Taste information is received by taste buds and transmitted to the hindbrain by special visceral sensory nerves, the taste nerves. The integrity of taste nerves is essential for the maintenance of taste buds in adult animals. However, a role for taste nerves in the ontogeny of taste buds, in the embryo and at early postnatal stages, has been controversial and is still unresolved. In this study, I establish in a definitive manner that embryonic taste bud formation is nerve-dependent in mouse, thus unifying mechanistically the maintenance/regeneration and ontogeny of these organs.

Parallel to this work, I re-examined the possibility (previously excluded by other authors) of a role for the transcription factor Foxg1 in epibranchial ganglion formation. I find that Foxg1 is essential for the differentiation of gustatory neurons in the geniculate ganglion. This novel role, together with previously described ones in the olfactory epithelium, otic placode and retina, unveils a striking physiological coherence of the functions of Foxg1 (outside its well established one in the cortex), as a master transcription factor for neurons involved in "special senses": vision, hearing, smell and taste.

Résumé

La composition chimique des aliments est perçue par les bourgeons du goût et transmise au cerveau postérieur par des nerfs viscérosensoriels particuliers, les nerfs du goût. L'intégrité de ces nerfs est essentielle au maintien des bourgeons du goût chez les animaux adultes. Cependant, leur rôle dans l'ontogénie des bourgeons, chez l'embryon et aux premiers stades postnataux, est controversé and reste non résolu. Dans cette étude, j'ai établi de façon définitive que la formation embryonnaire des bourgeons du goût dépend des nerfs gustatifs chez la souris, unifiant ainsi les mécanismes de maintien/régénération et d'ontogénie de ces organes.

En parallèle, j'ai réexaminé la possibilité (jusque-là exclue par d'autres auteurs) d'un rôle du facteur de transcription Foxg1 dans la formation des ganglions sensoriels épibranchiaux. J'ai découvert que Foxg1 est un déterminant des neurones gustatifs dans le ganglion géniculé. Ce nouveau rôle, de pair avec ceux décrits précédemment dans l'épithélium olfactif, la placode otique et la rétine, révèle une cohérence physiologique remarquable des fonctions de Foxg1 (en dehors de son rôle bien établi dans le cortex) en tant que facteur de transcription maître des neurones impliqués dans les « sens spéciaux » : vision, ouïe, odorat et goût.

Chapter I. Anatomy of the Peripheral Gustatory Pathways

Anatomy of taste organs 1.a Location and Morphology

The organs of taste perception are taste "buds", groups of fusiform neuroepithelial cells -the taste receptors -embedded in the epithelial lining of the mouth cavity, and whose shape is often compared to an onion, or a round bottle [START_REF] Latarjet | Traité d'anatomie humaine[END_REF], opening at the surface of the epithelium through a taste "pore". In mammals, most taste organs are situated on the tongue and on the soft palate, with some more in the oropharynx and epiglottis, and, in rodents, in the nasoincisor duct. In fish, depending on the species, an abundance of them can be found, in addition to the mouth, on the lips, barbels and even skin of the flanks all the way to the tail [START_REF] Ovalle | Surface morphology of taste buds in catfish barbels[END_REF].

On the tongue of mammals, the taste buds are located on small elevations or bumps of the epithelium, called papillae, at the center of which is a mesenchymal protrusion or "core". There are three morphological kinds papillae: fungiform, circumvallate and foliate (Figure 1). Fungiform papillae are shaped like small domes and harbor a single taste bud at the tip of the epithelial layer. Hundreds of fungiform papillae are located on the anterior two-thirds of the tongue, roughly aligned in parallel rows, in a particularly dense arrangement on the tip and on the edges [START_REF] Reiner | Genetic analysis of tongue size and taste papillae number and size in recombinant inbred strains of mice[END_REF]. The circumvallate papillae, found in the posterior third of the tongue (a single one in mice, and up to seven in humans, in a V-like linear arrangement), are much larger flattened dome-like structures, surrounded by a circular fold or wall (hence the name: "vallum" in Latin desginates a defensive wall), the dome and the wall being separated by a circular, or two semi-circular deep "trenches". The top surface of the dome is devoid of taste buds, but hundreds of them are found at the bottom of the trenches, in its outer and inner walls. Foliate papillae, located on the lateral edges of the posterior tongue are elongated folds separated by parallel trenches, which also house numerous taste buds. The quite different position of taste buds relative to the general structure of the papilla between fungiform, on one hand, and circumvallate/foliate on the other, might have something to do with their different embryonic origin: respectively ectodermal and endodermal.

The tongue is also covered with thousands of non-gustatory filiform papillae that have no role in taste per se but are involved in the sense of touch.

In the palate, taste buds are found in the nasoincisor duct, in a dens stripe at the junction of the hard and soft palate (the "Geschmacksstreifen") and scattered in the soft palate [START_REF] Miller | Distribution of the facial nerve to taste receptors in the rat[END_REF]. They are often said not to reside in papillae, but to be seated in the epithelium in the absence of any morphological specialization. In fact, a recent detailed analysis of the development of palatal buds [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF] clearly shows that they are also embedded in fungiform-like papillae, just less prominent than the lingual ones.

1.b Histology

In rodents, a functional mature taste bud consists of 50-100 cells arranged like the layers of an onion or the staves of a barrel, which open apically to a pore through which tastants can enter and come in contact with the taste cells. In anuran amphibians and some adult urodeles, taste organs have a distinct shape compared to all other vertebrates, much wider and with a much broader surface opened to the oral cavity, called "taste disks" [START_REF] Düring | The ultrastructure of taste and touch receptors of the frog's taste organ[END_REF][START_REF] Graziadei | The ultrastructure of frogs' taste organs[END_REF][START_REF] Zuwała | Morphological differentiation of taste organs in the ontogeny of Salamandra salamandra[END_REF]. Whether this distinct morphology underlies a different physiology, or reflects different developmental mechanisms is unknown.

Classically four types of cells are described, named Type I to Type IV (Figure 1E, Table1): Type I cells are spindle shaped cells, thought to function as glia (i.e. support cells). They tightly wrap around neighbouring taste cells, provide them with nutrition, and clear the extracellular space of certain metabolites.

They express nucleoside triphosphate diphosphohydrolase-2 (NTPDase2, a member of the family of calciumdependent ecto-ATPases), which hydrolyses the neurotransmitter adenosine 5-triphosphate (ATP) released by nearby cells. They also express the enzyme Glutamic acid decarboxylase (isoform GAD65), allowing them to synthesize GABA, which upon release, acts through GABA receptors and inhibits the secretion of ATP and other neurotransmitters by nearby taste cells [START_REF] Dvoryanchikov | GABA, its receptors, and GABAergic inhibition in mouse taste buds[END_REF].

Type II cells are polarized receptor cells, which express G protein-coupled receptors (GPCRs) for sensing bitter, sweet and umami taste modalities. There are no synapses between Type II taste cells and gustatory nerves [START_REF] Larson | The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves[END_REF]Roper, 2013). Instead, Type II cells communicate with nerve terminals using ATP as a fast neurotransmitter (degraded by NTPDase2 on the surface of Type I cells, see above) [START_REF] Finger | ATP signaling is crucial for communication from taste buds to gustatory nerves[END_REF], delivered intracellularly at focal sites from large mitochondria [START_REF] Romanov | Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex[END_REF] and released extracellularly through an ATP-permeable channel complex formed by a heteroduplex of the pore-forming subunits calcium homeostasis modulator I (CALHM1) [START_REF] Taruno | CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes[END_REF] and CALHM3 [START_REF] Ma | CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes[END_REF]. Type II cells comprise several sub-types based on their repertoire of membrane receptors. Generally, most Type II cells express one of two types of GPCRs: taste receptor type 1 (T1Rs, of which there are 3 subtypes, T1R1, TIR2 and T1R3) and taste receptor type 2 (T2Rs of which there are 40-80 subtypes) mainly association with bitter perception [START_REF] Chandrashekar | T2Rs function as bitter taste receptors[END_REF], but not both (Nelson et al., 2001). Either T1R2 or T1R3 is capable of binding sugars and thus transmit sweet stimuli. Other receptors (T2Rs, Gustducin or the combination of T1R1 and T1R3) respond to a variety of bitter substances. In addition, in most Type II taste cells, Ca2+ signaling molecules such as Gγ13, Phospholipase C-β2 (PLC-β2, a taste transducer related to the PLC signaling pathway [START_REF] Clapp | Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway[END_REF])) and Transient receptor potential channel M5 (TrpM5, a cationic channel gated by internal calcium stores [START_REF] Pérez | A transient receptor potential channel expressed in taste receptor cells[END_REF]) are required for sensing sweet, umami, and bitter tastants.

Type III cells are sour sensing cells. They are the only type of taste cells forming synapses with gustatory nerves. They express glutamate decarboxylase 67 (GAD67) -which allows them to synthesize GABA [START_REF] Dvoryanchikov | GABA, its receptors, and GABAergic inhibition in mouse taste buds[END_REF] -, Aromatic L-Amino Acid Decarboxylase (AADC) -which allows them to synthesize serotonin (5-HT) -, and polycystic kidney disease-like 2 /3(Pkd2l1 and Pkd1l3) -which are subunits of the PKDL ion channel that increases the response to sour tastants [START_REF] Sukumaran | Whole transcriptome profiling of taste bud cells[END_REF]. 5-HT is released by Type III cells and activates the gustatory neurons via 5-HT3a receptor [START_REF] Larson | The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves[END_REF]. In addition to sour, Type III cells are also excited by sweet, bitter and umami compounds, but indirectly, via ATP secreted by Type II cells (Roper, 2013). In line with the presence of conventional synapses with gustatory afferent nerves, Type III cells express genes typical of differentiated neurons, such as the neural cell adhesion molecule (NCAM) and the synaptosome-associated protein 25 (Snap25).

Finally, Type IV cells, are round shaped cells located in the basal region of a mature taste bud and are thought to be post-mitotic precursors that differentiate into all classes of taste receptor cells [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF].

Indeed taste buds constantly self-renew with an average lifespan of 10 to 14 days [START_REF] Beidler | Renewal of cells within taste buds[END_REF].

After birth, the classical marker for Type IV cells is sonic hedgehog (Shh) [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF]. The transcription factors Prox1, Hes6 and Mash1 are also expressed in the basal region of the mature taste buds, thus possibly in Type IV cells, although coexpression with Shh has never been assessed [START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF][START_REF] Seta | The bHLH transcription factors, Hes6 and Mash1, are expressed in distinct subsets of cells within adult mouse taste buds[END_REF].

Because they are undifferentiated precursor cells, and possibly of different subtypes, the use of the "Type IV" nomenclature for basal cells has been criticized [START_REF] Finger | Cell types and lineages in taste buds[END_REF][START_REF] Stone | Analysis of cell lineage relationships in taste buds[END_REF].

From taste buds to gustatory nerves, there were two known kinds of neurochemical messages: 1) 5'triphosphate (ATP) secreted by type II taste cells and acting on ionotropic P2X receptors (P2X2/P2X3) on gustatory nerves [START_REF] Finger | ATP signaling is crucial for communication from taste buds to gustatory nerves[END_REF]. 2) 5-HT released by type III cells and acting on taste fibers through 5-HT3 receptors [START_REF] Larson | The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves[END_REF]. Table 1: The molecular markers for taste bud progenitors and taste bud receptors [START_REF] Finger | Cell types and lineages in taste buds[END_REF] Linda A. Barlow, 2014). Hes6, Hes family BHLH transcription factor 6; NTPDase2, nucleoside triphosphate diphosphohydrolase-2; GAD65 and GAD67, glutamate decarboxylase 65 and 67; T1Rs and T2Rs, taste receptor type 1 and 2; PLC-β2, phospholipase C beta 2;

Anatomy of taste nerves

Taste organs are innervated by sensory fibers arising from neurons whose cell bodies reside in several cranial ganglia.

2.a. Cranial sensory ganglia, somatic and visceral, and their projections

Taste organs have a dual innervation: by visceral sensory neurons (that convey taste, the only conscious visceral sensation), and by somatic sensory neurons (that convey touch, pain and heat: not taste modalities, yet contributing to the sensation of taste) (Figure 2). Visceral sensory fibers to the taste buds emanate from Phox2b + neurons [START_REF] Ohman-Gault | The transcription factor Phox2b distinguishes between oral and non-oral sensory neurons in the geniculate ganglion[END_REF] of the geniculate ganglion (on the facial (VIIth) nerve), the petrosal ganglion (distal ganglion of the glossopharyngeal (IXth) nerve) and the nodose ganglion (distal ganglion of the vagus (Xth) nerve). Facial taste fibers carry the sense of taste from the nasoincisor duct, the "Geschmacksstreifen" and the soft palate through branches of the greater superior petrosal nerve (GSPN) (respectively the anterior, medial and posterior palatal nerves [START_REF] Miller | Distribution of the facial nerve to taste receptors in the rat[END_REF]), and from the fungiform papillae of the anterior two thirds of the tongue through the corda tympani (CT). Glossopharyngeal fibers carry taste from the foliate and circumvallate papillae through the lingual branches of the glossopharyngeal nerve, and the vagal fibers carry the taste from epiglottal buds through the superior laryngeal nerve [START_REF] Belecky | Postnatal development of palatal and laryngeal taste buds in the hamster[END_REF]) (and references therein). Somatic sensory fibers to the taste organs (and to their surrounding non-gustatory epithelium) travel in the lingual branch of the trigeminal (Vth) nerve [START_REF] Halsell | Gustatory and tactile stimulation of the posterior tongue activate overlapping but distinctive regions within the nucleus of the solitary tract[END_REF] (and references therein), in the lingual branches of the IXth nerve together with the taste fibers [START_REF] Tanaka | Intramucosal distribution of the glossopharyngeal sensory fibers of cats[END_REF] (and references therein), and the superior laryngeal branch of the Xth nerve together with the taste fibers [START_REF] Sato | Autoradiographic demonstration of the distribution of vagal afferent nerve fibers in the epiglottis of the rabbit[END_REF]. The ganglionic origin of the somatic sensory fibers in the glosspharyngeal and vagal nerves is likely to be the proximal ganglia of these nerves (respectively "superior" and "jugular"), since they are known, at least in mouse, to contain neurons with the genetic signature of somatic sensory (d'Autréaux et al., 2011); However, to our knowledge, the location of somatic sensory neurons projecting in the IXth and Xth nerves has never been directly studied in any species.

2.b. Innervation of the taste organs

Somatic and visceral neurites terminate in different ways within the taste organs. This was shown in two ways: in axolotls embryos, by injecting a GFP expression vector in a two cell stage embryos and later grafted its pharyngeal ectoderm (containing the placodes, from which visceral neurons arise, see below § II. 2) or its neural crest cells (from which somatic neurons arise, see § II. 2) into unlabeled embryos (Harlow & Barlow, 2007). The authors observed that neurons originated from placodes (i.e. visceral) exclusively innervated the taste bud itself (thus were "intragemmal"), whereas neurons from neural crest (i.e. somatic) only ended in the non-gustatory epithelium, displaying a fork-like shape at the periphery of the bud (thus were "perigemmal"). In mouse, a similar arrangement was demonstrated by using the "Brainbow" method of genetic tracing [START_REF] Zaidi | Innervation of taste buds revealed with Brainbow-labeling in mouse[END_REF] (see the fungiform papillae as an example in Figure 3).

2.c. Central projection of the sensory neurons

Through the gustatory nerves (GSPN, CT and IXth), taste information is transmitted to interneurons in the central nervous system that form the nucleus of the solitary tract (nTS), more precisely its rostral part which integrates taste sensory information and sends it to the cortex [START_REF] Whitehead | Distribution of synapses on identified cell types in a gustatory subdivision of the nucleus of the solitary tract[END_REF]. One fungiform papilla is innervated by several taste neurons (ranging from 3 to 14 in rat) (Krimm & Hill, 1998). One taste neuron innervates several fungiform papillae, but the numbers of papillae are variable depending on the stage (Zaidi and Whitehead, 2006).

The way specific tastes are transmitted to the brain is not completely understood. Two theories have gathered experimental arguments: the "labeled line coding" theory predicts that individual taste receptor cells, and the individual nerve fibers that innervate them are specialized for specific tastes (sweet, umami etc…), so that the different tastes are coded by dedicated neural pathways. On the other hand, the combinatorial code theory states that receptor cells can respond to different taste modalities (possibly with different thresholds) and that taste coding is provided by groups of neurons innervating these polymodal cells and /or several receptor cells types. This debate, which exceed the scope of this review is summarized in [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF].

Chapter II Ontogeny of the Peripheral Gustatory Pathways

Ontogeny of taste organs

The development of taste organs (which has been studied mostly in the rat and the mouse) is extremely protracted, beginning at around E12 in mouse and completed up to a month after birth [START_REF] Hall | Expression of sonic hedgehog, patched, and Gli1 in developing taste papillae of the mouse[END_REF]Hosley and Oakley, 1987). Moreover, it does not unfold at the same rate at different locations of the mouth cavity, palatal taste buds forming the first [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF], those in the circumvallate papilla the last (Hosley and Oakley, 1987). Finally, the cells are constantly replaced, with a global turnover of about 10 days [START_REF] Beidler | Renewal of cells within taste buds[END_REF][START_REF] Perea-Martinez | Functional cell types in taste buds have distinct longevities[END_REF]) [START_REF] Delay | Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage[END_REF], so that the development of taste buds, in a sense, never ends. These temporal aspects explain in part, technically and conceptually, many of the uncertainties concerning the mechanisms of taste organ development, and particularly of its relationship to innervation.

I will first describe the morphological events during taste bud development, then the concomitant changes in expression of the main genes documented during development; Then I will summarize the local, intraepithelial mechanisms known for taste placode and papilla development, and discuss in more detail the central question of the development of taste buds per se and its relationship to innervation, the focus of my experimental work. Finally, I will review the renewal of taste bud cells.

1.a. Morphological events

The sequence of morphological differentiation of taste organs can be divided in three phases (Figure 3).

In a first phase at around E12 in mice, local thickenings, called placodes, emerge in the originally homogeneous oral epithelium, and express a number of genes (see below). In a second phase, at around E14.5, fungiform and circumvallate taste placodes undergo an evagination (sometimes described as a sinking or invagination of the epithelium around them) [START_REF] Jitpukdeebodintra | Developmental patterning of the circumvallate papilla[END_REF][START_REF] Kim | Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development[END_REF][START_REF] Whitehead | Development of fungiform papillae, taste buds, and their innervation in the hamster[END_REF] and form a sort of dome, with a mesenchymal core, called a papilla. It is often considered that this phenomenon does not take place in the soft palate, although slight elevations can be detected which might correspond to papillae [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF]. Finally, at perinatal stages and later, onion-shaped taste buds form, made of elongated taste cells [START_REF] Mistretta | Development of fungiform papillae: patterned lingual gustatory organs[END_REF][START_REF] Mistretta | Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs[END_REF]: one at the apex of every fungiform or soft palate papilla, and about 150 at the bottom of the trenches of the circumvallate papilla.

In foliate papillae, the taste buds embedded in the trenches start appearing within 10 days after birth [START_REF] Miller | Proliferation of taste buds in the foliate and vallate papillae of postnatal hamsters[END_REF][START_REF] Oakley | Taste Bud Development in Rat Vallate and Foliate Papillae[END_REF] .

1.b. Gene expression events

The sequence of morphological changes is paralleled by the dynamic expression of a number of genes (Figure4), among which are signaling molecules, transcription factors and structural proteins. The following genes represent most of those documented during embryonic stages: Taste organs are born from epithelial cells which express cytokeratins as an important component of intermediate filaments. At least 20 cytokeratins are expressed in epithelial cells in a variety of combinations throughout the body. A subset of them is expressed specifically in taste bud cells: cytokeratins 7, 8, 18 and 19 [START_REF] Knapp | Keratins as markers of differentiated taste cells of the rat[END_REF] (and references within) and cytokeratin 20 in taste buds of the posterior third of the tongue [START_REF] Zhang | The distribution and origin of keratin 20-containing taste buds in rat and human[END_REF].

Cytokeratin8 (K8) is the most widely used marker of taste buds at postnatal stages. However, early in development, it is a general marker for the tongue epithelium, the so-called periderm, a simple epithelial monolayer that transiently covers all developing mammalian embryos [START_REF] Oakley | Building sensory receptors on the tongue[END_REF]) (and references therein). The transition of K8 from general epithelial marker to taste cell-specific marker is imperfectly assessed to this day. [START_REF] Mbiene | Distribution of keratin 8-containing cell clusters in mouse embryonic tongue: evidence for a prepattern for taste bud development[END_REF] described that K8 reactivity is already resolved in the pattern of the future taste buds at E13.5, thus before invasion of the epithelium by nerves, and this remains a key argument for the nerve independent formation of taste buds. However, this early restriction has been explicitly questioned by [START_REF] Oakley | Building sensory receptors on the tongue[END_REF] on the grounds that the dilution of the antibody (Troma-I) used by Mbiene and Roberts (1/2000), and even a ten-times lower dilution (1/200) does not give any signal in taste buds at any developmental stage in the hands "of others" (without reference). In this review, these authors claim instead that the first K8-positive precursors of taste cells appear at E17, but the 4 references provided either do not show anything at perinatal stages, or do so only at P0 -in the circumvallate [START_REF] Cooper | Functional redundancy and gustatory development in bdnf null mutant mice[END_REF] and the fungiform papillae [START_REF] Sun | Development of anterior gustatory epithelia in the palate and tongue requires epidermal growth factor receptor[END_REF] -or P1 -in the circumvallate [START_REF] Fan | Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors[END_REF]. At embryonic stages, the only convincing image of K8 expression, to my knowledge, is in the recent study of the palatal taste organs [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF]) that shows K8+ taste buds at E15, a stage at which they are said to "first appear" (no earlier stage being shown). It is difficult to extrapolate from these data to other regions of the oral cavity since the development of taste buds, as mentioned earlier, is not synchronous between different g-regions.

In sum, there is a shortage of data on the dynamic of K8 expression in taste organs, which I have tried to

remedy (see Results

). There is no data at all on the timing of expression of other keratins.

Keratin 14 is mostly expressed in the oral epithelium surrounding taste bud cells thus in a perigemmal posoiton), while only a few CK14-positive cells are found in the taste bud proper [START_REF] Asano-Miyoshi | Cytokeratin 14 is expressed in immature cells in rat taste buds[END_REF].

The Hogan's lab has used a CK14 CreER ;ROSA26LacZ line to trace CK14-expressing cells during taste buds maturation and in this way showed that CK14 labels at least some progenitors of the taste bud. [START_REF] Okubo | Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate[END_REF] (and see below).

Signaling molecules

Sonic hedgehog

One of the most precocious markers of taste papillae is Sonic hedgehog (Shh). Shh is expressed diffusely in the epithelium of the tongue primordia at E11.5, then restricted to the prospective sites of fungiform papillae at E12.5 [START_REF] Hall | Expression of sonic hedgehog, patched, and Gli1 in developing taste papillae of the mouse[END_REF] -most convincingly illustrated in [START_REF] Nakayama | Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo[END_REF]. In the palate, expression occurs somewhat later: it appears in the hard (bony) palate at E13.5 as transverse bands prefiguring the palatal ridges (or ruggae, which have a prehensile but no gustatory function), and in the soft palate at E14.5, in the punctate pattern of the future palatal taste buds. At the most anterior part of the soft palate (i.e. at the junction with the soft palate) a transverse band of Shh prefigures a linear arrangement of taste buds: the Geschmacksstreifen [START_REF] Nakayama | Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo[END_REF]. Finally, in mature taste buds (in practice, in the adult animal), Shh expression persists only in intragemmal round basal cells ("type IV") [START_REF] Miura | Co-expression pattern of Shh with Prox1 and that of Nkx2.2 with Mash1 in mouse taste bud[END_REF] interpreted as postmitotic precursors [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF]. To resolve the relationship between these different expression phases, a fate mapping study was performed with a tamoxifen inducible Shh::creER T2 , showing that Shh + cells traced by tamoxifen injection at E12.5, and even more so at E14.5, mostly give rise to intragemmal cells at P0. It was concluded [START_REF] Thirumangalathu | Fate mapping of mammalian embryonic taste bud progenitors[END_REF] that Shh expressing cells in fungiform papillae at E12.5-E14.5 mostly represent taste bud progenitors, which was added to the series of arguments for a nerve independent development of taste buds (see below). However, a few caveats should be mentioned: i) some non-taste epithelial cells are detected at P0 and in fact not quantified (the paper only quantifies epithelial clones completely outside taste papillae, which become dramatically rarer with injections at E14.5). ii) Taking into account the time of action of tamoxifen on the Cre ERT2 recombinase, and of the Cre ERT2 recombinase on the LacZ reporter allele, the tracing probably reveals cells that expressed Shh at around E15, which is after the arrival of nerve fibers in the epithelium [START_REF] Mbiene | Distribution of keratin 8-containing cell clusters in mouse embryonic tongue: evidence for a prepattern for taste bud development[END_REF].

In conclusion, from E11.5 to postnatal stages, there appear to be a progressive restriction of Shh expression, from the entire lingual epithelium to the taste placodes, then to the taste bud progenitors, and finally to its basal cells [START_REF] Liu | Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance[END_REF], the transition between these phases being hard to delineate so far. This transition is accompanied, at an undefined stage, by a dramatic change of function, from inhibitor of placode formation (see §1c) to essential signal for taste bud maintenance (see §1e).

BMPs

Bone morphogenetic proteins (BMP2, 4 and 7) participate in the spatial patterning of taste placodes.

BMP2 and BMP4 are expressed in regular spots on the anterior two third of tongue at around E12 (E14 in rat)

and then in the forming fungiform papillae. After E14 or E15, these two BMPs are gradually downregulated [START_REF] Jung | Shh, Bmp-2, Bmp-4 and Fgf-8 are associated with initiation and patterning of mouse tongue papillae[END_REF][START_REF] Zhou | Bone morphogenetic proteins and noggin: inhibiting and inducing fungiform taste papilla development[END_REF]. In the adult, BMP4 positive cells persist in the perigemmal region of fungiform and circumvallate papillae, i.e. adjacent to the basal part of taste buds. These BMP4 positive cells are hypothesized to correspond to slow cycling stem cells to differentiate into final taste bud cells [START_REF] Nguyen | Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice[END_REF]. BMP7 is expressed in the hard palate and tongue throughout embryogenesis. In hard palate, it is first evident at E14.5 and its expression persisted until E18.5. In the tongue, it is expressed widely throughout the tongue at E15.5 and then at E16.5 its expression restricted to the surround area of circumvallate papilla and the anterior part of the tongue [START_REF] Iwatsuki | Wnt signaling interacts with Shh to regulate taste papilla development[END_REF][START_REF] Liu | Wnt-beta-catenin signaling initiates taste papilla development[END_REF][START_REF] Zouvelou | Deletion of BMP7 affects the development of bones, teeth, and other ectodermal appendages of the orofacial complex[END_REF].

Transcription factors

Prox1 is a homologue of drosophila prospero homeobox gene. At E12.5, it is expressed in the fungiform taste placodes, at E14.5 in the apical epithelium of fungiform papillae and postnatally in basal cells [START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF], therefore in a pattern similar to Shh, as confirmed by double-labeling, except that at postnatal stages Prox1 labels only a subset of Shh + basal cells [START_REF] Nakayama | Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo[END_REF]. Functionally, prospero is required for cell cycle exit and differentiation of ganglion mother cells (Li and Vaessin, 2000). In mouse, Prox1

regulates the cell cycle of retinal progenitors (Dyer et al., 2003) and cochlear sensory hair cell neurogenesis [START_REF] Nishimura | Dynamic Expression of Sox2, Gata3, and Prox1 during Primary Auditory Neuron Development in the Mammalian Cochlea[END_REF], suggesting that it could have a role in the production of taste cells, but its function was never explored there.

Sox2 is a HMG-box transcription factor of the SoxB1 family. Its expression resembles those of Shh and

Prox1, except that it is consistently broader: At E12.5 it is detected at a high level in the placodes but also at a lower level throughout the tongue epithelium; at E14.5 a strong expression level is found in both Prox1 + (thus Shh + ) cells and in cells flanking the Prox1 + ones; and in the adult it is detected in perigemmal cells in addition to intragemmal ones [START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF]. By lineage tracing at perinatal or adult stages (using a K14-Cre ER [START_REF] Okubo | Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate[END_REF] or a Sox2-Cre ERT2 allele [START_REF] Ohmoto | Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain[END_REF] it was found that perigemmal Sox2 + cells (situated outside the taste bud itself, on its basal side) which PCNA + and PHH3 + (thus are dividing), contribute both to the taste buds and to nearby non-gustatory keratinized epithelial cells ("pore cells"), most clones that include taste cells also including non-taste ones. Thus perigemmal Sox2 + progenitor cells (or "stem" cells) appear bipotential, with two fates, gustatory and non-gustatory.

Proneural genes

Transcription factors (as well as receptor and ligands) related to the Notch signalling pathway are also upregulated during taste bud development.

Mash1 (Achaete-scute homolog 1) a proneural bHLH transcription factor. Is expressed after Shh and Prox1 at around E15 (first expression in isolated cells or clusters of a few cells in the soft palate at E16.5 in [START_REF] Nakayama | Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo[END_REF] and at the apical part of the circumvallate papilla from E14.5 in [START_REF] Seta | Notch-associated gene expression in embryonic and adult taste papillae and taste buds suggests a role in taste cell lineage decisions[END_REF]. At E18.5, Mash1 null mutants lack expression of aromatic L-amino acid decarboxylase (AADC) (a marker of type III cells in adults), in palatal taste buds and on the apical surface of the circumvallate papilla [START_REF] Seta | Mash1 is required for the differentiation of AADC-positive type III cells in mouse taste buds[END_REF] (but note that one of two locations documented in these studies, the apical side of the circumvallate papilla, does not give rise to taste buds in the adult).

Hes6 (Hes Family BHLH Transcription Factor 6) expression has also been documented in the apical side of the circumvallate papilla, and later at E18.5 in its trenches where taste buds will develop [START_REF] Seta | Notch-associated gene expression in embryonic and adult taste papillae and taste buds suggests a role in taste cell lineage decisions[END_REF].

Hes1, the receptors Notch1, Notch3 and Notch4, the ligands Dll1, Jagged1 and Jagged2 are also expressed at E18.5 in the trenches of the circumvallate papilla. And all these molecules are expressed in subsets of taste buds cells or perigemmal cells in the adult [START_REF] Seta | Notch-associated gene expression in embryonic and adult taste papillae and taste buds suggests a role in taste cell lineage decisions[END_REF][START_REF] Seta | The bHLH transcription factors, Hes6 and Mash1, are expressed in distinct subsets of cells within adult mouse taste buds[END_REF]. No functional data is available.

NeuroD, a basic helix-loop-helix transcription factor, first appears at postnatal day 3, and increases in number during postnatal development [START_REF] Suzuki | Expression of NeuroD in the mouse taste buds[END_REF].

Other genes have been examined only postnatally and mostly in adult taste buds. Those include all the markers of taste bud cells discussed in the anatomy section §I.1.b) plus two transcription factors:

Skn-1a (Pou2f3) is a POU homeodomain transcription factor specifically expressed in taste receptor and basal (progenitor) cells in the adult (the onset of expression has not been explored). It is essential for the generation of type II cell generation: Skn-1a mutants lose all type II receptor cells from taste buds (thus expression of the taste receptors T1Rs, T2Rs, Gustducin, Plcb2 and Trpm5) and have a concomitant increase in Type III cells (sensitive to sour) (thus upregulation of Pkd2l1, PKd1L3, Aadc and Snapp25) [START_REF] Matsumoto | Skn-1a (Pou2f3) specifies taste receptor cell lineage[END_REF]. As a consequence, Skn1a mutant mice lose sensations for sweet, bitter and umami tastes.

The transcription factor Nkx2.2 known for its role in the spinal cord and hindbrain [START_REF] Briscoe | Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling[END_REF] starts being coexpressed with Mash1 at postnatal stages ( at an undefined time, but posterior to P0.5) [START_REF] Miura | Co-expression pattern of Shh with Prox1 and that of Nkx2.2 with Mash1 in mouse taste bud[END_REF]. No functional data is available.

1.c. Mechanism of taste placode and papilla formation

Work on the formation of fungiform placodes and papillae have established that it occurs prior to innervation (see above) -and thus independently of nerves. The nerve independence of the formation of fungiform papilla morphogenesis was definitely proven to be nerve-independent in cultures of mouse tongue explants [START_REF] Mbiene | Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia[END_REF].

This observation directed the search for a mechanism to tongue intrinsic phenomena and five signaling pathways were discovered be involved and to interact in complex way: Shh, BMPs, retinoic acid, the Wnt-βcatenin pathway and the Fgf pathway (Figure5). i) Blocking Shh signalling, either pharmacologically or with blocking antibodies in embryonic tongue cultures, leads to the formation of supernumerary and oversized placodes and papillae (that overexpress Shh), in some cases eliminating the inter-papilla space and allowing taste placode formation in the "intermolar eminence", a normally placode-free region, [START_REF] Hall | Disruption of sonic hedgehog signaling alters growth and patterning of lingual taste papillae[END_REF][START_REF] Liu | Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance[END_REF][START_REF] Mistretta | Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium[END_REF][START_REF] Shahawy | Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid[END_REF]. Possibly because this role of Shh is so difficult to reconcile with its later one in taste bud maintenance and regeneration (see §1e), it was summarized in a recent review [START_REF] Mistretta | Tongue and Taste Organ Biology and Function: Homeostasis Maintained by Hedgehog Signaling[END_REF] by the broad and rather misleading statement "Shh is a principal regulator of fungiform papilla formation, patterning and differentiation". The evidence accrued so far shows that Shh plays a role, not in the "formation" of papillae per se (in fact, it inhibits it) but only in their patterning, by downregulating its own expression in the papilla surroundings and directing them to become a non-gustatory area. Of note, Shh expression in the oral cavity is associated with other non-gustatory placode-like specialization: the rugae of the bony palate and the dorsal surface of the circumvallate papilla (which does not produce taste buds, unlike the trenches) [START_REF] Welsh | Signaling integration in the rugae growth zone directs sequential SHH signaling center formation during the rostral outgrowth of the palate[END_REF] (and see Result chapter).

ii) The canonical Wnt-β-catenin pathway promotes the formation of placodes, both in size and in number, as demonstrated by pharmacological and genetic gain and loss-of-functions in embryonic tongues [START_REF] Liu | Wnt-beta-catenin signaling initiates taste papilla development[END_REF]. The Wnt pathway (more precisely the ligand Wnt10b) interacts with the Shh pathway in the following way: Wnt10b enhances Shh expression (together with placode formation)

while Shh inhibits Wnt10b expression, possibly accounting for the surround inhibitory effect of Shh on placode formation discussed above [START_REF] Iwatsuki | Wnt signaling interacts with Shh to regulate taste papilla development[END_REF].

iii) BMP7 is coexpressed in the placode/papilla. Its role was also investigated by gain and loss of function showing that it counteracts Wnt expression, and thus Shh expression in the placode. Its action is in turn inhibited by Follistatin secreted in the core mesenchyme of the papilla [START_REF] Beites | Follistatin modulates a BMP autoregulatory loop to control the size and patterning of sensory domains in the developing tongue[END_REF].

iv) Retinoic acid (RA) promotes taste placode formation, its activity being suppressed by Shh [START_REF] Shahawy | Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid[END_REF].

v) Mesenchyme-derived FGF10 counteracts Wnt signalling thus limits the size of the placodal field. [START_REF] Prochazkova | FGF signaling refines Wnt gradients to regulate the patterning of taste papillae[END_REF] Thus, a complex series of activators and inhibitors of papilla/placodal cell fate are expressed in the epithelium (and underlying mesenchyme) and manipulating these factors leads to alter spacing (and size) of these papillae/placodes. This suggests that the initial patterning of the placodes could result from a lateral inhibition phenomenon or, alternatively, from a reaction-diffusion (i.e. Turing) mechanism. Shh, Sox genes, BMPs and Wnts have been implicated in several reaction-diffusion systems. Strikingly, one of the best characterized Turing phenomenon occurs right next to soft palate and its taste buds, on the hard palate, for the specification of palatal ridges at regular intervals [START_REF] Economou | Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate[END_REF]. Those arise as linear placodes that share several markers with taste placodes ( [START_REF] Zhang | The distribution and origin of keratin 20-containing taste buds in rat and human[END_REF] and see below, the Results section). Of note, stripes and spots are easily interconvertible patterns during Turing phenomena [START_REF] Kondo | Reaction-diffusion model as a framework for understanding biological pattern formation[END_REF]. It is therefore tempting to hypothesize that the arrays of taste placodes on the tongue and the leopard spot-like arrangement of placodes on the soft palate also arise by a Turing phenomenon. This possibility has been alluded

to in [START_REF] Beites | Follistatin modulates a BMP autoregulatory loop to control the size and patterning of sensory domains in the developing tongue[END_REF] (the authors proposed that BMP7 and Follistatin "improve accuracy of a pattern forms by diffusion-driven instability") and a first model of this type, to explain the action of Fgf10 has recently been proposed in [START_REF] Prochazkova | FGF signaling refines Wnt gradients to regulate the patterning of taste papillae[END_REF].

Downstream of these signaling pathways must lie transcription factors. The only one for which a role has been documented is Sox2. The knock down of Sox2 in embryos [START_REF] Okubo | Sox2 is required for development of taste bud sensory cells[END_REF] or its conditional deletion [START_REF] Castillo-Azofeifa | SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis[END_REF]) [START_REF] Castillo-Azofeifa | SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis[END_REF] blocks both the formation of papillae and taste buds in them. Conversely, overexpression of Sox2 blocks the formation of filiform papillae, partially transformed in taste papillae (but without a taste bud), suggesting that the level of Sox2 determines the taste papillae versus filiform papillae fate (without it being sufficient for the differentiation of the taste bud itself). At least in adults, Sox2 lies downstream of Shh [START_REF] Castillo-Azofeifa | SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis[END_REF], but we just saw that the role of Shh is very different in adults and embryos.

The formation of circumvallate papillae differs somewhat from that of fungiform papillae. The trenches of the large dome-like structure arise by a clear invagination of the epithelium, at the bottom of which from the taste buds and open the duct of the associated salivary glands (von Ebner glands). Thus, the topographical relationship of taste buds to the papilla is completely different from fungiform papilla. And, unlike in fungiform papilla, the strong expression of some markers (Shh [START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF] but also others (see results section) on the dorsal surface of the papilla does not prefigure the formation of taste buds. Another difference is that nerve ablation such as in BDNF; NT-3KO entails an atrophy not only of the taste buds, like in fungiform papillae, but of the papillary structure itself [START_REF] Ito | Taste cell formation does not require gustatory and somatosensory innervation[END_REF].

1.d. Mechanism of taste bud formation (and its relationship to nerves)

Once the placodes and/or papillae have formed, taste buds differentiate in them. As mentioned earlier, this phase is difficult to study because it is very protracted and spans late embryonic and early pot-natal days.

Relatively few studies have examined this developmental window. For almost a century, the development of taste buds was examined via their striking dependency on their afferent nerves in adult animals: if taste nerves were cut, taste buds degenerated. If taste nerves were allowed to regrow, taste buds regenerated.

Among more than 200 studies (according to [START_REF] Castillo | Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium[END_REF]) demonstrating this property, the first was by Von Vintshgau and Hönigschmied on rabbit (Nervus Glossopharyngeus und Schmeckbecher, (1877) Archiv der Gesamten, Physiologie) (cited in [START_REF] Oakley | Building sensory receptors on the tongue[END_REF])), followed by the studies on the barbels of catfish [START_REF] Torrey | The relation of taste buds to their nerve fibers[END_REF] (Olmsted, 1920b), in dog (Olmsted, 1920a;[START_REF] Olmsted | Effects of cutting the lingual nerve of the dog[END_REF] in rats [START_REF] Guth | The effects of glossopharyngeal nerve transection on the circumvallate papilla of the rat[END_REF][START_REF] Segerstad | Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chordalingual nerve[END_REF], cats [START_REF] Guth | Taste buds on the cat's circumvallate papilla after reinnervation by glossopharyngeal, vagus, and hypoglossal nerves[END_REF], gerbil [START_REF] Cheal | Regeneration of fungiform taste buds: temporal and spatial characteristics[END_REF], hamster [START_REF] Whitehead | Persistence of taste buds in denervated fungiform papillae[END_REF] and mouse [START_REF] Guagliardo | Fungiform taste bud degeneration in C57BL/6J mice following chorda-lingual nerve transection[END_REF].

Indeed, regeneration of taste buds upon regrowth of their cut nerves has been understood historically as one of the first examples of embryonic induction (even before the role of the optic cup in inducing the lens discovered by Spemann, let alone before the Spemann organizer itself), and an example of a "trophic" action of nerves on their target, a classical concept back in the XIXth century (which "fell on deaf ears" as discussed in [START_REF] Jacobson | Developmental Neurobiology[END_REF]).

These studies suggested that the nerves transport and secrete a "neurohumoral substance" to maintain the integrity of taste organs (Olmsted, 1920a;[START_REF] Torrey | The relation of taste buds to their nerve fibers[END_REF]. In this context, a major question became whether this substance was specific to taste nerves, or a generic substance secreted by any nerve. This was addressed by experiments where taste papilla or tongue fragments were grafted ectopically so that no taste nerve could reinnervate them. The result was that some, but not any, non-taste nerve could do the job; for example, trigeminal afferents could rescue taste buds after grafting in the anterior chamber of the eye in the rat [START_REF] Gómez-Ramos | Taste buds in vallate papillae grafted to the anterior chamber of the eye[END_REF] and the newt [START_REF] Poritsky | The fate of taste buds in tongue transplants to the orbit in the urodele,Triturus[END_REF] (and references therein) but motor neurons or sympathetic ones could not substitute for taste nerves (e.g. [START_REF] Guth | Taste buds on the cat's circumvallate papilla after reinnervation by glossopharyngeal, vagus, and hypoglossal nerves[END_REF][START_REF] Zalewski | Combined effects of testosterone and motor, sensory, or gustatory nerve reinnervation on the regeneration of taste buds[END_REF])

These experiments on degeneration and regeneration echoed the fact that in many species, during development, the arrival of nerves precedes the appearance of mature taste buds [START_REF] Oakley | Building sensory receptors on the tongue[END_REF][START_REF] Sharaby | Initial innervation of the palatal gustatory epithelium in the rat as revealed by growth-associated protein-43 (GAP-43) immunohistochemistry[END_REF]. Reviews of the time thus often treat development and regeneration of taste buds in parallel, under the collective term of "taste bud formation" (e.g. [START_REF] Zalewski | Trophic functions of the neuron. VI. Other trophic systems. Neuronal and tissue specifications involved in taste bud formation[END_REF]). This seemed all the more justified since taste buds are constantly renewing [START_REF] Beidler | Renewal of cells within taste buds[END_REF] thus can be viewed as undergoing a permanent ontogeny, and taste bud regeneration after re-innervation as a reiteration of what happens during embryonic development, a proxy for their induction.

The first experimental study that addressed directly taste bud induction, as opposed to maintenance, in rodents (Hosley et al., 1987a;Hosley et al., 1987b) used as a model the circumvallate papilla, where taste buds practically all appear after birth (Hosley and Oakley, 1987). Thus, development in the strict sense could be studied with the usual postnatal technique of surgical interruption of nerves. The authors found that, bilateral destruction of the glossopharyngeal nerve at P3 -a stage at which there is virtually no mature taste bud in the circumvallate papilla -resulted in a complete lack of taste buds at P10, P15, and P21 and a 98% reduction at P90. However, this could have been due to continuous induction followed by continuous degeneration because of lack of trophic support throughout the protracted period of taste bud addition. The authors resorted to complicated experiments and quantifications to argue for the lack of induction: they compared the number of taste buds 90 days after unilateral glossopharyngeal avulsion (thus with no regeneration) made at P3 or at P75: the deficit was much bigger after the P3 avulsion than after the P75 one, despite a similar amount of residual nerve fibers from the contralateral nerve, leading the authors to argue that at P3 a different phenomenon was taking place, with a different threshold, than the failure of maintenance observed at P75: thus, failure of induction. However, this argument was indirect. It seems that a more radical demonstration would have been to

monitor the circumvallate papilla at closer intervals (every day or other day) after bilateral avulsion at P3. If no taste bud had ever appeared, the authors could have concluded that induction was impeded. In retrospect, the lack of this experiment might have been responsible for the failure of this crucial publication from cementing, to the degree it could have, the old idea that taste bud formation is nerve-dependent.

A few dissenting voices have regularly questioned the universality of the requirement for nerves in taste bud formation or maintenance. Some authors emphasized the persistence of a proportion of taste buds after denervation, notably in fungiform papillae of the anterior tongue [START_REF] Guagliardo | Fungiform taste bud degeneration in C57BL/6J mice following chorda-lingual nerve transection[END_REF]. However, due to nerve regeneration, the latest time point examined in that study was 20 days, leaving open the possibility (undiscussed in the paper) of a slower turnover rate in this region, and that given enough time, the 50% anterior taste buds apparently resistant to denervation would eventually disappear. More radically, Stone [START_REF] Stone | The origin and development of taste organs in salamanders observed in the living condition[END_REF] dissected fragments of the oral epithelium of a salamander larva before innervation, grafted them on its flank and obtained the morphological differentiation of taste organs. The paper did not address the possibility of innervation by trunk sensory nerves, which could have compensated for the lack of taste nerves. But the way in which Stone described the morphogenesis of the ectopic taste buds is indeed difficult to reconcile with the involvement of nerves: new taste buds arose by the splitting of peripheral cells from a preexisting bud. This "budding" of new taste organs from older ones was never reported, to our knowledge before or after, and in any species. Later, Wright made tongue-to-liver grafts in the adult newt [START_REF] Wright | TASTE ORGANS IN TONGUE-TO-LIVER GRAFTS IN THE NEWT, TRITURUS V. VIRIDESCENS[END_REF], arguing that no sensory nerve is likely to invade such grafts and claiming that no nerve was visible by silver stain. Yet taste bud persisted up to 8 months (it is unclear whether they degenerated before regenerating, or simply persisted). Both authors propose a critique of previous degeneration and regeneration studies by alluding to the possibility (which has not been validated later) that the disruption, by surgery, of the blood supply rather than the nerves, was causal.

These observations did not quite find their place in the general picture of taste bud ontogeny until they were repeated and extended by Barlow and[START_REF] Barlow | Embryonic taste buds develop in the absence of innervation[END_REF]. Unlike Stone, these authors verified the absence of nerves in the immediate vicinity of the ectopic taste buds, and they used two molecular markers to document the differentiation of taste buds cells: the calcium binding protein calbindin and 5HT.

Finally, they repeated the experiment in culture, consolidating the absence of nerve involvement.

Reconciling the developmental studies in amphibians and the long series of degeneration/regeneration studies in mammals and fish implied either a radical difference between amphibians and other animals, or that regeneration is a fundamentally different phenomenon from embryonic development: the former nerve dependent, the latter nerve independent, a notion regularly put forward to this day [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF][START_REF] Kapsimali | Developing a sense of taste[END_REF]. This interpretation implied the dismissal (Barlow and Northcutt, 1998) of the experiments on the rat circumvallate papilla mentioned above (Hosley et al., 1987a;Hosley et al., 1987b) on the account that they were made postnatally, while Oakley himself had mentioned the presence of "immature" taste buds at birth on the circumvallate papilla (Hosley and Oakley, 1987). Indeed, these immature taste buds, described as lacking a pore and having less elongated cells, were not documented in the induction/regeneration study, which left open the possibility that only their maturation but not their induction was nerve dependent.

In turn, the striking amphibian findings was followed by a series of developmental studies in rodents that attempted at rebuilding the case for a role of nerves in the induction of taste buds. In the late 90's and early 2000's, a flurry of genetic experiments monitored the formation of taste buds in pups in which cranial nerves had been developmentally damaged via their dependencies on neurotrophins, mainly Brain Derived Neurotrophic Factor (Bdnf). This abundant literature is difficult to summarize due to the diversity of experimental parameters:

i) different stages examined; ii) different criteria and markers to ascertain the presence of taste buds; iii) different types of taste bud studied (in the fungiform, foliate, or circumvallate papillae -remarkably never in the palate, despite the fact that their development is completed earlier); iv) the different neurotrophin or neurotrophin receptors whose knockouts are examined. Here is an attempt at summarizing the main findings:

The Bdnf KO was examined at the level of the vallate and fungiform papillae and taste buds were quantified on the basis of K8 staining [START_REF] Cooper | Functional redundancy and gustatory development in bdnf null mutant mice[END_REF], simple hematoxylin-eosin stain [START_REF] Mistretta | Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs[END_REF] or the presence of taste pores by SEM [START_REF] Nosrat | Lingual deficits in BDNF and NT3 mutant mice leading to gustatory and somatosensory disturbances, respectively[END_REF]. The three groups found a significant loss or atrophy of the papillae and decrease of the taste buds, although the figures differ somewhat:

i) For the vallate papilla: a reduction by 74% (at P7) and 90% (at P12) in the number of taste buds [START_REF] Cooper | Functional redundancy and gustatory development in bdnf null mutant mice[END_REF]; a 73% reduction at P15-P25 [START_REF] Mistretta | Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs[END_REF])); a circumvallate papilla "reduced in size" and containing "far fewer and less disturbed" taste buds than control (probably meaning "more disturbed"), but not quantified [START_REF] Nosrat | Lingual deficits in BDNF and NT3 mutant mice leading to gustatory and somatosensory disturbances, respectively[END_REF]. Both Oakley and Mistretta note that the deficit in taste buds was larger than could be explained by the atrophy of the papilla itself (i.e. was not a trivial consequence of papilla atrophy) and conversely was well correlated with the nerve density.

ii) For fungiform papillae: few anomalies in the papillae and taste buds at P7 or P12 in 1998 [START_REF] Cooper | Functional redundancy and gustatory development in bdnf null mutant mice[END_REF])) (but a 50% reduction of "normal taste buds" at P0 later by the same author [START_REF] Sun | Development of anterior gustatory epithelia in the palate and tongue requires epidermal growth factor receptor[END_REF]; 57% reduction of taste buds in fungiform papillae at P15-P25, substantially smaller in diameter with "remaining fungiform papillae selectively concentrated in the tongue tip region" [START_REF] Mistretta | Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs[END_REF]; a reduction by 80% of taste pores at P14 [START_REF] Nosrat | Lingual deficits in BDNF and NT3 mutant mice leading to gustatory and somatosensory disturbances, respectively[END_REF].

Most spectacularly, a later study by [START_REF] Nosrat | Lingual deficits in neurotrophin double knockout mice[END_REF]

in double

Bdnf /Nt3 KO at stage P0 found 85% reduction of fungiform papillae, and a lack of taste buds -presumably complete -as assessed by PGP-positive cells (however, this is a marker only of the infrequent Type III cells, not easy to distinguish from the nerves on the figures of the paper, even in the wild type). They interpreted the result as double requirement for gustatory and somatosensory innervation (the latter being responsive to Nt3). To complicate matters, however, the same authors later published two studies on double Bdnf/Nt4 KO [START_REF] Ito | Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4[END_REF] and double Bdnf/Nt3 KO [START_REF] Ito | Taste cell formation does not require gustatory and somatosensory innervation[END_REF] to show that at in the embryo incipient taste buds were normal in the knockouts.

Unfortunately, the only argument is the expression of K8 at a stage where it is not specific to taste buds (see discussion about markers).

In stark contrast, Fritzsch et al. [START_REF] Fritzsch | Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds[END_REF], examining the KO for the Bdnf receptor TrkB, and found many normal taste buds at E17.5 (not shown) and at P16

(unfortunately, the EM images are impossible to assess on the scanned papers that are available), but there is no quantification, only the notion that "the early differentiation of many taste buds appear to proceed along the spatiotemporal pattern as controls".

Liebl [START_REF] Liebl | NT4/5 mutant mice have deficiency in gustatory papillae and taste bud formation[END_REF] studied Nt4 KO mice and reported a 44% reduction of fungiform papillae at birth, getting worse at P60 and concluded that "these findings demonstrate that the Nt4 gene functions in the maintenance of fungiform gustatory papillae and raises the possibility for an earlier role in development."

Finally, Agerman et al [START_REF] Agerman | BDNF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development[END_REF] replaced Bdnf by NT3 (stage P7)

and found that NT3 cannot sustain taste bud innervation. Fungiform taste papillae were atrophic by P7 as assessed by SEM.

These studies were summarized by Oakley et al. as showing, in the aggregate, a dramatic loss in the circumvallate papilla and a "compromised" development of fungiform papillae [START_REF] Oakley | Building sensory receptors on the tongue[END_REF]. On the other hand, these studies were criticized by Northcutt and Barlow on the following grounds:

i) The authors highlighted the dissenting view of [START_REF] Fritzsch | Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds[END_REF] (see above).

ii) Only postnatal stages were examined, when the "first buds" or "incipient buds" are "already present". [In support of this point, the authors cite 4 references, only one of which, however, mentions immature taste buds at birth, and only on the circumvallate papilla: « 19.3 immature taste buds at birth and 17.7 in 1-day-old rat pups » (Hosley and Oakley, 1987) (the other papers either don't look at newborn pups or study only the taste papillae, not the buds). As mentioned before, it can indeed be considered a weakness of the ablation paper of 1987 (Hosley et al., 1987a) that these immature taste buds of the circumvallate were not examined.

In addition to these criticisms, one can find two intrinsic weaknesses in the neurotrophin studies:

i) The defects are always partial (except, possibly, for fungiform papillae of double Nt3/Bdnf KO [START_REF] Nosrat | Lingual deficits in neurotrophin double knockout mice[END_REF]), documented at birth, although superficially).

This leaves open the possibility that the defects reflect an ongoing formation combined with ongoing failure of maintenance, throughout the lengthy period of addition of taste buds in wild types.

ii) BDNF is expressed not only in the taste neurons of the geniculate ganglion but also in the taste bud cells themselves. Thus, there is potential for an autocrine or paracrine role of the neurotrophins inside the taste bud. This possibility was addressed by overexpression of Bdnf either throughout the mouth epithelium [START_REF] Krimm | Epithelial overexpression of BDNF or NT4 disrupts targeting of taste neurons that innervate the anterior tongue[END_REF] or in the muscle [START_REF] Ringstedt | Role of brain-derived neurotrophic factor in target invasion in the gustatory system[END_REF], which lead, simultaneously, to the misrouting of nerves and to taste bud atrophy or depletion. Since in these experiments a normal expression of Bdnf is maintained in the taste bud cells themselves, this argued, albeit indirectly, for a non-cell autonomous effect, thus for a nerve-dependency of taste bud development.

In conclusion, the field is right now in a confusing situation. In a 2004 review, Oakley stated: "diverse

[…] studies have reaffirmed that virtually all mammalian taste buds require innervation for their development" [START_REF] Oakley | Building sensory receptors on the tongue[END_REF]. But a 2010 paper could still be entitled "Taste cell formation does not require gustatory and somatosensory innervation" [START_REF] Ito | Taste cell formation does not require gustatory and somatosensory innervation[END_REF]. Linda Barlow, discoverer of the nerve independent formation of amphibian taste buds, and currently one of the most prolific authors in the field of taste bud development, conceded in 2013 that "in contrast to amphibians, differentiation of mammalian taste buds appears to require innervation because […] the majority of taste buds differentiates postnatally [START_REF] Kapsimali | Developing a sense of taste[END_REF] but reaffirmed 3 years ago that "in mammals, the embryonic development of taste buds, as well as papilla morphogenesis, are nerve independent [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF].

Part of these contradictions might be artificial or "semantic". The development of taste buds is a protracted process which straddles birth, starting with a placode at around E12.5 and lasting until the completion of cell differentiation, from the first post-natal days to several weeks after birth depending on locations. Thus, contrary to what Linda Barlow implies, the birth of the animal does not clearly separate two developmental stages of taste bud development, either collectively, across taste bud fields, or even for any individual taste bud.

From what stage then, can the taste bud be said to "form", to "differentiate", to "develop"? The "nerveindependence" school stresses the appearance of placodes (Shh + , Prox1 + , Sox2 + thickenings of the oral epithelium), calls it "induction" or "embryonic development of taste buds" [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF] and asserts that it is nerve-independent (which is not controversial, and which my data verify, see Result chapter). All the developmental events that follow and lead to a functional taste organ (an onion shaped organ made of 4 types of elongated cells that express a number of markers, open on the mouth cavity through a pore and receiving sensory innervation) are designated under varied terms such as "maturation" (El- [START_REF] El-Sharaby | Development and maturation of taste buds of the palatal epithelium of the rat: histological and immunohistochemical study[END_REF]Hosley and Oakley, 1987)), "elaboration of taste bud morphology" [START_REF] Barlow | A taste for development[END_REF], "differentiation of taste bud cells" [START_REF] Kapsimali | Developing a sense of taste[END_REF], or "papillary differentiation" (as opposed to its "formation") [START_REF] Hall | Disruption of sonic hedgehog signaling alters growth and patterning of lingual taste papillae[END_REF], are deemed "post-natal" [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF] (which is a simplification, see above) and are sometimes acknowledged [START_REF] Kapsimali | Developing a sense of taste[END_REF] to be nerve-dependent in mammals. But all these events could be considered as representing the true "formation" or even "induction" of taste buds (from placodes and papillae, which are only the sites at which they arise). Seen this way, nerves would be indeed very important for taste bud formation in mammals (there remaining, however, an irreconcilable difference between the rodent and amphibian models).

The controversy is not purely verbal: the dismissal or euphemizing of nerves as agent of the formation of taste buds has been influential enough, since the late 1990's that, to our knowledge, no one is looking for the "neurohumoral agent" required for taste bud formation that was postulated at the beginning of the XXth century [START_REF] Olmsted | Effects of cutting the lingual nerve of the dog[END_REF]. The focus has been almost entirely shifted to processes "intrinsic to the tongue" (Barlow and Klein, 2015) and very little work has been published on the role of nerves on taste bud development in the past 10 years [an exception is (Fei et al., 2014)].

Back in 1998, Northcutt and Barlow set out a clear agenda to resolve the controversy: "To test whether mammalian taste buds are induced by nerves, gustatory nerves must be prevented completely from reaching the epithelium where taste buds will reside, and the nerve-naive epithelium examined at birth, when taste buds first appear. If taste buds fail to form under these conditions, then we will be able to make a clear distinction between how mammals and amphibians generate taste buds (that is, mammalian taste buds are induced by nerves whereas those of amphibians are not). However, until this type of experiment has been performed, amphibian and mammalian studies cannot be compared directly ». This describes well my thesis project. I have reexamined the formation of taste buds, focusing on those that develop largely before birth (i.e. in the soft palate, never examined before), with cleaner genetic tools than before (which "completely prevent nerve from reaching the epithelium" since they prevent the birth of taste neurons in the first place), and -beyond the vague notions of "induction", "specification" or "maturation" -I monitored, with molecular markers, what differentiation events are nerve-dependent or independent in the embryos, up to the moment when taste buds are functional.

1.e. Mechanism of taste bud renewal and regeneration

As discussed above, taste buds are continuously renewed in about 10 days. By combining pulse-chase experiments with a thymidine analogue and labeling of different types of taste cells, the average life span of taste cells was determined to be 8 days for Type II cells, and 22 days for Type III cells (Type I cells could not be assessed for want of a cytoplasmic marker) [START_REF] Perea-Martinez | Functional cell types in taste buds have distinct longevities[END_REF]. Neither the lineage that leads to the production of these cells, nor the nature of the stem cells/progenitors are completely elucidated. The most careful and exhaustive proliferation study [START_REF] Sullivan | Stem and progenitor cell compartments within adult mouse taste buds[END_REF] found fast dividing cells both, around the taste bud (perigemmal) and inside the taste bud (intragemmal), the latter mostly round cells in a basal position (thus likely Type IV basal cells), but also a few fusiform ones in more apical positions (thus resembling Type I-III cells usually thought to be postmitotic). In addition, slowly dividing cells (by this criterion resembling "stem cells")

were found in both perigemmal and basal intragemmal locations. Thus, according to this description, there could exist a variety of progenitors, only some of which feature in the currently proposed developmental scenario outlined below.

Lineage tracing studies argue that during renewal, dividing perigemmal progenitor cells (or stem cells) that express CK14 [START_REF] Okubo | Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate[END_REF], Sox2 [START_REF] Ohmoto | Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain[END_REF] and, in the circumvallate papilla, Lgr5 [START_REF] Yee | Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue[END_REF] contribute, collectively, to both, non-gustatory epithelium and taste buds. Their immediate descendants in the taste buds would be Shh+ postmitotic basal cells [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF]. (These postmitotic precursors would be the source of Shh required for proliferation of their perigemmal progenitors, see §1e).

Eventually, Shh+ basal cells would switch off Shh and give birth to differentiated Type I, II and III cells. At every stage of this lineage it is unclear whether individual progenitors are unipotent or multipotent, since no clonal lineage tracing was done. The slowly or rapidly dividing intragemmal basal cells mentioned above and described in [START_REF] Sullivan | Stem and progenitor cell compartments within adult mouse taste buds[END_REF] but also [START_REF] Perea-Martinez | Functional cell types in taste buds have distinct longevities[END_REF] and [START_REF] Delay | Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage[END_REF], are not accounted for by this scenario, possibly because they are Shh-negative [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF].

Disrupting Shh signaling in adult tongues has been achieved through a variety of means: inducible deletion of Gli2 and/or Gli1, or expression of a dominant negative form of Gli2 [START_REF] Ermilov | Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling[END_REF], or inducible deletion or pharmacological blockade of Smothened [START_REF] Kumari | Recovery of taste organs and sensory function after severe loss from Hedgehog/Smoothened inhibition with cancer drug sonidegib[END_REF][START_REF] Mistretta | Tongue and Taste Organ Biology and Function: Homeostasis Maintained by Hedgehog Signaling[END_REF], leads to a massive disappearance of taste buds, often replaced by filiform papillae. Counter-intuitively, the constitutive activation of Shh by overexpression of an oncogenic form of Gli2 [START_REF] Liu | Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance[END_REF]) induces a similar phenotype, leading to the notion that taste bud renewal, thus maintenance, requires a balanced state of Shh signaling [START_REF] Mistretta | Tongue and Taste Organ Biology and Function: Homeostasis Maintained by Hedgehog Signaling[END_REF]. Finally, an intriguing observation was made on the action of Shh in adult tongue tissue. Conditional ectopic expression of Shh (in CK14+ keratinocytes, from a floxed Shh-IRES-YFP transgene, after injection of tamoxifen) produced ectopic mature taste cells, type II-IV, in non-gustatory parts of the lingual epithelium, that survive without any innervation for at least a month [START_REF] Castillo | Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium[END_REF]. The relationship of this de novo formation of taste cells to taste bud regeneration (and to normal development) is unclear for three reasons. Firstly, as discussed by the authors, the differentiation into type II-IV cells occurred despite their ongoing expression Shh, whereas expression is lost during differentiation in endogenous cells.

Secondly, (not discussed by the authors) even though there are signs of Shh signalling in the cells surrounding the recombined ones (e.g. Sox2 and Ptc1 upregulation), those cells are never recruited to become taste cells, since all differentiated taste cells express the recombined Shh-IRES-YFP reporter. In other words, taste cell differentiation appears in this experiment as a strictly cell autonomous effect of Shh (thus non-canonical or purely autocrine). Thirdly, the presence of differentiated cells a month after tamoxifen injection suggests either that the cells are abnormally long-lived, or that a subset of the transfected cells are progenitors, even though Shh+ dividing progenitors never occur normally [START_REF] Miura | Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells[END_REF]. Fourthly, as discussed by [START_REF] Mistretta | Tongue and Taste Organ Biology and Function: Homeostasis Maintained by Hedgehog Signaling[END_REF], these groups of taste cells do not have the general size and structure of a taste bud, and no possible taste detection function, since they are not innervated.

In conclusion, the role of Shh in fully differentiated or regenerating taste buds is strikingly different from the one in placodes: it promotes rather than inhibits the formation of taste organs. As stated above, nothing is known about the transition from one role to the other, during development.

As discussed above, and demonstrated in hundreds of publications, the nerve dependency of taste bud renewal is uncontroversial: this process is completely interrupted by the loss of innervation and resumes when nerves regenerate. As it turns out, the first event after denervation is a total loss of Shh in basal cells and consequently of Shh signalling in perigemmal cells (as assessed expression of target genes), which precedes the disappearance of the taste buds [START_REF] Miura | A strong nerve dependence of sonic hedgehog expression in basal cells in mouse taste bud and an autonomous transcriptional control of genes in differentiated taste cells[END_REF][START_REF] Miura | Shh and Ptc are associated with taste bud maintenance in the adult mouse[END_REF]. Conversely, an early sign of taste bud regeneration after re-innervation is the onset of expression of Shh in basal cells [START_REF] Miura | A strong nerve dependence of sonic hedgehog expression in basal cells in mouse taste bud and an autonomous transcriptional control of genes in differentiated taste cells[END_REF]. Thus, somehow, a nerve-derived signal lies upstream of the Shh signaling cascade.

In addition to basal cells, two papers have incriminated the nerves themselves as an additional source of Shh in adult taste buds. Genetic deletion of neuronal Shh, either by itself [START_REF] Lu | Neuronal delivery of Hedgehog directs spatial patterning of taste organ regeneration[END_REF] or in combination with suppression of epithelial derived Shh [START_REF] Castillo-Azofeifa | Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance[END_REF] leads to taste bud loss. However, this nervederived Shh cannot explain the classical phenomenon of taste bud loss upon never ablation, since the time course is much faster in the latter case (a few days as opposed to a month). As stated by Mistretta and Kumari "interactions between Hh signaling and innervation in taste bud maintenance, degeneration and regeneration is understudied". One could suggest that this is because the effect of innervation itself is understudied.

Ontogeny of taste neurons

Taste neurons are found in 3 cranial ganglia: geniculate, petrosal and nodose. The glia of the ganglia is derived from the neural crest, but the neurons are born in thickenings of the ectoderm lateral to the neural plate called neurogenic placodes. More precisely, taste neurons are born in the "epibranchial placodes" thus called because they are located dorsally at the bottom of each branchial cleft. The ontogeny of these placodes has been studied in chicken, zebrafish and mouse, to discover signals involved in the induction of the placodes and the transcriptional cascades taking place inside the placodes. I have uncovered the role of a transcription factor, Foxg1, previously known to be expressed in the placodes but thought not have any role.

Below I will only briefly review the complex ontogeny of epibranchial placodes to provide some context for my discovery.

Pan-placodal field

Epibranchial placodes are only one class of neurogenic placodes of the head, the other main ones being the olfactory, the dorsolateral (trigeminal and otic) and, in fish, the lateral line placodes. Trigeminal placodes give rise (together with the neural crest) to sensory neurons of the trigeminal ganglion (Vth), which sense touch, pain and temperature in the face and oral cavity. Otic placode generates neurons for the inner ear (cochlea and vestibule) that sense sound and balance. Olfactory placodes produce neurons which sense odors and pheromone signals. Epibranchial placodes produce sensory neurons that form the distal lobes of the ganglia of the VIIth (geniculate), IXth (petrosal) and Xth (nodosal) nerves, and sense taste as well as other, non-conscious visceral sensations among which blood pressure, blood osmolarity, blood oxygen, chemical composition of the alimentary canal, visceral distention.

All these placodes are proposed to arise by fragmentation and specialization of a larger crescent-shaped ectodermal territory at the neural plate stage, called the pan-placodal field, where all cells share a "ground state"

and competence to become placodal, a view championed notably by G. Scholsser [START_REF] Schlosser | Making senses development of vertebrate cranial placodes[END_REF]. The existence of this common population was established by fate mapping in teleosts, amphibians and amniotes embryos. Several signaling pathways (BMPs, Fgfs, Wnts and Wnt antagonists) and tens of transcription factors are known to be expressed -and in many cases have been functionally implicated -in the pan-placodal field.

This topic has been extensively reviewed in recent years [START_REF] Grocott | The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective[END_REF][START_REF] Moody | Transcriptional regulation of cranial sensory placode development[END_REF][START_REF] Schlosser | Making senses development of vertebrate cranial placodes[END_REF].

Epibranchial placodes

The three epibranchial placodes are located in a rostrocaudal sequence in the dorsal aspect and at the bottom of each branchial clefts in the vertebrate head. Neurogenesis in each placode occurs sequentially in mouse from embryonic day E8.5 to E10.5. Postmitotic [START_REF] Graham | Delamination of cells from neurogenic placodes does not involve an epithelial-to-mesenchymal transition[END_REF] neuronal precursors born in the first, second and third placodes delaminate, in a manner reminiscent of neurogenesis in Drosophila, and migrate inwardly and dorsally to form the geniculate, petrosal and nodose ganglia, appended, respectively, to the VII, IXth and Xth cranial nerves. The geniculate ganglion has formed by E10, while the petrosal and nodose ganglia form around E11.5-E12.5. At around the same time, the neural crest gives rise to the somatosensory neurons that will form the proximal ganglion of the same nerves (in the case of the geniculate the proximal and distal are fused (d'Autréaux et al., 2011)) and the glia of the distal ganglia (Le Douarin and Kalcheim, 1999). Some authors have argued that, for each nerve, the neural crest was involved in enabling and directing the inward migration of the corresponding placodal cells [START_REF] Begbie | Integration between the epibranchial placodes and the hindbrain[END_REF][START_REF] Freter | Cranial neural crest cells form corridors prefiguring sensory neuroblast migration[END_REF]. However, epibranchial ganglia form in a fairly normal way even after ablation of the neural crest [START_REF] Coppola | Epibranchial ganglia orchestrate the development of the cranial neurogenic crest[END_REF].

Genetic cascades

Several genes have been implicated in the specification of epibranchial placodes which have been studied in mouse, chicken and zebrafish.

Inductive signals

The endoderm of the pharyngeal pouches, but also the mesoderm, have been implicated in the specification of the overlying ectoderm into epibranchial placodes. The BMP signaling pathway participates in the differentiation of epibranchial placodes. In particular, Bmp7 induces epibranchial placode neurogenesis in chicken [START_REF] Begbie | Induction of the epibranchial placodes[END_REF]. In Zebrafish, Bmp mutants display a reduction of epibranchial neurons (manifested by fewer NeuD-positive cells in the placode). Also, loss and gain function experiments in chick and Xenopus embryos have involved Bmp4: placodal neurogenesis is inhibited by losing Bmp4 expression, while ectopic Bmp4 expression enhances it.

In zebrafish, a two-signal model was proposed, whereby the placode is induced by Fgf signals from the mesoderm, followed by a neurogenic phase enabled by Bmps and Fgfs from the endoderm of the pharyngeal pouch [START_REF] Nechiporuk | Specification of epibranchial placodes in zebrafish[END_REF].

Transcription factors

Eya, Six and Pax

The induction and formation of epibranchial placode and ganglia involves the transcriptional Eya/Six-Pax network. Eya1/2 encode protein tyrosine phosphatases that are expressed at the pan-placodal stage and continue to be expressed in all cranial placodes. Six4/5 bind directly to DNA and are expressed in the panplacodal region. Eya and Six together co-regulate the development of the placodes. Both Six and Eya mutant lie upstream of SoxB1 genes (such as Sox2) which together with Hes1/5 and Id genes allow proliferation of placodal cells. The down regulation of Six and Eya genes inaugurates the onset of expression of bHLH proneurla genes and thus, neurogenesis. Pax2 also plays role in the formation of the posterior palcodal field, common the epibranchial, otic and lateral line placodes [START_REF] Freter | Pax2 modulates proliferation during specification of the otic and epibranchial placodes[END_REF].

Neurogenin 2 (Neurog2)

The bHLH protein Neurog2 is the key proneural factor of epibranchial placodes. In mouse embryonic development, strong expression of Neurog2 is detected in the first (geniculate) placode at E8.5, in the second (petrosal) placode at E9.0 and in the third (nodosal) placode at E9.5. Although Neurog2 is not required for the formation of the placode itself, it is essential for the formation of the geniculate and petrosal ganglia [START_REF] Espinosa-Medina | Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors[END_REF][START_REF] Fode | The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons[END_REF], while it shares this determination function with Neurog1 in the nodose ganglion [START_REF] Takano-Maruyama | Differential contribution of Neurog1 and Neurog2 on the formation of cranial ganglia along the anterior-posterior axis[END_REF]. In neuronal precursors, Neurog2 activates a downstream cascade of bHLH differentiation genes, Neurog1, Math3, NeuroD and Nscl1, in sequence during their delamination from epibranchial placodes [START_REF] Fode | The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons[END_REF], whose functions have not been explored .

Phox2 genes

The paralogous homeobox genes Phox2a and Phox2b are largely specific for the autonomic reflex circuits of vertebrates [START_REF] Brunet | Phox2 genes -from patterning to connectivity[END_REF], of which the taste circuits have been considered a part, on anatomic grounds, since the early XXth century [START_REF] Herrick | An Introduction to Neurology[END_REF]. In line with this, Phox2a and Phox2b are expressed both, in epibranchial (thus viscerosensory) ganglia -including their gustatory part -and in the central target of these ganglia, the nucleus of the solitary tract [START_REF] Coppola | Phox2b expression in the taste centers of fish[END_REF][START_REF] Dauger | Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways[END_REF]. They are also expressed in the efferent neurons that respond reflexively to gustatory information, such as the preganglionic and ganglionic neurons for salivation [START_REF] Espinosa-Medina | Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors[END_REF][START_REF] Pattyn | Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b[END_REF][START_REF] Pattyn | The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives[END_REF].

Phox2a is switched on in parallel with Neurog2 in the epibranchial placodes and in its absence epibranchial ganglia are atrophic, which might be the cause of the early post-natal death of Phox2a knockouts [START_REF] Morin | Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a[END_REF]. After delamination, the ganglion cell precursors switch on Phox2b, the paralogue of Phox2a [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]. In the geniculate ganglion, Phox2b expression by gustatory neurons has been directly

verified by tracing the axons with a Phox2b::Cre allele and a reporter gene [START_REF] Ohman-Gault | The transcription factor Phox2b distinguishes between oral and non-oral sensory neurons in the geniculate ganglion[END_REF], the Phox2b -neurons of the geniculate being derived from the neural crest, and bieng somatosensory (to the external ear). In the absence of Phox2b, epibranchial ganglia are also atrophic [START_REF] Pattyn | The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives[END_REF] and the residual neurons switch their molecular profile, as well as their mode of projections to the central nervous system, to that of somatosensory neurons: Phox2b thus acts as a somatosensory to viscerosensory identity switch (d'Autréaux et al., 2011). The peripheral projections have not been traced, but in Phox2b KO the greater superficial petrosal nerve, to taste buds of the soft palate (see §2a, anatomy of taste nerves) is consistently missing, the main branch of the glossopharyngeal nerve is atrophic, while the corda tympani is sometimes missing and sometimes thinner.

It is tempting to hypothesize, but yet unexplored, that the latter, when present, comprises exclusively somatosensory fibers to the taste buds.

Foxg1

Forkhead box G1 (Foxg1, previously called Brain Factor 1 (BF-1)), is a winged-helix transcription factor, first characterized for its expression in the central nervous system [START_REF] Hatini | Expression of winged helix genes, BF-1 and BF-2, define adjacent domains within the developing forebrain and retina[END_REF][START_REF] Tao | Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain[END_REF], and its requirement for the development of cerebral hemispheres [START_REF] Li | Characterization of the structure and function of the gene for transcription factor BF-1, an essential regulator of forebrain development[END_REF][START_REF] Xuan | Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres[END_REF]. Foxg1

regulates the proliferation of neural progenitors and, in the knockout, a massive reduction of cells lead to an atrophic telencephalon. Foxg1 is also involved in the development of the olfactory epithelium where its expression regulates Sox2-positive cell proliferation and differentiation [START_REF] Duggan | Foxg1 is required for development of the vertebrate olfactory system[END_REF]. Rather preliminary studies have also been made on its role in the otic placode where it is involved in sensory cristae differentiation and possibly their sensory innervation, although the exact cellular compartment of action is incompletely resolved [START_REF] Hwang | Foxg1 is required for proper separation and formation of sensory cristae during inner ear development[END_REF][START_REF] Pauley | Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear[END_REF]. In the epibranchial placodes, Foxg1 expression was first revealed by analyzing a knockin of LacZ in the Foxg1 locus [START_REF] Hatini | Dynamics of placodal lineage development revealed by targeted transgene expression[END_REF]. However, examination of the homozygote embryos did not reveal the massive disruption of proliferation observed in the olfactory placode and it was concluded (on the basis of data not shown) that Foxg1 was dispensable for cranial sensory neurogenesis.

This presumed absence of role in the epibranchial placodes, and the relatively superficial analysis of its role in the otic placode, might explain that Foxg1 barely features in general reviews on placodal development, and mostly for its involvement in the anterior placodal field [START_REF] Moody | Transcriptional regulation of cranial sensory placode development[END_REF][START_REF] Schlosser | Making senses development of vertebrate cranial placodes[END_REF][START_REF] Schlosser | Early embryonic specification of vertebrate cranial placodes[END_REF].

An unpublished transcriptomic study from our group showed Foxg1 to be upregulated in the epibranchial ganglia of Phox2b knockouts, thus showing, at the very least, a genetic interaction between Foxg1 and a bona fide determinant of these placodes. This inspired me to reexamine the question of its role in epibranchial ganglion formation. The data is presented in the second part of the Result section.

RESULTS

My experimental work has addressed two very different questions: the controversial relationship of taste bud differentiation to taste bud innervation. And the undescribed role of the transcription factor Foxg1 in the differentiation of taste neurons. The first project is now in the form of an article that I will submit before my PhD defense. The second one remains preliminary. I will present the two projects in succession.

RESULTS I. TASTE BUD FORMATION DEPENDS ON TASTE NERVES

ABSTRACT

It has been known for more than a century that, in adult animals, the maintenance of taste buds depends on their afferent nerves. However, the initial formation of taste buds was shown to be nerve-independent in amphibians and evidence to the contrary in mammals has been endlessly debated, mostly due to indirect and incomplete means to impede innervation during the protracted perinatal period of taste bud differentiation. Here, by genetically ablating, in mice, all somatic (i.e. touch) or visceral (i.e. taste) neurons for the oral cavity, we show that the latter but not the former are absolutely required for the formation of their target organs, the taste buds.

RESULTS

Experimental disruption of taste organ innervation

I first devised a way of completely interrupting taste bud innervation in the embryo. Placodes are epithelial thickenings within which taste buds will eventually develop. Tongue placodes progressively change shape to become papillae with a mesenchymal chore, and palatal placodes do it also but to a much lesser extent [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF]. Several genes are expressed in the placodes and papillae before the formation of taste buds, whose expression I examined.

The most precocious marker identified so far is the signaling molecule Shh, soon followed by the transcription factor Sox2. Both are expressed widely in the tongue epithelium until they become restricted to taste placodes in a punctuated pattern around E12.5 [START_REF] Liu | Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance[END_REF][START_REF] Nakayama | Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo[END_REF][START_REF] Thirumangalathu | Fate mapping of mammalian embryonic taste bud progenitors[END_REF]. Two days later, the transcription factors Prox1, Mash1 and Hes6 are expressed in the placodes, Mash1

and Hes6 being restricted to just a few cells [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF][START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF].

I examined all these markers in the soft palate and tongue of Neurog1/Neurog2 KO and found them all present at E14.5 (Figure 3 and4). The expressions of Shh and Sox2 was actually stronger in the palate of the double KO than in wild type (Figure 3), while the expressions of Prox1, Mash1 and Hes6 was essentially unchanged.

WT

Neurog1/2KO

Localized epithelial Shh expression also occurs in the hard palate, in stripes which become elevated and eventually give rise the palatal ridges or "rugae palatinae", bony ridges involved in food grasping. The pattern of Shh in this region arises by a Turing mechanism [START_REF] Economou | Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate[END_REF][START_REF] Lee | Shh signaling is essential for rugae morphogenesis in mice[END_REF]. These rugae receive innervation from the trigeminal ganglion. I found that, in addition to Shh, the incipient rugae, despite the fact that they never give rise to taste buds, share with the taste placodes the expression of Sox2, Prox1, Mash1 and Hes6

(Figure 5 left panel) and that, like in placodes and fungiform papillae, this expression occurs independently of innervation (Figure 5 right panel).

Thus, soft palatal and tongue taste papillae together with hard palatal rugae appear as related epithelial specializations, undergoing, to various extents, a morphological elevation and sharing a gene expression program. It would be interesting to investigate whether taste placodes on the tongue and soft palate also arise by a Turing mechanism (a first indication can be found in [START_REF] Prochazkova | FGF signaling refines Wnt gradients to regulate the patterning of taste papillae[END_REF].

Thus, the induction of early markers of fungiform and soft palate taste placodes (and palatal rugae) occurs and is maintained until E14.5 in the absence of any sensory innervation. The circumvallate papilla receives its visceral innervation from the petrosal ganglion and its somatic innervation from the superior ganglion (see Introduction) both traveling in the glossopharyngeal nerve. We previously found that in Neurog2 KO the superior ganglion is atrophic, probably in non-cell-autonomous fashion (data not shown). In line with this, the innervation of the circumvallate papilla was essentially absent in Neurog2

KO and the papilla failed to form in both, the single Neurog2 and double Neurog1/2 KO (Figure 6). Expression of Shh in the placode occurred normally in the Neurog2 and Neurog1/2 KO, in line with the fact that its onset precedes the arrival of nerves [START_REF] Iwatsuki | Wnt signaling interacts with Shh to regulate taste papilla development[END_REF][START_REF] Lee | Shh signaling is essential for rugae morphogenesis in mice[END_REF][START_REF] Liu | Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance[END_REF] Thus, unlike the fungiform and soft palate papillae, the circumvallate papilla requires visceral innervation for both papillary morphogenesis and the expression of the placodal/papillary markers Sox2, Mash1, Prox1 and Hes6. These results corroborate and extend the previous observation of circumvallate papilla atrophy in neurotrophin knockouts [START_REF] Ito | Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4[END_REF] . Note: quantification of the loss of immature taste buds and of K8+ cells at E18.5 remains to be done.

Visceral sensory innervation is required for the differentiation of taste receptor cells

I then examined the expression of late differentiation markers by taste bud cells. The time of the terminal differentiation of taste bud cells differs at different locations. In the tongue it is at one week postnatally for fungiform papillae [START_REF] Barlow | Progress and renewal in gustation: new insights into taste bud development[END_REF], and from P1 to P21 for the circumvallate papilla (Hosley and Oakley, 1987;[START_REF] Okubo | Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate[END_REF] and just before birth for the soft palate [START_REF] Rashwan | Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium[END_REF]. Since Neurog1 or

Neurog2 KO die at birth, I was largely restricted, for the detection of differentiated taste bud cells, to the soft palate. (As it happens, soft palate taste buds have never been examined in knockouts of neurotrophins, despite the fact that, being fully differentiated just at around birth, they are perfectly suited to address the question of embryonic development of taste buds). To optimize the detection of late differentiation markers, I prolonged gestation by one or two days using Proligestone (Delvosterone™), an analogue of progesterone, which blocks delivery, and surgically delivered the embryos at E20.5 (corresponding to P1 or P2 for the natural pregnancy of B6D2 dam).

I examined the following markers of taste bud cells in the palate at E20.5:

-Cytokeratin 8 (K8) which labels all taste buds cells in mature taste buds, although the exact time of onset of expression is debated [START_REF] Oakley | Building sensory receptors on the tongue[END_REF], In wildtype pups, all taste buds had the classical onion or barrel shape, made of elongated cells, most of them positive for K8 and KCNQ1, while a fraction of them were positive for the other markers (Fig. 10).

-

WT

Neurog2KO Neurog2KO

In stark contrast, in Neurog2 KO, there was a dramatic reduction both, in the number of taste buds and in their size as assessed by K8 expression on cryosections of the soft palate: 88.9% fewer K8 + positive clusters per hemi palate (Chart1A), whose average size was reduced by 88% (Chart1B). Overall, the area occupied by K8+ taste bud cells in the soft palate was reduced by 98.3% in the Neurog2 mutants. Moreover, the few remaining cells tended to be smaller than in the wild type and round rather than elongated (Fig10 A-E and F-J).

Only about half of the residual atrophic clusters contained cells -usually one -expressing KCNQ1, NTPDase2, Gustducin or Trpm5 and none contained Pkd2L1-positive cells (Figure 10 K-O).

In Neurog1 mutants, which are deprived of somatosensory innervation, taste buds were very similar in number and size to wild type animals. Occasionally two or three taste bud clusters grew next to each other (Figure 11G and I) which was rarely observed in wild types. All the markers tested were expressed in a manner comparable to wild type (Figure 11 A-E).

Thus, visceral innervation is absolutely required for the formation of taste buds at the time of birth.

However, the differentiation of individual taste cells toward a terminal Type1, II or III fate is not completely blocked as evidenced by the occasional differentiated cell. Rather, it is the contingent of cells recruited to form a taste bud which is drastically diminished, in most cases abolished, suggesting that a main action of the nerves is to maintain, or produce a pool of progenitors.

Note: I will test the possibility that proliferation of perigemmal progenitors is affected in Neurog2 knockouts by combining Sox2 immunofluorescence and detection of the proliferation marker Ki67.

At what stage is the formation of taste bud anlage disrupted in the absence of innervation? (in progress)

My work, so far, shows that taste buds are missing at birth in the soft palate of Neurog2 KO. Although this finding is sufficient to resolve the twenty years old controversy about whether the embryonic formation of taste buds is nerve-dependent, it begs the question of the precise stage of development which is disrupted in the absence of nerves. This is a difficult question because little is known about taste bud development during the interval that separates the formation of the placode and that of the taste bud. One could even say that there is no precise histological or molecular definition of what a taste bud anlage really is.

Two events have been proposed to mark the appearance of taste buds, which both involve the restriction of a general epithelial marker to the putative taste cell precursors:

1) The restriction of Shh expression to taste cell precursors. This criterion is very hard to use, for the following reasons:

i) It is operational (experimental) and retrospective: the diagnosis that Shh-positive cells at a given developmental stage are all taste cell precursors can only be made by lineage tracing.

ii) The process of restriction is progressive and the precise stage at which the restriction occurs is elusive and hard to define for technical reasons (e.g. the time of action of the inducible Cre recombinase) with even a 24 h precision.

iii) The criterion is not absolute since several locations in the oral cavity express Shh in a restricted fashion, yet never give rise to taste buds: the dorsal surface of the CIRCUMVALLATE papilla, and the rugae.

2) The onset of K8 in taste cell precursors after a first phase of widespread expression in the "periderm". The second criterion is straightforward because these two phases of K8 expression look different, peridermal expression being superficial to the ectoderm, while expression in the ectoderm is throughout the apico-basal thickness. However, as discussed in the introduction, no good record of the precise dynamics of K8 expression is available. Thus, I am currently establishing a time course of K8 expression in the palate and tongue, in wild types, Neurog1 and Neurog2 mutants (Figure 12).

Conclusion and Discussion

In conclusion, my findings resolve a more than twenty-year debate about the requirement of nerves for the embryonic formation of taste buds. They corroborate previous findings based on neurotrophin KO, which however, failed to resolve the issue, due to their incomplete phenotypes. The embryonic formation of taste buds depends on their afferent taste nerves, just like their permanent renewal, or regeneration after degeneration, does.

The exact cellular events that are dependent of the nerve remain to be established. In fact, my findings underlie how little we understand about the emergence of taste buds from taste placodes during development. Most molecular events that were previously highlighted as stepping stones on the path to taste bud formation (onset of cytokeratin 8 expression, as well as Mash1, Prox1, Sox2 and Hes6) certainly occur in the absence of nerves but are shared with regions of the ectoderm that never develop taste buds (the palatal ridges and the dorsal surface of the circumvallate papilla) and are not followed by taste bud formation in the absence of visceral nerves. If they are required for taste bud formation (which is unclear at this moment) they constitute a differentiation program that aborts in the absence of nerve.

One possible scenario, is that after the taste placode has attracted the taste nerve (through Bdnf expression) the nerve promotes, in the placode, either the formation, or the maintenance, or the proliferation, or all of the above, of taste bud progenitors, which are yet to be molecularly characterized in the taste placodes beyond their likely expression of Shh. In the absence of nerves, an occasional Shh + cells of the placode would be able to act as such a progenitor and give rise to a small clone of taste cells, as I observed in Neurog2 KO at E20.5. However, the formation of the organ itself with its characteristic number and arrangement of cells, never occurs, presumably by almost immediate exhaustion of the pool of progenitors.

Taste bud formation thus appears as resulting from the action of nerves on a nerve-independent placode, likely involving a collaboration between nerve-derived and epithelium-intrinsic signals. The action of the nerve is presumably exerted through a secreted factor, the "neurohumoral substance" postulated a century ago, the quest for which I hope my data will encourage. One way to search for it could be by comparative transcriptomics between visceral and somatic neurons -since the latter are clearly deprived of it -looking for candidate secreted trophic factors. A more direct way would be to test in vitro fractions of supernatants from visceral sensory neurons on tongue cultures. A caveat is that to my knowledge there is, so far, no culture system which is sufficiently long-term to test the differentiation of taste organs. The discovery of this factor is clearly complicated by the fact that it is most likely not to be sufficient to trigger taste bud formation and to require the presence of a placode, thus of an already organized embryonic structure. It might be possible to exploit organoids to provide such structures [START_REF] Aihara | Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid[END_REF].

RESULTS II.

My work on the role of Foxg1 in the differentiation of taste neurons remains in a preliminary stage and will eventually be described in an article entitled:

FOXG1 IS A DETERMINANT OF TASTE NEURONS INTRODUCTION

The roles of Foxg1 in telencephalic development have been well studied [START_REF] Xuan | Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres[END_REF].

Foxg1 is also expressed in head placodes: the otic placode, where its ablation leads to morphological and innervation defects of the cochlea and vestibule [START_REF] Hwang | Foxg1 is required for proper separation and formation of sensory cristae during inner ear development[END_REF][START_REF] Pauley | Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear[END_REF], the olfactory placode where its KO leads to complete agenesis of the olfactory epithelium [START_REF] Duggan | Foxg1 is required for development of the vertebrate olfactory system[END_REF]) [START_REF] Kawauchi | Foxg1 promotes olfactory neurogenesis by antagonizing Gdf11[END_REF], and the three epibranchial placodes where it was claimed that there was no phenotype in the Foxg1 KO, although none of the epibranchial placodes was documented in the figures [START_REF] Hatini | Dynamics of placodal lineage development revealed by targeted transgene expression[END_REF]. Transcriptomic data on epibranchial ganglia in wild-type versus Phox2b knockout mice (data not shown from our lab) revealed that Foxg1 was upregulated in Phox2b KO, which was an incentive to reexamine the case of Foxg1 function in epibranchial placodes.

RESULTS

Expression of Foxg1 in the epibranchial placodes and cranial ganglia

During embryogenesis, Foxg1 is expressed in the placodes at the bottom of each epibranchial cleft (the placodes from which will form the geniculate ganglion (pVII), the nodose ganglion (pIX) and the petrosal ganglion (pX), between E8.5 to E10.5 [START_REF] Hatini | Dynamics of placodal lineage development revealed by targeted transgene expression[END_REF] and Figure1A,B,C. Phox2a, a determinant of cranial ganglia is also expressed in the neuronal progenitors of the epibranchial placodes [START_REF] Morin | Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a[END_REF]. I compared the patterns of expression of Foxg1 and Phox2a by in situ hybridization combined with immunohistochemistry: Foxg1 and Phox2a expressions coincided in the epibranchial placodes regions, (Figure Perspective on these findings Thus, neurogenesis still takes place in the epibranchial placodes of Foxg1 mutant. However, the overexpression of Neurog2

suggests that the balance between proliferation and differentiation is disrupted. It will be necessary to examine proliferation rates as well as expression of several genes downstream of Neurog2 in epibranchial placodes, such as Neurog1, Math3, NeuroD. mutant, the geniculate ganglion looked smaller and less condensed, and some of its cells looked like they had started migrating along the facial nerve (VIIth) (Figure 3 A, B and C). As to the petrosal ganglion, it was either absent or fused with the nodose ganglion, although the phenotype was incompletely penetrant (Figure 3 C).

However, the combined volume of the petrosal and nodose ganglia was not significantly different in Foxg1 mutant compared to wild type (Chart 1).

At E11.5, The projections of the epibranchial ganglia showed several defects. The geniculate ganglion consistently lost its projection to the palate (the greater superficial petrosal nerve, GSPN), while the chorda tympani (another branch of gVII, which projects to the anterior two thirds of the tongue) appeared thinner than in wild type and was occasionally absent; The main branch of gIX (glossopharyngeal nerve) was atrophic and disorganized (Figure 3D, E and F)

Foxg1KO

WT The relative volume of IX/X ganglia

Since the projection of the geniculate ganglion to the palate was missing, but the projection to the anterior tongue (the chorda tympani) were preserved to an extent, I sought to assess more precisely the degree to which Foxg1 affects the neurons that project to the taste buds on the anterior two thirds of the tongue. Since taste organs are innervated by both, somatosensory and visceral sensory neurons, I looked for a way of distinguishing the two.

First, I tried to directly visualize fibers derived from Foxg1 positive neurons in the fungiform taste buds, which could have provided an easy way of assessing their state in the Foxg1 KO. Unfortunately, the Cre transgene in the Foxg1::Cre background is expressed in a massively ectopic fashion, sometimes ubiquitously [START_REF] Hébert | Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures[END_REF], which I could verify (data not shown), preventing a specific visualization of the projections of Foxg1-positive neurons. A novel Foxg1::Cre transgene has since been published, but the Cre being knocked in the 3' untranslated portion of the gene and expressed from an IRES sequence, this allele does not allow inactivation of Foxg1 [START_REF] Kawaguchi | Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line[END_REF].

Second, since Foxg1 is expressed in visceral (taste) but not somatic (touch) neurons, I tested several established markers presumably specific for each type of fibers. TrkB has been reported to be expressed in the geniculate ganglion, therefore possibly in visceral sensory fibers (Krimm et al., 2013) although the geniculate contains a somatosensory part (d'Autréaux et al., 2011). However, I found that it is also expressed in a subset of trigeminal ganglion cells, therefore in somatosensory neurons. (Figure 4 A and B). Trpv1, a nociception receptor, has been considered as a general marker of somatosensory neurons. However, I found it expressed only in a subset of trigeminal neurons. (Figure 4C and D) so that negative fibers in the taste buds could still have a trigeminal origin. The purinergic receptors P2X2 and P2X3 have been used as markers of visceral sensory neurons (and mutants for these molecules lose the sensation of taste [START_REF] Ishida | P2X(2)-and P2X(3)-positive fibers in fungiform papillae originate from the chorda tympani but not the trigeminal nerve in rats and mice[END_REF][START_REF] Larson | The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves[END_REF].

However, as for TrkB, I found P2X2 and P2X3 also expressed in the trigeminal ganglion (data not shown).

Therefore, I did not find any general and specific marker for somatosensory or viscerosensory fibers which could have allowed me to assess the state of the latter by immunofluorescence staining in Foxg1 KO. Finally, the morphology of the residual innervation was abnormal, most of the fibers apparently not able to penetrate the epithelium of the apex of fungiform papillae and with abnormal trajectories (Figure 6).

Conclusion and Discussion

Thus, Foxg1 is transcriptional determinant of the visceral sensory neurons for taste buds in the soft palate and anterior tongue.

My results should inspire a substantial revision of our view of Foxg1 function during neural development. Apart from its role in the telencephalon, Foxg1 has been presented in several reviews as a marker of anterior placodes (adenohypophyseal and olfactory) and a determinant of the latter only [START_REF] Schlosser | Early embryonic specification of vertebrate cranial placodes[END_REF].

This failed to take into account Foxg1 function in the otic placode [START_REF] Pauley | Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear[END_REF]. Now, in addition, I show that it has a crucial role in epibranchial ganglionic precursors. Overall, these data suggest that Foxg1 has less to do with a role in anteroposterior organization of the placodal field than with neuronal specification in several sensory neurogenic placodes.

In addition to the three placodes cited, Foxg1 is involved in the morphogenesis of the retina, which develops not from a placode, but as an evagination of the telencephalon [START_REF] Fotaki | Foxg1 is required to limit the formation of ciliary margin tissue and Wnt/β-catenin signalling in the developing nasal retina of the mouse[END_REF][START_REF] Smith | The Transcription Factor Foxg1 Promotes Optic Fissure Closure in the Mouse by Suppressing Wnt8b in the Nasal Optic Stalk[END_REF][START_REF] Tian | Foxg1 regulates retinal axon pathfinding by repressing an ipsilateral program in nasal retina and by causing optic chiasm cells to exert a net axonal growth-promoting activity[END_REF]. In contrast, Foxg1 has no expression in one of the head placode, the trigeminal, which conveys general somatosensory information. Thus, the role of Foxg1 in sensory neurogenesis extends beyond the placodes, and the most inclusive correlate of Foxg1 action is not developmental but physiological: it is a master gene for neurons involved in "special senses" -these specialized sensory modalities (taste, olfaction, smell and vision) processed by head organs, and opposed to the "general senses" of touch, pain and heat processed by the trigeminal and dorsal root ganglia.

α-Trpm5, guinea pig, 1 in500, (Liman's lab, USC)

For Shh and Karetin 8 immunoreations, sections underwent antigen retrieval treatment in citrate buffer at 90 ℃ for 10min. For Pkd2L1 immunoreation, tissue should be fixed in paraformaldehyde for 1hour.

Secondary antibodies: Neurogenin1 (Neurog1) [START_REF] Ma | neurogenin1 Is Essential for the Determination of Neuronal Precursors for Proximal Cranial Sensory Ganglia[END_REF]: the partial fragment of the Ngn1 ORF was replaced by GFP, the gene encoding green fluorescent protein, and a PGK-neo cassette.

Foxg1Cre (BF-1) [START_REF] Hébert | Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures[END_REF]: the Foxg1 locus was targeted by cre.

Animal treatment

To delay the birth of Neurog1 and Neurog1/2 KO pups, 100ul diluted Delvosteron (MSD) solution (Delvesteron: Nacl(0.9%)=1 :1) was subcutaneously injected to the pregnant mouse (the16.5 days of pregnancy).

Pups were taken on the 20.5 days counted by the embryonic stage.

Imaging

Tissue processed by in situ hybridization was photographed under bright field with 20X object (Leica microscope). Tissues processed by immunofluorescence were photographed under 63X (zoom in 2 times) and 25X with a SP5 confocal microscope (Leica).

Statistical analyses

Résumé

La composition chimique des aliments est perçue par les bourgeons du goût et transmise au cerveau postérieur par des nerfs viscérosensoriels particuliers, les nerfs du goût. L'intégrité de ces nerfs est essentielle au maintien des bourgeons du goût chez les animaux adultes. Cependant, leur rôle dans l'ontogénie des bourgeons, chez l'embryon et aux premiers stades postnataux, est controversé and reste non résolu. Dans cette étude, j'ai établi de façon définitive que la formation embryonnaire des bourgeons du goût dépend des nerfs gustatifs chez la souris, unifiant ainsi les mécanismes de maintien/régénération et d'ontogénie de ces organes.

En parallèle, j'ai réexaminé la possibilité (jusque-là exclue par d'autres auteurs) d'un rôle du facteur de transcription Foxg1 dans la formation des ganglions sensoriels épibranchiaux. J'ai découvert que Foxg1 est un déterminant des neurones gustatifs dans le ganglion géniculé. Ce nouveau rôle, de pair avec ceux décrits précédemment dans l'épithélium olfactif, la placode otique et la rétine, révèle une cohérence physiologique remarquable des fonctions de Foxg1 (en dehors de son rôle bien établi dans le cortex) en tant que facteur de transcription maître des neurones impliqués dans les « sens spéciaux » : vision, ouïe, odorat et goût.

.

Mot-Clé

Organe du goût, Neuron du goût, Développement, Foxg1 et Souris

Abstract

Taste information is received by taste buds and transmitted to the hindbrain by special visceral sensory nerves, the taste nerves. The integrity of taste nerves is essential for the maintenance of taste buds in adult animals. However, a role for taste nerves in the ontogeny of taste buds, in the embryo and at early postnatal stages, has been controversial and is still unresolved. In this study, I establish in a definitive manner that embryonic taste bud formation is nerve-dependent in mouse, thus unifying mechanistically the maintenance/regeneration and ontogeny of these organs.

Parallel to this work, I re-examined the possibility (previously excluded by other authors) of a role for the transcription factor Foxg1 in epibranchial ganglion formation. I find that Foxg1 is essential for the differentiation of gustatory neurons in the geniculate ganglion. This novel role, together with previously described ones in the olfactory epithelium, otic placode and retina, unveils a striking physiological coherence of the functions of Foxg1 (outside its well established one in the cortex), as a master transcription factor for neurons involved in "special senses": vision, hearing, smell and taste.

Key Words

Taste organ, Taste neuron, Development, Foxg1 and Mouse

Figure1:

  Figure1: The schematic representation of the anatomy of taste organs A: the distribution of taste organs on the tongue B: Fungiform papilla C: Circumvallate papilla (of human) C: Foliate papilla D: Four type cells within the taste bud (Adapted from[START_REF] Daikoku | Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat[END_REF];[START_REF] Yarmolinsky | Common sense about taste: from mammals to insects[END_REF];[START_REF] Mistretta | Tongue and Taste Organ Biology and Function: Homeostasis Maintained by Hedgehog Signaling[END_REF]

Figure 2 :

 2 Figure 2: The schematic view of the gustatory neural circuits (Information adapted fromSun et al., 2015;[START_REF] Oakley | Building sensory receptors on the tongue[END_REF]. Somatosensory neurons from trigeminal ganglion travel with taste neurons from geniculate ganglion through greater superficial petrosal nerve innervate taste buds on soft palate and through chorda tympani innervate taste buds of fungiform papillae and foliate papillae. Somatic and visceral sensory fibres traveling along with glossopharyngeal nerve innervate circumvallate papillae. All these gustatory information are carried to the hindbrain via the visceral sensory afferents.

Figure 4 :Figure 3 :

 43 Figure 4: The Chronology of gene expression in fungiform during the embryogenesis. (Details see Introduction

Figure 5 :

 5 Figure 5: Model illustrating how BMP7 acts to regulate SOX2 expression, Wnt-β-catenin signaling and SHH expression in developing fungiform papillae of the anterior tongue. (Adapted from (Beites et al., 2009))

Figure 1 :Figure 2 :

 12 Figure 1: Innervation of the soft palate at E16.5 in Neurogenin mutants. Immunostainings on floating sections showing sensory nerves to the soft palate stained in green by Tuj1. A and A': Innervation (somatic and visceral) of the soft palate in wild type (WT). B and B': Somatic sensory innervation of soft palate in Neurog2 KO. C and C': Visceral sensory innervation of the soft palate in Neurog1 KO. D and D': loss all sensory innervation of the tongue and palate in double Neurog1/2KO. A', B', C' and D' close ups (63 x, zoomed x 2) of A, B, C and D ( 20X), Scale bar=10µm.

Figure 3 :Figure 4 :

 34 Figure 3: Expression of early placodal genes in the soft palate is nerve-independent. In situ hybridization, immunohistochemistry or immunofluorescence at E14.5 for Shh, Sox2, Mash1 or Hes6 (blue), Prox1 (green). Tuj1staining of nerves appears in brown (IHS) or red (IF). Expressions of all the 5 genes is maintained in double Neurog1/2 mutant.

Figure 5 :

 5 Figure 5: Expression of early placodal markers in the rugae of the hard palate. In situ hybridization, immunohistochemistry or immunofluorescence at E14.5 for Shh, Sox2, Mash1 or Hes6 (blue), Prox1 (green). Tuj1staining of nerves appears in brown (IHS) or red (IF). Expressions of all the 5 genes is maintained in double Neurog1/2 mutant.

  (Figure 7A'-C' for Neurog2 KO, and Figure 8A' for Neurog1/2 KO)), but subsequently, at E14.5, Shh was upregulated to a lesser extent in the mutants (Figure 7 and 8), and Sox2, Mash1, Prox1 and Hes6 were either not expressed or only in a few cells (Figure 8 for Neurog1/2 KO, and data not shown for Neurog2 KO). The placode never developed into a papilla.

Figure 6 :Figure 7 .

 67 Figure 6: Innervation of the circumvallate papilla at E16.5 Immunostaining on 100 µm floating sections for b3-tubulin, with Tuj1 (in green) in the indicated genotypes. Morphogenesis of the circumvallate papilla is heavily compromised in Neurog2 KO. And completely prevented in Neurog1/2 KO.

Figure 8 :

 8 Figure 8: Expression of Mash1, Prox1 and Hes6 in the circumvallate taste papilla is nerve-dependent. Compared to the expression pattern of Shh and Sox2 in WT (A-B), Mash1, Prox1 and Hes6 (C-E) are expressed in the subsets on the dorsal surface of circumvallate papillae at E14.5 but their expression are lost in Neurog1/2KO (A'-E').

Figure 9 .

 9 Figure 9. Visceral sensory innervation is essential for the formation of immature taste buds at E18.5. Expression of Shh at E18.5 is often, but not always, downregulated in soft palate and fungiform papillae of Neurog2 mutant. At this stage papillae of the soft palate are occupied by large collections of K8+ cells in the wild type. In Neurog2 KO, very few cells are K8 and correspond to the Shh+ cells.

  The voltage-gated potassium channel KCNQ1, expressed in most or all taste bud cells, -The ectoenzyme NTPDase2 (expressed on Type I cells), -The G protein Gustducin (expressed on Type II cells), -The Transient receptor potential cation channel subfamily M member 5 (Trpm5, expressed on Type II cells), -Plcb2 (expressed on Type II cells), -Pkd2l1 (expressed on Type III cells.

Figure 10 :

 10 Figure 10: The presence of palatal taste buds at E20.5 depends on visceral sensory nerves. Immunofluorescence at P2 (E20.5) for cytokeratin 8 (K8), β3-tubulin, and KCNQ1 or NTPDase2 or Gustducin or Trpm5 or Pkd2L1. A-E: WT. F-J: Neurog2KO harbor few taste buds, each containing few K+ cells. K-O: KCNQ1, NTPDase2, Gustducin and Trpm5 are expressed in some of the residual cells, but not Pkd2L1.

Figure 11 :

 11 Figure 11: The presence of taste buds at E20.5 does not depend on somatic sensory nerves. Immunofluorescence at P2 (E20.5) for cytokeratin 8 (K8), β3-tubulin, and KCNQ1 or NTPDase2 or Gustducin or Trpm5 or Pkd2L1 in WT (A-E) and Neurog1 KO (F-J)

Figure 12 :

 12 Figure 12: Time course of cytokeratin8 expression in the palate and tongue of wild type embryos. Immunofluorescence for cytokeratin8 (K8) in red (close ups in B&W in lower right), Shh in green and Tuj1 in blue at E13.5, E14.5 and E16.5. Taste placodes are localized by Shh staining. In the hard and soft palates (respectivelyA, E, I and B, F, J) the expression of K8 is restricted to a superficial layer of the epidermis (possibly the periderm) at E13.5, then extends throughout the thickness of the epithelium at the level of the placodes at E14.5, and starts being downregulated in the hard palate at E16.5. In fungiform papillae (C, G and K), K8 expression is switched on ubiquitously at E14.5 and restricted to a subset of placode Shh + cells by E16.5. In the circumvallate papilla (D, H, L), K8 expression is throughout of the epidermis from E13.5 to E14.5 (D and H) and becomes salt-and-pepper at E16.5 (L).

Figure 1 :

 1 Figure 1: Foxg1 expression in epibranchial placodes and visceral sensory ganglia. A-C Wholemount embryos hybridized with Foxg1, showing the geniculate (pVII), petrosal (pIX) and nodose (pX) epibranchial placodes. C shows a magnified view of A. D-J Foxg1 in situ hybridization combined with Phox2a or Phox2b immunohistochemistry E10.5 (D-F), E11.5 (G-H) and E13.5 (J). Double Phox2a+/Foxg1+ cells are delaminating from the pVII (D), pIX (E) and pX (F) and migrating to form the ganglia. (G-H) At E11.5, the three visceral sensory ganglia are formed by a mixture of Foxg1+ and Foxg1) cells, all of them expressing Phox2b.(J) the situation is unchanged in the nodose and petrosal ganglia, now partially fused, at E13.5

Figure 2 :Figure 3 :

 23 Figure 2: Neurogenesis in the nodose placodes of Foxg1 KO. Neurogenesis of the epibranchial placode was tested by the expression pattern of neurogenin2 (Neurog2) in blue. The migrating placodal cells are stained by phox2a in brown. In Foxg1 mutant, there is a massive increase of Neurog2+ cells.

Figure 4 :

 4 Figure 4: Expression of putative markers for visceral or somatic sensory neurons in the trigeminal ganglion. (A and B) TrkB a marker of visceral sensory neurons is expressed in a subset of trigeminal, thus somatosensory neurons. (C and D) Trpv1, marker of somatosensory neurons is not expressed in all the neurons of the trigeminal ganglion.

Figure 5 :Figure 6 :

 56 Figure 5: Innervation of the soft palate at E16.5 in Neurog1, Foxg1 and Neurog1/Foxg1 mutants Immunofluorescence on floating sections showing sensory innervations of soft palate with the Tuj1 antibody (in green). (A): in Foxg1 mutant, which lack a GSPN projection, the somatosensory fibers remain in the epithelium of soft palate. (B): in Neurog1 mutant, visceral sensory fibers remain in the epithelium of soft palate. (C) in Neurog1//Foxg1 mutant, the soft palate has lost all its sensory innervation.

  α-rabbit A488, 1:500, Jackson Immunoresearch Laboratories(#711-545-152) α-mouse Cy5, 1:500, Jackson Immunoresearch Laboratories (#715-175-150) α-goat A647, 1:500, Jackson Immunoresearch Laboratories (#705-606-147) α-Rat Cy3, 1:500, Jackson Immunoresearch Laboratories (#712-165-153) α-guinea pig A488, 1 in 500, Jackson Immunoresearch Laboratories (#706-545-148)Transgenic Mouse lineNeurogenin2 (Neurog2)[START_REF] Fode | The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons[END_REF]: the complete protein coding sequence located in the second exon of the gene Ngn2 were replaced of CreERT2.

  Perspective on these findingsMy findings on Foxg1 can also be useful for my main project about taste bud development. Since the Foxg1 knockout is lacking any visceral innervation of the soft palate, I will examine palatal taste bud development in these mutants, where I expect to see the same phenotype than in the Neurog2 knockouts. If this is the case, it will answer the potential criticism that the Neurog2 KO phenotype is cell autonomous, through some transient, undetected expression of Neurog2 in the taste organ anlage.

	100%
	80%
	60%
	40%
	20%
	0%

Chart 1: Relative volume of the distal IX/X ganglia. There are no significant different between WT and Foxg1 mutants.

E and F for pIX and pX). Subsequently, the double positive neuronal precursors delaminate from the placodes and migrate to form the ganglia (gVII, gIX and gX) (Figure1 G and H). During the migration process, Foxg1 is downregulated is some cells, while Phox2b, the paralogue of Phox2a[START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]) is switched on. In the fully formed ganglia (around E9.5 for the geniculate and E10.5 for the petrosal and nodose) Foxg1 positive cells represent only a subset of visceral cranial sensory neurons (Figure1G, H and J). Consistent with my findings, a transcriptomic study showed that, in adults, all Foxg1 neurons of the geniculate ganglion are Phox2bpositive(Dvoryanchikov et al.

, 2017).

Acknowledgements

I dedicate my four-year PhD thesis to my parents, mum Jie LIU ( 刘杰) and dad Hanjie FAN (范汉

C

The total volume of entire fungifomr papillae (µm 3 )

Neurog1KO

Neurog1/Foxg1

MATERIAL AND METHODS

Histology

Mouse embryos or pups of different stages (E12.5, E14.5 or E20.5 (postnatal stage P1 or P2)) were fixed in 4% paraformaldehyde (PFA) overnight, then dissected into pieces. For cryostat section, tissue were embedded in 7% gelatine and 15% sucrose mix and stocked at -80℃, then sectioned (20µm) in sagittal plane for examining palates and anterior tongues and in coronal plane for examining circumvallate papillae. For floating section, tissue were embedded in 3% agarose and cut (100 µm) with Leica Vibratome. For wholemount immunofluorescent staining, samples were treated with 3DISCO protocol adapted from Ali et al. [START_REF] Ertürk | Three-dimensional imaging of solvent-cleared organs using 3DISCO[END_REF].

In Situ Hybridization and immunofluorescence were performed as described in [START_REF] Coppola | Epibranchial ganglia orchestrate the development of the cranial neurogenic crest[END_REF] and All Charts were drawn with PRSM software.