↗ Abp1 ↗ B3galt2 ↗ Cd84 ↗ Acp5 ↗ Batf2 ↗ Cd8a ↗ Adam12 ↗ Bcl3 ↗ Cdh15 ↗ Adamts7 ↗ BC013712 ↗ Cdk6 ↗ Adamtsl3 ↗ Bdkrb1 ↗ Cebpb ↗ Adora3 ↗ Birc3 ↗ Cebpd ↗ Adra2a ↗ Btla ↗ Chad ↗ Adssl1 ↗ C1ra ↗ Chi3l1 ↗ Aifm3 ↘ C1s ↗ Chi3l3 ↗ Akna ↗ C3 ↗ Chka ↘ Aldh1a1 ↗ C5ar1 ↗ Cilp ↗ Aldh3a1 ↗ Cacna1h ↗ Clec12a ↗ Alox12 ↗ Calb2 ↗ Clec4n ↗ Alox5ap ↗ Caskin1 ↗ Clec5a ↗ Amica1 ↗ Casp4 ↗ Cntfr ↘ Angptl4 ↗ Ccl7 ↗ Col13a1 ↗ Antxr2 ↗ Ccl8 ↗ Col16a1 ↗ Apbb1ip ↗ Ccl9 ↗ Col22a1 ↗ Apobr ↗ Ccno ↗ Col3a1 ↗ Apod ↗ Ccr2 ↗ Col5a3 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Col8a2 ↗ Ecm1 ↗ Gbp7 ↗ Comp ↗ Egln3 ↗ Gcnt4 ↗ Coro1a ↗ Ehf ↗ Gda ↗ Coro6 ↘ Elf3 ↗ Gfi1 ↗ Cp ↗ Entpd1 ↗ Gfod1 ↗ Cryab ↗ Epha3 ↗ Gjb2 ↗ Csrnp1 ↗ Ero1l ↗ Gjb3 ↗ Cst7 ↗ Evi2a ↗ Gjb4 ↗ Ctsc ↗ Evi2b ↗ Glipr1 ↗ Ctsk ↗ Eya2 ↗ Glipr2 ↗ Ctss ↗ F10 ↗ Gm11428 ↗ Ctsw ↗ F3 ↗ Gm266 ↗ Cxcl16 ↗ F5 ↗ Gm6682 ↗ Cxcr4 ↗ Fam71f2 ↗ Gngt2 ↗ Cxcr6 ↗ Fam83a ↗ Gpr133 ↗ Cybb ↗ Fas ↗ Gpr171 ↗ Cyp26a1 ↗ Fcer1g ↗ Gpr18 ↗ Cyth4 ↗ Fcrlb ↗ Gpr88 ↗ Cytip ↗ Fes ↗ Gpx3 ↗ D730048J04Rik ↗ Fgf7 ↗ Grap2 ↗ Dcstamp ↗ Fgl2 ↗ Grem1 ↗ Ddit4l ↗ Fgr ↗ Gyk ↗ Ddn ↘ Flt3 ↗ H2-Ab1 ↗ Dennd1c ↗ Fmnl1 ↗ H2-D1 ↗ Dgat2 ↗ Fos ↗ H2-Eb1 ↗ Dock2 ↗ Fut7 ↗ H2-K1 ↗ Dok3 ↗ Fxyd2 ↗ H2-Q10 ↗ Dpt ↗ Fxyd5 ↗ H2-Q4 ↗ Dram1 ↗ Fyb ↗ H6pd ↗ Dtx4 ↗ Gabrb3 ↗ Hand2 ↘ Dusp1 ↗ Galr2 ↗ Hapln4 ↗ E2f8 ↗ Gdf10 ↗ Hk3 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Hmha1 ↗ Irf4 ↗ Ly9 ↗ Hrh2 ↗ Isg20 ↗ Lyz2 ↗ Htra4 ↗ Itgae ↗ Mal ↗ Hvcn1 ↗ Itgal ↗ Map3k8 ↗ Ibsp ↗ Itgax ↗ Mepe ↗ Icam1 ↗ Itgb2 ↗ Mmp10 ↗ Ifi47 ↗ Itgb3 ↗ Mmp12 ↗ Ifitm5 ↗ Jdp2 ↗ Mmp13 ↗ Ifitm6 ↗ Junb ↗ Mmp25 ↗ Igfbp4 ↗ Klf2 ↗ Mmp9 ↗ Igfbp7 ↗ Klk1 ↗ Mpo ↗ Igj ↗ Klrk1 ↗ Ms4a4d ↗ Igsf6 ↗ Krt23 ↗ Mst1r ↗ Igtp ↗ Krt7 ↗ Mt3 ↗ Ikzf1 ↗ Laptm5 ↗ Muc1 ↗ Il10ra ↗ Lat ↗ Muc6 ↘ Il13ra2 ↗ Lat2 ↗ Mxd1 ↗ Il17f ↗ Lck ↗ Myo1f ↗ Il18r1 ↗ Lcp1 ↗ Myo1g ↗ Il1f9 ↗ Lcp2 ↗ Naip2 ↗ Il1r1 ↗ Lilrb3 ↗ Napsa ↗ Il1rap ↗ Litaf ↗ Ncf1 ↗ Il1rl1 ↗ Lpl ↗ Ncf4 ↗ Il1rn ↗ Lrg1 ↗ Nckap1l ↗ Il27ra ↗ Lrrc25 ↗ Neurl3 ↗ Il2rg ↗ Lrrc32 ↗ Nfam1 ↗ Il4ra ↗ Lrrc55 ↗ Nfe2 ↗ Il6 ↗ Lsp1 ↗ Nfkb2 ↗ Inpp5d ↗ Ltbp2 ↗ Nfkbie ↗ Irak2 ↗ Ly6a ↗ Nfkbiz ↗ Irak3 ↗ Ly6e ↗ Ngp ↗ Irf1 ↗ Ly6i ↗ Nlrc5 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Nmb ↗ Plcb2 ↗ S100a8 ↗ Nnmt ↗ Plcg2 ↗ S100a9 ↗ Nod2 ↗ Pmaip1 ↗ Samhd1 ↗ Nptx1 ↗ Podn ↗ Sash3 ↗ Npy ↗ Podnl1 ↗ Sele ↗ Ntn1 ↗ Ppp1r16b ↗ Selp ↗ Oas3 ↗ Ppp1r3d ↗ Selplg ↗ Oasl1 ↗ Prss22 ↗ Sema4a ↗ Ocstamp ↗ Prtn3 ↗ Sept1 ↗ Orm1 ↗ Psca ↗ Serpinb1a ↗ Oscar ↗ Psd4 ↗ Serpine1 ↗ Otop2 ↘ Psmb8 ↗ Serpine2 ↗ P4ha3 ↗ Psmb9 ↗ Serping1 ↗ Padi2 ↗ Pth1r ↗ Sfpi1 ↗ Pappa2 ↗ Ptpn7 ↗ Sfrp2 ↗ Parp10 ↗ Ptprc ↗ Sfrp4 ↗ Parvg ↗ Rab20 ↗ Sgms2 ↗ Pax3 ↗ Rab32 ↗ Sh2d3c ↗ Pcsk6 ↗ Rac2 ↗ Shc2 ↗ Pde1b ↗ Ramp1 ↗ Shisa3 ↗ Pdzk1ip1 ↗ Rasal3 ↗ Siglece ↗ Pfkfb3 ↗ Rasgrp1 ↗ Six2 ↗ Pglyrp4 ↗ Rasgrp4 ↗ Sla ↗ Pi16 ↗ Rbp4 ↗ Slamf7 ↗ Pik3ap1 ↗ Rel ↗ Slamf8 ↗ Pik3cg ↗ Relb ↗ Slc16a3 ↗ Pik3r5 ↗ Retnlg ↗ Slc2a1 ↗ Pilra ↗ Rimklb ↘ Slc2a3 ↗ Pim1 ↗ Rin3 ↗ Slc36a2 ↗ Pion ↗ Rinl ↗ Slc37a2 ↗ Pla2g7 ↗ Rsad2 ↗ Slc39a4 ↗ Plau ↗ S100a4 ↗ Slc45a3 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Slc5a1 ↗ Tnfrsf1b ↗ 2010001M09Rik ↗ Slfn8 ↗ Tnfrsf26 ↗ 2210020M01Rik ↗ Soat2 ↗ Tnfsf11 ↗ 2310046A06Rik ↗ Socs3 ↗ Tnfsf14 ↗ 2610109H07Rik ↗ Sp100 ↗ Tnfsf8 ↗ 2610528A11Rik ↗ Spink5 ↗ Tnfsf9 ↗ 2700038G22Rik ↘ Spn ↗ Tnip1 ↗ 5330426P16Rik ↗ Spon1 ↗ Traf1 ↗ 5830416P10Rik ↗ Spp1 ↗ Treml2 ↗ 6330406I15Rik ↗ Srpx2 ↗ Trim30a ↗ 9230116N13Rik ↘ Sst ↗ Trpm2 ↗ 9430031J16Rik ↗ St6galnac5 ↗ Tyrobp ↗ A2m ↗ Stac3 ↘ U90926 ↗ A4galt ↗ Stambpl1 ↗ Ubd ↗ A530013C23Rik ↗ Stat4 ↗ Upk1b ↗ A930013F10Rik ↘ Steap4 ↗ Upp1 ↗ AA467197 ↗ Stk17b ↗ Vegfc ↗ AB124611 ↗ Susd2 ↗ Was ↗ AF251705 ↗ Svep1 ↗ Wisp2 ↗ AW112010 ↗ Syk ↗ Wnt2b ↗ Abp1 ↗ Tac2 ↘ Xylt1 ↗ Acp5 ↗ Tap1 ↗ Zbp1 ↗ Acsl1 ↗ Thbs2 ↗ Zc3h12a ↗ Actr3b ↗ Thy1 ↗ Adam12 ↗ Tlr2 ↗ Adamtsl3 ↗ Tlr4 ↗ Adcy1 ↗ Tm4sf19 ↗ Adra2a ↗ Tmem119 ↗ Adssl1 ↗ Tmem154 ↗ Agt ↗ Tmprss11bnl ↗ Aifm3 ↗ Tnfaip8l2 ↗ Akna ↗ Tnfrsf11b ↗ Aldh1a1 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Aldh3a1 ↗ C1s ↗ Cdk6 ↗ Alox12 ↗ C3 ↗ Cebpd ↗ Alox5ap ↗ C5ar1 ↗ Celsr3 ↗ Angptl4 ↗ Cacna1h ↗ Chad ↗ Ankk1 ↗ Calb2 ↗ Chi3l1 ↗ Antxr2 ↗ Caskin1 ↘ Chi3l3 ↗ Apbb1ip ↗ Casp4 ↗ Chka ↘ Apobr ↗ Ccl17 ↗ Chst7 ↗ Apod ↗ Ccl7 ↗ Cilp ↗ Apol6 ↗ Ccl8 ↗ Clca4 ↗ Arg1 ↗ Ccl9 ↗ Clec12a ↗ Arhgap25 ↗ Ccno ↗ Clec4n ↗ Arhgap9 ↗ Ccr2 ↗ Clec5a ↗ Arhgdib ↗ Ccr7 ↗ Clec7a ↗ Arl11 ↗ Cd109 ↗ Cntfr ↘ Atf3 ↗ Cd177 ↗ Col13a1 ↗ Atp10a ↗ Cd247 ↗ Col16a1 ↗ Atp2a3 ↗ Cd28 ↗ Col22a1 ↗ Atp6v0d2 ↗ Cd300lb ↗ Col3a1 ↗ Atp8b1 ↗ Cd300ld ↗ Col5a3 ↗ Atp8b4 ↗ Cd33 ↗ Col8a2 ↗ B2m ↗ Cd37 ↗ Comp ↗ B3galt2 ↗ Cd48 ↗ Coro1a ↗ B430306N03Rik ↗ Cd6 ↗ Coro6 ↘ Bace2 ↗ Cd68 ↗ Cp ↗ Batf2 ↗ Cd72 ↗ Cryab ↗ Bcl3 ↗ Cd80 ↗ Cst7 ↗ Bdkrb1 ↗ Cd84 ↗ Ctsc ↗ Birc3 ↗ Cd8a ↗ Ctsk ↗ Btk ↗ Cd96 ↗ Ctss ↗ Btla ↗ Cdc42ep2 ↗ Ctsw ↗ C1ra ↗ Cdh15 ↗ Cxcl16 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Cxcr4 ↗ Evi2a ↗ Gjb3 ↗ Cxcr6 ↗ Evi2b ↗ Gjb4 ↗ Cyba ↗ Eya2 ↗ Glipr1 ↗ Cybb ↗ F10 ↗ Glipr2 ↗ Cyp26a1 ↗ F3 ↗ Gm11428 ↗ Cyp2f2 ↗ F5 ↗ Gm266 ↗ Cyth4 ↗ Fam25c ↗ Gm6682 ↗ Cytip ↗ Fam26f ↗ Gmip ↗ D730048J04Rik ↗ Fam83a ↗ Gngt2 ↗ Dcstamp ↗ Fas ↗ Gpr132 ↗ Ddit4l ↗ Fcer1g ↗ Gpr133 ↗ Ddn ↗ Fcgr4 ↗ Gpr171 ↗ Dennd1c ↗ Fcrlb ↗ Gpr18 ↗ Dgat2 ↗ Fes ↗ Gpr88 ↗ Dnahc6 ↗ Fgf7 ↗ Gpr97 ↗ Dock2 ↗ Fgl2 ↗ Gprc5d ↗ Dok3 ↗ Fgr ↗ Gpx3 ↗ Dpt ↗ Flt3 ↗ Grap2 ↗ Dram1 ↗ Fmnl1 ↗ Grem1 ↗ Dtx4 ↗ Fos ↗ Gsx1 ↘ Dusp4 ↗ Fxyd2 ↗ Gyk ↗ Dusp5 ↗ Fxyd5 ↗ H2-Ab1 ↗ E2f8 ↗ Fyb ↗ H2-D1 ↗ Ecm1 ↗ Gabrb3 ↗ H2-Eb1 ↗ Eepd1 ↗ Galr2 ↗ H2-K1 ↗ Egln3 ↗ Gbp7 ↗ H2-Q10 ↗ Ehf ↗ Gcnt4 ↗ H2-Q4 ↗ Emid1 ↗ Gda ↗ H2-T24 ↗ Entpd1 ↗ Gfi1 ↗ H6pd ↗ Epha3 ↗ Gfod1 ↗ Hand2 ↘ Ephb2 ↗ Gfra4 ↗ Hapln4 ↗ Ero1l ↗ Gjb2 ↗ Havcr2 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Hk3 ↗ Ipcef1 ↗ Litaf ↗ Hmha1 ↗ Irak2 ↗ Lpl ↗ Hrh2 ↗ Irak3 ↗ Lrg1 ↗ Htra4 ↗ Irf1 ↗ Lrrc25 ↗ Hvcn1 ↗ Irf4 ↗ Lrrc32 ↗ Ibsp ↗ Irf5 ↗ Lrrc55 ↗ Icam1 ↗ Isg15 ↗ Lsp1 ↗ Ifi27l2a ↗ Isg20 ↗ Ltbp2 ↗ Ifi44 ↗ Islr2 ↗ Ly6a ↗ Ifi47 ↗ Itgae ↗ Ly6e ↗ Ifitm5 ↗ Itgal ↗ Ly9 ↗ Ifitm6 ↗ Itgam ↗ Lyz2 ↗ Igfbp4 ↗ Itgax ↗ Mal ↗ Igfbp7 ↗ Itgb2 ↗ Map3k8 ↗ Igj ↗ Itgb3 ↗ Mepe ↗ Igsf6 ↗ Itgbl1 ↗ Mfsd4 ↗ Ikzf1 ↗ Itk ↗ Mmp12 ↗ Il10ra ↗ Jdp2 ↗ Mmp13 ↗ Il13ra2 ↗ Junb ↗ Mmp25 ↗ Il1f9 ↗ Klf2 ↗ Mmp8 ↗ Il1r1 ↗ Klrk1 ↗ Mmp9 ↗ Il1rap ↗ Krt1 ↗ Mpo ↗ Il1rl1 ↗ Krt7 ↗ Ms4a4d ↗ Il1rn ↗ Laptm5 ↗ Mst1r ↗ Il21r ↗ Lat ↗ Mt3 ↗ Il27ra ↗ Lat2 ↗ Muc1 ↗ Il2rg ↗ Layn ↗ Mxd1 ↗ Il34 ↗ Lck ↗ Myo1f ↗ Il4ra ↗ Lcp1 ↗ Myo1g ↗ Il6 ↗ Lcp2 ↗ Naip2 ↗ Il7r ↗ Lilrb3 ↗ Napsa ↗ Inpp5d ↗ Liph ↗ Ncf1 ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Ncf4 ↗ Pcsk6 ↗ Pth1r ↗ Nckap1l ↗ Pde1b ↗ Ptpn7 ↗ Neurl3 ↗ Pdzk1ip1 ↗ Ptprc ↗ Nfam1 ↗ Pf4 ↗ Ptprcap ↗ Nfkb2 ↗ Pfkfb3 ↗ Rab20 ↗ Nfkbie ↗ Pglyrp4 ↗ Rab32 ↗ Nfkbiz ↗ Pi16 ↗ Rac2 ↗ Ngp ↗ Pik3ap1 ↗ Ramp1 ↗ Nkg7 ↗ Pik3cg ↗ Rarres1 ↗ Nlrc5 ↗ Pik3r5 ↗ Rasal3 ↗ Nlrp12 ↗ Pilra ↗ Rasgrp1 ↗ Nmb ↗ Pim1 ↗ Rasgrp4 ↗ Nnmt ↗ Pion ↗ Rbp4 ↗ Nod2 ↗ Pla1a ↗ Rel ↗ Nptx1 ↗ Pla2g7 ↗ Relb ↗ Npy ↗ Plau ↗ Retnlg ↗ Nsg2 ↘ Plcb2 ↗ Rimklb ↗ Ntn1 ↗ Plcb4 ↗ Rin3 ↗ Oas2 ↗ Plcg2 ↗ Rinl ↗ Oas3 ↗ Pmaip1 ↗ Rnf149 ↗ Oasl1 ↗ Podn ↗ Rsad2 ↗ Ocstamp ↗ Podnl1 ↗ S100a4 ↗ Orm1 ↗ Pou2f2 ↗ S100a8 ↗ Oscar ↗ Ppp1r16b ↗ S100a9 ↗ Otop2 ↗ Ppp1r3d ↗ Samhd1 ↗ P4ha3 ↗ Procr ↗ Sash3 ↗ Padi2 ↗ Prss22 ↗ Sbno2 ↗ Padi4 ↗ Prtn3 ↗ Scnn1a ↗ Pappa2 ↗ Psca ↗ Scrt1 ↗ Parp10 ↗ Psd4 ↗ Sele ↗ Parvg ↗ Psmb8 ↗ Selp ↗ Pax3 ↗ Psmb9 ↗ Selplg ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Sema4a ↗ Socs3 ↗ Tnfrsf1b ↗ Sema6b ↗ Sp100 ↗ Tnfrsf26 ↗ 37135 ↗ Spink5 ↗ Tnfsf11 ↗ Serpine1 ↗ Spn ↗ Tnfsf9 ↗ Serpine2 ↗ Spon1 ↗ Tnip1 ↗ Serping1 ↗ Spp1 ↗ Traf1 ↗ Sfpi1 ↗ Srpx2 ↗ Treml2 ↗ Sfrp2 ↗ Sst ↗ Trim30a ↗ Sfrp4 ↗ St6galnac5 ↗ Trpm2 ↗ Sgca ↗ Stac3 ↘ Tyrobp ↗ Sgms2 ↗ Stambpl1 ↗ Upk1b ↗ Sh2b2 ↗ Stat4 ↗ Upp1 ↗ Sh2d2a ↗ Steap4 ↗ Ush1c ↘ Sh2d3c ↗ Stk17b ↗ Vegfc ↗ Shc2 ↗ Susd2 ↗ Vnn1 ↗ Shisa3 ↗ Svep1 ↗ Was ↗ Siglece ↗ Syk ↗ Wisp2 ↗ Six2 ↗ Tac2 ↘ Wnt2b ↗ Sla ↗ Tacstd2 ↗ Xylt1 ↗ Slamf7 ↗ Tap1 ↗ Zbp1 ↗ Slamf8 ↗ Thbs2 ↗ Zc3h12a ↗ Slc12a1 ↘ Thy1 ↗ Slc16a3 ↗ Tiparp ↗ Slc28a3 ↗ Tlr2 ↗ Slc2a1 ↗ Tlr4 ↗ Slc36a2 ↗ Tm4sf19 ↗ Slc37a2 ↗ Tmem119 ↗ Slc39a4 ↗ Tmem154 ↗ Slc45a3 ↘ Tmem173 ↗ Slc5a1 ↗ Tmprss11bnl ↗ Slfn8 ↗ Tnfaip8l2 ↗ Soat2 ↗ Tnfrsf11b ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN 2610109H07Rik ↗ Ctsc ↗ Igj ↗ AA467197 ↗ Ctsk ↗ Il10ra ↗ AW112010 ↗ Ctss ↗ Il1f9 ↗ Acp5 ↗ Cxcl1 ↗ Il1rn ↗ Adam12 ↗ Cxcr4 ↗ Il2rb ↗ Alox5ap ↗ Cybb ↗ Il2rg ↗ Antxr2 ↗ Cyp26a1 ↘ Il4ra ↗ Apod ↗ Cytip ↗ Irak3 ↗ Arg1 ↗ Dcstamp ↗ Irf1 ↗ Arhgdib ↗ Dock2 ↗ Itgam ↗ Atp6v0d2 ↗ Dusp1 ↗ Itgb2 ↗ B2m ↗ Ecm1 ↗ Itgb3 ↗ Bcl3 ↗ Egln3 ↗ Laptm5 ↗ C3 ↗ F10 ↗ Lcp2 ↗ C5ar1 ↗ Fcer1g ↗ Lilrb3 ↗ Cass4 ↗ Fgl2 ↗ Litaf ↗ Ccl22 ↗ Fgr ↗ Lrg1 ↗ Ccr7 ↗ Fxyd5 ↗ Ltbp2 ↗ Cd109 ↗ Gjb2 ↗ Ly6e ↗ Cd163l1 ↗ Gjb3 ↗ Lyz2 ↗ Cd3e ↗ Gm11428 ↗ Mepe ↗ Cd68 ↗ Gpr35 ↗ Mmp12 ↗ Cebpd ↗ Gpx3 ↗ Mmp13 ↗ Chi3l1 ↗ H2-Ab1 ↗ Mmp3 ↗ Col13a1 ↗ H2-Eb1 ↗ Mmp9 ↗ Col3a1 ↗ H2-K1 ↗ Mt3 ↗ Col8a2 ↗ Hvcn1 ↗ Mxd1 ↗ Comp ↗ Ibsp ↗ Myo1f ↗ Coro1a ↗ Icam1 ↗ Myo1g ↗ Coro6 ↘ Icos ↗ Ncf1 ↗ Cp ↗ Ifitm5 ↗ Ncf4 ↗ Csf3 ↗ Igfbp4 ↗ Nfkbiz ↗ Grooved vs. Smooth GENE UP/DOWN GENE UP/DOWN GENE UP/DOWN Oscar ↗ Selplg ↗ Stk17b ↗ Pappa2 ↗ Serpine1 ↗ Stx11 ↗ Pcsk6 ↗ Sfpi1 ↗ Syk ↗ Pla2g7 ↗ Sfrp2 ↗ Tac2 ↘ Prss27 ↗ Sfrp4 ↗ Tarm1 ↗ Psmb8 ↗ Sgms2 ↗ Thy1 ↗ Ptafr ↗ Sh2d2a ↗ Tlr2 ↗ Ptprc ↗ Slc16a3 ↗ Tmem119 ↗ Rac2 ↗ Slc2a6 ↗ Tnfrsf1b ↗ Ramp1 ↗ Slc37a2 ↗ Traf1 ↗ S100a4 ↗ Slfn1 ↗ Tyrobp ↗ S100a8 ↗ Socs3 ↗ Upp1 ↗ S100a9 ↗ Spn ↗ Wfdc2 ↗ Samhd1 ↗ Spon1 ↗ Zap70 ↗ Samsn1 ↗ Spp1 ↗ Zc3h12a ↗ Sash3 ↗ Srpx2 ↗ ↗ Selp ↗ Stac3 ↘ ↗
Suppl. Tab. 4. List of the genes differentially expressed in the presence or the absence of aplastic groove. All the genes listed above are differentially expressed with a FC≥2. Genes highlighted in yellow are the genes of interest to conduct further studies on the ageing of the incisor growth region. In black is the list obtained analyzing the raw data, in blue the one obtained analyzing the Sox2-normalized data, and in grey is the list obtained analyzing the Fgf10-normalized data. In bold are the genes found in at least 2 of the 3 analyses.

DISCUSSION

Our study aimed at performing a wide analysis to monitor age-related changes in gene expression profiles in the tooth growth region, and in the entire incisor, as well as gathering large-scale data on the setting of the most frequent age-related abnormality seen on the mouse upper incisors. From theses wide analyses, we want to address the precise role of candidate genes we found involved

Relevance of both strategies

The analysis of the growth region transcription profile has a theoretical advantage over the one of entire incisor transcription profile because we avoid the risk of diluting the signal from the stem cell niches. However, the LCM performed to micro-dissect the growth region has two main biases. Sectioning prior to the micro-dissection has to be done on fresh tissues, without any treatment that may impact the quality of the RNAs contained in the tissue. The incisor enamel is a very hard matrix (mineralized at 96%), and even when sectioning the growth region, with is the less mineralized part of the tooth, it is difficult to ensure reproductive slicing. The second bias is that the micro-dissection has to be performed without a complete histological staining, and thus without a clear demarcation of the area of interest.

The direct consequence on the biological relevance of the dataset is the risk of not having the same ratio of epithelial and mesenchymal cells sampled. Along with this risk is the possibility that we did remove the entire structure, leading to a potentially incomplete picture of the region in terms of the represented cell types. The third bias was that given the small quantity of RNA obtained by sample, we were forced to combine different samples of the same cohort, leading to the introduction of a supplementary source of heterogeneity.

Although there is no clear strain-dependent or age-dependent relationship between the B6-6, B6-12, B6-18, CD1-6, CD1-12, and CD1-18 samples, actors involved in the maintenance of the stem cell niches (namely Sox2 and Fgf10) are not differentially expressed.

The relatively small list of genes which expression profile varies along with ageing might be attributed to the small quantities extracted given the size of the biological samples. The use of two normalization methods here helped us to address part of the variability, and to remain cautious with the differentially expressed genes found.

As stated above, working with entire incisors might dilute the information from the growth region, but it also allows a better reproducibility working on replicates. Lastly, it gives us access to the transcriptomic information from the transit-amplifying and the differentiation zones, which are successively found when following the growth direction of the incisor. This explains why more differentially expressed genes were retrieved from our second analysis, but can also constitute an explanation for the non-congruence of the differentially expressed gene lists between the two experiments. Overall, the approach working on entire incisors seems to be suitable to our biological question, and especially to the molecular understanding of the groove phenotype.

What is our top 20 genes of interest?

Several groups of genes stood out during our analyses. The first one is composed of factors involved in hair development. Fgf7, also known as keratinocyte growth factor, as well as Krt7 and Krt6a, which are coding for structural proteins that are found in hair, skin and nails make up this group (Gu and Coulombe, 2007;Sandilands et al., 2013). Fgf7 is normally expressed just around the epithelial cervical loops (Bei and Maas, 1998). Because one of the age-related defects we highlighted is the growth of hair out of the incisor dental cavity, it would be interesting to address changes in cell fate in the ageing niche. Special attention was paid during the dissection to avoid the contamination of the dissected sample and we consider such contamination by tissues outside of the studied area unlikely. All three genes might be implicated in the differentiation of hair from the upper incisor growth region. One last gene could be added to this group because of its action in cell fate commitment: Gfi1 (Shroyer et al., 2005). Further studies on the possible link between these differentially expressed gene likely to help specify the molecular network driving cell fate.

Two genes have been associated with various mineralization defects: Mmp20 with amelogenesis imperfecta, and Dspp with dentinogenesis imperfecta (Sreenath et al., 2003;Wright et al., 2009). Both diseases consist in the accumulation of defective traits of the enamel and the dentin, and notably in their color. Changes in color and in the enamel mineralization rate might be similar to the dysplasias seen in mice from six months on.

Another metalloprotease has been found differentially expressed: Mmp25. It has been mainly

been associated with the development and the closure of the secondary palate (Brown and Nazarali, 2010), and since the incisor growth region is not in direct contact with the secondary palate, this indicates an unexpected gene detection.

Wnt signaling pathway is an actor of tooth development (Liu et al., 2008). As we said earlier, the mesenchyme in-between the cervical loops is competent to Wnt signaling pathway, which help regulating the development of the correct number of incisors (Fujimori et al., 2010). Although epithelial do express some Wnt genes, it has been proven that the Wnt/β-catenin signaling pathway is not directly regulating epithelial stem cells (Suomalainen and Thesleff, 2010). Recently, the role of this pathway in tissue renewal and regeneration has also been reviewed (Clevers et al., 2014). Sost is a member of Wnt signaling pathway, as well as Grem1, Sfrp4, Wisp2, and Wnt2b ( Xu et al., 2000;Semënov et al., 2005;Bovolenta et al., 2008;Katoh and Katoh, 2009;Katoh, 2011). Interestingly, the list of differentially expressed genes encompasses one of the rare cases where the expression level is higher in older mice (Wnt2b).

Among the genes highlighted in the study are also several mediators of cell migration or proliferation. This is the case of Hand2 (specific to mesenchymal cell proliferation), Glipr2, Podn, and Cxcr4 (Murdoch, 2000;Xiong et al. 2009, 2;Huang et al., 2013;Hutter et al., 2013). Hrh2, Ibsp and Spp1 can also be clustered in this category because of their involvement in epithelial cell morphogenesis, and in biomineral tissue development (Kobayashi et al., 2000;Vaes et al., 2006). Since the continuous growth is a very dynamic process that implies a tightly regulated balance between cell proliferation and migration, the information provided by those actors could shed some light on the ageing of these cellular processes.

Lastly, our interest has also been drawn to Cyp26a1, which is coding for an enzyme acting notably on the retinoic acid pathway (Tahayato et al., 2003). Retinoic acid pathway has been documented to act on tooth development [START_REF] Jones | The effect of retinoic acid on mouse mandibular molar development in vitro, using alkaline phosphatase as a molecular indicator of differentiation[END_REF]. Cyp26a1 expression is promoted in the grooved incisor group, and it would be interesting to characterize its role in the development of aplastic grooves.

Correlation between the presence of a groove and a strong immune response

With 135 genes involved in immune response being up-regulated in grooved incisor group compared to similar age mice with smooth incisors, the correlation between the immune response and the development of a groove in old mice is highly marked. It is impossible right now to decipher which one causes the other, but we could imagine that the absence of any enamel within the aplastic grooves facilitates infections.

Mammalian organisms can develop a two-phase immune response (Janeway et al., 2001). The first step is called innate immune response and implicates mostly members of the NF-κB pathway (Hatada et al., 2000). In our gene list, we identified many chemokines, complement genes, metalloproteases, interleukins, and histo-incompatibility complex genes.

To a lesser extent, we also identified some actors of the adaptive immune response (mainly Toll-like receptors and interferons) (Iwasaki and Medzhitov, 2004). This orientates the type of inflammation to a process that would be causes by bacterial exposure.

Interestingly, when Robins and Rowlatt described age-related abnormalities in laboratory mice (1971), they noticed a great hair impaction in the gingival tissues, and hypothesized that the accumulation of hair from grooming may be causing cyst. In their model, the cysts would be the cause of the physical disruption of the incisor growth region, leading to the formation of enamel grooves. We tend to be more cautious, firstly because no exaggerated hair impaction was detected in the mouse colony we monitored. Our transcriptomic analyses confirm the existence of an immune response, but the histological analyses we performed when characterizing the upper incisor ageing did not reveal traces of inflammation or infiltration.

CONCLUDING REMARKS

We have provided here a frame to further investigate incisor stem cell niche ageing. By addressing the questions of the experimental design and by trying to overcome the biases we encounter from sample preparation to library generation, we have tackled one key process: cellular ageing. Genes become differentially expressed as the mice grow old, and important transcriptomic changes are associated with the occurrence of age-related defects, such as an important immune response and expression of genes involved in non-dental ectodermic appendages.

B.4 CONCLUSIONS

The interest for incisors in research is multifaceted. From an evolutionary point of view, these teeth are an indicator of the level of diversity displayed in this mammalian order, and attest to the acquisition of a complex morphology. Moreover, because of their continuous growth properties, incisors are models in cell biology and genetics.

The existing variability of rodent incisors is especially visible when looking at the color and the ornamentation of their vestibular side. From this statement, I investigated changes that are set at the scale of the animal lifetime by addressing the occurrence of age-related changes in the mouse upper incisors.

As the mouse grows older than six months, its upper incisors indeed start to display mineralization defects, as well as signs of stem cell niche disruption. Among the abnormal phenotypes seen in the mineralized tissues, the occurrence of a vestibular groove is observed the most frequently. The comparative monitoring of two mouse line (CD1 "swiss" and C57Bl/6N) showed that inbred mice display more severe phenotypes, their enamel groove often becoming completely aplastic.

From the very precise growth pattern of the rodent incisors, which originates from the presence of stem cell niches fueling the organ renewal throughout the lifespan of the animal, we can deduct that abnormalities are set within the growth region. Histological analyses showed that the stem cell niches can be directly disrupted, and the defects can be traced on serial sections.

Because stem cell ageing is the major concern for the homeostasis of numerous organs, and more globally for organisms, we are eager to understand its molecular determinants. In order to further investigate the plausible age-related changes in gene expression profiles, I have set the basis for molecular studies by making use of the powerful NGS technologies.

Several biological functions appear down-regulated in ageing incisor, for example cell proliferation and migration, or mineralization process. On the contrary, innate immune response appears strongly promoted in the grooved incisors when compared to the smooth ones. Dental stem cells may not be the most commonly used model in cell biology, but their morphological and molecular disruption will greatly contribute to the understanding of stem cell ageing.

CONCLUSION & PERSPECTIVES

he study of the FGF-activated ERK-MAPK pathway reaffirmed the crucial role of the Rsk gene family in craniofacial, and especially in dental development. We showed that Rsk2 -/Y mice only moderately recapitulate the human Coffin-Lowry syndrome. The comparison of their dental phenotype to the one of Spry1 -/-, Spry2 -/-, and Spry4 -/-mice allowed us to specify on the potential evolutionary role of the pathway during murine rodent evolution. The atavisms found in the mutant mice studied in the course of this project confirm the importance of the whole pathway and precise the role of some of its members. Indeed, the occurrence of supernumerary teeth in three of these mutants highlights that these genes could have played a role in the reduction of the dental formula during the transition from pre-muroid to muroid rodents. This reduction is likely to have remodeled the mesial part of the 1 st molars as indicated by the numerous mesial rearrangements in the mutant mice. On the distal part of the 1 st molars, only the Sprouty gene family could have been an actor of the evolutionary reduction of the posterocone cusps. Bringing Tg(KRT14-Spry4)#Krum mice to the picture, we conclude that delay in forming a proper cap-staged tooth germ impacts tooth mineralization, tooth proportions and cusp arrangement, highlighting the role of FGF signaling pathway in the correct timing of tooth developmental sequence.

Results found in the study of the ERK-MAPK pathway highlight what has been stated

for decades: despite an extremely reduced and specialized dentition, mice are a good model to investigate evolutionary changes in the shape and tooth number determination. The FGFactivated ERK-MAPK pathway is not the 1 st pathway that has been intensively studied from T human pathology to global tooth evolution: the work of the Eda pathway reached a similar extent [START_REF] Grüneberg | The molars of the tabby mouse, and a test of the "single-active Xchromosome"hypothesis[END_REF][START_REF] Miller | The dentitions of tabby and crinkled mice[END_REF]Charles et al., 2009;[START_REF] Sadier | The ectodysplasin pathway: from diseases to adaptations[END_REF].

Of course, the evolutionary role of the FGF-activated ERK-MAPK pathway could always be further specified using gain of function or conditional mutants, but the most interesting outcome of this research is linked with the development of new tools to study gene regulatory networks. Indeed, we entered an era of data computing in which we could and should link the great amount of tooth development actors worked on so far. All the phenotypic information scientists collected in the dental evo-devo field could only help us improve the drawing of these networks.

Working on Tg(KRT14-Spry4)#Krum specimens also addressed the question of the sequential formation of mouse molar cusps. This is a more fundamental work, but it could be applied to the variations of cusp pattern seen in humans [START_REF] Scott | The anthropology of modern human teeth: dental morphology and its variation in recent human populations[END_REF] or even linked with abnormalities observed in patients. Our understanding of the phenomenon would be greatly improved by expanding research on the formation of enamel knots. To date, enamel knots are characterized by some specific gene expression patterns and by the non-proliferating state (Vaahtokari et al., 1996;Jernvall et al., 1998;[START_REF] Laurikkala | Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot[END_REF]. Focusing on the cell biology level, we could set up lineage tracing experiment at the scale of tooth development in order to understand the specification of the primary enamel knot, and of the secondary enamel knots in forming a tooth. All the rodents do not display the same molar crown shape, and a comparative approach including rodents with various dental morphologies seems adequate to address this diversity.

The review of incisor diversity in rodent has brought out the potential use of these teeth in the study of shape evolution, as well as unresolved acquisition of a grooved enamel surface and a differential coloration of the enamel layer. Interestingly, the grooved enamel surface is found in WT mice, but only in specimens aged of at least six months. It appears that some age-related defects appear in both ageing mice and ageing sand rats. The exhaustive list of these age-related defects set in the specimens studied encompasses an increased number of enamel mineralization defects, the development of enamel grooves (one to two, aplastic or not), the growth of ectopic hair from the tooth socket, an increased occurrence of dens in dentes and odontomas, as well as the development of dentin grooves. The enamel grooves are the most frequent defect, being noticed in 50% of the monitored mice. The realization of an in vivo monitoring of mouse upper incisors ageing over approximately one year and a half not only provided an accurate list of the defects, but it also specified their timing of occurrence from six months on. Monitoring the morphometrical properties of the incisors also showed a constant increase in the mesio-distal length of the upper incisors. A further insight into the histology of the growth region in the ageing specimens revealed the occurrence of abnormal folding and proliferation events that affect the specific organization of the incisor stem cell niches. In an attempt to address the global molecular disruption of the ageing stem cell niches, we unraveled the differential expression of some genes in our region of interest, including markers of hair development, regulators of cell proliferation, differentiation and migration, as well as members of the Wnt signaling pathway. The comparison of gene expression profiles between smooth incisors and grooved incisors of the same age revealed an important immune response in the case of grooved incisors.

In performing this research, we provided a starting point for the study of the impact of ageing in the dental stem cell niches. The ultimate goal of every scientist working on the ageing of a specific stem cell niche is to be able to fully characterize and eventually counteract those mechanisms regardless of the type of niche. We should firstly focus on this very same model, and investigate the specific role of our genes of interest. The joint study of these gene expression patterns and of genetically engineered animals will help us understand the extent of the age-related modifications. We will eventually aim at setting an in vitro model of dental stem cell ageing from all the in vivo data collected.

The question of cell fate could be addressed in trying to characterize the hair growing out of the incisor socket. Due to the difficulty of spotting this rare phenotype on ageing mice, we could start with the investigation on mutant murine lines displaying ectopic hair growth from their tooth sockets, before going back to the ageing model. Given the resemblance between the incisor stem cell niches and the hair bulge, we are confident that investigations on the differentially expressed genes belonging to the Wnt pathway will also help us understand how the fate of dental stem cells can be changed in the course of ageing.

Lastly, in order to address the link between the occurrence of aplastic enamel groove and the large innate immune response, we plan on scrutinizing the upper incisor phenotype of immunodeficient mouse models. The current availability of several lines deficient for various actors of this type of immune response will help us understanding the causality of the phenomenon and the possible predominance of some actors of the NF-κB immune response.

An interesting complementary approach would consist in performing a new RNA-seq experiment with 18-month-old mice displaying non-aplastic enamel groove, and to compare the extent of the immune response.

By addressing two levels of the rodent dentition dynamics, I have worked on classic dental evo-devo, but I have also stated the potential of the mouse incisors to become a new model of stem cell ageing. My contributions hopefully proved that we still have a lot to learn from the mouse, and that the golden age of tooth evo-devo is not over yet.

  FIG. 1: Simplified view of dental evolution in mammals FIG. 2: X-ray microtomographic view of a mouse skull

Fig. 2 :Fig. 3 :Fig. 6 :Fig. 5 :Fig. 2 :

 23652 Fig. 2: Molar tooth proportions in the Spry1 -/-, Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice Fig. 3: Abnormal phenotype in the Spry1 -/-, Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice Fig. 4: ST phenotype and surface in the Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice Suppl. Fig. 1: Details of some shared mutant features in the Rsk2 -/Y mice Fig. 5: Mutant dentition recapitulates the evolution of murine tooth characters

FIG. 1 :

 1 FIG. 1: Simplified view of dental evolution in mammals. Phylogenies are simplified from Bininda-Edmonds et al. 2007. (A) Phylogeny and maximal dental formula in mammalian infraclasses. (B) Phylogeny and maximal dental formula of the various eutherian superorders. (C) Euarchontoglire order Phylogeny and maximal dental formula in the various euarchontoglire orders. Irectangles: incisors, Ctriangles: canines, Pblack circles: premolars, Mwhite circles: molars.

FIG. 2 :

 2 FIG. 2: X-ray microtomographic view of a mouse skull. Incisors (yellow) and molars (pink) are separated by a toothless gap called diastema. Scale bar: 0.5 mm.

  FIG. A.1: Mouse first lower molar development. The dental germ undergoes successive histomorphogenetical modifications leading to formation of a mineralized matrixes-covered tooth ready to erupt. D Ep: dental epithelium; D Mes: dental mesenchyme; C Mes: condensed mesenchyme; D P: dental papilla; pEK: primary enamel knot; sEKs: secondary enamel knots.

  advantage of the availability of mutant mice for upstream regulator of RTKs(SPRY, yellow in FIG. A.2), and for a downstream effector kinase (RSK2, orange in FIG.A.2) to compare their adult dental phenotypes. By examining the presence of common phenotypes, I address plausible evolutionary role of the cascade in building the dentition of modern mice. I start with a conventional approach on knockout (KO) mice to characterize Rsk2 action on craniofacial development, and more specifically on tooth formation. Then, I compare the Spry1, Spry2, and Spry4 mutant molar phenotypes to the one of Rsk2 -/Y mice. In order to perfect our understanding of the role of ERK-MAPK cascade members, I lastly investigate molar phenotype in transgenic mice over-expressing the Spry4 gene in the oral epithelium.

FIG. A. 2 :

 2 FIG. A.2: Simplified view of the ERK-MAPK cascade. Growth factors binding their receptor (RTK) trigger the activation of the phosphorylation cascade down to the final activation of the pathway effectors. This pathway regulates most of the cellular processes, including gene expression, cell cycle progression, cell motility and metabolism. Our interest is drawn to the Sprouty family (Spry) of RTK inhibitors (yellow), and to one of the effector kinase RSK2 (orange).

FIG. A. 3 :

 3 FIG. A.3: Variation of molar shape and number in Rsk2 -/Y and Rsk1,2,3 null mice analyzed by X-ray microtomography. All molar rows are similarly oriented (top corresponds to mesial, and left to lingual side). On the left are WT molars, other rows are mutant molars as indicated. Arrowheads point to ST; dottend ellipses show the reduction of the mesial-most affected cusp. Numbers refer to specimen ID. Scale bar: 0.7 mm.

FIG. A. 5 :

 5 FIG. A.5: Variation of M 1 root slope in Rsk2 -/Y mice. Vestibular side of the molars is facing. On the top is a WT M 1 , then the gradient of slope is presented in the mutants. Dotted lines help visualizing the inclination from the tip of the first chevron to the basis of the main root. Scale bar: 0.5mm

Fig. 2 :

 2 Fig. 2: Molar tooth proportions in the Spry1 -/-, Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice. First molars are coloured in green, second molars in pink, and third molars in blue. Filled forms represent the WT for each background, squares represent the Spry1 -/-mutants, Spry2 -/- mutants without (blanked) or with (dot) ST, Spry4 -/-mutants without (blanked) or with (dot) ST and Rsk2 -/Y mutants without (blanked) or with (dot) ST.

Fig. 3 :

 3 Fig. 3: Abnormal phenotype in the Spry1 -/-, Spry2 -/-, Spry4 -/-and Rsk2 -/Y mutant mice. (A) WT dental rows; (B) Spry1 -/-dental rows; (C) Spry2 -/-dental rows; (D) Spry4 -/-dental rows; (E) Rsk2 -/Y dental rows. Coloured arrowheads correspond to the features displayed in the Fig. 2.

Fig. 4 :

 4 Fig. 4: ST phenotype and surface in Spry2 -/-, Spry4 -/-and Rsk2 -/-mutants. ST range from small rounded monocuspid teeth to large multicuspid teeth. Right column indicates the mean surface in all the ST-displaying mutant mice, error bars represent the standard deviation. V and M respectively point towards the vestibular and mesial directions. Scale bar: 0.4mm.

Suppl. Fig. 1 :

 1 Details of some shared mutant features depicted in the Rsk2 -/Y mice. (A) The disconnection of the lingual-most cusp of the M1 1 st chevron. (B) Variation of the M1 mesial root inclination in mutant mice. Note the straightening of the slope in Rsk2 -/-mutants.

  ST occurs very similarly to what is observed in Sprouty and Rsk2 mutants. Thus, modulation of the Wnt signaling pathway upstream of the ERK-MAPK pathway could mimic the effect of loss of Sprouty gene or Rsk2 function, which might explain phenotypic similarities in the occurence of ST in both pathways.

Fig. 5 :

 5 Fig. 5: Mutant dentition recapitulates the evolution of murine tooth characters. (A) The dental phenotype arising from the loss of function of Spry2, Spry4 and Rsk2 genes mimics the reduction of tooth number in muroid rodent evolution. (B) Spry2 -/-M 1 mimics Progonomys debruijni M 1 .

  in rodents. These genes could have played a pivotal role in the reduction of the dental formula and in the rearrangement of the mesial parts of the first molars during the transition from premuroid to muroid rodents prior to 45 Ma. They also migt have acted in the disappearance of the posterocone and in the connections of the lingual cusps to the chevrons during the transition from basal murines to Mus auctor between 9.2 and 6.5 Ma. Both morphological transformations happened independently, separated by about 35 Ma, suggesting that the premolar loss might have been triggered by Rsk2 gene dosage augmentation. The posterocone would then have been triggered by an increase in Sprouty gene expression. Finally, an interesting issue remains open, which is the potential role of Sprouty or Fgf genes in the morphogenesis of the connections between lingual cusps and central cusps of the M 1

Fig. 1 :

 1 Fig. 1: Most prevalent phenotypes in the K14-Spry4 mice compared to the phenotype of their WT littermates. M (mesial), V (vestibular), and L (lingual) refer to the orientation of the molar row. (A) Upper WT molar row, occlusal (left) and mesial (right) views. (B) Upper transgenic molar row, occlusal (left) and mesial (right) views. Black arrowhead points to the irregularities of the tooth neck, pink arrowhead to small enamel pits or irregularities. (C) Lower WT molar row, occlusal (left), vestibular (center), and lingual (right) views. Note the very smooth tooth neck displayed at the limit of the enamel layer. (D) Lower transgenic molar row, occlusal (left), vestibular (center), and lingual (right) views. Orange arrowhead points the reduction of the mesio-vestibular cusp, while blue arrowheads show the reduction of the distal-most cusps of both M 1 and M 2 . On the vestibular and lingual view, note the indentations of the tooth neck. Scale bar: 0.75 mm.

Fig. 2 :

 2 Fig. 2: Additional cusp defects in the transgenic M 1 and M 1 . (A) Upper WT molar row phenotype. (B) From left to right, transgenic M 1 also display a connection between the two lingual-most cusps (4%), a disconnection of the lingual cusp of the 1 st chevron (2%), a partial vestibular crest (2%), and a splitted and individualized 1 st chevron central cusp (2%). (C) Lower WT molar row phenotype. (D)From left to right, transgenic M 1 also display a ectopic connection of the distal-most part of the tooth (4%), a bigger mesio-lingual cusp (4%), a splitted mesio-lingual cusp (2%), and cingular cusps (2%). Scale bar: 0.75 mm. M and L arrows respectively points towards the mesial and lingual directions.

Fig. 3 :

 3 Fig. 3: Comparison of the molar surface in the transgenic to the molar surface of their WT littermates. The tooth surface was measured in occlusal views. WT tooth are depicted with filled hexagons, transgenic molars with blanked hexagons; error bars represent the measurement standard deviation. (A) Measurements in the upper rows show that transgenic M 1 and M 2 display a smaller surface (t-test, p-value<0.05). (B) Measurements in the lower rows show all three transgenic molars have a surface smaller than the WT one (t-test, p-value<0.05).

Fig. 4 :

 4 Fig. 4: Comparison of transgenic and WT first molar histo-morphogenesis from the bud to prior to the bell stage. CL invagination (black arrowheads) and pEK (dotted circles) formation are delayed in the transgenic cohort. First picture row is displayed at the same scale than in E13.0 WT M 1 ; second picture row is displayed at the same scale than in E14.5 WT M 1 . Scale bars: 100 µm.

Fig. 5 :

 5 Fig. 5: Abnormal shape of the transgenic molar germ cervical loops. (A) WT embryo at E16.5. (B) Transgenic embryo at E16.5. Note the irregularly large aspect of the cervical loop. (C) Close up on the defect. Scale bars: 100 µm.

Fig. 6 :

 6 Fig. 6: Shh gene expression variations in the lower tooth germ. Note the apparent weaker staining in the lower transgenic genes, likely due to the smaller region identified as pEK. Dotted lines help visualizing the tooth germ.

Fig. 8 :

 8 Fig. 8: Fusion of the upper and lower jaws in the transgenic mice. The penetrance of the phenotype is 8%. (A) Milder fusion phenotype, with the maxillary and mandibular shelves still visible. (B) More severe fusion with a proliferating event sealing the upper and lower jaws together. Scale bars: 500 µm.

  FIG. B.1: Development of the mouse lower incisor. The dental germ undergoes successive histomorphogenetic stages, similar to what is observed during molar development, except that the incisor germ rotates towards distal direction. D Ep: dental epithelium; D Mes: dental mesenchyme; C Mes: condensed mesenchyme; D P: dental papilla; pEK: primary enamel knot.

  FIG. B.2:Incisors are asymmetric teeth.. Enamel is only displayed on the vestibular (or labial) side. The asymmetry results from histological asymmetry between the labial cervical loop (laCL) and the lingual cervical loop (liCL). Epithelial stem cells (ESCs) are located in the laCL, next to the loosely arranged stellate reticulum cells (SRCs), while mesenchymal stem cells (MSCs) are more dispersed within the pulp. Ameloblast lineage (AMB lin.) derives from ESCs which will differentiate as the exit the niche. Odontoblast lineage (ODB lin.) arises from the MSCs found in the pulp, more precisely from the neurovascular bundle.

  muroid rodents tend to have an insertion mesially to the upper molar row (FIG. B.3A), whereas some subterranean rodents like the Bathyergidae have their growth region set more distally (FIG. B.3B, C).

FIG. B. 3 :

 3 FIG. B.3: Differential insertion of the upper incisors in various rodent species. Sagittal view of 3 rodent skulls, with the upper incisors colored in red, and the growth region pointed with a white arrowhead. (A) Psammomys obesus (Myomorpha, Muridae, Gerbillinae; specimen from the livestock of Deakin University, Australia); (B) Bathyergus suillus (Hystricomorpha, Hystricognathi, Bathyergidae, Bathyerginae; MNHN ); (C) Heliophobius argentiocinereus (Hystricomorpha, Hystricognathi, Bathyergidae, Bathyerginae; MNHN). Scale bars: 5mm. For the MNHN specimen collection number, please refer to the annex 4.

  FIG. B.4 (following page): Presence of enamel groove across the rodent phylogeny. (A)Schematized phylogeny of the three most representative rodent suborders (Sciuromorpha, Myomorpha and Hystricomorpha) at the subfamily level (adapted fromFabre et al., 2012). It shows in red the convergent occurrence of the grooved enamel surface. (B) Rodent phylogeny at the suborder level (fromFabre et al., 2012). (C-G) Inventary of the presence of smooth enamel surface (smooth), grooved enamel surface (grooved), and unscrutinized genera (unknown). Numbers in brackets indicate the total number of genera within the rodent suborders, or within the rodent families.

  FIG. B.5: Diversity of grooved incisor phenotype among the order Rodentia. (A) Gerbillus sp. displays one central groove whereas Psammomys obesus does not display any. (B) Geomys bulleri (left) and Geomys bursarius (left) do not display the same number of grooves. (C) In the Otomyinae subfamily, the groove displayed in not localized at the same place (from left to right Myotomys unisulcatus, Paratomys brantsii, and Otomys irroratus). (D) The vestibular grooves are never aplastic, as it is well visible in Thryonomys swinderianus. Arrowheads point out the presence of grooves, the white dotted line draws the limit between the enamel and dentin layers. Scale bars: 1 mm (except in D 2.5 mm). All specimens except Psammomys obesus and Thryonomys swinderianus (personal collections) come from the MNHN (Paris), the NHL (London) or the MRAC (Tervuren) collections. For collection numbers, please refer to annex 4.

(

  FIG. B.6: Inter-and intra-specific variation in the color of the rodent incisor enamel. (A) Interspecific color variation in the suborder Myomorpha, from left to right: Psammomys obesus, Microtus arvalis, Eliurus minor, Nesomys rufus. Scale bars: 1 mm. (B) Intra-specific color variation between two Mus musculus littermates, left is 22 days-old, right is 54 days-old. (C) Intra-individual color variation between the upper and the lower incisors of a same Mus musculus specimen. Scale bars: 0.5 mm. Mus musculus specimens were collected from our livestock, other specimens (except Psammomys obesus) come from the MNHN collections. For collection numbers, please refer to annex 4.

Fig. 1 .

 1 Fig. 1. Mouse incisors are asymmetric teeth that continuously grow throughout the lifespan of the animal. (A) Incisors are asymmetric teeth displaying enamel only on their vestibular (labial) side. The growth region is circled, and usually spotted by the presence of two cervical loops on the labial (laCL) and the lingual (liCL) sides of the tooth. (B) Epithelial stem cells (ESCs) are contained within the stellate reticulum population (SRCs) in the laCL. As the ESCs move out from the niche, they differentiate into the ameloblast lineage (AMB lin.). Mesenchymal stem cells (MSCs) are dispersed within the pulp, and they form the odontoblast lineage (ODB lin.) as they differentiate.

Fig. 2 .

 2 Fig. 2. Mouse upper incisors display various age-related abnormalities from about six months of age onwards. (A) Phenotypic aspect of the visible part of upper incisors in the mice that were monitored in vivo. From left to right, incisors display: a smooth enamel surface, an enamel dysplasia (green patch), a groove in the enamel layer, a hair growing out of the tooth cavity, a large and aplastic groove, two aplastic grooves. The last two defects were only found in inbred mice. Scale bars: 250 µm. (B) Chronology of the occurrence of the defects. 1/3 of the mice end up with a combination of defects, which are most frequently defects on the enamel layer.

Fig. 4 .

 4 Fig. 4. X-ray microtomography allows a better characterization of the possible age-related abnormalities found in mice. Histogram shows the proportion of incisors showing defects in each age group. Colors correspond to the frame of the phenotypes pictured on the right. Grey: normal incisor; dark purple: one enamel groove; orange: enamel dysplasia; blue: dentin fold; yellow: combination of defects; light purple: two enamel grooves. The proportion of defects increases with the age of the considered group, and the enamel groove are here also the most frequent defect displayed. m. month; I. incisive. Scale bar: 500 µm.

Fig. 5 .

 5 Fig. 5. Psammomys obesus also displays age-related abnormalities on its upper incisors. (A) 3month-old specimen displaying a normal upper incisor phenotype. (B) 12-month-old specimen displaying signs of growth region disruption (red arrowheads), a probable odontoma (black arrowhead), and a dentin groove (yellow arrowhead). (C) 18-month old specimen dislaying a dentin groove (yellow arrowhead). Scale bar: 3 mm.

Fig. 6 .

 6 Fig. 6. Upper incisor elongation continuously progresses as the mouse ages. Filled circles represent the evolution of the mesio-distal length of C57Bl/6N depending on the mouse age. Blanked circles show the same progression in CD1 "swiss" mice. Scale bars display the measurement standard deviation. The elongation follows a linear regression in both genetic backgrounds. C57Bl/6N: y=0.00167x+0.53455 (R²=0.9642, p-val<2.2e-16); CD1 "swiss": y=0.001279x+0.542190 (R²=0.9698, p-val<2.2e-16).

Fig. 7 .

 7 Fig. 7. A fold in the ameloblast layer causes enamel groove. (A, B) Transverse sections in the right and left incisors of a 18-months old C57Bl/6N mouse. (C, D) Transverse sections of the same specimen, only more proximally towards the growth region. All the sections were stained using Masson's trichrome (in red is the enamel, in blue is dentin). Scale bars: 100 µm.

Fig. 8 .

 8 Fig. 8. Abnormalities of tissue organization can be detected in the growth region and the ameloblast layer even when the protruding phenotype appears normal. (A) 12 month-old C57Bl/6N specimen displaying an hyperplastic laCL (yellow dotted curve) and a fold affecting the odontoblast, dentin and ameloblast layers (yellow arrowheads). (B). 6-month-old CD1 "swiss" specimen displaying an abnormal duplication of the ameloblast layer originatin from an abnormal cell proliferation (bottom right hand corner). The normal histology is framed in green. Scale bars: 100 µm.

Fig. 1 .

 1 Fig. 1. Schematized organization of the upper incisor growth region, and age-related defects. (A) Incisors are asymmetric teeth displaying enamel only on their vestibular (labial) side. The growth region is circled. (B) The growth region is constituted of the labial and lingual cervical loops (laCL and liCL), and the in-between pulp. Epithelial stem cells (ESCs) are contained within the laCL. As the ESCs move out from the niche, they differentiate into the ameloblast lineage (AMB lin.). Mesenchymal stem cells (MSCs) are dispersed within the pulp, and they form the odontoblast lineage (ODB lin.) as they differentiate. (C) Mouse upper incisors are affected by ageing. Defects are set in the growth region an become visible as the tooth grows. From left to right are displayed a normal (smooth) incisor, one displaying enamel dysplasia (orange), one displaying a vestibular enamel groove (purple), one with a hair growing out of the tooth socket (gray), and one aplastic vestibular groove (no enamel left). Scale bars: 0.5 mm

Fig. 4 .

 4 Fig. 4. [following page] Genetic variation and differentially expressed genes depending on the specimen age and phenotype. These data refer to the entire incisor transcriptomic analysis. Each column is organized similarly with (A) PCA plot of the variability in the specimen gene expression profile, the percentage indicate the contribution of the principal axes of variation; (B) heatmaps and dendrogram indicating the relationships between the samples based on their gene expression; (C) correlation graphs where black dots represent the filtered genes, pink dots represent the differentially expressed genes, and blue dots represent those with a FC≥2. 1 st column is the raw data, 2 nd the Sox2-normalized data, and 3 rd the Fgf10-normalized data.

  This part of my work focuses on a study realized in collaboration with a team of the Developmental Biology and Stem Cells department from the IGBMC (Illkirch, France) published earlier this year in PLoS One (see annex 2: Laugel-Haushalter et al., 2014). The

study aims at addressing the role of Rsk2 gene in mouse craniofacial development in order to question the relevance of Rsk2 -/Y and Rsk1,2,3 null mutants as models for the X-linked craniofacial disorder called Coffin-Lowry syndrome (OMIM #303600). Mutations in RSK2

Fig. 1: Dental character matrix. [

	previous page] On the first column are displayed WT
	upper (top) and lower (bottom) molar rows, with coloured arrowheads pointing to the
	localization of the most frequent defects seen in the 4 mutant backgrounds. Each character is
	listed with the occurrence frequency in all the mutant mice observed. (c1-10) are defects of
	the upper mutant postcanine teeth. (c1) is the occurrence of a ST; (c2-4) are M 1 1 st chevron
	defects, respectively lingual cusp disconnection, straight mesial cusp and absence of the
	vestibular cusp; (c5) is the presence of an extra lingual cusp between the 1 st and 2 nd
	chevrons; (c6) is the disconnection of the lingual cusp of the M 1 2 nd chevron; (c7) is the
	occurrence of a lingual crest; (c8) is the occurrence of an extra distal cusp; (c9) is the
	disconnection of the mesio-lingual cusp of the M2; (c10) is the connection of distal cusps.
	(c11-17) are defects of the lower mutant postcanine teeth. (c11) is the occurrence of a ST;
	c(12) is the absence of the mesio-vestibular cusp; (c13) is the display of very symmetric
	mesial-most cusps; (c14) is the split of the mesio-vestibular cusp; (c15) is the occurrence of
	an extra mesial cusp; (c16) is the strong disconnection of the mesial cusps of the M 1 ; (c17) is the abnormal connection of the 2 nd and 3 rd lophs of the M 2 .

  Devo of Vertebrate Dentition, Institute of Functional Genomics of Lyon, ENS de Lyon, CNRS UMR 5242, University of Lyon 1, 46 allée d'Italie, 69364 Lyon cedex 07, France Sequencing platform, Institute of Functional Genomics of Lyon, ENS de Lyon, CNRS UMR 5242, University of Lyon, 46 allée d'Italie, 69364 Lyon cedex 07, France § Corresponding author: laurent.viriot@ens-lyon.fr

	1 §
	1 : Evo-

2

:

Number of differentially expressed genes in the growth region transcriptomic analysis.

  Mmp20 has being associated with amelogenesis imperfecta and could play a role in the age-related increase in mineralization defects. Sost is a member of Wnt signaling pathway. Incisor mesenchyme is competent to Wnt signaling Each sub-table specifies the comparison realized, as well as the number of genes of interest revealed working on raw, Sox2-normalized or Fgf10-normalized count tables. DE: differentially expressed, FC≥2: fold change superior or equal to 2.

	Growth region analysis		Growth region analysis
	12 months vs. 6 months	DE+FC≥2	18 months vs. 6 months	DE+FC≥2
	raw data	17	raw data	21
	Sox2-norm.	0	Sox2-norm.	0
	Fgf10-norm.	0	Fgf10-norm.	4
	Tab. 1.

Number of differentially expressed genes in the entire incisor transcriptomic analysis.

  Each sub-table specifies the comparison realized, as well as the number of genes of interest revealed working on raw, Sox2-normalized or Fgf10-normalized count tables. DE: differentially expressed, FC≥2: fold change superior or equal to 2.

			Entire incisor
	18 months vs. 6 months	DE+FC≥2	grooved vs. smooth	DE+FC≥2
	raw data	28	raw data	442
	Sox2-norm.	31	Sox2-norm.	502
	Fgf10-norm.	6	Fgf10-norm.	145
	Tab. 2.

months vs. 6 months

ACKNOLEDGEMENTS

We thank E. Maître for her help in the morphological description of the teeth. We thank A. Hanauer, V. Fraulob and B. Schuhbaur for providing, expanding and genotyping respectively the Rsk2 -/Y mouse colony, and Nicolas Strauli and Sarah Alto for help with the Sprouty mouse breeding. We are grateful to A. Langer and P. Dolle. We also thank V. Lazzari for his critical reading of the manuscript. This work was funded by grants from the University of Strasbourg, the Hôpitaux Universitaires de Strasbourg (API, 2009-2014, Development of the oral cavity: from gene to clinical phenotype in Human to ABZ), the IFRO, and EU-ERDF A27 (Oro-dental manifestations of rare diseases to ABZ), the ENS de Lyon (to LV), and the NIH (R01 DE021420 to ODK). Support for this research was also provided by the RMT-TMO Offensive Sciences initiative, INTERREG IV Upper Rhine program http://www.genosmile.eu. CONTRIBUTIONS L.V and O.D.K conceived the project. P.M and C.C designed the analyses, which P.M and C.C performed. P.T conducted the ESRF imaging of Sprouty mouse dental rows. V.L.H and A.B-Z provided the Rsk2 -/Y mice, while A.J bred the Sprouty null mice. P.M, C.C, A.B-Z, O.D.K and L.V wrote the manuscript.

ACKNOLEDGEMENTS

We thank Nicolas Strauli and Sarah Alto for help with the Sprouty mouse breeding. We also thank S. Pantalacci for sharing Shh plasmid with us. We are grateful to J. Richman for critical discussions over the project. This work was funded by grants from the ENS de Lyon (to LV), and the NIH (R01 DE021420 to ODK).

ACKNOWLEDGEMENTS

We are grateful to the PBES animal facility (UMS3444, Lyon, France), and to B. Pain for insightful discussion on the topic. This research was funded by a grant from the ENS de Lyon (to LV and CC).

ACKNOLEDGEMENTS

We are grateful to F. Chatonnet for computing a DESeq2 function for the IGFL use. We thank M. Sémon for insightful discussions about the classical analysis workflow. We also would like to thank C. Rey, from the ProfileXpert platform (ViroScan 3D, www.viroscan3D.com, Lyon, France), for performing the LCM experiment, as well as B. Jost and M. Phillips from the IGBMC Microarray and Sequencing platform (member of France Génomique program).

Disruption of the incisor stem cell niches in ageing mice

Pauline Marangoni, Ludivine Bertonnier-Brouty, Cyril Charles, Laurent Viriot 1* Team Evo-Devo of Vertebrate Dentition, Institute of Functional Genomics of Lyon, ENS de Lyon, CNRS UMR 5242, University of Lyon 1, 46 allée d'Italie, 69364 Lyon cedex 07, France § Corresponding author: laurent.viriot@ens-lyon.fr ABSTRACT Rodent incisors continuously grow throughout their lifespan due to the presence of adult stem cells at their proximal end. The addition of new tissues from this growth region balances the abrasion generated when the animal gnaws. Although the surface of mouse incisors is normally smooth, upper incisors tend to display irregularities originating from the growth region as the animal gets older than its life expectancy in the wild. Performing in vivo monitoring on CD1 "swiss" mice and C57Bl/6N mice, we provided a live phenotypic analysis of some of these defects. X-ray microtomography, morphometrical tools and histological staining were used to assess incisor morphology. We detected mineralized tissue anomalies from about six months of age, most frequently vestibular enamel grooves. The abnormality proportion only increases from that point onwards. Upper incisors were shown to continuously widen as mice age. A fold in the enamel secreting layer was linked with enamel irregularities, and hyperplastic epithelial stem cell niche with modifications of incisor geometry. Our work provides an accurate description of the abnormality setting up to address age-related stem cell niche disruption.

KEY WORDS

Stem cell ageing, incisor, groove, mouse, sand rat, chrono-morphology, histology ABSTRACT Stem cell ageing is a growing concern because of the negative impact it has on organ homeostasis. Mouse incisors are an example of continuously growing organ, what is enabled by the presence of several stem cell niches at their proximal end. From six months on, ageing affects the phenotype of the mouse upper incisors only. By developing two complementary transcriptomic approaches, we address the question of the molecular disruption of the ageing stem cell niches. We show that by analyzing gene expression profiles from the microdissected stem cell niches or from the entire incisor, we manage to detect age-related changes in the gene expression levels. The general rule appears to be a global down-regulation of the genes normally expressed in the incisor and in its growth region. We also show by comparing the transcriptome of incisors at the same age, but displaying a smooth or a grooved vestibular surface, that a large immune response is set up in the grooved incisor cohort. Together, these data confirm that age-related transcriptomic changes are detectable. Further examination of selected candidate genes based on their known implication in various developmental processes or signaling pathways will help establishing a causal link between these defects and the phenotypic defects we previously reported.

KEY WORDS

Stem cell ageing, RNA-seq, micro-dissection, enamel groove Annex 3

Control of the incisor number by Sprouty genes