Understanding developmental mechanisms in evolution is crucial to apprehend the diversifi cation of organismal forms [START_REF] Alberch | Ontogenesis and morphological diversifi cation[END_REF][START_REF] Darwin | The origin of species by means of natural selection[END_REF][START_REF] Smith | Developmental constraints and evolution: a perspective from the Mountain Lake conference on development and evolution[END_REF]. In mammals, changes occur during all development phases (prenatal and postnatal). Postnatal growth plays an essential role in the acquisition of the adult shape. During this period, the craniofacial complex undergoes many changes in functional constraint forcing the different tissue to accommodate while adjusting, along the growth and at the adult stage, to a certain level of functional performance. These different developmental interactions respond to several infl uencing factors such as molecular, genetic and cellular processes but also the environment. The latter will play on these interactions, but in different ways between the prenatal and postnatal phases, as gestational environment and environment at birth are different. In this context, taking into account the existing diversity in mammals, variation in time and space of developmental processes within an anatomical context will infl uence the mapping from genotypes to phenotypes [START_REF] Debat | Mapping phenotypes: canalization, plasticity and developmental stability[END_REF][START_REF] Alberch | Developmental constraints in evolutionary processes[END_REF], Salazar-Cuidad et al. 2003[START_REF] Milocco | Is evolution predictable? Quantitative genetics under complex genotype-phenotype maps[END_REF][START_REF] Hallgrimsson | Let's face it-complex traits are just not that simple[END_REF]. Physical constraints undergone by organisms in development could be similar in a certain range of anatomical contexts, and would structure in a comparable way the phenotypic variation by infl uencing the spatialization of underlying developmental processes.

These processes lead to discontinuities and directionalities in the space of shape [START_REF] Alberch | Ontogenesis and morphological diversifi cation[END_REF][START_REF] Gerber | Not all roads can be taken: development induces anisotropic accessibility in morphospace[END_REF][START_REF] Salazar-Ciudad | Why call it developmental bias when it is just development?[END_REF]. They induce biases in the phenotypic outcomes of random mutations [START_REF] Alberch | Developmental constraints in evolutionary processes[END_REF], Halgrimsson et al. 2006[START_REF] Uller | Developmental plasticity and evolutionary explanations[END_REF] and therefore in the evolutionary trajectories [START_REF] Kavanagh | Developmental bias in the evolution of phalanges[END_REF][START_REF] Uller | Developmental bias and evolution: A regulatory network perspective[END_REF]. During mammalian postnatal growth, major biomechanical changes, in response to change in diet (i.e. weaning) and behavior infl uence the spatialization of tissue modeling and remodeling [START_REF] Zelditch | Modularity of the rodent mandible: integrating bones, muscles, and teeth[END_REF][START_REF] Zelditch | Epigenetic interactions: the developmental route to functional integration[END_REF].

Changes in muscle strain, tooth growth, development of sensory organs etc. led to convergences in the developmental processes imposing some directionality in the ontogenetic trajectories despite a diversifi ed anatomical context. Consequently, these changes constrained the evolution of ontog-enies and adult shape variation (Hallgrimsson & Hall 2011). These developmental biases result in some parallelism between developmental and evolutionary trajectories [START_REF] Alberch | Size and shape in ontogeny and phylogeny[END_REF][START_REF] Gould | Ontogeny and Phylogeny[END_REF][START_REF] Webster | Evolutionary modifi cations of ontogeny: heterochrony and beyond[END_REF]. Still, early shape differentiation has a profound effect on the directionality and intensity of shape changes during ontogeny [START_REF] Goswami | Do developmental constraints and high integration limit the evolution of the marsupial oral apparatus?[END_REF][START_REF] Conith | The infl uence of divergent reproductive strategies in shaping modularity and morphological evolution in mammalian jaws[END_REF]. Developmental processes and their degree of freedom infl uence the phenotypic production, questioning the importance of evolution of the ontogenetic trajectories in shaping the diversity of organismal shapes.

For testing the interactions between the developing organism and its environment and their potential evolutionary consequences, Rodentia is a good example of broad diversity in all aspects and thus a study group of choice. It is a very diverse and disparate mammal order, in which changes can be observed on a large scale. The mandible and cranium present a wide variety of forms across this order. Rodents present also wide diversity in diets, behavior and ecology. In addition, it includes model organisms that can be easily reared in laboratory following a precise experimental confi guration to test the effects of diverse sources of variation.

Generalities on the group

The order Rodentia is taxonomically very diverse, with over 2500 living species among 34 families (Fig. 1.2). All along this manuscript, the classifi cation followed is that proposed by Wilson et al. 2016. Rodents form the richest order of mammals in terms of taxa. Indeed, 40% of known mammal species belong to the Rodentia (Lacher et al., 2016). They occur in all continents and most oceanic islands, the most noticeable exception being Antartica. Their size range is very wide, from 3g for the African pigmy mouse (Nannomys) to 60kg for the capybara (Hydrochoerus hydrochaeris).

However, most species are small to medium in size (15g to 15 kg). The majority of rodents have a hairy body, some are fully or partially covered with spines. They have a wide range of ecologies, from arboreal to burrowing, from aquatic to desert, and are found in most environments throughout the world. This multitude of sizes and habitats implies the existence of very diverse diets (frugivorous, herbivorous, insectivorous, omnivorous, granivorous, etc., [START_REF] Nowak | s Mammals of the World[END_REF]. This diversity is closely linked to the morphology of the masticatory apparatus (skull, mandible, muscles).

The fi rst occurrence in the fossil record is described during the Paleocene (-60 to -55 Ma, [START_REF] Hartenberger | Description de la radiation des Rodentia (Mammalia) du Paléocène supérieur au Miocène; incidences phylogénétiques[END_REF][START_REF] Van Itterbeeck | Woodland in a fl uvio-lacustrine environment on the dry Mongolian Plateau during the late Paleocene: Evidence from the mammal bearing Subeng section (Inner Mongolia, PR China)[END_REF]. Principal adaptive radiation of most of the modern families took place in the Paleocene/Eocene, these families were then well established in the Early Oligocene [START_REF] Vianey-Liaud | Possible evolutionary relationships among Eocene and lower Oligocene rodents of Asia, Europe and North America[END_REF].

Craniofacial anatomy

Cranial anatomy

Lower view (Fig. 1.3) -The skull of rodents is generally elongated and broad, with a laterally compressed anterior part. The nasals extend to the anterior part of the snout, spilling outwards through the nasal openings. The premaxillae extend across the median part of the rostrum. They inhabit the incisor alveolus, which extends a little further posteriorly in adult specimens, sometimes to the squamosal (Hydrochaerus). In inferior view, the palatine foramens are located at the contact of the premaxillae and maxillae, and form two large elongated slits. The maxillae extend posteriorly to the contact with the squamosal (Hydrochaerus and Cavia), but in most species end at the pterygoid.

The palatine is most often with parallel lateral margins, but in some cases may be reduced anteriorly, with the rows of teeth converging forward (Spalax and Myocastor). The pterygoids are placed behind the palatine, and bear posteriorly projecting apophyses. They are fused early to the basysphenoid. The tympanic bulla is formed by a bony bulla pierced by the auditory meatus, exiting to the outside of the ear canal. The basioccipital, placed on the posterior part of the skull in inferior view, bears at each of its lateral ends the paraoccipital processes. The supraoccipital has occipital condyles on its lower part.

Superior view (Fig. 1.4) -The frontals are large, with a length of one quarter to one third of the total length of the skull. The parietals are positioned behind the frontals. The interparietal joins the parietals and supraoccipital and is sometimes absent in some burrowing rodents.

Lateral view (Fig. 1.5) -The zygomatic arch includes the maxilla, jugal and squamosal. In addition to containing the inner tips of the incisors, the maxilla supports the jugal teeth (molars and premolars). Its height therefore varies according to the structure of the roots. The maxilla has a strong zygomatic apophysis perforated by the infra-orbital foramen, which varies greatly in size depending on the species but can reach dimensions as large as the orbit. The jugal is positioned laterally to the orbit but can also sometimes extend to its anterior part. The posterior end of the jugal is placed under the zygomatic process of the squamosal. The posterior part of the zygomatic arch consists of the squamosal. f: frontal, i: incisor, iof: infra-orbital formaen, it: interparietal, j: jugal, mtb: mastoid part of the tympanic bulla, mx: maxilla, na: nasal, o: occipital, oc: occipital condyle, ocr: occipital crest, pa: parietal, pmx: premaxilla, poa: paraoccipital apophysis, pta: pterygoid apophysis, zam: zygomatic apophysis of the maxilla, scr: squamosal crest, so, supraoccipital, sq: squamosal, tb: tympanic bulla, zam: zygomatic apophysis of the maxilla, zas: zygomatic apophysis of the squamosal, zw: zygomatic window.

Mandibular anatomy

The mandible is the bone under the skull with which it is articulated. It is composed of two hemimandibles, which are therefore two bones connected by their anterior part (Fig. 1.6). The size and shape of the various mandibular processes vary according to the size and placement of the muscles attached to them. The coronoid process can be very high for example in the genus Ondatra to completely absent in Ctenodactylus [START_REF] Potapova | Morphofunctional transformations of the jaw muscles in rodent evolution[END_REF]. Consequently the curvilinear length of the lunate notch will vary accordingly. The condylar process allows the mandibular articulation in the squamosal and is most often of large size. The angular process, placed posteroinferiorly on the mandible, is very variable in shape and length. The lower incisor and its alveolus extend over a large part of the mandible and may extend to the condylar process. -Hystricognaths, which have a strong angular apophysis that is inwardly projecting, while their condyloid apophysis is outwardly directed.

-Sciurognaths, which, unlike hystricognaths, have an angular process directed outwards and a condyloid process directed inwards.

Tooth anatomy

Rodents have continuously growing incisors with a reinforced enamel band on the anterior surface. The canines are absent, leaving large diastemas separating the incisors from the jugal teeth.

This anatomy is directly related to the gnawing function of the incisors.

Rodents exhibit a high degree of dental diversity, based on tooth pattern and growth habit associated with crown height. There are two main dental formulae: I 1 / 1 , C 0 / 0 , P 0 / 0 , M 3 / 3 and I 1 / 1 , C 0 / 0 , P 2 / 2 , M 3 / 3 (Wilson et al., 2016), in some species, only a fourth premolar persists in the dental row. The pattern on the occlusal surface varies, as does the number of cusps (Fig. 1.8). The four main types of tooth pattern for molars are: bunodont (Mus), lophodont (Chinchilla), lopho-bunodont (Nectomys), prismatic (Lemmus) and loxodont (Hydrochoerus).

Masticatory muscle anatomy

The morphology of the craniofacial complex varies according to the arrangement of the masticatory muscles of rodents. Three skeletal parts are closely related to the arrangement of this muscle complex (zygomasseteric complex): the infraorbital foramen, the zygomatic arch and the mandible.

The zygomasseric complex is a complex of four muscles: the masseter, the temporalis, the pterygoid and the digastricus.

The masseter has two distinct parts: the lateral masseter and the medial masseter. The lateral masseter extends from the lower border of the zygomatic arch to the anterior part of the angular process. It is itself divided into two parts, one anterior and superfi cial, one posterior and deep. The medial masseter attaches to the medial side of the zygomatic arch or to the inner wall of the orbit.

It also attaches to the mandibular bone above the insertion of the lateral masseter. It is divided into two parts, one anterior and one posterior.

The temporalis is smaller than the masseter. It inserts at the level of the squamosal on the occipital crest and in the interorbital region. Its main function is to ensure the closure of the jaw.

The pterygoid separates into an internal and an external branch. The medial branch is short and thick, it attaches to the pterygoid fossa (and sometimes to the orbit) and is inserted behind the medial side of the angular process of the mandible. The lateral is a small muscle extending from the pterygoid area to the medial side of the condylar process.

The digastricus extends from the paraoccipital process to the lower edge of the mandible.

It allows the retraction and lowering of the mandible.

The infraorbital foramen allows the passage of the medial masseter muscle. The shape and size of this foramen is very variable. Depending on the different insertions of the muscles, four infraorbital structures can be found (Brandt 1855, Wood 1965, Fig. 1.9):

-Protogomorph, with a small to medium infraorbital foramen, a reduced masseter and a predominant temporalis.

-Sciuromorph, with a very small infraorbital foramen and a lateral masseter inserted in the anterior part of the orbital arch -Myomorph, which is intermediate between the two previous types. The infra-orbital foramen is narrow at the base and widened in its upper part.

-Hystricomorph, with a highly developed, almost circular infraorbital foramen.

Tissue growth

Bone modeling and remodeling

During life and thus during growth, bone undergoes modeling and remodeling. Bone modeling is defi ned as the formation of bone by osteoblasts on a given surface [START_REF] Allen | Bone modeling and remodeling[END_REF], while bone remodeling is the result of resorption of the bone synchronized with formation (by osteoblasts, Table 1.1). In this manuscript, I will talk about bone remodeling, as it treats of postnatal development and the majority of the primary bone is already present in the organism [START_REF] Lee | A computational analysis of bone formation in the cranial vault using a coupled reaction-diffusion-strain model[END_REF]. Bone is a dynamic tissue [START_REF] Zaidi | Skeletal remodeling in health and disease[END_REF]) that must maintain the stability and integrity of the organ (Proff & Römer 2008). Bone remodeling is an active process by which the bone can change in shape,size and microstruccture in response to physiologic infl uences and mechanical strains maintaining strength and mineral homeostasis [START_REF] Katsimbri | The biology of normal bone remodelling[END_REF]. This process relies on the balance between bone resorption and deposition. These two functions must be coupled in time and space to ensure the stability of bone mass [START_REF] Rucci | Molecular biology of bone remodelling[END_REF]. Bone resorption is performed by osteoclasts, some large multicleated cells [START_REF] Teitelbaum | Genetic regulation of osteoclast development and function[END_REF]. Bone deposition is made by osteoblasts, which are specialized bone-forming cells. They produce bone by synthesis and secretion of type I collagen [START_REF] Katsimbri | The biology of normal bone remodelling[END_REF]. Bone strength, size and shape depends on various factors as bone mass, geometry and microstructure that varies during life. During this thesis, the molecular aspects of this process will not be discussed. However, it is an important factor to keep in mind as these mechanisms are directly involved in shape changes during growth.

Masseter muscle growth

The muscle fi ber is produced from myoblasts, which are stem cells that differentiate into myotubes and then into muscle fi bers, made of myosin and actin. During the development of the masseter muscle, the differentiation of myoblasts occurs the most actively between the 13 th embryonic day and birth for mice. The activation of muscles fi bers in mouse, begins at the 15 th embryonic day and continue after weaning [START_REF] Yamane | Expression of myogenic regulatory factors during the development of mouse tongue striated muscle[END_REF][START_REF] Yamane | Embryonic and postnatal development of masticatory and tongue muscles[END_REF][START_REF] Saito | Effects of diet consistency on the expression of insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins during the development of rat masseter muscle soon after weaning[END_REF].

Around weaning, the membrane proteins responsible of muscle fi bers synthesis are progressively eliminated [START_REF] Saito | Effects of diet consistency on the myosin heavy chain mRNAs of rat masseter muscle during postnatal development[END_REF], Yamane et al. 2004[START_REF] Yamane | Embryonic and postnatal development of masticatory and tongue muscles[END_REF].

Tooth growth

Growth can be continuous or fi nite, so molars can be brachyodont, mesodont, hypsodont to hypselodont (Lazzari et al. 2015, Fig. 1.10). The growth of these teeth mechanically constrains the growth of other tissues as it implies a different volume occupied by the teeth in the mandible and skull. This is why this factor is important to consider during growth and certainly plays a role in bone remodeling. It is therefore integrated into this work, particularly in Chapter 2.

Objectives

The skull is a highly integrated structure, architecturally complex, as it is composed of many skeletal elements, and functionally, as it is involved in various tasks essential to the organism.

At the same time, and somewhat paradoxically, this unit is highly evolvable and presents a great diversity of forms. The basis of craniofacial shape variation and its control are as much, related to the additive effects of genes as to their epigenetic and context-specifi c interactions during development. In this manuscript, epigenetics is defi ned by the "Sum of the genetic and nongenetic factors acting upon cells to control selectively the gene expression that produces development and evolution" (Hallgrimson & Hall 2011). These epigenetic interactions during growth will respond to mechanical and other stimuli between the ossifi cation centers, the tissues and organs making up the head. In particular, these interactions control the spatialization and intensity of bone remodeling in response to other tissues strain. They will thus compensate and coordinate the growth of the different tissues and organs in order to acquire and/or maintain certain functions, such as the occlusion between the upper and lower jaws. These epigenetic interactions are thus essential to the normal development of the skeleton in general and the skull in particular. By responding to changes in forces and movements, they will be a potential driver of microevolutionary changes (and thus macroevolutionary changes) at the morphological level favoring adaptive directions of variation and generating new functional covariations between traits. Despite this central role, the importance of these interactions in the expression of inter-specifi c differences and in the longer term in the dynamics of clades remains poorly understood. Analysis of the tempo of adult disparity acquisition during ontogeny is a key element in understanding the differential fi lling of the shape space by clades. The level of disparity within a clade generally appears to be already present in the young and the evolution of ontogenetic trajectories would little affect this level of disparity. The early postnatal stages would therefore appear to be more fl exible than the late stages, which are subject to greater functional constraints. Thus, in sciuridae, a relatively conservative clade, the disparity in mandibular shape seems to be generated prenatally and maintained by compensation for ontogenetic variation (Zelditch et al. 2016). On the other hand, rodents as a very diversifi ed group in term of species, dental types, muscles architectures, diet, ecologies etc… is likely to present an increase in mandibular and cranial disparity between juvenile and adult changes, it is therefore crucial to pay attention to ontogenetic changes. These changes appear to be concomitant with tooth eruption and the acquisition of mastication; radically different between clades. A disparity established prenatally would suggest a secondary, compensatory role for epigenetic processes during growth.

On the contrary, the post-weaning establishment of inter-specifi c differences would imply a central role for epigenetic processes in the expression of specifi c and potentially the integration within the variation of the craniofacial complex of ecological and behavioral differences between taxa.

To begin this manuscript (Fig 1.11), I will take a broad view to obtain a macroevolutionary picture of postnatal mandibular development in rodents, in order to map general growth patterns between and within clades (Chapter 2). Subsequently, I will focus on the different developmental processes undergone by the craniofacial complex, this time on a fi ner taxonomic and temporal scale. For this purpose, I will describe the different experimental techniques envisaged and implemented (Chapter 3). Postnatal development will then be described in three model species (Chapter 4). Finally, in these three species, the role of mechanical constraints during growth will be questioned using variations in diet (Chapter 5). Chapter 2 -Development and divergence of the mandible shape at a macroevolutionary scale

Rodents represent a very diverse order and therefore harbour a multitude of different morphologies and life history traits. This mammalian order is therefore a very good playground for studying macroevolutionary divergence. Many studies deal with morphological diversity by taking into account only adult specimens [START_REF] Hautier | Patterns of covariation in the masticatory apparatus of hystricognathous rodents: implications for evolution and diversifi cation[END_REF][START_REF] Wilson | Commonalities and evolutionary divergences of mandible shape ontogenies in rodents[END_REF][START_REF] Zelditch | Relationships of diversity, disparity, and their evolutionary rates in squirrels (Sciuridae)[END_REF]. The starting point of this axis is to compare postnatal developmental trajectories in a larger number of taxa, in order to reveal patterns of growth at a macroevolutionary scale. I therefore sought to collect both adult and juvenile (pre-weaning) specimens in order to compare the disparity at these two stages to observe developmental dynamics on a large scale.

Two strategies were available to me. The fi rst would have been to restrict the sampling to a suborder and sample a large number of individuals per species. This strategy implies sampling more stages per species, in terms of age (adding subadults, adults, ageing adults), allometry, and more individuals per stage in order to establish more accurate trajectories. It could have been applied to the Myomorpha, as the individuals are numerous in collections because they come from trapping and breeding. The other strategy is the one I have chosen, i.e. to try to cover as much of the rodent phylogenetic tree as possible by sampling more species but fewer individuals per taxon.

By taking this approach, it is possible to cover a greater diversity of life history traits. To provide information on morphological changes occurring between birth and adulthood, juvenile stage was taken as youngest as possible to make sure to be before weaning. In the same time, it ensures the covering of an important portion of postnatal development. As weaning is both dietary and behavioral, it is spread out in different ways in different species, so it is important to cover as much of the development as possible. The main diffi culty of this approach is to fi nd juvenile specimens in good condition. Indeed, many collection specimens are either already sub-adult, too fragile or deteriorated (due to incomplete ossifi cation at a young age). Due to the diffi culty of obtaining juvenile specimens in good condition, it was not possible to include Castorimorpha and Anomalomorpha in this study. The same applies to Sciuromorpha, for which only two species could be sampled, but these allow us to compare our results with those of Zelditch et al. (2016), whose study focused on Sciuridae. A lagomorph, Oryctlagus cuniculus, was used to root the phylogenetic tree of the sampled species (Fig. 2.1).

Classically, life history traits taken into account in morphological studies are locomotion [START_REF] Hautier | Patterns of covariation in the masticatory apparatus of hystricognathous rodents: implications for evolution and diversifi cation[END_REF], Munoz 2021[START_REF] Samuels | Skeletal indicators of locomotor adaptations in living and extinct rodents[END_REF], habitat [START_REF] Camargo | Cranial shape predicts arboreal activity of Sigmodontinae rodents[END_REF], Durao et al. 2019) and more specifi cally for studies concerning the craniofacial complex, diet [START_REF] Hautier | Patterns of covariation in the masticatory apparatus of hystricognathous rodents: implications for evolution and diversifi cation[END_REF][START_REF] Samuels | Cranial morphology and dietary habits of rodents[END_REF]. This seems logical given the importance of the diet, since the cranium and mandible must allow the animal to eat. However, it seemed appropriate to me, when dealing with the development of the adult form of these bones, to take into account factors inherent to growth itself. In other words, I wished to assess the traits surrounding the fi rst stages of life, and more specifi cally to test the impact of gestation duration, weaning age and number of litters per year for each species. Indeed, these factors are usually considered in single species studies (behavioral and immunity studies, Wolff & Sherman 2008) but rarely in broad taxonomic studies. Chapter 3 -Ontogenetic changes and plasticity across Myomorpha: experimental design

In the previous chapter, ontogenetic shape change of the mandible was observed at a large taxonomic scale but with only two stages. As the ontogenetic trajectories are non-linear, it was therefore essential to provide information on the change in morphology during growth at a fi ner scale. The most feasible solution was therefore to breed on model organisms whose traits are well known in the literature. The vast majority of the laboratory-raised rodents are Myomorpha (except Guinea pig), which was also a very positive point, as these are the ones in which I was able to observe the greatest magnitude of change between the two stages studied. This is why three species of Myomorpha over the four classic Myomorpha models (the fourth being Rat) were selected in order to describe the morphological changes between birth and adulthood (sexual maturity) through weaning. Thus, the following chapters of this manuscript will focus on a much smaller taxonomic scale but at a fi ner resolution along the growth path to be placed, this time, from the point of view of developmental processes.

As development involves various tissues, I have sought to follow bone growth, muscle growth and morphological change. It was therefore necessary to test various ways of monitoring tissue changes. For bone, I tried to follow the growth quantitatively with fl uorochrome markings and qualitatively with the observation of bone formation/resorption structures by SEM observation. For muscle, I tried to follow the evolution of the quantity of proteins composing the muscle fi ber (actin/myosin) by Rt-qPCR. Finally, morphological changes were monitored by geometric morphometrics. Depending on the species, the number of mothers and offspring varies. Gravid mice and hamsters were obtained from registered suppliers (University of Burgundy facility for mice and Janvier Labs for hamsters), while for gerbils two males and fi ve females (Charles River) were needed in order to carry out mattings at the central animal facility. For the mice, six pregnant dams were received and gave birth to litters ranging from four to seven pups. For hamsters, six pregnant dams gave litters ranging from seven to thirteen pups. Five female gerbils were mated with two males, resulting in litters of fi ve to nine pups. The total sample size was therefore forty mice, sixty-one hamsters and forty-two gerbils. Details of the specimens can be found in Annexe 2.

Experimental design

One individual per litter was sacrifi ced at 7 and 14 day old. After weaning, at 21 days for mice and hamsters or 30 days for the gerbils, the remaining individuals were divided into two groups with different diets. The fi rst group was fed with pellets (Safe ® A04) to match the conventional diet. The second group was fed pellets that were transformed into soft food by dipping in water. The pellets were moistened for 6h in the fridge to obtain a slurry to represent a less mechanically demanding diet. Individuals were collected at 35 and 63 days. Moms were used as 100 days control and so were only fed with conventional food.

Fluorochromes study

Material and methods

Three different fl uorochromes were selected in order to be able to recognize the different markers more easily (Table 3.1). These different markers were chosen for their suitability for administration by injection and their dosages are well known in the literature. Three different colors of fl uorescence were chosen in order to easily recognize the stages of bone development at the time of microscopic observation.

Tetracycline (C 22 H 24 N 2 O 8 ) is classically used as a bacteriostatic antibiotic [START_REF] Chopra | Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance[END_REF]. It is also a yellow dye that binds to developing bones and teeth by clumping with calcium on the ossifi cation front [START_REF] Puranen | Reorganization of fresh and preserved bone transplants: an experimental study in rabbits using tetracycline labelling[END_REF]. Its molecular weight is 444.44 g/mol.

Alizarin (C 14 H 8 O 4 ) is a red dye from the madder root. It binds to bone during its formation by chelating calcium [START_REF] Myers | Alizarin and tetracycline binding by bone mineral[END_REF]. Its molecular weight is the lowest of the three fl uorochromes used at 342.253 g/mol.

Xylenol orange (C 31 H 32 N 2 O 13 S) is a dye of the triarylmethan family (Horobon, 2020). It is the most molecularly heavy fl uorochrome of the three with 760.59 g/mol.

Table 3.1. Properties and dosage of administered fl uorochromes.

These products are in powder form and were mixed with PBS (Fisher Bioreagents) as advised by the European Directive 2010/63/EU for injection. As recommended by the same directive, individuals were injected at 0.02mL of solution per g of animal, intraperitoneally using 25G 0.5mm x 16mm needles (BD Microlance™ 3).

During development, bone undergoes multiple pulses of mineralization and remodeling, in response to various stimuli as hormone fl uctuation or mechanical constraint [START_REF] Kovar | Near-infrared-labeled tetracycline derivative is an effective marker of bone deposition in mice[END_REF]. These episodes can be labelled by polychrome labelling (Pautke et al. 2005). Polychrome fl uorescent labelling is a standard technique used classically to characterize the growth of the bone [START_REF] Harris | A microscopic method of determining rates of bone growth[END_REF][START_REF] Frost | Tetracycline-based histological analysis of bone remodeling[END_REF][START_REF] Lee | Migration and differentiation of nuclear fl uorescencelabeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice[END_REF]. The principle consists in using calcium-binding fl uorochromes which settle in the sites of mineralization. By injecting several fl uorochromes (different colors) in intra-peritoneal injection, the sequence of bone growth can be observed. As I aimed to characterize the pattern and timing of mandible growth during postnatal development, I tried to inject these fl uorochromes during growth at specifi c ages, in order to see preferred sites of bone growth and their evolution.

The specimens used in the fi rst instance were as follows (Table 3.2): Table 3.2. Pilot specimens used for fl uorochromes observations.

Once the skulls were recovered, they were embedded in LR White resin (Sigma-Aldrich), vacuum bagged for 24 hours and cured for 12 hours at 40°C. Once the plots were formed, they were sawn with a microtome (Leica SP 1600) to a thickness of 300 μm. This thickness was chosen after testing in order to ensure the integrity of the section while allowing its observation. Throughout the process, the samples were kept in a dark box between two manipulations in order to preserve the fl uorescence of the fl uorochromes.

Observations of these slides were carried out at Ifsttar (Institut français des sciences et technologies des transports, de l'aménagement et des réseaux), on a ZEISS Axio Scope SIP microscope. Several excitation beams were tested (DAPI, GFP, RFP, YFP) and only the use of the DAPI fi lter allowed the observation of the three fl uorochromes.

In light of the results obtained on the pilot specimens (described below) and taking into account the time required to prepare the samples, it was decided to limit the observation analysis to the sole pilot individuals.

The results were uneven across sections and specimens. On most sections, tetracycline markings are visible but discontinuous. Alizarin markings are only visible on some sections (Fig. 3.2 A-B) and no xylenol orange markings are visible. Therefore, growth rates could not be estimated from the obtained sections and images.

Why it probably did not work

There are several likely explanations for the negative results. Firstly, the individuals were injected intraperitoneally (IP). This means that they were injected into the peritoneum, which is the semi-permeable membrane lining the viscera and abdominal wall (Lichtenstein, 2011). It extends above the blood and lymphatic capillary beds, so fl uids injected into this cavity can be rapidly reabsorbed [START_REF] Baek | Evaluation of a novel technique for intraperitoneal injections in mice[END_REF]. This type of injection is easier to perform than an intravenous injection in small organisms but also has some disadvantages such as risks of intra-intestinal injection, which poses little risk to the animals but limits the uptake of fl uorochromes. There are also some risks of peritonisis, hemorrhage secondary to blood vessel perforation or accidental organ trauma [START_REF] Guarnieri | Considering the risks and safety of intraperitoneal injections[END_REF], which are dangerous for the animal's condition, but none of the above-mentioned pathologies has been observed. There is therefore a risk that the injected products have been poorly administered, resulting in poor absorption of the fl uorochromes, especially given the high incidence of poor injection even among experienced practitioners, failure rate is reported to be of the order of 10-20 % (Gaines Das & North 2007). Such failure rates suggest that treatment by IP must be monitored by ultrasound scan to validate the administration especially in design with longitudinal treatments as it was used here.

Secondly, although great care was taken to keep the specimens in a dark environment, the entire sawing process had to be carried out in daylight, which could alter the fl uorescence ability of the fl uorochromes attached to the bone. This step can take up to a full day for a block, which is not necessarily negligible. In order to test this hypothesis, a small portion of the three fl uorochromes has been exposed to daylight for one month. The lower intensity of fl uorescence observed is the tetracycline one which is well visible on our specimens, this effect seems not to explain the pour observation of alizarin and the absence of xylenol.

Thirdly, the individuals had to be kept in a freezer while waiting to be cleaned by the dermestid beetles. Fluorochromes are usually stored at room temperature for tetracycline and alizarin, at 5°C for xylenol orange. This waiting stage could therefore have deteriorated the fl uorochromes. Tetracycline is highly resistant to freezing [START_REF] Okombe | Detection des residus d'antibiotiques dans les denrees alimentaires d'origine animale commercialisees a lubumbashi en Republique Democratique du Congo[END_REF]. To test this factor, a small portion of the products were kept at -20° during one month. In this case too, the lower intensity of fl uorescence observed is the tetracycline one. This factor does not seem to explain my observations. Finally, xylenol orange has a much higher molecular weight than the other two molecules used. Thus, it is less easily assimilated, which could be the reason for its absence in the observed tissues.

Table 3.3. Fluorescence intensity measured in μW/cm² on the three different fl uorochromes in different confi gurations of conservation: a control one, after on month spent in a freezer at 20°C, after one month at the daylight.

If it had worked

The succession of different colored markings at specifi c ages was intended to be able to recognize sites of greater or lesser ossifi cation. Indeed, the interval between two markings would have made it possible to measure the rate of bone growth over a given period of time. The initial idea was to be able to digitally mark the ossifi cation fronts (fl uorescent lines) and then reconstruct a model to visualize the ossifi cation accelerations at given locations in 3D. It would then have been possible to compare these data with the results obtained on the scans presented in chapter 4 to see if the areas of deformation and growth of the mandible were directly consistent with bone remodeling.

Bone microstructure

Adult morphology results from multiple and complex processes including bone growth and remodeling. The bone must accommodate to enable the growth of the individual both allometrically and functionally. The bone is formed and resorbed multiple times by specialized cells coordinated to respond to the extension of bone, but also to different movement undergone by the different skeletal elements between them and/or with the soft tissues (e.g., relocation, primary and secondary displacements, and rotations, [START_REF] Freidline | Exploring modern human facial growth at the micro-and macroscopic levels[END_REF]. It therefore seemed appropriate to try to document this bone remodeling on our specimen. In this way, we could have mapped the areas of bone resorption and formation. Subsequently it would have been possible to compare the arrangement of these histological remodeling zones with the ones observed on 3D models.

A mandible and a skull of a 15-day-old mouse was observed using SEM to explore the surface of the bone (Figure 3.3). The age of the specimen was chosen because at this stage, the skull undergoes a strong growth. We compared the structures observed to the one seen in the literature [START_REF] Martinez-Maza | Brief communication: Identifi cation of bone formation and resorption surfaces by refl ected light microscopy[END_REF][START_REF] Blackett | Squirrels-an overview of common clinical presentations and important diseases[END_REF]. Unfortunately, the structures were very diffi cult to assess to resorption or formation. Indeed, the benchmarks present in the literature have been developed on other groups. It would have been necessary to develop our own repository to apply to our specimens, which was not possible during this work. This is why I have abandoned these observations for this thesis work in order to focus on geometric morphometrics.

Myosin study

Material and methods

During development, the various tissues must coordinate to obtain a functional masticatory apparatus [START_REF] Mcneill | Occlusion: what it is and what it is not[END_REF]. The growth of muscles is thus as important as that of the bone, especially the masseteric complex as it is the one involved in structuring the craniofacial complex. The contraction of a single muscle fi ber involves the motor unit within each myofi bril, called the sarcomere. The sarcomere is composed of two types of fi laments: the actin and the myosin protein fi laments. The myosin fi laments show a greater variety than the actin fi laments. The plasticity of the muscle fi bers is linked to the isoform diversity of the myosin heavy chain (MHC) which is determinant in the contractile properties of the muscle [START_REF] Mahdavi | Sarcomeric myosin heavy chain gene family: organization and pattern of expression[END_REF]). The MHC isoforms preferentially expressed in masseter muscles are MHC-IIa (MyH2) and MHC-IIb (MyH4) associated to the actin [START_REF] Mahdavi | Sarcomeric myosin heavy chain gene family: organization and pattern of expression[END_REF]. Heterogeneity in muscle structure allows adaptability to different developmental [START_REF] Monemi | Fetal myosin heavy chain increases in the human masseter muscle during aging[END_REF][START_REF] Raadsheer | Masseter muscle thickness in growing individuals and its relation to facial morphology[END_REF] and functional demands [START_REF] Auluck | A three-dimensional in vitro model system to study the adaptation of craniofacial skeletal muscle following mechanostimulation[END_REF] during postnatal development. As muscle development is linked to the functioning of the masticatory apparatus, I wanted to test whether the expression of these two myosin isoforms (Myh2 and Myh4) and actin were expressed in the same way in the two food groups on mouse specimens (Table 3.3), as the primers for this species were already available. Masseter biopsies were taken immediately after death and stored in RNAlater solution (Invitrogen) at -20°C. The expression of myosin and actin in the masseter biopsies was assayed by RT-qPCR. Total RNA was extracted from masseter biopsies with Trizol® (Ambion by life technologies) and quantifi ed by optical density at 260 nm using NanoDrop 2000 (Thermo Scientifi c). RNA was transcribed into complementary DNA (cDNA) using Moloney murine leukemia virus (M-MLV) reverse transcriptase, random primers (0.5 μg/μg RNA, Promega) and recombinant RNasin® Plus RNase Inhibitor (Promega). The qPCR was then carried out in 96-well plates in order to quantify the cDNAs using a Master mix PowerUpTM SYBRTM Green (Applied biosystems). mRNA expression was determined using the 2-ΔCt method after GAPDH (ΔCt) subtraction. The sense and antisense primers used (Eurogentec) are listed in table 3.4.

Why it probably did not work

Unfortunately, the housekeeping gene (GAPDH) primer mix turned out to be contaminated, so normalized results are not reliable. In addition, we cannot normalize the amount of myosin by actin since this protein also enters into the composition of the muscle fi ber. The amount of RNA extracted from the samples was not suffi cient to make replicas, so it is not possible to perform new analysis on these biopsies.

If it had worked

It was expected that the amount of myosin and actin expressed would be lower in specimens that had been fed a soft diet. If this had been the case, this could have meant that the production of the proteins that make up the muscle fi bers has a close link with the mechanical load applied to these same muscles. Thus, this production would not be entirely linked to protein signaling triggered during development.

If no difference had been observed between the different diet groups, it would have meant that the mechanical constraint does not modify the quality of the muscle fi bers during development. The role of mechanical strain will be more discussed in chapter 5.

Acquisition of 3D models and landmarks

Bones were cleaned using dermestid beetles except those of 7-day-old juveniles, the skull being poorly ossifi ed and likely be too damaged by the insects. The cranium and mandibles were scanned by μCT scan (Bruker Skyscan 1174) at 50kV and 800 μA (exposition of 5000ms for the 7-day-old specimens, 3000ms for the others). They were then reconstructed using AvizoR9.2 (FEI systems).

On the mandibles, fourteen landmarks were digitized using Digit3DLand R package 0.1.3 [START_REF] Laffont | Digit3Dland : Digitalization of 3D landmarks on mesh[END_REF], together with 22 curve semilandmarks along fi ve distinct curves (with two semilandmarks on the coronoid process curve, three along the two lunar notch curves, seven on the curve between postcondylar and angular processes, and ten on the masseter ridge). An atlas was created from a mouse specimen with the PseudoLMGenerator module of the SlicerMorph package [START_REF] Rolfe | SlicerMorph: An open and extensible platform to retrieve, visualize and analyse 3D morphology[END_REF]) on 3D Slicer 4.11 [START_REF] Fedorov | 3D Slicer as an image computing platform for the Quantitative Imaging Network[END_REF], with fi ve patches of surface semi-landmarks totalizing 256 semi-landmarks (Fig. 3.5). This atlas was transferred on all models using the ProjectSemiLM module. Curve and surface semilandmarks were then slid along their tangent by minimizing the bending energy and back-projected on the 3D surfaces (Gunz et al. 2005) u sing the Morpho R package 2.9 (Schlager 2017). A pilot study to evaluate this pipeline was done on a set of 28 mice from a AJxB6 backcross (28 day old, Maga et al 2015). These mice were digitize at 55 landmarks from which 23 were paired landmarks and 9 unpaired. The non-rigid alignment using the coherent point drift algorithm was done on and values ranging from 1 to 10 to evaluate the variation in landmarking according to the deformation process. The root mean squared error (RMSE) between the predicted right landmarks and the original ones was computed as the Euclidean distance between the two landmark conformations standardized by the original centroid size. This RMSE ranges from 0.02 to 0.033. After merging the original left side and the predicted right side, the individuals were also superimposed using a generalized full Procrustes analysis with bilateral symmetry [START_REF] Klingenberg | Shape analysis of symmetric structures: quantifying variation among individuals and asymmetry[END_REF]. The principal components analysis on the symmetric component shows a homogeneity of the predictions but some systematic bias compared to the manual shapes. This bias has been well recognized in the fi rst attempts of automatic landmarking [START_REF] Devine | A registration and deep learning approach to automated landmark detection for geometric morphometrics[END_REF], Navarro & Maga 2016[START_REF] Percival | The effect of automated landmark identifi cation on morphometric analyses[END_REF], but here, the Procrustes distance between the median of the predicted symmetric shapes and the manual shape is always lower than between two individuals.

Chapter 4 -Postnatal development of the craniofacial complex

The aim of this chapter is to study the two constitutive units of the cranium, the skull and the mandible, to compare their timing and rate of development during the postnatal stages. To this end, the postnatal trajectories of three model species, the hamster, the mouse and the gerbil, are followed on both the mandible and the skull. The monitoring of shape changes during postnatal development has never been documented taking into account the whole craniofacial complex to my knowledge. Furthermore, this monitoring is realized by comparing three phylogenetically close species in order to compare the plastic behavior of these bone structures at both intra-and inter-specifi c levels.

The two units being very closely related since they are anatomically linked and has to maintain functions, one would expect the skull and the mandible to follow similar timing and rate of development. Nevertheless, these two elements are not totally constrained by the same tissue interactions and therefore should not present the same developmental constraints [START_REF] Richtsmeier | Phenotypic integration of neurocranium and brain[END_REF]). Thus, it is conceivable that these two structures could show different timing in ontogeny on the condition that they remain suffi ciently integrated to maintain some effi ciency in their common functions.

The fi rst part of this chapter is composed of the study of mandible shape ontogeny, which was accepted to Royal Society Open Science on March 21th, 2022. The second part refers to the study conducted on the skull, completing the data for the entire craniofacial complex. The third part will discusses the morphological integration of these two anatomical structures.

Functional constraints channel mandible shape ontogenies 4.2 Skull postnatal development

The development of the skull has mainly been studied in model organisms such as the mouse or the rat, in order to understand the processes of ossifi cation and suturing of the different bones that make up the skull. These studies have mainly focused on the protein and mineralization processes of the bone [START_REF] Holleville | BMP signals regulate Dlx5 during early avian skull development[END_REF], Martinez-abadias et al. 2013, Motch Perrine et al. 2014, Percival and Ritchtsmeier 2013[START_REF] Wan | BMP signaling in skeletal development[END_REF]. They have mostly monitored the prenatal stages (Barbeito-Andrés et al. 2016), pathological cases (Motch Perrine et al. 2017), or growth of individual bones and not of the whole cranium shape [START_REF] Maga | Postnatal development of the craniofacial skeleton in male C57BL/6J mice[END_REF].

The morphological description of the structures in formation is mostly done by histological images [START_REF] Lee | A computational analysis of bone formation in the cranial vault in the mouse[END_REF], as it is quite diffi cult to obtain clean and resolved tomographic images of these very young stages (prenatal and fi rst postnatal days, as cartilage is more diffi cult to capture with X-rays than bone). However, thanks to advances in X-ray tomography, it is becoming increasingly possible to obtain 3D models of young and therefore not fully ossifi ed stages. These protocols needs a substantial preparation time, like DiceCT (Diffusable iodine-based contrast-enhanced Computed Tomography) which requires several days of sample processing before scanning [START_REF] Callahan | A guide for optimal iodine staining and high-throughput diceCT scanning in snakes[END_REF], often longer scanning times under high energy intensity. Last but not least, these protocols require a real expertise together with an important segmentation time in order to differentiate the different structures, which could look very alike [START_REF] Cox | Microcomputed tomography of craniofacial mineralized tissue: A practical user's guide to study planning and generating quality data[END_REF], sometimes it even needs the help of deep learning pipeline (Zheng et al. 2022). That is why for this study, it was preferred to still clean the bones before scanning for adult specimens and to treat only young specimens with ethanol.

Material and methods

The ontogenetic study of the skull used the same material and methods that were used for the mandible in the ROS manuscript. Here, for the skull, only landmarks were applied , which does not allow a description of the shape change as precise as for the mandible where semi-landmark patches were applied. It was not possible to place patches, as the skulls of the younger stages were not fully sutured. This is a current limitation on skull studies [START_REF] Toussaint | A landmark-free morphometrics pipeline for high-resolution phenotyping: application to a mouse model of Down syndrome[END_REF], and the skull was mapped with a signifi cant number of landmarks spread as much as possible over the whole unit. The fi rst two components of the PCA cumulates 60.7% of the total shape variance (Fig. 4.1 A). The gerbil and mouse trajectories stay close in the 7-day-old and 14-day-old stages and then diverge gradually. The hamster trajectory appears more distant on the two fi rst components in relation to an important shape difference from the fi rst stage.

Results

After accounting for shape differences between species using parallel transport, the 7-dayold juveniles are grouped together by construction (Fig4.1 B). Between 7 and 14 days, gerbil and hamster's trajectories are pretty close. The 14-day-old stages are spread along PC2 separating the trajectories of the three species. The trajectories of the two murids (gerbils and mice) are getting closer from the third stage and are separated on PC2 with the cricetid trajectory (hamsters). Shape changes along PC2 correspond to an extension of the palatine and basisphenoid ( The amount of shape changes along the ontogeny is similar between the three species (path distance equal to 0.33 ± 0.08 for gerbil and hamster, 0.27 ± 0.01 for mouse). The timing of these changes is also similar with a maximum between 7-day-old and 14-day-old and then a progressive decrease until 63 day-old (Fig 4.1 F). At the fi rst stage rate of shape changes is comprised between 0.023 for hamsters and 0.015 for mouse, this rate is then divided by fi ve for gerbil and hamster, by three for mouse at the second stage. At the third stage (35 to 63-day-old), this rate is signifi cantly reduced (0.0010 for hamster to 0.0017 for mouse) and tends to 0.00088 (± 0.00007) at the fourth stage.

Gerbils and hamsters share similar directions of shape changes (Fig 4.1 G), starting with an angle of 36°and then accumulate differences progressively. Mice present a different direction from the fi rst stage (with a θ of 63.9° with gerbils and 62.7° with hamsters). At the third stage, mouse and hamster's trajectories are getting slightly closer (58°) which is close to the θ at the same stage for gerbils and hamsters (52°). In general, the three trajectories are quite different since 35-day-old.

The directions of shape changes (Fig 4 .1 H) within species prior (7 to 14 days) to the weaning phase (14 to 35 days) are different to the trajectory during weaning for all three species, more for gerbils (α = 107°) than for hamsters (α = 86°) and mice (α = 76°). On the contrary, the directions after weaning (35 to 63 days) are more similar for gerbils (α = 76°) and hamsters (α = 56°) than for mice (α = 92°).

Discussion

Our results show that skull shape appears to be subfamily dependent from the start, with murids and cricetids being distant in the fi rst two components (PCA). No general pattern emerges from the fact that these shapes are already partly established at the fi rst stage. Weaning does not seem to have a signifi cant impact on the change in skull shape, the development of which fi nally takes place quite early in the postnatal ontogeny.

The skull and mandible therefore do not behave in the same plastic way during postnatal development. Indeed, they do not respond to the same constraints. The mandible develops at the same time as the teeth and muscles. The skull, for its part, must also synchronize its growth with that of the brain and the various sensory organs it contains [START_REF] Richtsmeier | Phenotypic integration of neurocranium and brain[END_REF]. This hypothesis is supported by the anatomical regions modifi ed during development. The expansion of the parietal may be linked to the brain growth. The nasal undergoes an extension to allow the occlusion of the incisors with those of the mandible as well as the development of the entire olfactory region. The modifi cation of the squamosal region is certainly linked to the accommodation with the growth of masseter muscles and the expansion of mandible's processes.

Furthermore, it seems that the changes undergone by the two units do not occur with the same timing. Indeed, the trajectory shift visible on the gerbil mandible, due partly to allometric effect, is not present in the skull. On the other hand, the large amount of shape changes present in the fi rst stage (7 to 14 days) of the mouse mandible is not observed in the skull. There is thus a certain desynchronization between the developmental rates of the mandible and the skull.

Covariation between the skull and the mandible

After looking independently at the skull and the mandible, the question of their integration within the craniofacial complex arises. Thus, it is proposed in this section to look at the covariation of these two units. The methods of analysis of covariation gain in performance with large samples.

It has been chosen here to look at this signal not by species but rather by combining them to obtain a Myomorpha signal.

Material and methods

To study the morphological integration of the skull and the mandible, a partial least-squares analysis (PLS) was performed with the Morpho R package 2.9 (Schlager 2017). The per-species perage sample sizes are too small to allow to get good estimates of the covariance matrices for these 15 groups (species age groups) considering that integration metrics (rv-coeffi cient, etc) could be very sensible to small sample sizes [START_REF] Fruciano | Resampling-based approaches to study variation in morphological modularity[END_REF]. Therefore, the sample was pooled according to the expected marginal means of the species age groups, and the covariation between the two anatomical units was studied on this pooled sample of 81 individuals. It means that the observed patterns of covariation correspond to the main pattern of integration conserved along the ontogeny and across all species. The skull and the mandible appear to covariate slightly (Rv = 0.22, p = 0.004). The main pattern as represented by the fi rst PLS axes (Fig. 4.2) accounts for 22.2% of the total squared cross-covariance (r = 0.59, p = 0.31). This pattern appears roughly consistent during the ontogeny and across species according to the age-based ellipses which are about the same. Two additional PLSs explain more than 10% of the covariation (PLS2 = 18.3%, r = 0.56, p = 0.03, PLS3 = 11.5%, r = 0.52, p = 0.09) and four additional present a percentage higher than 5%.

The most remarkable anatomical differences between negative and positive values of the fi rst PLS axes as a function of age are described in the following table.

Table 4.1. Main anatomical differences along the fi rst PLS axes as function of age.

Discussion

The two units composing the craniofacial complex seem to slightly covariate. Indeed, the covariance is spread over several PLS axes, meaning that the pattern of covariation depends on many factors. However, although slight, covariation patterns emerge between the skull and the mandible, regardless of species or age. Indeed, throughout development and as feeding and behavioral changes occur, the craniofacial complex must remain functional. Without this cohesion between the two units, the animal would limit its chances of individual survival, especially in a wild context [START_REF] Blackett | Squirrels-an overview of common clinical presentations and important diseases[END_REF][START_REF] Miller | Natural History and Medical Management of Squirrels and Other Rodents[END_REF][START_REF] Sainsbury | Oral disease in free-living red squirrels (Sciurus vulgaris) in the United Kingdom[END_REF]. The covariation of the different structure is essential to maintain appropriate size and shape across ontogeny and ranges of phenotypic variations (Hallgrimsson et al. 2007). It has been observed that mutation on cartilage synthesis gene can alter covariance in mouse's skull by increasing the variance in cartilage formation and so the variance in brachymorph phenotype (Hallgrimsson et al. 2009). Thus, while covariation may be altered by changes in the development of a single tissue type, this covariation may be more sensitive if several structures are considered as they have to coordinate to maintain a functional craniofacial complex. It has also been proposed that the growth of the different parts of the skull does not necessarily take place in a coordinated manner, but that this process can occur via compensatory interactions that correct locally disproportionate growth [START_REF] Zelditch | Developmental regulation of skull morphology II: ontogenetic dynamics of covariance[END_REF]. Shape is thus actively regulated by developmental plasticity of the bone. Thus potentially allowing accommodation to the various internal or external factors regulating and/or forcing bone remodeling. The shape is therefore both canalized and rapidly adaptable to any change.

Conclusions

After birth, mandible and skull are modifi ed with different timing. The mandible seems to be sensitive to different factors as gestational time, weaning but is also partly phylogeny dependent. After an early shape divergence between subfamilies, changes seem to be driven by muscular loading and other mechanical stresses arising around weaning. The skull, on its part, shows also an early shape divergence between subfamilies but does not undergo major changes during weaning. This unit is constrained by more tissues interactions than the mandible and must therefore follows a different development in terms of timing, although these two units must interconnect to ensure the viability of the individual. Nevertheless, the skull and the mandible covariate slightly throughout development, which is consistent with the need to maintain a functional head.

Chapter 5 -Plasticity in postnatal development

In mammals, weaning is a key moment, characterized by transitions in feeding behaviors and loading in the feeding apparatus. As shown in the previous chapter, the skull and the mandible do not exhibit the same response to these changes. During this period, the tissues will have to accommodate to the various mechanical changes related to the modifi cation of diet and behavior.

The chewing muscles respond to the chewing forces and movements and develop to meet this need, as well as the need to gnaw or other adult behaviors. Thus, if the tissues must adapt to various stresses, one may wonder if this response varies according to changes in the stresses applied to the whole craniofacial complex during weaning.

In natural setting, this variation in strain may occur depending on availability of different food types in relation to seasons, dispersion in new environments, or at larger scale related to climate changes. The expression of the adult shape would thus be sensible to over-strain or under-strain, and the shape variation between spatial and/or temporal populations would partly relate to this plastic response. These variations in constraints could therefore lead to responses at different scales. At the individual scale, the organism may show developmental plasticity in relation to availability of resources (food type at weaning); when at the population scale, an evolution of the shape in response to environmental forcing through plasticity may occur by phenotypic accommodation [START_REF] Jane | Phenotypic accommodation: adaptive innovation due to developmental plasticity[END_REF][START_REF] Nijhout | Genetic assimilation and accommodation: Models and mechanisms[END_REF].

To assess the importance of strain variation on the adult shape, and thus of the plastic potentiality of the CF complex, the effect of diet could easily be controlled in animal experiments. The animals used in this study were therefore divided into two groups, one fed with conventional pellets (Hard diet) and the other with pellets boiled by hydration (Soft food). This type of experimentation is classically used to inform the plasticity of the cranium and the mandible. However, these studies have concerned pathological cases such as dystrophy [START_REF] Renaud | Epigenetic effects on the mouse mandible: common features and discrepancies in remodeling due to muscular dystrophy and response to food consistency[END_REF][START_REF] Spassov | Congenital muscle dystrophy and diet consistency affect mouse skull shape differently[END_REF], focused on the adult shape [START_REF] Menegaz | Ontogenetic and functional modularity in the rodent mandible[END_REF], informed the reaction of the tissues by changing the diet during the study [START_REF] Mitchell | More challenging diets sustain feeding performance: Applications toward the captive rearing of wildlife[END_REF] or sought to apply over-stressing [START_REF] Mavropoulos | Masticatory demands induce region-specifi c changes in mandibular bone density in growing rats[END_REF]Odman & Kiliaridis 2010), and in my knowledge none has been realized explicitly in a comparative setting.

Previous studies have shown that soft diet results in structural and morphological changes in the craniofacial complex due to functional load differences [START_REF] Mavropoulos | Rehabilitation of masticatory function improves the alveolar bone architecture of the mandible in adult rats[END_REF][START_REF] Mavropoulos | Normal masticatory function partially protects the rat mandibular bone from estrogen-defi ciency induced osteoporosis[END_REF][START_REF] Chen | Sex differences in chondrocyte maturation in the mandibular condyle from a decreased occlusal loading model[END_REF][START_REF] Dias | Infl uence of food consistency on growth and morphology of the mandibular condyle[END_REF][START_REF] Utreja | Cell and matrix response of temporomandibular cartilage to mechanical loading[END_REF][START_REF] Ödman | The effect of retraining hypofunctional jaw muscles on the transverse skull dimensions of adult rats[END_REF]. At structural level, this type of diet leads to a decrease in bone volume, to bone thickness alveolar, bone trabecular volume and subchondral condylar bone volume (Odman et al., 2008;[START_REF] Mavropoulos | Rehabilitation of masticatory function improves the alveolar bone architecture of the mandible in adult rats[END_REF][START_REF] Anderson | Adaptive plasticity in the mouse mandible[END_REF], when at anatomical level, mandibles develop shorter processes [START_REF] Mavropoulos | Effect of different masticatory functional and mechanical demands on the structural adaptation of the mandibular alveolar bone in young growing rats[END_REF][START_REF] Anderson | Adaptive plasticity in the mouse mandible[END_REF]).

In the present study, the goal is to (1) document the effect of diet during postnatal development and thus assess the role of plasticity in the establishment of the craniofacial shape along ontogenetic trajectories, (2) to compare if this plasticity effect varies among species. Here I am looking at whether there are inter-specifi c differences, as the anatomies of the three species are different. Indeed, the plastic response could depend on the specifi c anatomical setting and therefore be dependent of the systematics. Several scenarios are possible. Either the same timing of growth between the skull and the mandible is found in order to keep a complete cohesion between these two units; it has been shown previously in this manuscript that it was not the case in hard food diet. However, a less important constraint could slow down the forcing on the two structures and thus allow a better synchronized developmental timing, as it was shown in the previous chapter, the skull and the mandible can have different growth timings, while keeping a functional cohesion. It would thus be expected that, although most of the postnatal growth has already passed by the time of weaning, there would be a modifi cation of the ontogenetic trajectory, notably that the vector lengths in soft diet group are potentially shorter than in hard food group. On the other hand, it would also be expected that inter-specifi c divergence would be less in soft food specimens, as the adult shape may be less expressed due to the lack of mechanical forcing applied by the muscles on the bone reducing the infl uences of differentiated adult behaviors. Anatomically speaking, the structures associated with the establishment of the masticatory muscles (ascending ramus processes) could be less developed in soft food specimens. Thus, the plasticity of the craniofacial complex could provide a rapid response to environmental factors, in modulating the mechanical performance and thus reducing the potential of maladaptation, the bone physiology allowing an almost instantaneous response to the availability of food resources.

Material and Methods

The specimens used in this study were bred in the central animal facility of the University of Burgundy (Project APAFIS#18405-2019011014262528). From weaning onwards, some individuals were fed with hard food pellets, others with pellets mixed with water. The specimens used were those corresponding to the post-weaning stages (35 and 63 days old). No dental problems, weight loss or different weight gain between diets were observed in either group during the experiment (Fig 5.1), meaning that the constraint applied by diet is weak.

The landmarks used on the mandible and the skull are the same as those used in the previous chapter as well as the statistical shape analysis. The ontogenetic trajectories were parallel transported prior to analysis; this time the 35-day-old expected marginal means of each species were superimposed.

Results

For the mandible, the fi rst two components of the PCA cumulate 72,53% of the total shape variation (Fig 5 .2A). The 35-day-old are grouped together. Two major directions of development are represented. In one hand the trajectory of hamster projects well along PC1; in the other hand, the trajectory followed by mice and gerbils projects mostly along PC2. However, the directionality of the trajectories does not vary between soft and hard within each species. Moreover the soft and hard growth vectors within each species are of similar norms, indicating the same amount of change regardless of the associated diet. The ANOVA analysis reveals a diet effect on shape changes as well as a small age × diet effect which refl ects the duration of the treatment (Table 5.1). During the postweaning development, specimens fed with hard diet develop longer and more robust condylar and angular processes . The coronoid process is slightly longer also in hard diet fed specimens. In general, the entire posterior part of the mandible is less extended in soft diet fed specimens. The diastema is thicker in hard diet fed shape. Table 5.1. ANOVA analysis on mandible.

Table 5.2. ANOVA analysis on skull.

Concerning the skull, the fi rst two components of the PCA cumulate 65,59% of the total shape variation (Fig 5 .2B). The 35-day-old are opposed to the 63-day-old along PC1. As for the mandible, growth vectors are similar in direction and norm within each species. The ANOVA analysis reveals no effect of the diet on shape changes. There are few anatomical differences between the skull shapes associated with the two diets (Fig 5 .2 E-G). Hard diet fed specimens show a very slightly more infl ated cranial vault (parietal and interparietal).

Discussion

The results of this study show similar growth vectors between the different diets, both in the skull and the mandible. Although most of the postnatal growth had already occurred before weaning, it was expected that the vectors associated with the soft diet would be shorter than for the hard diet. Indeed, the masticatory muscles being less solicited in the soft diet, the constraints are lower, so the bone could have been less forced to adapt to the new chewing constraint induced by weaning [START_REF] Renaud | Epigenetic effects on the mouse mandible: common features and discrepancies in remodeling due to muscular dystrophy and response to food consistency[END_REF] On the other hand, there is a notable difference in the shape of the mandible. The different processes of the ascending ramus are shorter than in the hard regime, thus reducing the whole posterior region of the mandible. However, a strong development of this part of the mandible is associated with the implementation of the major constraints applied by the masticatory muscles [START_REF] Menegaz | Ontogenetic and functional modularity in the rodent mandible[END_REF]. For the skull, the only difference observed is very small, the cranial vault is very slightly more infl ated in the hard diet specimens. This observation was also made by Menegaz et al. in 2010 on rabbits. Specimens were reared in the laboratory and kept as free as possible from easily gnawed objects in order to obtain results as characteristic as possible of the diet. For example, the incisor length of individuals reared on a soft diet could not have been maintained well after 63 days without intervention on the incisors. In contrast, in the wild, individuals have elements to gnaw on. Thus, in such an environment, the mandible would have the plastic capacity to respond to the resource constraints of the environment, while at the same time being able to gnaw without worrying about maintaining incisors of a normal size, which would not present a health risk for the animal.

Here a net difference in plasticity between the skull and the mandible has been shown, and only the mandible exhibits a diet effect. Once again, it is important to note that the mandible is composed of fewer different tissues and could therefore more easily undergo a much greater bone remodeling in relation to bone loading than the skull. The mandible will then modify itself in accordance with the more or less strong need to create a lever capable of handling a more or less hard food and to support the tension applied by the muscles. At this stage, moreover, the skull has already seen the almost complete development of its internal structures (brain, olfactory and auditory organs), which are not compressible. There is therefore a desynchronization of the two units forming the craniofacial complex. Moreover, the areas impacted by food changes on the skull may be more localized at the muscle insertions which could not have been well captured with landmarks only.

The mandible allows for a slight adaptation to the diet and thus to the availability of resources. It is a possible rapid adaptation of the mandible to the environment and to the resources, as it allows to modify leverage in bite force without involving weight change of individuals. This plastic response could underline the adaptation of populations in aligning the mandible shape to the need in performance. This effect is observed regardless of the species in this study. It may possibly be extended to close species within Myomorpha and may highlight a possible mechanism of parallel evolution.

Conclusion

In this experiment, it was shown that the difference in diet after weaning had no effect on the timing of acquisition of adult shape, as the growth vectors were of similar length. However, anatomically speaking, the bony structures associated with muscle development are shorter on the mandibles associated with the soft diet. On the other hand, only slight differences are observed on the skull and no signal is found to associate them with the diet. Thus, the plasticity associated with the diet would only be carried by the mandible. However, this plasticity allows a rapid response to the availability of food resources and does not jeopardize the cohesion between the skull and mandible, allowing a functional craniofacial complex to be maintained and enabling the survival of individuals.

Conclusions

During this thesis, I fi rst took a broad view through the macroevolutionary scale, in order to map general growth patterns of postnatal mandibular development in rodents, between and within clades. In order to provide precise information on the different processes undergone by the craniofacial complex during postnatal development, the other part of this work focused on a fi ner taxonomic and temporal scale. For this purpose, three model rodent species belonging to the same suborder (Myomorpha) were sampled at fi ve different growth stages. Finally, in these three species, the role of mechanical constraints during growth was questioned using variations in diet General postnatal growth patterns of the mandible were fi rst mapped between and within the main clades of rodents to provide a macroevolutionary picture of the postnatal mandibular development. Rodentia is a very diverse and disparate mammal order, in which postnatal changes can be observed on a large scale. This study was focused on Hystricomorpha and Myomorpha which exhibit diverse life-history traits among species. Gestation and weaning are shorter in Myomorpha, which generally give birth to large litters several times a year, involving a short prenatal development and a rapid and therefore mechanically brutal weaning. Hystricomorpha give birth to small litters a few times a year. Parental care is therefore different, with longer behavioral weaning and less brutal dietary weaning, over a much longer period. The potential for postnatal plasticity could likely be different between the two s uborders. A wide variety of diets is also found within these two groups. Changes in diet might correlate with evolutionary changes in muscle attachment, direction of mastication and mandible shape [START_REF] Álvarez | Deep changes in masticatory patterns and masseteric musculature confi gurations accompanied the eco-morphological evolution of cavioid rodents (Hystricognathi, Caviomorpha)[END_REF]. They also show diverse infraorbital morphologies, muscles attachment and types of molar growth. We observed trajectories between juvenile (before weaning) and adult stages. The results highlighted that the main evolutionary and developmental patterns appear to be along orthogonal directions of the shape space. Also, most of the shape divergences arise along postnatal directions of growth. Nevertheless, differences between suborders are raised. In Hystricomorpha, most of the development is apparently taking place during the gestational period as it presents short magnitude of changes in long postnatal periods and most trajectories are genus-specifi c. For Myomorpha, most of the development occur postnatally as it presents a much stronger magnitude of changes over a shorter growth period, also postnatal trajectories of Myomorpha subfamilies are closer to one another. Juvenile mandibles present similarities among suborders within rodents, with a fl attened shape. Mandible shape then diversifi es during growth in relation to muscle and tooth development, with epigenetic interactions coordinating the changes in the alveolar and muscle-bearing regions. During this study, only the mandible was considered but if we compare to the different studies conducted on the skull, we can highlight some differences between the two units constituted by the craniofacial complex. The skull is a composite unit constituted by multiple bones, and this assemblage is constrained by the development of teeth, facial muscles and internal organs. This complex entanglement of many tissues may explain the low skull disparity in relation to lineage diversifi cation described in Rodentia [START_REF] Alhajeri | A phylogenetic test of adaptation to deserts and aridity in skull and dental morphology across rodents[END_REF], or in the adaptive radiation of some families [START_REF] Maestri | The ecology of a continental evolutionary radiation: Is the radiation of sigmodontine rodents adaptive?[END_REF], or the relatively overlapping growth pattern of the skull observed among rodent clades [START_REF] Wilson | Commonalities and evolutionary divergences of mandible shape ontogenies in rodents[END_REF]). The higher complexity of skull development is likely to reduce its dependence on muscle and tooth development and this difference in complexity probably explains the contrast observed between these two skeleton elements of the head.

In the fi rst study, the mandible shape change was analyzed at a large taxonomic scale but considering only two stages of the postnatal growth: an early phase after birth and the adult stage. Ontogenetic trajectories are well-known to be non-linear [START_REF] Green | Developmental nonlinearity drives phenotypic robustness[END_REF][START_REF] Mitteroecker | Advances in geometric morphometrics[END_REF]. It was therefore essential to provide information on the change in morphology during growth at a fi ner scale using several subadult stages. Moreover, as the mandible is tightly link with the skull, it is essential to look at these changes on the entire craniofacial complex. The two units are closely related because anatomically linked. One would expect the skull and the mandible to follow similar timing and rate of development. However, these two elements are not totally constrained by the same tissue interactions as said before and therefore should not present the same developmental constraints. It is conceivable that these two structures could show different timing in ontogeny on the condition that they remain suffi ciently integrated to maintain some effi ciency in their common functions. So, I followed these trajectories on three model organisms, all three belonging to Myomorpha. After birth, a differential timing of shape changes is observed between mandible and skull. The mandible seems more sensible to different factors as gestational time, weaning and appears phylogeny-dependent. This bone undergoes an early shape divergence between families. After, changes seem to be driven by muscular loading and other mechanical stresses arising around weaning. The skull shows also an early shape divergence between subfamilies but does not undergo major changes during weaning. Despite this difference in developmental timing, the skull and the mandible covary slightly throughout development and maintain a functional head.

The changes in the mechanical constraint during the activation of the masticatory apparatus does not seem to infl uence the mandible and the skull in the same way. The idea was to document the effect of diet during postnatal development, thereby assessing the role of plasticity in the establishment of the craniofacial shape and comparing whether this plasticity effect varies among species. Thus, an experiment was designed to contrast two levels of this constraint by applying a classic diet (hard food) to the control group from weaning onwards and a sub-contractive diet (soft food) to another group. Several hypotheses were exposed. The timing of growth could be synchronized between the two structures in soft diet, an under-strain being less forcing on the mandible, despite a desynchronization already observed in hard diet. It was also expected that although most of the postnatal growth has already passed by the time of weaning, there would be a modifi cation of the ontogenetic trajectory, notably that the vector lengths in soft diet group could be potentially shorter than in hard diet group. Another hypothesis was that inter-specifi c divergence would be less in soft diet group, as the adult shape may be less expressed due to the lack of mechanical forcing. Anatomically speaking, the structures associated with the establishment of the masticatory muscles (ascending ramus processes) were expected to be less developed in soft food specimens. The dif-ference in diet after weaning appears to have no effect on the timing of acquisition of adult shape. As expected, the bony structures associated with muscle development are shorter on the mandibles associated with the soft diet. Only slight differences were observed on the skull and no interaction with diet was found. Thus, the plasticity associated with the diet seems to only be carried by the mandible. This plasticity allows a rapid response to the availability of food resources as long as a cohesion between the skull and mandible is maintained, ensuring a functional craniofacial complex and survival of individuals.

Along this thesis manuscript, a difference in postnatal development between taxonomic groups and between the skull and the mandible was observed. The mandible seems to be more plastic than the skull, the latter is constrained by more tissues interactions than the mandible and must therefore follows a different development in terms of timing. But this obviously does not preclude the cohesion of these two units ensuring the viability of the individual. These elements allow a rapid plastic response of the organism to variations in the type of food available from weaning. Thus, postnatal trajectories of the mandible could be infl uenced throughout growth by the organismal environment (food, behaviors…) allowing mechanisms such as phenotypic accommodation to contribute to the evolutionary responses of populations.

Perspectives

Throughout this thesis, scientifi c objectives to decipher the causal mechanisms of the craniofacial disparity onset and experimental setups were intertwined. Several experimental attempts were not successful, but could be improved without a doubt, allowing postnatal growth to be monitored by several proxies. Different perspectives on prenatal and soft tissue developments have also emerged to get an integrative view of the craniofacial development.

Regarding experimental improvement, the observation of growth by fl uorochrome markings could be enhanced by having the injections checked by monitored ultrasound scan. As temperature and light do not seem to have a signifi cant effect on the fl uorescence capacity of the injected products, it does not seem relevant in the fi rst instance to limit freezing and light exposure. Secondly, concerning the observation of microstructures of bone resorption and formation on the surface by SEM imaging, it would fi rst be necessary to establish an atlas of these structures. This would require an important image compilation coupled with histological samples to decipher whether the structures are bone resorption or formation, and this should be done in a close model organism to the species under study to reduce anatomical differences of bones and soft tissues.

Unfortunately, the attempt to estimate myosin and actin expression was not successful due to contamination of the housekeeping gene to normalize the results. As the samples did not provide a suffi cient amount of protein, this experiment could not be reproduced and will have required to replicate the animal breeding. In addition, only mouse samples were processed due to the availability of primers. This estimation of the expression of genes characteristic of muscle growth could be applied, with the design of new specifi c primers, to other laboratory species.

On a macroevolutionary scale, I was able to highlight that in the Hystricomorpha, the adult shape was already well established at birth in most of the species studied but it was not the case for Myomorpha. It would therefore be interesting to look at the development this time before birth. Studies on prenatal development are generally conducted on model organisms such as the mouse or the rat, based on 2D measurements [START_REF] Sone | Fetal growth and development of the coypu (Myocastor coypus): Prenatal growth, tooth eruption, and cranial ossifi cation[END_REF][START_REF] Wilson | Comparison of prenatal and postnatal ontogeny: cranial allometry in the African striped mouse (Rhabdomys pumilio)[END_REF] or histological observations [START_REF] Frommer | Prenatal development of the mandibular joint in mice[END_REF][START_REF] Struthers | The prenatal skull of the Canadian porcupine (Erethizon dorsatus)[END_REF]. Moreover, these studies are conducted on pathological models (Barbeito-Andrès et al. 2016, Motch Perrine et al. 2014), or on the effect of intra-uterine stress, particularly in toxicology [START_REF] Giglio | Effect of prenatal ethanol exposure on the growth of rat mandible skeletal units[END_REF], Ismail and Janjua 2001[START_REF] Shen | Effect of prenatal alcohol exposure on bony craniofacial development: a mouse MicroCT study[END_REF]. There is therefore a lack of data, to my knowledge, on the prenatal monitoring of healthy organisms. It would therefore be possible to look at in-utero development in the various suborders, to characterize the ontogenetic timing and rates. As Hystricomorpha are born with a quasi-adult shape, their prenatal growth could present analogies with the postnatal growth of Myomorpha.

This thesis was mainly focused on bone development, it would also be possible to study the development of the muscles (masticatory and lingual), especially as different groups of rodents are defi ned by their infra-orbital and therefore musculofascial structure. In order to obtain an accurate picture of development, it is essential to take into account the different tissues that make up the craniofacial complex and thus to know the scenario of their setup. To do this, it is necessary to be able to characterize the muscular tissues very early in the growth process, which can be experimentally challenging. Indeed, masticatory muscles and their architecture are classically described via dissections or only on adult specimens for 3D reconstructions [START_REF] Cox | Digital dissection of the masticatory muscles of the naked molerat, Heterocephalus glaber (Mammalia, Rodentia)[END_REF][START_REF] Cox | Reviewing the morphology of the jaw-closing musculature in squirrels, rats, and guinea pigs with contrast-enhanced microCT[END_REF], Cox et al. 2012, Ginot et al. 2018a, b). This characterization of muscles could be considered fi rst at the macroevolutionary scale, although it is conditioned by the availability of collections of the different stages of interest on potentially wild species. This study could also be conducted on a fi ner scale, on the three laboratory species already used in this manuscript. Attempts at iodine labelling of a 7-day-old juvenile mouse (DiceCT, Fig. 6.2) have already been made with results that assure me that this part may be feasible. High-resolution episcopic microscopy [START_REF] Geyer | High-resolution episcopic microscopy (HREM)-simple and robust protocols for processing and visualizing organic materials[END_REF] can also be added and confronted to the results obtained by iodine labelling to facilitate soft tissue recognition. Throughout the different aspects of this manuscript, I realized that there was a great lack of data on chewing movement. Only few data are available and emanate from direct visual observations [START_REF] Gorniak | Feeding in golden hamsters, Mesocricetus auratus[END_REF] or extrapolation from the wear of the dental occlusal surface [START_REF] Charles | Dental microwear in relation to changes in the direction of mastication during the evolution of Myodonta (Rodentia, Mammalia)[END_REF][START_REF] Coillot | Correlated changes in occlusal pattern and diet in stem Murinae during the onset of the radiation of Old World rats and mice[END_REF]. This aspect was especially interesting when studying variable diets. It has already been shown that some rodents like the hamster could adapt this movement depending on whether they were feeding on pellets or seeds [START_REF] Gorniak | Feeding in golden hamsters, Mesocricetus auratus[END_REF]. Indeed, this chewing movement could intuitively infl uence the way the masticatory muscles function and then their development.

During this type of study, it might therefore be appropriate to equip the cages with a camera, or, to a different extent, to fi lm the chewing of the individuals in a given time. These images could be processed with deep learning approach like DeepLabCut [START_REF] Mathis | DeepLabCut: markerless pose estimation of user-defi ned body parts with deep learning[END_REF][START_REF] Nath | Using DeepLabCut for 3D markerless pose estimation across species and behaviors[END_REF]) and compared to obtain an overview of chewing and mastication behavior. Abstract: The skull is, architecturally and functionally, a strongly integrated and complex structure. At the same time and somehow paradoxically, this unit is highly evolvable and presents a high diversity of shapes. Epigenetic interactions in response to mechanical stimulations will compensate and coordinate the growth of the different organs constituting the head, in order to acquire and/or to maintain certain functions. These interactions will allow the normal development by controlling the bone remodeling. In spite of its central role, the importance of these interactions in the expression of differences between species and at remains poorly understood.

The project of the PhD thesis aims I) at studying the onset of the craniofacial disparity in rodents during development, and II) at estimating the importance of the epigenetic processes during this postnatal growth.

Université Bourgogne Franche-Comté 32, avenue de l'Observatoire

Figure 1 . 1 .

 11 Figure 1.1. Schematic diagram of the feedback loop between genetic and epigenetic levels which characterize developmental processes (After Albertch 1991, Oster & Albertch 1982).

Figure 1 . 2 .

 12 Figure 1.2. Number of families, genera and species per suborder in Rodentia. Lagomorpha are present as an outgroup.

Figure 1 . 3 .

 13 Figure 1.3. Cranial anatomy of rodents in inferior view (Rattus rattus). apf: anterior palatine foramen, bo: basioccipital, bs: basisphenoid, eac: external auditory canal, fmg: foramen magnum, gf: glenoid fossa, i: incisor, iof: infraorbital foramen, j: jugal, mx: maxilla, na: nasal, oc: occipital condyle, pal: palatine, pmx: premaxilla, poa: paraoccipital apophysis, pt: pterygoid, pta: pterygoid apophysis, sq: squamosal, tb: tympanic bulla, zam: zygomatic apophysis of the maxilla, zar: zygomatic arch, zas: zygomatic apophysis of the squamosal, zw: zygomatic window.

Figure 1 . 4 .

 14 Figure 1.4. Cranial anatomy of rodents in superior view (Rattus rattus). f: frontal, iof: infra-orbital formaen, it: interparietal, j: jugal, mx: maxilla, na: nasal, ocr: occipital crest, pa: parietal, pmx: premaxilla, poas: post-orbital apophysis of the squamosal, scr: squamosal crest, so: supraoccipotal, sq: squamosal, zam: zygomatic apophysis of the maxilla, zar: zygomatic arch, zas: zygomatic apophysis of the squamosal, zw: zygomatic window.

Figure 1 . 5 .

 15 Figure 1.5. Cranial anatomy of rodents in lateral view (Rattus rattus). eac: external auditory canal,

Figure 1 . 6 .

 16 Figure 1.6. Mandibular anatomy in rodents in labial (A) and lingual view (B). ap: angular process, corm: mandibular corpus, cp: condylar process, crp: coronoid process, di: diastema, lm: line of the mylohyoid, ln: lunar notch, mf: masseter fossa, pn: posterior notch, rc: ramus crest, rf: ramus fossa, tp: tuberosity of the pterygoid, vrmf: ventral ridge of masseter fossa.

Figure 1

 1 Figure 1.7. Mandibular structures in rodents (After Courant, 2000). A) Hystricognath. B) Sciurognath. Red arrow: orientation of the condylar process, blue arrow: orientation of the angular process.

Figure 1 . 8 .

 18 Figure 1.8. Patterns of tooth occlusal surface. A) lopho-bunodont. B) bunodont. C) lophodont D) buno-selenodont. E) prismatic. (Modifi ed from Lazzari et al. 2015).

Figure 1

 1 Figure 1.9. Infra-orbital structures in rodents (After Courant, 2000). A) protogomorph. B) sciuromorph. C) myomorph. D) hystricomorph. slm: superfi cial lateral masseter (red arrow); dlm: deep lateral masseter (blue arrow); mam: medial anterior masseter (green dotted arrow); mpm: medial posterior masseter (yellow dotted arrow).

Figure 1 .

 1 Figure 1.10. Hypsodonty index. A) brachyodont. B) mesodont. C) hypsodont. (Modifi ed from Lazzari et al. 2015).

Figure 1 .

 1 Figure 1.11. Diagram representing the plan followed throughout this manuscript.

Figure 2 . 1 .

 21 Figure 2.1. Phylogeny of rodents (After Lacher et al. 2016). Sampled families are shown in dark, corresponding genera are added in brackets.

  Three different species were bred (Fig 3.1), the mouse (Mus musculus: BALB/c), the golden hamster (Mesocricetus auratus: RjHan:AURA) and the Mongolian gerbil (Meriones unguiculatus: Crl:MON). These animals were bred in the central animal facility of the University of Burgundy (Dijon) and were the subject of a project application reviewed by the ethical committee (Project APAFIS#18405-2019011014262528).

Figure 3 . 1 .

 31 Figure 3.1. Experimental design.

Figure 3

 3 Figure 3.2. Section of the right hemi-mandible, observed by fl uorescence microscopy, of individuals. A) G-KMH-1. B) M-EFS-2. C) position of the section in the mandible.

Figure 3

 3 Figure 3.3. SEM observation of test specimen.

Figure 3 . 4 .

 34 Figure 3.4. Quantifi cation of the different genes depending on the age, the sex or the diet.

Figure 3 . 5 .

 35 Figure 3.5. Landmarking on the mandible, dark blue dots are landmarks, light blue dots are curves landmarks, orange, yellow, pink, purple and green dots are patch-landmarks. A) in lateral external view. B) in lateral internal view.

Figure 3 . 6 .

 36 Figure 3.6. Skull landmarkings. A) From the left to the right, in upper view, lower view, lateral view; yellow dots are upaired landmarks, blue dots are paired landmarks, red dots are mirrored landmarks. B) Pipeline used to mirror paired landmarks on skulls.

Figure 4

 4 Figure 4.1. A) First two principal components (PC) of the variation in the skull shape across ages and taxa. B-C-D-E) Analyses of postnatal trajectories after parallel transport on the common reference average of the expected marginal means at 7 days). B) First two parallel transported principal components (PC). C-E) Shape changes of the skull along PC2 after parallel transport; C) in lower view; D) in upper view; E) in lateral view. F) Magnitude (in Procrustes unit per day) of the growth vectors of shape changes between two consecutive developmental stages normalized by the age difference. G) Angles between growth vectors of two species at the same developmental interval. H) Angles between the 14-35 days vector and the 7-14 days (prior) or the 35-63 days (after) growth vectors of each species. Error bars represent sampled standard errors.

  Fig 4.1 C) in inferior view. In superior view (Fig4.1 D), development is marked by the expansion of the parietal and the nasal. In lateral view (Fig 4.1 E), one can see the development of the squamosal.

Figure 4 . 2 .

 42 Figure 4.2. Main pattern of covariation between the mandible and the skull. A) First PLS axes. Individuals are colored according to their age. Ellipses correspond to 68% of the data (1 ). B) First PLS axes. Individuals are colored according to their species. C-H) Skull and mandible shape corresponding to the negative values of PLS axes according to the ages. I-N) Skull and mandible shape corresponding to the positive values of PLS axes according to the ages. C & I) Skull in dorsal view. D & J) Skull in lateral view. E & K) Skull in ventral view. F & L) Mandible in dorsal view. G & M) Mandible in lateral internal view. H & N) Mandible in lateral external view.

Figure 5 . 1 .

 51 Figure 5.1. Weights measured during rearing of mice, hamsters and gerbils; at 35 and 63 days of age according to diet.

Figure 5 . 2 .

 52 Figure 5.2. Analyses of post-weaning trajectories after parallel transport on the common reference (average of the expected marginal means at 35 days). A) First two parallel transported principal components (PC) on mandibles. B) First two parallel transported PCs on skulls. C-D) Shape changes between 35 and 63 day-old of the mandible with hard diet effect mapped on soft diet mean shape. C) Mandible in lateral internal view. D) Mandible in lateral external view. E-G) Shape changes between 35 and 63 day-old of the skull with hard diet effect mapped on soft diet mean shape. E) Skull in lateral view. F) Skull in upper view. G) Skull in lower view.

Figure 6 . 1 .

 61 Figure 6.1. Diagram representing the conclusions.

Figure 6 . 2 .

 62 Figure 6.2. DiceCT tests on a 7-day-old mouse specimen (EasyTOM S 150, RX solutions, 5.8 μm, 80kV, 50 μA). A-B) Different slides. C) positions of the slides.

Titre:

  Développement postnatal et évolution du complexe craniofacial chez les rongeurs Mots clés : Développement, Morphométrie géométrique, Epigénétique, Craniofacial, Rongeurs, Croissance Résumé : Le crâne est une structure fortement intégrée, complexe architecturalement et fonctionnellement. En même temps et paradoxalement, cette unité est très évolvable et présente une très grande diversité de formes. Les interactions épigénétiques lors de la croissance vont permettre de répondre aux stimulations mécaniques, de compenser et coordonner la croissance des différents organes composant la tête afin d'acquérir et/ou de maintenir certaines fonctions. Elles vont permettre un développement normal en contrôlant la remodélisation osseuse. Malgré ce rôle central, l'importance de ces interactions dans l'expression des différences entre espèces et à plus long terme dans la dynamique des clades reste peu comprise. Le projet de thèse vise à étudier I) la mise en place de la disparité craniofaciale chez les rongeurs au cours du développement et II) d'évaluer l'importance des processus épigénétiques lors de cette croissance postnatale. Title : Postnatal development and evolution of the rodents' craniofacial complex Keywords : Development, Epigenetic, Geometric morphometrics, Craniofacial, Rodents, Growth

Table 1 .

 1 1. Characteristics of modeling and remodeling in bone (After Allen & Burr 2014).

Table 3 .

 3 3. Specimens used for myosin study.

Table 3 .

 3 4. Sequences of primer used for myosin, actine and GAPDH genes.

Conclusions & PerspectivesConclusions & Perspectives

Annexes

Annexe 2 -Specimens used during experiments