Ludivine Bertonnier-Brouty
email: ludivine.bertonnierbrouty@ens-lyon.fr

Laurent Viriot

Thierry Joly

Cyril Charles

Morphological features of tooth development and replacement in the rabbit Oryctolagus cuniculus

Keywords: Tooth development, mammalian tooth replacement, diphyodonty, 3D-reconstructions, rabbit teeth

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The signaling pathways involved in tooth development and morphogenesis are highly studied in mice [START_REF] Balic | Tissue Interactions Regulating Tooth Development and Renewal[END_REF], fishes [START_REF] Rasch | An ancient dental gene set governs development and continuous regeneration of teeth in sharks[END_REF], or reptiles [START_REF] Whitlock | Biology of tooth replacement in amniotes[END_REF]. Regarding tooth replacement [START_REF] Bertin | Current perspectives on tooth implantation, attachment, and replacement in amniota[END_REF], a majority of fish and reptiles have the ability to replace their teeth continuously throughout their life (polyphyodonty) contrary to the majority of mammals that replace their teeth only once (diphyodonty). In mammals, most odontogenesis studies use the mouse as a model species and mouse tooth development is well described from its morphogenesis to the genetic mechanisms involved. Tooth development is commonly divided into five stages: the placode, the bud, the cap, the bell, and finally the maturation stages. At first, a dental placode appears from the oral epithelium by thickening. Next, at the bud stage, the dental epithelium invaginates, followed by a condensation of the dental mesenchyme.

Then, the epithelium starts to fold, forming the cap stage; this stage marks the beginning of histo-differentiation. The tooth cuspidogenesis starts at the bell stage. Cells begin to secrete dentin and enamel layers at the late bell stage. In the maturation stage, the tooth gets fully mineralized. Because the mouse has no dental replacement, the mechanisms of tooth replacement in mammals are poorly known. Some studies have been done in the ferret [START_REF] Järvinen | The role of the dental lamina in mammalian tooth replacement[END_REF][START_REF] Jussila | Initiation of teeth from the dental lamina in the ferret[END_REF], the shrew [START_REF] Järvinen | The taming of the shrew milk teeth[END_REF], the fruit bat [START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF] and the minipig [START_REF] Wang | Morphology and chronology of diphyodont dentition in miniature pigs, Sus Scrofa[END_REF][START_REF] Wang | Tracking diphyodont development in miniature pig in vitro and in vivo[END_REF]. Altogether, these studies have characterized the morphological changes involved in tooth replacement: the initiation of tooth replacement begins by a budding of the replacement dental lamina at the lingual part of the tooth stalk. Then the replacement dental lamina plunges into the mesenchyme and the tooth morphogenesis begins. Moreover, the first molecular observations of tooth replacement mechanisms in mammals have been done in the ferret Mustela putorius furo [START_REF] Järvinen | The role of the dental lamina in mammalian tooth replacement[END_REF][START_REF] Jussila | Initiation of teeth from the dental lamina in the ferret[END_REF]. However, few developmental stages were studied due to the difficulty to obtain ferret embryos at the correct stage and the presence of seasonal estrus. The shrew Sorex araneus is limited as a model because their deciduous teeth never erupt. In the fruit bat Eidolon helvum, 3D reconstructions and histological sections are good illustrations of mammalian tooth replacement but authors indicated that this species is no longer available for experimental purposes [START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF]. Tooth development and replacement has also been described in the minipig Sus scrofa, whose teeth size and morphology are pretty similar to human teeth [START_REF] Wang | Morphology and chronology of diphyodont dentition in miniature pigs, Sus Scrofa[END_REF]. However, at least 100 days are necessary from the appearance of the replacement dental lamina to the mineralization of the replacement tooth in the minipig. So, the slow development, the size of the animal and ethical concerns are significant obstacles to study tooth development in the minipig. For now,

any model can answer both morphological and molecular questions about mammalian tooth replacement, and previous studies are restricted to some parts of dental development. We thus decided to use the European rabbit (Oryctolagus cuniculus) as a new model to study tooth development and replacement. The rabbit is already an important model for biomedical research [START_REF] Bosze | Application of rabbits in biomedical research: a review[END_REF] and the whole genome has been sequenced (OryCun2.0, 2009). The rabbit dentition is diphyodont with a replacement of some incisors and all premolars [START_REF] Horowitz | Deciduous dentition in the rabbit (Oryctolagus cuniculus). A roentgenographic study[END_REF]. They possess two upper and one lower incisors, three upper and two lower premolars and three upper and lower molars on each side.

Deciduous premolars have a limited growth with root formation whereas permanent teeth are continuously growing [START_REF] Sych | Heterochrony in the development of vestigial and functional deciduous incisors in rabbits (Oryctolagus cuniculus L.)[END_REF]. Associated with this continuous growth, each permanent tooth in the rabbit is supposed to possesses a pool of mesenchymal and epithelial stem cells in its growing part, as in all hypselodont species [START_REF] Tummers | Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species[END_REF].

Rabbit dental development has been studied during the 70's and 80's, Horowitz and collaborators (1973) completing a chronology of deciduous tooth eruption using radiography, giving information on the mineralized part of the teeth. Navarro carried out histological studies of the postnatal development in maxillary (1975) and mandibular cheek teeth (1976).

However, the deciduous incisors and premolars are completely mineralized at birth and thus Navarro et al. do not document their development. [START_REF] Hirschfeld | Incisors of the Rabbit: Morphology, Histology, and Development[END_REF] and [START_REF] Ooë | Développement embryonnaire des incisives chez le lapin (Oryctolagus cuniculus L.). Interpretation de la formule dentaire[END_REF] have studied the incisor development in embryos, but nothing has been reported for the cheek teeth development. [START_REF] Michaeli | The cheek teeth of the rabbit: morphology, histology and development[END_REF] gave a simplified chronology of rabbit cheek tooth development during embryogenesis without separating each tooth and with histological illustrations only for the post-natal stages. All these studies result in a composite and fragmental chronology of the tooth development in rabbit. The purpose of our work is to supply a complete report of the development of all deciduous and permanent teeth in the rabbit. We decided to study rabbits from 12 days post fertilization to 4 days post-natal in order to obtain a complete chronology of tooth development from the initiation of the deciduous tooth to the mineralization of the replacement teeth. Using histological studies and 3D-reconstruction of soft tissues, we present the developmental and morphological characteristics of rabbit teeth.

Material & Method

Samples

Embryos and newborn rabbit samples used for histology and X-Ray microtomography were obtained through collaboration with a standard production for feeding recorded at the Biology Departmental livestock establishment (EDE) n°38044102. We worked on common rabbit breeding regularly crossed with wild rabbits to ensure genetic mixing. The gestation period for a rabbit is about 31 days. We collected embryos at 12,14,16,18,20,22,24,26 and 28 days post fertilization (dpf) and rabbits at 0, 1, 2, 3 and 4 days post-natal (dpn). 3 to 21 embryos were analyzed for each developmental stage. For juvenile and adult rabbits, observations were made on 230 specimens of Oryctolagus cuniculus from the Muséum National d'Histoire Naturelle (MNHN, Paris, France). Age of the specimens were not indicated so we used the skull size as proxy, our sampling contained skull size from 36.65 to 108.8mm. In the 230 specimens, only four still had their deciduous teeth. Each tooth is specifically named: we use 'I', 'P' and 'M' for incisor, premolar and molar respectively; the deciduous teeth are indicated with a 'd'; the tooth number is in exponent for the upper teeth and in index for the lower teeth.

Histology

After dissection of the upper and lower jaws, samples were fixed overnight in 4% PFA. Samples were then rehydrated and fixed in Holland Bouin solution during 1 day. Jaws were demineralized for 1 to 7 days in Morse's solution (10% sodium citrate + 22% formic acid) or in RNase-free 12.5% EDTA + 2.5% PFA during 6 weeks, according to the subsequent experiment to be performed. Then, jaws were embedded in paraffin and serially sectioned with a Leica microtome (7 μm thick slices). Sections were carried out in parasagittal or frontal plane. For histological purpose, samples were dewaxed in xylene, and then colored via an optimized Masson's trichrome protocol with aniline blue, hemalum and fuschin.

In situ hybridization

Specific probes for Osteocalcin and K14 have been designed using the rabbit genome available online (OryCun2.0, 2009). Each probe has been sequenced before use, and then probes were synthesized with RNA-dig labeling nucleotides. Classical mouse in situ protocol has been optimized to be compatible with rabbit tissues. Tissues were treated with HCl 0.2M, with proteinase K 5µg/ml and then incubated with TEA-Acetic anhydride. Hybridization step were done at 62°C. Sections were whashed in a warm washing buffer (1X SSC and 50% formamide) and in MABT solution. After 2 hours in a blocking solution, slides were covered of anti-Dig-AP-antibody solution at 4°C overnight. Sections were washed with MABT and NTMT with teramisol. Sections were stained with BM purple solution and mounted in Aquatex.

X-ray microtomography and 3D reconstruction

Conventional X-ray microtomography allows reconstructing 3D models of mineralized tissues such as bony head and teeth, thus providing a non-invasive access to internal structures. Using phosphotungstic acid (PTA) staining, that binds heavily to various proteins and connective tissue [START_REF] Metscher | MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues[END_REF]; we were able to detect soft tissues by X-Ray microtomography. After fixation, half-head samples were stained in a solution 0.3% PTA in 70% ethanol between 1 week and 8 months according to the size of the sample. Oldest specimens were demineralized to better detect the soft tissue staining. Once the PTA contrast staining was complete, samples were embedded in paraffin and radiographed using a Phoenix Nanotom S (GE Measurement and Control), which was set up with a tungsten source X-ray tube operating at 100 kV and 70 µA for the mineralized tissues and 60kV and 70µA for the soft tissues. The Phoenix datosx2CT software was applied to gather radiographies in a reconstructed 3D volume, in which the voxel size ranged from 1 to 12 μm depending on the size of the specimen. Post-treatment of 3D volumes, including virtual sectioning as well as sub-volume extractions, was performed using VG-studio max software. Surface smoothing was done with Meshlab software. Incisors hole positions were detected using VG-studio max software and frontal sections were done at each hole using a rotation section plane to respect the natural curvature plane of the incisors. We studied 59 holes from ten incisors (6 individuals); the two remaining incisors were used for histology purposes. The centroid of the incisor section was drawn using ImageJ software and mesio-distal and linguo-vestibular axes were positioned in the incisor section. An angle was calculated to determine the hole position, with the mesial position at 0° (and 360°), the vestibular at 90°, the distal at 180° and the lingual position at 270°. Plots were made using R software and the ggplot2 library.

Results

Chronology of incisor development and replacement

We have observed the establishment of the incisors on the upper and lower jaws. The dI1 and the dI2 incisors in the upper and lower jaws appear synchronous in their development.

Rabbits have another incisor in the maxillary, named the dI 3 , which is replaced during ontogeny. We will focus here on the description of the upper incisors (Fig. 1), since we do not observe dental replacement in the lower ones. All our 3D reconstructions of the epithelial parts of the incisors are available in Morphomuseum (3D Dataset will remain locked until publication of the main article, Bertonnier-Brouty, [START_REF] Bertonnier-Brouty | 3D reconstructions of dental epithelium during Oryctolagus cuniculus embryonic development related to the publication "Morphological features of tooth development and replacement in the rabbit Oryctolagus cuniculus[END_REF].

The epithelium is thicker and the mesenchyme begins to condensate around it. At this stage, right and left placodes are not connected to each other. Two days later, it is still impossible to name the incisors, the lower and upper incisors are at the bud stage, the left and right buds seem now connected lingualy. At 18 dpf, all the upper incisors are linked to each other and to the oral epithelium by an epithelium bond, supposed to be a dental lamina rest. As the three incisors follow each other in a mesio-distal plane they are identifiable. From this stage, we can follow the development of each tooth individually.

Vestigial incisor dI 1

At 18 dpf, the first upper incisor is already mineralized with dentin surrounded by dental soft tissues very close to the oral epithelium. This dI 1 is a vestigial tooth; it will not give rise to a functional incisor (Fig. 2,A). At 24 dpf, the dI 1 has passed through the oral epithelium and is now trapped in the oral mucosa (blue triangle in Fig. 1, Fig. S1). At 28 dpf, the dI 1 is shed. dI 2 At 18 dpf, the dI 2 is at the cap stage, linked mesialy to the other dI 2 and in rotation. As in mouse [START_REF] Mucchielli | Correlation of asymmetric Notch2 expression and mouse incisor rotation[END_REF], the dI 2 rotates antero-posteriorly and become parallel to the long axis of the jaws, contrary to molars that stay perpendicular to the jaw axis (Fig. 1).

At 20 dpf, the dental lamina that links the teeth together is completely disorganized and the two dI 2 are no longer linked. The dI 2 has already secreted a thin layer of dentin and the 3D reconstructions show that the vestibular and lingual cervical loops are well formed. These structures are supposed to contain the stem cells that will allow the continuous growth of the tooth during the animal's entire life. At 22 dpf, the teeth are completely separated. At 24 dpf, the dI 2 occlusal surface is very close to the oral epithelium and highly mineralized. The dI 2 presents enamel in its entire mineralized surface. We observed a big cervical loop at the vestibular side and a very small one at the lingual part and this incisor has a fold on its vestibular surface on the mesial side. At 26 dpf, the dI 2 is close to erupt. The secretion of enamel at the vestibular and lingual part of the tooth continues. At 28 dpf, the dI 2 is erupting.

At birth, the unworn dI 2 presents enamel in the entire erupted occlusal surface. In direction to the growth zone, we detect enamel in the vestibular and the lingual part, and then the enamel production in the lingual part stops and enamel is secreted only in the vestibular part of the tooth. At 4 dpn, the dI 2 are worn in their occlusal surface, showing the dentin. In the juvenile, around 1 month post-natal, we observed that the dI 2 present enamel only in the vestibular surface, so the entire part with enamel in the lingual and vestibular position is already worn (Fig. 2,A). In adult, the dI2 is ever-growing. We observed that the dI 2 and the I 3 do not have the same shape at the occlusal surface, the dI 2 has a sharper beveled edge form compared to the I 3 , linked to the gnaw ability of the dI 2 . dI 3 At 18 dpf, we observed the dI 3 in a transitional stage between bud and cap at the distal position of the dI 2 . At the vestibular part of the dI 3 , we observed an epithelium structure distally oriented that is probably a dental lamina rest (Fig. 1, pink triangle). At 20 dpf, the dI 3 , still connected to the dI 2 , is now in a cap stage and rotates antero-posteriorly. At 22 dpf, dI 3 is still linked to the oral epithelium but separated to the dI 2 . We observed a lingual budding of the replacement dental lamina in the dI 3 (Fig. 1, black triangle). At 24 dpf, the dI 3 is already well mineralized and now perfectly orientated compared to the working position. The dI 3 presents small cervical loops at the vestibular and lingual part of the tooth. We detect the replacement dental lamina of the dI 3 attached to the antero-lingual part of the tooth, already parallel to the oral epithelium. At 26 dpf, the cervical loops of the dI 3 are reduced; we observed the start of the root formation. The replacement dental lamina has grown but is still linked to the dI 3 . At 28 dpf, the dI 3 has erupted to the oral epithelium but still do not exit of the oral mucosa. The dI 3 is the only incisor that develops roots; the pulpal cavity seems to close. The I 3 is in a bud stage and no longer linked to the dI 3 . At birth, the dI 3 present enamel in the entire occlusal surface. The I 3 continues its development in a cap stage. At 4 dpn, the permanent I 3 begins to mineralize, the roots of the dI 3 are still in mineralization. The dI 3 is worn in the occlusal surface, showing the dentin. In the juvenile, the roots of the dI 3 have finished mineralizing and we observed root resorption. The I 3 is now well mineralized and will soon replace the dI 3 (Fig. 2,A). Interestingly, the I 3 is entirely covered by enamel in juveniles on the while the dI 2 have enamel only in the vestibular part of the tooth. In adult, the I 3 is covered by enamel in the vestibular and lingual part of the tooth (Fig. 2A, Fig. S2).

Specificity of the rabbit incisors: dentin holes in the newborn incisors

The dI 2 incisor mineralizes at 24 dpf, a thin and continuous layer of dentin is secreted by the odontoblasts. Enamel is first secreted in all the surface of the dI 2 , then restricted to the vestibular and lingual sides to finally be secreted only in the vestibular part of the tooth (Fig. S2). In samples from 24 dpf to 28 dpf, the dentin layer of the dI 2 is continuous; no uneven formation of dentin is detected. However, we observed dentin defects in newborn rabbits. In rabbits from 0 to 4 dpn, we detect the presence of holes in the dentin of the dI 2 , opening the pulp cavity at specific locations, the lower incisors are not affected (Fig. 3, A-D).

Older samples, from juveniles to adults, do not have holes in their incisors, indicating a mechanism of tooth repair.

To study the appearance and repair of the holes, we identified 59 holes in 10 dI 2 incisors from six individuals from 0 to 4 dpn. We precisely localized each hole in frontal section of the incisor by calculating an angle in function of the incisor centroid and the mesiodistal and linguo-vestibular planes (Fig. 3,A). We observed that the majority of the holes are at the distal side of the tooth (73%), and some in the mesial part (27%). No hole is observed in the vestibular or lingual part of the incisor. By comparing the repartition of the holes in right and left incisors of a same individual, we conclude that holes are independently localized in the two incisors on the proximo-occlusal axis; we did not observe any symmetry (Fig. 3,B).

Moreover, the number of holes is variable from one tooth to another, even in the same individual. Interestingly, on the 3 and 4 dpn rabbits, we observed a reparation system by the presence of dentin and cells inside the hole (Fig. 3,D). This reparation seems very efficient as the holes appear at birth and are repaired two days later. We decided to use Osteocalcin in situ staining as a marker for the dentin formation process (Fig. 3,E). We observed that the cells in the holes are odontoblasts still expressing Osteocalcin, so still in a secretion stage. These secreting odontoblasts, coming from the pre-existing odontoblast layer, could explain the presence of new dentin in the holes. Keratin 14 staining indicates the epithelial cells; we wanted to check if the holes could deform the layer of epithelial cells. We detect in the incisor a perfect layer of ameloblasts that is not disrupted by the presence of the hole that are formed at the mesial or distal edges of the enamel layer (Fig. 3,F).

Therefore, these holes are present only in newborns at the mesial or distal limits of the enamel layer, are repaired by the pre-existing odontoblasts and no longer observed during adulthood. Holes seem thus to occur in a very short period of development around or just after the birth of the animal.

Chronology of cheek teeth development and replacement

We observed the establishment of the cheek teeth on the upper (Fig. 4) and lower (Fig. 5) jaws. We focus here our description on the mechanisms of tooth replacement. The steps of the tooth replacement are illustrated in the figure indicating all the tooth tissues (Fig. 6). All our 3D-reconstructions of the epithelial parts of the cheek teeth are available in Morphomuseum [START_REF] Bertonnier-Brouty | 3D reconstructions of dental epithelium during Oryctolagus cuniculus embryonic development related to the publication "Morphological features of tooth development and replacement in the rabbit Oryctolagus cuniculus[END_REF].

At 12 dpf, the oral epithelium is regular in thickness; we detect no morphological trace of tooth development initiation. Two days later, the oral epithelium has thickened in the upper and lower jaws, forming the dental lamina (Figs. 4,5). The dental lamina thickens continuously over a large part of each mouth quadrant and the mesenchyme seems condensed around it, forming the dental placode. In contrast to humans [START_REF] Juuri | The Biology Underlying Abnormalities of Tooth Number in Humans[END_REF], the rabbits do not have a unique continuous dental lamina around the jaw arch but distinct dental laminas for the right cheek teeth, the incisors and the left cheek teeth. At 16 dpf, we observed the budding of the dental lamina with a high condensation of mesenchyme around it. At this stage, it is still impossible to name the teeth, the epithelium being continuous in thickness and shape over the entire length of the bud, we never detect individual tooth buds. In the upper jaw, the budding tooth row is rounded, following the vestibular commissure, and measures 1.3mm long from the mesial to distal part of the bud (Fig. 4). In the lower jaw, the budding tooth row is straighter and smaller with only 0.8 mm long (Fig. 5).

From 18 dpf on, we can identify the teeth and so we will now described the development of each tooth individually. Rabbit cheek teeth have a thin and long dental stalk compared to the mouse or the fruit bat [START_REF] Dosedělová | Fate of the molar dental lamina in the monophyodont mouse[END_REF][START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF]. We observed that the development of the dP4 is the fastest, followed by the dP3, dP 3 and dP 4 that are synchronous and then by the dP 2 so we described tooth development following these categories. For the molars, the M1 is the first to develop and then the development timing follows the mesio-distal organization plan. The lower molar begins their development before the upper molar. dP 2 -P 2 At 18 dpf, the dP 2 is at a transition stage between bud and cap (Fig. 4). The tooth is connected to the oral epithelium and to the followed tooth by the interdental lamina. At the mesial part of the dP 2 , the interdental lamina is present in almost all the diastema length with an oral epithelium link only in the mesial and distal extremities. At 20 dpf, the dP 2 is in a cap stage. The interdental lamina at the mesial part has disappeared but is still present in the distal part of the dP 2 . The dental lamina stalk begin to detach from the oral epithelium. At 22 dpf, the dP 2 is in a bell stage. Initiation of the replacement begins with an epithelial bud from the dental lamina in the lingual part of the dP 2 , called the replacement dental lamina (also named successional lamina). At 24 dpf, the dP 2 is completely detached from the oral epithelium. The replacement dental lamina, still connected to the dP 2 , plunges into the mesenchyme by keeping a lingual orientation (Fig. 4). The interdental lamina is now very thin but still connects the replacement dental lamina of the dP 2 to the replacement dental lamina of the dP 3 . At 26 dpf, the dP 2 begins its mineralization. The dental replacement lamina of the dP 2 is still growing in a lingual direction. At 28 dpf, the dP 2 have the crown completely mineralized and just begins to mineralize at the root level. The P 2 is in a cap stage and is localized in a distolingual plane compared to the dP 2 . At birth, the dP 2 is completely mineralized from the occlusal surface to the roots and the P 2 is still in a cap stage. Four days later, the P 2 is now at the bell stage and begins to move from its lingual position to apical to the dP 2 . Then, around 1 month old, the rooted dP 2 is pushed by the P 2 (Fig. 2,B). The dP 2 has a completely worn crown surface and the roots begin to reduce, the P 2 is ready to erupt. P 2 is hypselodont (with an unlimited growth) whereas the dP 2 was hypsodont. In adult, the P 2 display a complex shape with numerous folds filled with cementum at the mesial part of the tooth. dP 3 -P 3 / dP3-P3 / dP 4 -P 4

At 18 dpf, the dP3, dP 3 and dP 4 are at the cap stage. The teeth are linked to the oral epithelium and connected to each other with the interdental lamina (Fig. 4). At 20 dpf, the dP3, dP 3 and dP 4 are in a bell stage (Figs.4,5). Initiation of the replacement begins in the lingual part of the teeth; we detected the replacement dental laminas. The tooth replacement begins in the dP 4 and we observed a shift in the initiation of replacement with a gradient of development from the distal premolar towards the most mesial premolar. The interdental lamina continues to link all the teeth together but the dental lamina begins to be detached from the oral epithelium. At 22 dpf, the dP 3 , dP3 and dP 4 are at the bell stage. All the premolars display a replacement dental lamina in the lingual side, and in the dP 4, we can see that this dental lamina begins to dive in the mesio-lingual direction. Each upper and lower tooth is still connected to the others but dental lamina is completely detached from the oral epithelium. At 24 dpf, the premolars have finished their morphogenesis and now mineralize by secreting dentin and enamel. The replacement dental laminas are highly invaginated into the mesenchyme and are still attached to some elements of the interdental laminas. The replacement dental laminas from the dP 3 and dP 4 grow in a mesial orientation whereas the replacement dental lamina of the dP3 grows towards the base of the deciduous tooth while maintaining its lingual orientation. The interdental lamina is resorbed around the dP3, detached of the dP 3 , broken between the dP 3 and the dP 4 , and very thin between the other upper teeth. At 26 dpf, the deciduous premolars are now highly mineralized in their occlusal surface. The replacement dental laminas are budding to give the replacement teeth. The P3 is developing at the lingual basis of the dP3, the P 3 in a mesio-distal plane of the dP 3 and the P 4 in a mesio-lingual plane of the dP 4 . At 28 dpf, the dP3, dP 3 and dP 4 are mineralized to the roots. The permanent premolars are all in the cap stage, localized at the basis of the deciduous teeth in different positions: The P3 is localized at the basis of the dP3 in the lingual side, the P 3 is in a mesio-vestibular plane compared to the dP 3 and the P 4 is in a mesio-lingual plane compared to the dP 4 . At birth, the deciduous premolars are well mineralized with dentin, enamel and cementum. The P3, P 3 and P 4 are in a bell stage. The P3 begins to migrate from its lingual position to under the dP3 whereas P 3 and P 4 are still in their developing position. At 2 dpn, the P 4 begins to mineralize followed two days later by the P3 and P 3 . These permanent premolars are now positioned under their deciduous teeth (Fig. 2,B,C, Fig. S1). Then, around 1 month old, the rooted deciduous teeth dP3, dP 3 and dP 4 are pushed by the permanent premolars (Fig. 2,B). The deciduous premolars have a completely worn crown surface and the roots begin to reduce, the permanent teeth are ready to erupt. All the permanent cheek teeth are hypselodont. In adult, the P 3 and P 4 have a molarized bilophodont shape with a lingual fold filled with cementum, whereas the P3 has a complex shape with more folds at the mesial part of the tooth (Fig. 2,B,C).

dP4-P4

At 18 dpf, the dP 4 is at the cap stage. The dP4 is connected to the oral epithelium and to the other teeth of the row by the interdental lamina (Fig. 5). 2 days later, the dP4 is at the bell stage and the initiation of the replacement has begun at the lingual part of the tooth, where we detect the replacement dental lamina. At 22 dpf, the dP4 is the first cheek tooth to mineralize. The tooth is detached from the oral epithelium, but the replacement dental lamina is still connected to the deciduous tooth. At 24 dpf, the replacement dental lamina is detached from the dP4 that is detached to the other teeth. The replacement dental lamina plunges into the mesenchyme keeping the lingual orientation. At 26 dpf, the P4 is budding from the replacement dental lamina at the lingual basis of the dP4 crown. At 28 dpf, the dP4 is completely mineralized and the P4 is at the cap stage, localized at the basis of the dP4 in the lingual side. At birth, the P4 is the first permanent cheek tooth to mineralize and begins to migrate from its lingual position to below the fully mineralized dP4. Two days later the permanent tooth is well-localized bellow the dP4. At 4 dpn, the dP4 is the first cheek tooth to erupt (Fig. 2,C). Then, around 1 month old, the dP4 has a completely worn crown surface and the roots begin to reduce, the P4 is ready to erupt. P4 is hypselodont with a continuous growth.

In adult, the P4 has a molarized bilophodont shape (Fig. 2,C).

M1-M 1

At 18 dpf, the M1 is in a bud to cap stage transition and the M 1 in a bud stage (Figs.4,5). At 20 dpf, the M1 and M 1 are in a cap stage. At 22 dpf, the M1 is at the early bell stage and the M 1 is in the cap stage. We observed an epithelial bud at the lingual part of the M1 and M 1 , indicating a transient rudimentary successional lamina. At 24 dpf, The M 1 and M1 are in a bell stage and the lingual buds are no more visible. Molars are connected distally to the M 2 and M2 but only the M 1 is still connected to a premolar mesialy. At 26 dpf, the M1 begins its mineralization and the M 1 finishes its morphogenesis. At 28 dpf, the M1 and M 1 have the crown completely mineralized, with dentin, enamel and cementum. Then, in juveniles and adults, we observed that the molars are hypselodont with a bilophodont shape (Fig. 2,B,C).

M2-M 2

At 22 dpf, we observed the budding of the continual lamina at the distal part of the tooth row that will give the M 2 and M2 by serial addition of molars (Figs.4,5). At 24 dpf, the M2 and M 2 are in a cap stage, and these teeth are still attached mesialy to the interdental dental lamina. At 26 dpf, the M2 and M 2 are in the cap stage and are the only teeth connected with the remains of dental and interdental lamina. At 28 dpf, the M2 is now detached from the dental lamina and is in a bell stage. The M 2 , in cap stage, is still linked to the dental lamina.

At birth, the M2 begins to mineralize its occlusal surface whereas the M 2 is in bell stage. At 2 dpn, the M 2 and M2 are mineralizing with dentin, enamel and cementum (Fig. 2,B,C). Then, in juveniles and adults, we observed that the molars are hypselodont with a bilophodont shape.

M3-M 3

At 28 dpf, we observed a completely separated dental lamina that is budding in the distal part of the lower tooth row that will gives the M3 (Fig. 5). In the upper row, we also detect the budding of the dental lamina in the distal part of the M 2 that will give the M 3 (Fig. 4). At 2 dpn, the M3 is at the cap stage, followed two days later by the M 3 . Then, in a juvenile around 1 month old, we observed that the M 3 is well mineralized but not erupted (Fig. 2,B). In adult, the M3 and M 3 are highly reduced compared to other molars. Among 188 observed adult Oryctolagus cuniculus specimens, two lacked the upper third molars with no sign of loss, indicating that this tooth can sometimes not be fully developed.

To summarize, the development of the maxillary teeth is late compared to the mandible counterparts and all the teeth of a same row are not synchronized. In the upper and lower rows, the dP4 is the first forming tooth, that will be followed by the dP3, and then, only in the maxillary, by the dP 2 (Fig. 7). The development of replacement teeth follows the same order. Regarding the molars, the M1 is the first to develop, followed by the M2 and finally the M3.

Discussion

We show here the morphology (Fig. 6) and the chronology of rabbit teeth development(Fig. 7). Using 3D reconstructions, we were able to clearly identify the different steps of tooth development and replacement. Our study contains more developmental stages and we separated the results for each tooth, allowing us to obtain a complete and more precise chronology than the ones available in the literature [START_REF] Hirschfeld | Incisors of the Rabbit: Morphology, Histology, and Development[END_REF][START_REF] Michaeli | The cheek teeth of the rabbit: morphology, histology and development[END_REF][START_REF] Navarro | Histological Study on the Postnatal Development and Sequence of Eruption of the Maxillary Cheek-Teeth of Rabbits (Oryctolagus cuniculus)[END_REF][START_REF] Ooë | Développement embryonnaire des incisives chez le lapin (Oryctolagus cuniculus L.). Interpretation de la formule dentaire[END_REF]. 3D-reconstructions from the placode stage lead to the conclusion that the canine and first premolars dP 1 and dP1-2 are never detected in rabbit embryos. These results suggest a complete loss of these teeth in rabbits, contrary to vestigial tooth rudiments in mice (homologous to the dP3 and dP4), detected in early tooth development [START_REF] Sadier | Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning[END_REF][START_REF] Viriot | Evolutionary Implications of the Occurrence of Two Vestigial Tooth Germs During Early Odontogenesis in the Mouse Lower Jaw[END_REF]. However, some observations still need to be clarified, as the epithelium link between the vestigial incisor dI 1 and the dI 2 . This relationship was already problematic for [START_REF] Ooë | Développement embryonnaire des incisives chez le lapin (Oryctolagus cuniculus L.). Interpretation de la formule dentaire[END_REF], who discussed the localization of the dI 2 compared to the dI 1 . In mammals, replacement teeth begin their development at the lingual part the deciduous predecessor [START_REF] Järvinen | The role of the dental lamina in mammalian tooth replacement[END_REF]. Here the dI 1 and dI 2 incisors are in a mesio-distal plan and not in a vestibulo-lingual plan, so it seems more similar to serial development with an interdental lamina between deciduous incisors. However, the rabbit dI 2 has the specificity ofbeing the only tooth to be deciduous and ever growing. For other teeth, the ever-growing ability appears only in permanent teeth. [START_REF] Hirschfeld | Incisors of the Rabbit: Morphology, Histology, and Development[END_REF] concluded that due to its ever-growing capacity the so-called dI 2 is the permanent tooth coming from the dI 1 . More data on the precise dynamics of the tooth development between 16 and 18 dpf are needed to fully understand the nature of the so-called dI 2 . Reconstructions of the concerned structures during the transition from bud to separated teeth in 3D should help to conclude on the links between these incisors.

One particularity of incisor development in rabbits is the appearance of holes that occur only in newborns. The cause of these holes is unknown. These tooth defects have been observed in all our samples, indicating a common phenomenon. We did not find in literature any description of systematic presence of tooth defects at one specific age in rabbits or other species. In this study, we worked on common rabbit breeding regularly crossed with wild rabbits to ensure genetic mixing, limiting the possibility that these dental abnormalities are specific to one genetic selection in a closed breeding. Some hypotheses can be considered to explain the hole formation, as a possible mechanical compression due to the development of adjacent teeth or bone mineralization or the result of molecular signals. It could be interesting to identify which mechanical forces at birth could be responsible for tooth fractures without morphologically affecting the other tissues. These causes could be purely mechanical (development of the bones or the other upper incisors) or molecularly facilitated (involvement of signaling pathways involved in root resorption for example). The observed reparation system, which systematically takes place in the first days of an animal's life, indicates an ability of quick tooth repair, probably due to the high secretion capacity of the odontoblasts in the ever-growing incisor. This speed in tooth repair is incompatible with the classical reparative dentin that require differentiation and migration of new odontoblasts from a pulpal progenitor cell [START_REF] Moses | Immunohistochemical study of small integrinbinding ligand, N-linked glycoproteins in reactionary dentin of rat molars at different ages[END_REF]. So here, another type of tertiary dentin could close the hole, the reactionary dentin that is formed from a pre-existing odontoblast.

The 3D-reconstructions of the rabbit premolars indicate that the permanent upper premolar begins its morphogenesis in a mesio-vestibular plane compared to its deciduous tooth, so in the opposite side in contrast to the other teeth. [START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF] presented in the fruit bat different modalities of tooth replacement throughout the jaw. They showed, using 3D-reconstructions, that the canine, the second and the third premolar do not have the same modalities of replacement due to the relative position of the deciduous and permanent teeth.

In rabbit, the initiation of the tooth replacement always begins by a growth of the dental lamina at the lingual position of the tooth that will give the replacement dental lamina as in the ferret or fruit bat [START_REF] Jussila | Initiation of teeth from the dental lamina in the ferret[END_REF][START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF]. Then, the separation between the first generation of teeth, the oral epithelium and the replacement dental lamina can vary. First, the teeth lose their connection with the oral epithelium. Variations are then observed; the replacement dental lamina can first be separated from the tooth or from the interdental lamina.

Concerning the tooth position of the second generation compared to the first it also depends on the teeth as in the fruit bat. For the lower premolars and the upper incisor, the permanent teeth develop directly on the lingual side of the deciduous teeth. For the upper cheek teeth, the position of the second-generation development varies for each premolar. The tooth replacement begins at the lingual part of the deciduous tooth, but then the replacement dental lamina plunges and develops in different orientation depending on the tooth. The P 2 is localized in a disto-lingual plane compared to the dP 2 , the P 3 is in a mesio-vestibular plane compared to the dP 3 and the P 4 is in a mesio-lingual plane compared to the dP 4 . Indeed, the P 2 and P 3 develop on the same frontal plan, with one lingualy and the other vestibulary. Then, all the permanent teeth migrate under the associated deciduous teeth. Compared to the fruit bat or the ferret [START_REF] Järvinen | The role of the dental lamina in mammalian tooth replacement[END_REF][START_REF] Popa | Complex patterns of tooth replacement revealed in the fruit bat ( Eidolon helvum )[END_REF], the rabbit dental replacement lamina seems to plunge much deeper into the mesenchyme before it begins to initiate its permanent tooth morphogenesis, as observed in the minipig [START_REF] Wang | Morphology and chronology of diphyodont dentition in miniature pigs, Sus Scrofa[END_REF].

During rabbit molar development, we observed buddings at the lingual part of the first molars that quickly disappear. Same tooth shape has been observed in the mouse; a rudimentary successional dental lamina (RSDL) is visible in the lingual part of the M1 tooth germ [START_REF] Dosedělová | Fate of the molar dental lamina in the monophyodont mouse[END_REF]. The RSDL in mouse as in the rabbit does not have odontogenic potential [START_REF] Popa | Revitalising the rudimentary replacement dentition in the mouse[END_REF]. However, by stabilizing the Wnt/ βcatenin signaling in the RSDL, it is possible to obtain a new tooth [START_REF] Popa | Revitalising the rudimentary replacement dentition in the mouse[END_REF]. The molars can be considered as deciduous teeth that are never replaced [START_REF] Luckett | An Ontogenetic Assessment of Dental Homologies in Therian Mammals[END_REF]; the study of this budding observed in the rabbit with the molecular information obtained in mutant mice could identify why these teeth are not replaced.

In this study, we have shown that the rabbit seem to be a good model in dental research. Rabbits are already used in odontological studies but we showed here that they are also useful for studying tooth morphogenesis. Studies of tooth development and replacement in rabbits could allow understanding the mechanisms of mammalian tooth replacement. We showed that common molecular experiments can be done in rabbit tissues as in situ hybridization and rabbits can also be a relevant model to use more advanced techniques as CRISPR/Cas9 technology [START_REF] Yan | Generation of multi-gene knockout rabbits using the Cas9/gRNA system[END_REF]. Compared to other species used to study tooth replacement (the ferret, the fruit bat, the shrew and the minipig), rabbit embryos are easy to get, with large litters of embryos and a pretty short gestation time (31 days). The main disadvantage to the study of the rabbit compared to the other models already proposed is an incomplete dentition with the lack of the canines. However, rabbits have also ever-growing permanent teeth, allowing study of other issues such as dental stem cells maintaining in addition to mammalian dental replacement.

Conclusion

We provided here the description of the complete histo-morphological chronology of the tooth development and replacement in rabbit. It can be considered as a new dataset that allows a better understanding of the dental replacement among mammals. The 3Dreconstructions gave useful information about the geometric organization of the dental replacement and the precise knowledge of the dental development and replacement chronology in this species is a starting point that will allow further studies of gene functions at specific time windows. Development of the dI 1 , dI 2 , dI 3 and I 3 from 14 to 28 days post-fertilization (dpf). Indicated in blue, the vestigial incisor; in pink, the interdental lamina; in black (dotted line), the dental tissues; in black (arrow), the replacement tissues. M, mesial; L, lingual; O, occlusal. (D) Tooth nomenclature: "I", "P" and "M" for incisor, premolar and molar; the deciduous teeth are indicated with a "d"; the tooth number is in exponent for the upper teeth and in an index for the lower teeth. In blue the vestigial incisor, in red all the deciduous teeth that will be replaced. Indicated in blue, enamel layer in the dI 2 ; in yellow, enamel layer in the dI 3 ; in red, enamel layer in the I 3 . The white dotted line indicates the frontal section plane illustrated in the white rectangle. V, vestibular; M, mesial. Scale bar: 1mm.

Figure legends

Fig. 1 .

 1 Fig.1. 3D-reconstructions of the epithelial part of the upper incisors during development.

Fig. 2 .

 2 Fig.2. 3D-reconstructions of the mineralized part of the rabbit teeth. (A) Mineralized tissues

Fig. 3 .

 3 Fig.3. Hole repartition in the upper incisor. (A) Distribution of the holes in 10 upper incisors from 0 to 4 days post-natal (dpn). (B) Distribution of the holes in the two dI 2 from a same

Fig. 4 .

 4 Fig.4. 3D-reconstructions of the epithelial tissues and histology in rabbit upper cheek teeth

Fig. 5 .Fig. 6 .Fig. 7 .

 567 Fig.5. 3D-reconstructions of the epithelial tissues and histology in rabbit lower cheek teeth

Fig. S1 .

 S1 Fig.S1. Histological sections of the rabbit upper incisors and lower cheek teeth. (A) Frontal

Fig. S2 .

 S2 Fig.S2. Views of the 3D reconstructions of the upper incisors mineralized tissues and virtual

Acknowledgements

We thank Nicolas Goudemand and all the members of his team for their help and support. We thank all the members of the "Evolution of vertebrate dentition" for the helpful advices and comments. We thank Joanne Burden for help in the final preparation of the manuscript. We thank Cécile Callou, who is responsible for the collection of the lagomorphs at the Museum National d'Histoire Naturelle (MNHN) of Paris, for giving us access to these collections and for her help during our visit. We are grateful to Mathilde Bouchet-Combe, from the SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENSL, UCBL1), who helped for X-Ray microtomographic analyses. The authors declare no conflict of interest

Author contributions

L.B.B: contributed to design, to acquisition, analysis, and interpretation, drafted manuscript.

L.V: contributed to conception, to analysis and interpretation, critically revised manuscript.

T.J: contributed to interpretation, critically revised manuscript. C.C: contributed to conception and design, to analysis and interpretation, critically revised manuscript.


Gaëlle Muraille

Simon Tricard
email: tricard@insa-toulouse.fr.

Edwin A Baquero

Benjamin Chekroun

Delphine Lagarde

Xavier Marie

Bruno Chaudret

Céline Nayral
email: cnayral@insa-toulouse.fr

Fabien Delpech
email: fdelpech@insa-toulouse.fr

Medium effect on Cd3P2 quantum dots photoluminescence and addition of Pt nanoparticles: Inner filter effect and screening phenomena

Introduction

Quantum dots (QDs) are promising objects due to their wide range of applications, ranging from light-emission to photodetection, solar cells, light emitting diode or tunable emitters for biolabeling [1][2][3][4][5] . In the case of applications in solution (i.e. temperature probes, fluorescence resonance energy transfer, etc.), it is crucial to know the limiting conditions of use. Indeed, numerous parameters may affect the photoluminescence (PL) of QDs such as the concentration of ligands [6][7][8] , the dilution 9,10 , the presence of other chemical compounds [11][12][13] , and may hinder the use of fluorescence properties as a quantitative tool. In particular, the consequences of the Inner-Filter Effect (IFE) have to be seriously taken into account to avoid misleading interpretations 14,15 . This concentration-dependent effect can be described as the combination of: (i) a Primary Inner Filter Effect (PIFE) due to the fact that concentrated solutions act as a real filter preventing light going through the solution, and (ii) a Secondary Inner Filter Effect (SIFE) due to the possible reabsorption of the emitted light by the solution. When the concentration is high enough, IFE results in a decrease of the luminescence intensity compared to more diluted samples. SIFE occurs when the absorbance of the sample overlaps its luminescence. The QD absorption in the emission range then implies a decrease of the luminescence intensity, and thus a distortion of the band shape. Indeed, the emission of given QDs can be reabsorbed by other QDs with a lower energy band gap (larger ones), which can then subsequently undergo radiative transition (and emit lower energy photons) or non-radiative relaxation. This phenomenon leads to an apparent decrease of the emission of the smaller QDs (at higher energy) and an increase of the emission of the larger ones (at lower energy). As a result, in presence of SIFE, the global PL emission wavelength is red-shifted while the QD concentration increases. Even if such effects are sometimes considered as "obvious", very few systematic studies have been carried out in depth [7][8][9] . We can mention the works of Zhang et al. 16 on colloidal carbon QD solutions or of Mei et al. on solutions of ZnSe QDs 9 , this latter evidencing that the optimal QD density to maximize the PL intensity was related to QD spacing and thus to the QD size. More recently, Noblet et al. 14 investigated the concentration effect on CdTe QD solutions to develop a model based on the IFE. They demonstrate that the concentration dependent behaviour of QD fluorescence can be fully understood by the IFE, precluding any hypothesis of dynamic quenching. Despite reabsorption is generally considered as limiting 15,17 , some recent studies take advantage of reabsorption appearing at high concentrations to build optical sensors based on the IFE 18,19 , which requires then a deep understanding of this effect for the optimization of such systems 20 .

A current concern focuses on combining QDs and metallic nanoparticles (NPs) to study energy transfer between them 13,[21][22][23] . The goal of such an approach would be to control the transfer of the exciton -generated by light absorption in the QDs -or of the excited electron or hole from the QDs to the NPs, by a suitable engineering of the relative energy levels. QDs would act as antennas to harvest light energy and thus activate the physical or chemical activity of NPs, e.g. in charge transport or catalysis. A challenge in this field is to overcome the low energy transfer efficiency from or to the QDs due to their small size 24 . In solution, a simple dilution can affect the PL of QDs, by lowering the concentration of ligands, of quenchers and increasing the distance between objects. It is thus crucial to precisely know phenomena affecting the PL of QDs and to differentiate reabsorption, screening and quenching effect.

We have chosen to study the combination of platinum (Pt) NPs and cadmium phosphide (Cd3P2) QDs. Cd3P2 is an interesting material because of its small band gap of 0.55 eV (at 300 K) 25 and its large exciton Bohr radius (18 nm), which make it suitable for systems emitting from the visible to the near infrared [26][27][28] , including the whole sunlight range of emission. Ultra-small (< 2 nm) Pt NPs are air stable and have demonstrated to show Coulomb blockade at room temperature, phenomenon that makes them promising nanoelectrodes to study charge transport at the nanoscale 29 .

In this context, we first present the reabsorption effect of Cd3P2 QDs and Cd3P2@ZnS core-shell QDs. The consequence of addition of Pt NPs on the PL of Cd3P2 QDs is then investigated.

Experimental method

Materials:

Cadmium acetate (Cd(OAc)2, ≥99.99%), octylamine (≥99.0%), tris(dibenzylideneacetone)diplatinum(0) (Pt2(dba)3, ≥98.0%) and tris(trimethylsilyl)phosphine ((TMS)3P, ≥98.0 %) were purchased from Strem, ethylene sulphide (C2H4S, ≥98.0 %), hexadecylamine (C16H35N, ≥98.0 %) and pentane (CH3(CH2)3CH3, ≥98.0 %) were purchased from Aldrich. Ethanol (EtOH), tetrahydrofuran (THF) and toluene (RPE, Analysis grade) were purchased from Carlo Erba. Ethanol and toluene were dried and degassed before use. All manipulations were carried out under argon atmosphere using Schlenk tubes and vacuum line techniques or in glovebox with anhydrous solvent purged under argon before using.

Synthesis of Cd3P2

QDs: In a typical low-temperature synthesis, 0.692 g (3.0 mmol) of Cd(OAc)2 and 1.702 g (7.05 mmol) of hexadecylamine were mixed with 8 mL of toluene and loaded into a 50 mL three-necked flask. The solution was heated to 35°C and 2 mL of a (TMS)3P solution (0.5 mol.L -1 in toluene, 1 mmol) was injected. After injection the colour of the solution changed from yellow to battle-grey. The reaction was maintained at 35°C for 20h. The same protocol is used to form Cd3P2@octylamine taking octylamine instead of hexadecylamine with the same ratio.

Synthesis of Cd3P2@ZnS QDs: In a typical low-temperature synthesis, 5 mL of Cd3P2 QDs (0.125 mmol based on P) and 165.3 mg (0.3740 mmol) of freshly prepared zinc acetate dioctylamine (Zn(OAc)2(OAm)2) in 3.9 mL of toluene were mixed and loaded into a 50 mL three-necked flask.

The solution was heated to 35°C and 1.10 mL of ethylene sulfide solution (0.36 mol.L -1 in toluene, 0.369 mmol) was injected. The reaction was maintained at 35°C for 20h. After, the solution was kept in the glovebox at -30 °C. The same procedure is used to shell either Cd3P2@octylamine or Cd3P2@hexadecylamine.

Synthesis of Pt nanoparticles: a solution of tris(dibenzylideneacetone)diplatinum(0) in tetrahydrofuran (THF) at 4.12 mM is prepared and introduced in a fisher-porter. Then a pressure of 1 bar of carbon monoxide (CO) is applied for 3 min. After that, the fisher-porter is closed and left under magnetic stirring for 30 min. The THF is evaporated and 15 mL of pentane is added under vigorous stirring. The magnetic stirring is stopped after a few minutes and the nanoparticles precipitate. The supernatant is removed and 3 mL of THF is added under stirring.

This washing step is repeated twice.

Addition of Pt nanoparticles:

The QDs were first dried from toluene solution and then dispersed in THF. Solutions with different QDs/Pt NPs ratios are obtained by adding different concentrations of Pt NPs and keeping the same concentration of QDs, in THF. A solution of pure QDs is prepared in the same way (ratio 1:0) in order to be able to compare properly.

Samples characterization

TEM analysis: samples for TEM analysis were prepared by evaporation of a drop of the colloidal solution deposited onto a carbon-covered copper grid. TEM analysis were performed at the "Centre de MicroCaractérisation Raimond Castaing" on a JEOL JEM 1400 electron microscope operating at 120kV with a point resolution of 4.5 Å. The size distributions were determined by measuring by hand the diameter of ca. 320 particles using Image J software. The mean diameter and the standard deviation are calculated from these values.

UV-Visible Absorption measurement: absorbance spectra were measured in solution by using a Perkin Elmer lambda 35 scanning spectrometer with 2 mm-wide cells.

Photoluminescence (PL) measurement: continuous wave cw-PL spectra were recorded in solution using a xenon lamp as excitation source. The white light is dispersed by a monochromator and filtered at 500 nm. The PL emission is collected at 90° with respect to the excitation beam, dispersed by a monochromator and recorded using a UV-vis Hamamatsu photomultiplier tube. Acquisition is performed by the FelixGX software and all the spectra were recorded with the same parameters. The detection range is limited to wavelengths below 800 nm, so the signal cannot be detected entirely for several solutions. In order to determine the full width at half maximum (FWHM), the curves were fitted with the GaussAmp function of Origin, the correlation coefficient was checked for each curve (R 2 > 0.995).

Determination of absolute quantum yield: Quantum yield measurements were performed at room temperature using an integrating sphere (Labsphere). The calculations were performed following a procedure already reported 30 . Briefly the quantum yield was calculated as the ratio of the number of emitted photons and the number of absorbed photons: the number of emitted photons was obtained by subtracting the signal of the solvent to the signal of the sample in the range of emission and by integrating the resulting signal. The number of absorbed photons was obtained by subtracting the signal of the solvent to the signal of the sample around the peak of excitation and by integrating it. The luminescence parts of the curves were fitted with a GaussAmp function on Origin software before the calculation of the integrated emission.

Time-resolved photoluminescence (TRPL) experiments:

TRPL spectra were performed at room temperature using a mode-locked frequency-doubled Ti-sapphire laser with a 1.5 ps pulse width and a wavelength of 430 nm. A pulse picker was used to reduce the repetition rate from 80 MHz to 0.4 MHz. The non-resonant TRPL is dispersed by a spectrometer and then recorded using a Hamamatsu S20 photocathode streak camera with an overall temporal resolution of ~ 50 ns.

The excitation average power is kept at ~ 20 µW @ 0.4 MHz with a laser spot diameter of ~ 2 mm.

Results and discussion

The evolution of PL with the concentration has been investigated on Cd3P2 and Cd3P2@ZnS QDs, synthesized accordingly to published protocols 31 . Transmission electron microscopy (TEM) images of the samples are shown in Figure 1. The mean diameter of the roughly spherical particles is centred around 2.6 nm (standard deviation SD ± 0.7 nm) for the cores and 3.6 nm (SD ± 0.9 nm) for the shelled QDs. The QD concentration effect is studied by preparing a set of successive diluted samples. The concentration of the samples is expressed on the basis of the introduced (TMS)3P which is the limiting agent (the concentrations of (TMS)3P, going from 18.8 to 0.10 mmol.L -1 , are given in Table 1 from C1 to C18). The optical absorption at 500 nm, chosen as a concentration marker, has been systematically measured and reported (see Table 1). All graphs depending on the concentration presented in this paper are traced as a function of absorbance. The range of application of the Beer-Lambert law is limited to the very low concentrations, from C9 to C18 (see Figure S1).

Dilution study

First of all, the integrity of the Cd3P2 QDs in solution and upon dilution has been investigated (C1, the stock solution of Cd3P2 QDs, is diluted up to a factor of 188 to obtain the sample C18).

Absorption spectra are presented in Figure S2a. The absorbance decreases upon dilution as expected, and normalized absorption spectra at the different concentrations overlap perfectly (Figure S2b). The constant shape of the absorption spectrum upon dilution indicates that the size distribution of the QDs is unmodified, and thus their integrity preserved.

Continuous wave (cw)-PL spectra of solutions of Cd3P2 QDs at the different concentrations are presented in Figure 2a. Upon dilution, fluorescence spectra exhibit a shape significantly dependent to concentration. The PL intensity first increases to reach a maximum for C6 and then decreases (Figure S3). For sake of clarity, absorption and PL spectra of three different concentrations are traced in Figure 2b, evidencing the overlap between absorption and emission. The small Stokes shift comes from reabsorption phenomena (SIFE), since the emission of the smallest QDs can be reabsorbed by the largest ones. Upon dilution, this results in a blueshift of ~ 55 nm (from 770 to 715 nm) within the range of the considered concentrations (Figure 2c) while at the same time, the FWHM increases from 62 to reach a maximum at 87 nm (Figure 2d). The impact of the reabsorption on the FWHM is of main importance because this value is commonly used to evaluate the size-dispersity of suspensions of QDs 4,34,35 . However, the correlation between the FWHM and the size distribution can be applied only when using highly diluted solutions. The same argumentation is valuable for the PL emission wavelength (Figure 2c). These values are representative of the sample only when the SIFE is avoided. Concerning Cd3P2 QDs, the emission wavelength indeed continues to decrease upon dilution even for highly diluted solutions, showing that IFE are still present at such concentrations. Unfortunately, it was not possible to measure significant signal in absorbance and PL anymore with the same acquisitions parameters at more diluted solution. Cd3P2 QDs show such strong IFE that it was not possible to observe any concentration range where the FWHM and the emission wavelength is stabilized. TRPL measurements (Figure 3) provide information on the temporal evolution of the optical transitions. Contrary to what observed in cw-PL experiments (Figure 2a), a linear decrease of the time-integrated PL intensity upon dilution is observed while the emission wavelength remains stable, the slight blue-shift observed between C3 and C8 is not significant in TRPL (Figure 3a). We assume that the reabsorption phenomena are not efficient enough as the overall QDs population is still in a bleach state during most of the emission lifetime. Transient absorption measurements have indeed demonstrated that exciton bleach recovery in II-VI QDs occurs in similar time scale as the PL decay time measured here (≈135 ns) [START_REF] Jin | Dicksonb and Tianquan Lian[END_REF] .

Luminescence spectra of different dilutions of core-shell QDs are recorded under the same conditions as mentioned above. The spectra are presented in Figure S4a, and the evolution of the maximum PL intensity of both, core and core-shell QDs, is presented in Figure S4b. The coreshell system exhibits the same behavior as the Cd3P2 cores: upon dilution, the intensity first increases and then decreases. The evolutions of the absorption spectra (see Figure S5), of the emission wavelength (Figure 4a) and of the FWHM (Figure 4b) show the same trend as for the Cd3P2 cores. The emission wavelength blue-shift, between the highest and lowest concentration of Cd3P2@ZnS, is evaluated to ~50 nm. The shape of absorption spectra remains unchanged upon dilution, indicating that the average size and the size distribution of the QDs are stable (see Figure S5).

Quenching and screening effect

Recent reports focus on hybrid structures to study the energy exchange between two different kinds of nanoparticles 13,[21][22][23] . In this context, Pt NPs were chosen as model systems to form mixed assemblies of metallic NPs with Cd3P2 QDs. A process where Pt NPs (~1-3 nm) are naked (i.e. only stabilized by the solvent THF, and by carbon monoxide coming from the synthesis process) was used 37 . The interest of such naked NPs is the absence of extra ligands; we are thus completely sure that no exchange phenomena will compete with the octylamine ligand stabilizing the QDs, which will thus remain chemically unmodified -excess of octylamine from the QD solution will probably coordinate to the Pt surface. Pt NPs were added to Cd3P2 QDs stabilized with octylamine at different ratios. A solution of QDs with an absorbance of 0.1 was prepared, thus fixing the concentration of QDs. The concentration of the Pt NPs varied to obtain different QDs: Pt NPs ratios (1:0 -1:0.1 -1:1 -1:10). The ratios correspond to the molar ratios Cd3P2:Pt, taking into account the quantity of introduced (TMS)3P/2 -which is the limiting agent for the Cd3P2 QD synthesis -and the quantity of introduced Pt atoms. TEM images were realized to check the homogeneity of these different mixtures (Figure 5). For all ratios, both the QDs and the Pt NPs are well dispersed and uniformly mixed. To study the effect of the Pt NPs on the optical properties of QDs, absorption spectra of the different mixtures were measured and are presented in Figure S7. The broad peak around 600 nm related to the QDs (curve 1:0) disappears progressively with the addition of Pt NPs while the absorbance at higher energies increases. PL intensity and quantum yield of mixtures were measured (Figure 6). They both decrease to be null at a ratio of 1:10 so the presence of Pt NPs implies a decrease of the QDs fluorescence. The question is thus now to determine if the loss of PL intensity is the consequence of a quenching by Pt NPs, or the result of a screening by the metallic NPs. In the case of static quenching, a complex is formed at the fundamental state. This complex should have a different absorption than the addition of Pt NPs alone and of QDs alone. On The absorption spectra of the mixture and the mathematical addition of independent spectra have the same trend and superimpose almost perfectly. This observation does not evidence the formation of a complex at the fundamental state. In this particular case, no specific interaction is demonstrated between the Pt NPs and the QDs, so that neither dynamic nor static quenching can be stated. The decrease of luminescence is most probably the consequence of absorption of the incident and emitted light by the Pt NPs. This demonstration aims at warn the community that a decrease of QDs luminescence by the addition of a coproduct in solution is not necessarily the consequence of a quenching effect. Indeed, one should always double-check first that it is not the result of a simple screening effect, which is not systematically done in every article.

Conclusion

As a conclusion, the IFE explains the behaviour of the PL upon dilution of Cd3P2 and Cd3P2@ZnS QDs solutions. A systematic study has never been performed on such QD so far. For each type of QDs, the FWHM, the intensity and the emission wavelength are concentration-dependent, while the decay time of PL remains constant in our measurements conditions. The characteristics of the IFE upon dilution are first an increase and then a decrease of the PL intensity, a blue-shift of the emission wavelength and a widening of the FWHM. As a consequence, in such a case, the maximum PL intensity is not representative of the emitting core size and the FWHM is not representative of the size distribution at any concentration. Concerning the mixture of Pt NPs and Cd3P2 QDs, the decrease of the PL intensity by adding Pt NPs is not caused by a quenching but by a simple screening of the emission of QDs by Pt NPs. In a nutshell, this report stresses that PL measurements can give important information on the nature of a colloidal suspension, but one should be aware that several experimental pitfalls can be encountered on PL interpretation: reabsorption for a FWMH narrowing and not a narrower size distribution, reabsorption for a blue-shift and not a size decrease, reabsorption for an intensity decrease and not a QD denaturation, reabsorption for a shift in emission wavelength after shelling and not a decrease of the core size, screening effect for a co-product addition and not a quenching effect.

Ideally, concentration of the QDs is such an important parameter for PL measurements that information about suitable concentrations for spectral testing should be systematically investigated in any study. Taking all these considerations into account, Cd3P2 QDs are promising elements for using luminescence as a tool for studying response to external parameters (light, temperature, chemical compounds, etc.), if they are studied in proper conditions, being aware of the previous limitations. Experiments in that sense are currently under investigation. Inset: zoom on the 450-800 nm range

Figure 1 .

 1 Figure 1. TEM images of a) Cd3P2 and b) Cd3P2@ZnS

Figure 2 .

 2 Figure 2. a) PL spectra of Cd3P2@HDA -b) Normalized PL and absorption spectra of Cd3P2@HDA, red vertical dashed lines represent the overlap zone -c) Evolution of the FWHM as a function of the absorbance at 500nm

Figure 3 .

 3 Figure 3. a) Time-integrated TRPL spectra of Cd3P2@hexadecylamine Inset: Evolution of total PL Intensity as a function of absorbance -b) PL dynamics of Cd3P2@hexadecylamine integrated over the full spectral range at

Figure 4 .

 4 Figure 4. a) Evolution of the emission wavelength and b) Evolution of the FWHM of Cd3P2 and Cd3P2@ZnS upon dilution TRPL decay times were found to be around 60-90 ns for the shorter and 185-215 ns for the

Figure 5 .

 5 Figure 5. TEM images of Cd3P2 and Pt NPs (ratio Cd3P2:Pt NPs): Pt NPs present a higher contrast than Cd3P2 QDs

Figure 6 .Figure 7 .

 67 Figure 6. a) PL spectra and b) Quantum yield of mixtures of Cd3P2 and Pt NPs

Figure 8

 8 Figure 8 absorption spectra of Pt NPs (0:1), of QDs (1:0), of the mixture (1:1) and of the

Figure 8 .

 8 Figure 8. Absorption spectrum of Pt NPs (0:1), of QDs (1:0), of the mixture of NPs and QDs (1:1) and of the mathematical addition of absorption spectra of NPs and QDs (0:1 + 1:0)

Figure S1 .Figure S3 .FigureFigure S7 .

 S1S3S7 Figure S1. Evolution of the absorbance of Cd3P2@HDA QDs at 500 nm with the concentration of the limiting

Table 1 . Corresponding concentrations of (TMS)3P/2 with the absorbance at 500 nm

 1

	Notation	Corresponding concentration	Absorbance at
		of (TMS)3P/2 (mmol.L -1 )	500 nm
	C1	18.8	4.3
	C2	12.5	3.6
	C3	9.38	2.7
	C4	6.26	1.9
	C5	4.69	1.5
	C6	3.13	1.2
	C7	2.35	0.83
	C8	1.56	0.65
	C9	1.17	0.55
	C10	0.98	0.45
	C11	0.78	0.35
	C12	0.69	0.30
	C13	0.59	0.25
	C14	0.49	0.19
	C15	0.39	0.13
	C16	0.29	0.10
	C17	0.20	0.06
	C18	0.10	0.03

Acknowledgments

This work was supported by the Université Paul Sabatier, the Région Occitanie, the CNRS, the Institut National des Sciences Appliquées of Toulouse, the Programme Investissements d'Avenir under the program ANR-11-IDEX-0002-02, reference ANR-10-LABX-0037-NEXT, and the European Commission for ERC Grant No GA694159 -MONACAT. E.A.B. is grateful to Marie Curie Actions and Campus France for a PRESTIGE post-doc fellowship (FP7/2007-2013) under REA grant agreement no. PCOFUND-GA-2013-609102.

E-mail: cnayral@insa-toulouse.fr, fdelpech@insa-toulouse.fr, tricard@insa-toulouse.fr.