1. Developmental Biology
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Taste bud formation depends on taste nerves

  1. Di Fan
  2. Zoubida Chettouh
  3. G Giacomo Consalez
  4. Jean-François Brunet  Is a corresponding author
  1. Institut de Biologie de l’ENS (IBENS), Inserm, CNRS, École normale supérieure, PSL Research University, France
  2. East China Normal University, China
  3. San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Italy
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Cite this article as: eLife 2019;8:e49226 doi: 10.7554/eLife.49226

Abstract

It has been known for more than a century that, in adult vertebrates, the maintenance of taste buds depends on their afferent nerves. However, the initial formation of taste buds is proposed 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 proper formation of their target organs, the taste buds.

https://doi.org/10.7554/eLife.49226.001

Introduction

Taste buds are onion-shaped clusters of 60–100 taste receptors and support cells, embedded in epidermal papillae and distributed in a punctate pattern in the tongue and soft palate epithelia. They sense nutrients in the oral cavity and transmit taste information to the termini of sensory neurons, through conventional (Finger et al., 2005) and non-conventional (Ma et al., 2018; Romanov et al., 2018) synapses. Taste receptors and their support cells have a limited life span of 8 to 20 days depending on cell types (Perea-Martinez et al., 2013) and are constantly renewed from progenitors situated outside (Okubo et al., 2009; Ohmoto et al., 2017; Perea-Martinez et al., 2013) and, for a small minority, inside (Perea-Martinez et al., 2013) (and references therein) the taste bud. This process depends on the sensory afferents as demonstrated by the degeneration and regeneration of taste buds triggered, respectively, by the degeneration and regeneration of their surgically severed nerves (Oakley and Witt, 2004). While nerve-dependency has been uncontroversial for more than a century concerning the maintenance of taste buds in adult animals (Jacobson, 1991), it is still debated concerning their initial development in the embryo. Mature taste buds have been observed to form in the absence of nerves in amphibians (Barlow et al., 1996). In mammals, the case for nerve-dependency of taste bud development was mostly made through genetic invalidations of neurotrophins or their receptors, which hamper, to various extents, the outgrowth of sensory afferents towards the taste buds and simultaneously entail large losses of taste buds on the tongue at postnatal stages (Oakley and Witt, 2004). However, the protracted development of lingual taste buds, most of which mature after birth, left open the possibility that their postnatal loss reflected a failure of ‘maintenance’ or ‘maturation’ after a process of nerve-independent embryonic development (Barlow and Northcutt, 1998; Kapsimali and Barlow, 2013). To resolve this decades-old controversy, we completely blocked sensory innervation of the oral cavity in mouse by genetically preventing the birth of cranial sensory neurons, and examined the formation of taste buds — including palatal ones, whose development is almost entirely embryonic.

Results and discussion

Taste organs (taste papillae and their resident taste buds) of the anterior tongue and soft palate are innervated by somatic sensory neurons (for touch and pain) located in the trigeminal ganglion and visceral sensory neurons (for taste) located in the geniculate ganglion (Watson et al., 2012). The circumvallate papilla receives innervation from visceral sensory neurons in the distal (petrosal) ganglion of the glossopharyngeal nerve, while its somatosensory ones, projecting in the same nerve (Frank, 1991) (and references therein) are probably located in the proximal (‘superior’) ganglion. To suppress innervation of taste organs, we exploited the developmental dependency of cranial sensory neurons on the proneural transcription factors Neurog1 and Neurog2. As early as day 9.5 of embryonic development (E9.5), the knockout of Neurog1 blocks neuronal differentiation in the trigeminal, superior and jugular ganglia (Ma et al., 1998) which harbor somatic sensory neurons. Inactivation of its paralogue Neurog2, expressed in epibranchial placodes, blocks the formation of the geniculate and petrosal ganglia, which harbor visceral sensory neurons (Fode et al., 1998). In each single Neurog1 and Neurog2 knockout at E16.5, the epithelium of the tongue and of the soft palate retained nerve fascicles at regularly spaced locations (corresponding to the taste organs) from, presumably, visceral or somatic sensory fibers, respectively (which thus navigate to their target independently of each other) (Figure 1—figure supplement 1). In double Neurog1/Neurog2 knockouts, which lose all sensory innervation of the head (Espinosa-Medina et al., 2014), all nerve fibers had disappeared from the epithelium and underlying lamina propria of the palate and tongue, as expected (Figure 1—figure supplement 1).

Taste organs differentiate from taste placodes, specialized patches of the oral epithelium, thickened by apico-basal elongation of the cells, which transform into dome-shaped papillae with a mesenchymal core — most dramatically at the site of the single circumvallate papilla, on a smaller scale for fungiform papillae of the tongue, and also in the palate albeit less conspicuously (Rashwan et al., 2016) — and express a number of markers. Expression of the signaling molecule sonic hedgehog (Shh), after a diffuse phase throughout the oral epithelium, resolves around E12.5 into a punctate pattern corresponding to taste placodes, which also start expressing Prox1 and high levels of Sox2 (Thirumangalathu et al., 2009; Nakayama et al., 2008; Liu et al., 2013; Okubo et al., 2006). Two days later, Shh, Sox2 and Prox1 were still expressed in the same pattern in the placodes — by then about to become papillae (hereafter placodes/papillae) — (Figure 1A,B, Figure 1—figure supplement 2), and the transcription factors Ascl1 and Hes6 (Seta et al., 2003) were switched on in just a few cells (Figure 1B, Figure 1—figure supplement 2). All these markers were expressed in a normal pattern in the soft palate and anterior tongue of double Neurog1/2 knockouts at E14.5, except for Sox2 whose expression was stronger and expanded in the palate (Figure 1C, Figure 1—figure supplement 2). Expression of these markers was not restricted to taste placodes/papillae but also occurred in the incipient ridges (rugae) of the hard palate (which never give rise to taste buds) and this expression was also essentially unchanged in Neurog1/2 knockouts (Figure 1A,D). Between E14.5 and E16.5, a cluster of cells in each taste papilla or ruga had switched on cytokeratine 8 (CK8) in both wild type and Neurog1/2 KO (Figure 1B,D, Figure 1—figure supplement 2). Thus, fungiform and palatal taste papillae (whose morphology and, as we show here, gene expression program are similar to that of palatal rugae) are epithelial specializations that form in the absence of any nerve, in agreement with prior observations of fungiform placodes/papillae development in cultured tongue explants (Farbman and Mbiene, 1991Mbiene et al., 1997Hall et al., 2003). The single circumvallate papilla of the posterior tongue stood in contrast. In the wild type, it displayed the same gene expression events as fungiform and palatal papillae on its dorsal surface (Figure 1A,E). However, in this case, as in rugae, expression of Shh, Sox2, Hes6, Ascl1, Prox1 and CK8 does not prefigure the later differentiation of taste bud cells, which takes place after birth, mostly in the semi-circular trenches, not at the dorsal surface. In the Neurog1/2 knockouts at E14.5 (a day after arrival of nerve, thickening of the placode and onset of Shh expression (as previously observed on tongue explants [Mistretta et al., 2003] and see Figure 1—figure supplement 3A), the sparse expression of Hes6, Ascl1 and Prox1 was preserved, but Shh and Sox2 was not upregulated and morphogenetic events leading to papilla formation were stalled (Figure 1F), corroborating and extending a previous observation of circumvallate papilla atrophy in Brain-Derived Neurotrophic Factor/Neurotrophin three double knockouts (Ito et al., 2010). A similar phenotype was obtained in single Neurog2 KO (Figure 1—figure supplement 3A,B), which lack petrosal ganglia (Figure 1—figure supplement 3C). Therefore, the circumvallate papilla, already known to differ from fungiform papillae by its ontogenetic requirement for an Fgf10 signal from the mesenchyme (Petersen et al., 2011), also differs from both fungiform and palatal papillae by requiring its afferent nerve for its formation.

Figure 1 with 3 supplements see all
Soft palate taste papillae as well as hard palate rugae, but not the tongue circumvallate papilla form without innervation.

(A–F) Combined immunohistochemistry for β-III tubulin (Tuj1, brown) and in situ hybridization (blue) for the indicated probes (top panel and left four columns), or immunofluorescence for Prox1 or CK8, combined with immunofluorescence for β-III tubulin and a counterstain with DAPI (two right columns) in wild type (A,B,E) and double Neurog1/2 KO (C,D,F) at E14.5 or E16.5 as indicated. In the circumvallate papilla, markers are expressed on the dorsal surface (white arrowhead for CK8) but not at the lower part of the trenches (red arrowhead in the right column), where taste buds will develop after birth. CK8 is also expressed in flattened cells of the periderm (asterisks). For every probe two animals were examined. CP : circumvallate papilla; FP : fungiform papilla ; PP : palatal papilla ; R : ruga. Scale bars: 20 μm.

https://doi.org/10.7554/eLife.49226.002

By E18.5, in wild type embryos, taste buds had begun to form, as onion shaped clusters of many cells expressing CK8, spanning the height of the oral epithelium, to different extents in different locations. From this developmental stage on, we will designate these taste bud anlagen, irrespective of their size and degree of maturation, as ‘CK8-positive (CK8+) cell clusters’. In the palate, they almost reach their mature size and structure by E18.5 (Rashwan et al., 2016) (Figure 2A). In Neurog2 knockouts, that is in the absence of visceral nerves, there was a dramatic deficit in both the number of CK8+ cell clusters detected in every other section (43% fewer) in the palate (this number can be taken as an approximation of the number of taste buds in the palate, see Materials and methods) and their cross-sectional area (66% smaller) so that the overall area occupied by CK8+ cells was 80% smaller than in wild types (Figure 2A). The latter measure, on two-dimensional projections of confocal stacks through 20 μm-thick sections, likely underestimates the extent of the deficit. To rule out that the massive shortfall in taste cells in Neurog2 KO at E18.5 reflected developmental delay rather than failure, and to circumvent the neonatal death of Neurog2 KO, we prolonged pregnancies by 2 days with Delvosteron delivered to the dams, and examined the embryos at E20.5 (equivalent to post-natal day 2 (P2) for the normal pregnancy of C57BL/6 mice). The degree of atrophy was even more pronounced at E20.5 than at E18.5, due to a decrease in the number of CK8+ cell clusters (now 89% fewer than in wild type) and a stagnation of their average size (while their wild type counterparts had enlarged), so that the total area occupied by CK8+ cells in the soft palate was smaller by 96% relative to wild type (Figure 2B). CK8+ cell clusters in the tongue, which are harbored in fungiform papillae and are still immature at this stage in wild type, were similarly atrophic in the mutants (Figure 2—figure supplement 1). Those of the circumvallate papilla were absent (Figure 2—figure supplement 1). To verify that the taste bud phenotype of Neurog2 knockouts does not stem from a cell-autonomous action of Neurog2 in taste placodes (through some undetected transient expression there), we devised a second genetic strategy to prevent visceral innervation of the soft palate. We found that ablating the transcription factor Foxg1, expressed in epibranchial placodes (Hatini et al., 1999), causes defects in epibranchial ganglia, and a fully penetrant (n = 5) lack of the greater superficial petrosal nerve as early as E11.5 — thus of the visceral innervation to the soft palate (Figure 2—figure supplement 2). In Foxg1 KO embryos at E18.5 the palatal taste buds were atrophic in a manner comparable to Neurog2 KO embryos (Figure 2—figure supplement 2). In contrast to visceral (taste) innervation, somatic (touch) innervation was dispensable for taste bud formation, as demonstrated by the normal number of palatal CK8+ cell clusters and total area occupied by CK8+ cells in E20.5 Neurog1 knockouts (Figure 2C).

Figure 2 with 2 supplements see all
Formation of palatal taste buds requires visceral but not somatic innervation.

Typical examples of CK8+ cell clusters detected by immunofluorescence in wild type and Neurog2 or Neurog1 KO, at E18.5 (A) or E20.5 (B,C), and quantification of the number of clusters throughout the soft palate on alternate sections, and overall surface (in μm2) occupied by CK8+ cells (in alternate sections) throughout the soft palate. *: p<0.05, ***: p<0.001, ****: p<0.0001 and ns: p>0.05; error bars presented as mean ± SD (n = 3 for A, C and n = 4 for B). The individual results for each animal are represented by dots. Scale bar: 20 μm.

https://doi.org/10.7554/eLife.49226.006

At E20.5 (P2), many palatal taste buds in wild types are fully mature and probably functional (Rashwan et al., 2016). In line with this, we could detect type I, II and III cells recognizable by the expression, respectively, of the ecto-ATPase Entpd2, the Gustducin α-chain Gnat3 or the cation channels Trpm5 and Pkd2l1, all cells sharing the expression of the potassium channel Kcnq1 (Matsumoto et al., 2011) (Figure 3A). In Neurog2 knockouts, expression of each marker was missing in many of the residual CK8+ clusters (Figure 3B), but present, except for Pkd2l1, in one or two cells of others (Figure 3C). Neurog1 KO pups had a normal complement of differentiated cells in their fully formed buds (Figure 3D). The cells in the Neurog2 mutants that display one marker or other of mature taste receptors argue that taste nerves are indispensable, not for the differentiation of CK8+ cells into taste bud cells (by the criterion of the markers tested), but for the constitution, maintenance or proliferation of the pool of progenitors required for bud formation, possibly reminiscent of the role of parasympathetic nerves in the organogenesis of salivary glands (Knox et al., 2010). In line with this, high Sox2 expression in perigemmal cells (i.e. closely apposed to the taste buds), a hallmark of taste cell progenitors (Okubo et al., 2006; Ohmoto et al., 2017), was massively reduced in Neurog2 KO (although a few residual CK8+ cells were themselves Sox2+) (Figure 4A), together with that of the proliferation marker Ki67 (Figure 4B). We could not detect any sign of cell death at E16.5 or E18.5 in or around the taste cell clusters of wild type or mutant pups by the TUNEL reaction or immunofluorescence against Caspase 3.

Taste bud cell differentiation is impeded, but not always, in the absence of visceral innervation.

Sections through taste buds in the soft palate of wild types (A), Neurog2 KO (B,C) and Neurog1 KO (D) at E20.5 (P2), immunostained with the indicated antibodies. Kcnq1, Entpd2, Gnat3 and Trpm5 are occasionally detected in residual CK8+ cells of Neurog2 KO embryos. In Neurog1 KOs, taste buds contained the normal complement of markers. Scale bar: 20 μm.

https://doi.org/10.7554/eLife.49226.010
Taste nerves are required for Sox2 expression and cell proliferation around taste bud anlagen.

(A,B) Immunofluorescence against CK8 and Sox2, or CK8 and the proliferation marker Ki67 on representative CK8+ cell clusters in the soft palate from wild type, and Neurog2 KO, at E18.5. The graphs show the counts of Sox2High and Ki67+ cells inside or within two cell diameters of CK8+ cell clusters. **: p<0.01; error bars presented as mean ± SD (for Sox2High cells, a sample of 72 wild type and all (n = 100) mutant clusters were counted, on three animals; for Ki67+ cells, a sample of 71 wild type and all (n = 101) mutant clusters were counted on three animals). The individual results for each animal are represented by dots. Scale bar: 20 μm.

https://doi.org/10.7554/eLife.49226.011

The scattered residual CK8+ cell clusters of aneural oral epithelia resembled, by their small size yet expression of terminal differentiation markers, those induced by ectopic expression of Shh (Castillo et al., 2014). Such clusters might thus represent the endpoint, in the absence of subsequent innervation, of taste bud organogenesis triggered by local Shh (endogenous to the taste placodes, or ectopically induced in the epithelium). Along the same lines (and barring a myriad of possible species differences), the most parsimonious reconciliation of our finding with the apparently opposite one in axolotl would be that the ectopic aneural taste buds obtained in that species are much smaller than normal ones — as appears from Figure 2B versus Figure 4D of Barlow et al. (1996); or that in axolotl, taste buds would be so much smaller than in mouse as to not require a pool of progenitors.

All in all, we show that formation of taste buds in the mammalian embryo is as dependent on innervation as their renewal and regeneration are in the adult. In line with this, the time between the arrival of nerves and taste bud formation (2 to 3 days in the mouse palate; Rashwan et al., 2016) is in the same range as that between re-innervation and taste bud regeneration in gerbils (Cheal and Oakley, 1977) — the only species, to our knowledge, where the lag between regrowth of nerves and reformation of taste buds was measured. In light of our results, ‘maintenance’, in the case of taste buds, can be interpreted as an ongoing ontogeny. The developmental role that we document for nerves is neuron-type specific: exerted by visceral sensory neurons, derived from epibranchial placodes, not by somatosensory neurons, derived from the neural crest. This specificity makes less likely the possibility, that we cannot exclude however, that the nerve associated cells (Schwann cell precursors and Schwann cells) would have the inducing role, rather than the nerve fibers themselves. At the molecular level, nerve-derived Shh has been implicated in the maintenance of adult taste buds (Castillo-Azofeifa et al., 2017; Lu et al., 2018), with a proposed action, however, too slow and too limited to underlie the dramatic effect of nerve cutting on the integrity of taste buds — or on expression of Shh in taste bud cells (Miura et al., 2004). The nerve-derived trophic ‘hormone-like’ substance(s) postulated a century ago (Olmsted, 1920) to induce taste buds — in the adult for their continuous renewal, and, as we show here, in the embryo for their ontogeny — are likely yet to be discovered.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyAnti- Neurofilament (mouse monoclonal)Hybridoma BankHybridoma Bank (#2H3)RRID:AB_531793one in 500
AntibodyAnti-Gustducin (Gnat3) (goat polycolonal)MyBioSourceMyBioSource (#MBS421805) RRID:AB_10889192one in 500
AntibodyAnti-Entpd2 (rabbit polycolonal)http://ectonucleotidases-ab.com/(Bartel et al., 2006)RRID:AB_2314986one in 500
AntibodyAnti-Pkd2l1 (rabbit polycolonal)MilliporeMillipore (#AB9084)
RRID:AB_571091
one in 1000
AntibodyAnti-Prox1 (rabbit polyclonal)MilliporeMillipore (#AB5475) RRID:AB_177485one in 1000
AntibodyCytokeratin8 (Troma-I) (rat monoclonal)Developmental Studies Hybridoma Bank (DSHB)RRID:AB_531826one in 400
AntibodyAnti-Kcnq1 (rabbit polyclonal)MilliporeMillipore (#AB5932) RRID:AB_92147one in 1000
AntibodyAnti-Ki67 (rabbite polycolonal)abcamabcam (#ab15580) RRID:AB_443209one in 200
AntibodyAnti-Sox2 (goat polyclonal)R and D SystemsR and D Systems (#AF2018) RRID:AB_355110one in 500
AntibodyAnti-Trpm5 (guinea pig polyclonal)Obtained from ER Liman’s lab, USCone in 500
AntibodyAnti-βIII Tubulin (Tuj1) (mouse monoclonal)CovanceCovance (#MMS-435P) RRID:AB_2313773one in 500
AntibodyAnti-Phox2b (Rabbit polyclonal)Pattyn et al., Development, 124,4065–4075 (1997)one in 500
AntibodyDonkey anti-goat A488Thermo FisherThermo Fisher (#A-11055) RRID:AB_2534102one in 500
AntibodyDonkey anti- guinea pig A488Jackson Immunoresearch LaboratoriesJackson Immunoresearch Laboratories (#706-545-148)one in 500
AntibodyDonkey anti- mouse A488InvitrogenInvitrogen (#A-21202) RRID:AB_141607one in 500
AntibodyDonkey anti- rabbit A488Jackson Immunoresearch LaboratoriesJackson Immunoresearch Laboratories (#711-545-152)one in 500
AntibodyDonkey anti-rat Cy3Jackson Immunoresearch LaboratoriesJackson Immunoresearch Laboratories (#712-165-153)one in 500
Commercial kitVectastain Elite ABC KitsVector LaboratoriesVector Laboratories, PK-6101 and PK-6012
Chemical compoundDAB (3,3’-Diaminobenzidine)SigmaSigma (#SLBP9645V)
Commercial reagentProligesterone (Delvesterone)MSD Animal HealthDelvosteron: NaCl (0.9%)=1:1

Histology

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In situ hybridization and immunochemistry on sections or wholemounts have been described (Coppola et al., 2010). Immunofluorescence on cryostat or vibratome sections was performed as previously described (Espinosa-Medina et al., 2014). Wholemount immunofluorescent staining was made of cleared embryos following the 3DISCO protocol (Ertürk et al., 2012) and imaged with an Sp8 confocal microscope. 3D reconstructions were performed using the IMARIS imaging software.

Mouse embryos (E11.5, E12.5, E14.5 and E16.5) or 4% paraformaldehyde (PFA)-perfused pups (at P0 (E18.5) and P2 (E20.5)) were fixed in 4% PFA overnight at 4°C, and the tongue or palate was dissected out. For cryostat sections, tissues were embedded in 7% gelatine/15% sucrose and sectioned at 20 μm. For vibratome sections, tissues were embedded in 3% agarose (in PBS) and cut at 100 μm.

Immunohistochemical reactions were done with the Vectastain Elite ABC Kits (PK-6101 and PK-6012; Vector Laboratories) and color revealed by DAB (3,3’-Diaminobenzidine).

For anti-Pkd2l1 and anti-Sox2 immunoreactions, light fixation was required with 4% PFA at 4°C for 3 hr.

Antisense RNA Probes used were Hes6 (obtained from Ryoichiro Kageyama), Ascl1 (obtained from François Guillemot), Shh (obtained from Andrew McMahon) and Sox2 (obtained from Dr. Robin Lovell-Badge).

Transgenic mouse lines

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Neurog1/2 KO were bred by intercrossing single Neurog1 and Neurog2 heterozygous knockouts, then Neurog1/2 double heterozygotes.

All lines were maintained by crossing with C57BL/6 x DBA/2 F1 mice.

Animal treatment

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Proligestone (Delvosteron) was used to prolong the gestation of Neurog1 and Neurog2 KO mice. At E16.5, 100 μL of diluted Delvosteron (MSD Animal Health) solution (Delvosteron: NaCl (0.9%)=1:1) was injected subcutaneously in each pregnant mouse. Pups were surgically delivered on day 20.5 of embryonic development.

Statistical analyses

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For the quantification of CK8+ cell clusters, one cryosection out of two through the entire soft palate was analyzed in Neurog1 and Neurog2 KO embryos, wild types serving as controls. This raw number was used as an estimate of the number of CK8+ cell clusters in the palate, without multiplying by two and performing the Abercrombie correction. Indeed, all the clusters we counted in the wild type spanned the whole height of the epithelium and/or had a visible pore, de facto excluding objects that would be tangentially sectioned. This amounts to a systematic loss of ‘caps’, an important source of errors in the Abercrombie correction (Hedreen, 1998). Our method makes it next to impossible that clusters (with an average width of 28 μm (E18.5) and 38 μm (E20.5), see Source data 1) would be counted more than once on alternate 20 μm sections. On the other hand, a few clusters were probably lost, if they were centered on one of the uncounted section. The accuracy of our method is verified by the fact that we found the same number of clusters at E20.5 and E18.5 despite the 36% increase in average diameter, and also that we found (not shown) the same number of clusters on alternate 30 μm sections (as opposed to 20 μm).

For each genotype, all CK8+ clusters (if there were less than 10), or up to ten (if there were more) were imaged, for the wild type in 2 to 3 sections around the midline of the soft palate (where taste buds were the densest), for the mutants, in one out of two sections throughout the palate. Confocal imaging was performed on a Leica SP5 microscope or a LSM 880 Airyscan Zeiss microscope. The surface occupied by each CK8+ cluster was automatically outlined and measured with the Fiji software, and the mean cross-sectional area was calculated for each genotype. To calculate the entire surface occupied by CK8+ cell clusters on alternate sections of one half of soft palate, the mean value of the calculated surfaces of CK8+ cell clusters was multiplied by the raw, uncorrected number of CK8+ clusters. Statistical analysis was performed using unpaired two-tailed t-test. Results are expressed as mean ± SD. All graphs were performed with GraphPad Prism software. For the reason that we did not count ‘caps’ of wild type CK8+ clusters (see above), the decrease in total area is likely underestimated.

References

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    Embryonic taste buds develop in the absence of innervation
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    (1996)
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Decision letter

  1. Jeremy Nathans
    Reviewing Editor; Johns Hopkins University School of Medicine, United States
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  3. Igor Adameyko
    Reviewer; Karolinska Institutet, Sweden

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Taste bud formation depends on taste nerves" for consideration by eLife. Your article has been reviewed by Marianne Bronner as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Igor Adameyko (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As you will see, all of the reviewers were impressed with the significance and novelty of your work, but each reviewer also had specific and useful comments for improving the manuscript. Specifically, there are suggestions relate to the quantification of the data (reviewers 1 and 2), the over-simplification of the Title and Abstract (reviewers 2 and 3), and the Discussion section (all three reviewers).

I am including the three reviews at the end of this letter. I appreciate that the reviewers' comments cover a range of suggestions for improving the manuscript. We trust that most of these can be accommodated in a reasonable period of time. We look forward to receiving your revised manuscript.

Reviewer #1:

This is a nifty short report on genetic ablation of either somatosensory or visceral sensory neurons and the effects on development of taste buds. The work is generally clear and well-written and offers a modicum of new information on the neural dependency of the sensory end organs for taste.

A major obstacle to clarity in this work is the lack of a clear definition of how the authors use the term "Taste buds". As the MS describes, development in this system proceeds from non-innervated placodes through some intermediate stages culminating in a mature taste bud. The terminology used in this work seems inconsistent, sometimes referring to the intermediate stages as "taste anlage", but never clearly defining the term. Does a taste anlagen contain elongate, differentiated taste cells? When does a small collection of elongate taste cells become a "taste bud"? For example, Figure 2 shows elongate differentiated taste cells in Neurog2 KO animals, but the caption suggests that taste buds do not form in this KO line. Since the bar graphs of this figure report on number of taste buds observed, it is essential that the authors define what structure was counted as a taste bud. If in fact the authors are counting as taste buds these small collections of differentiated elongate cells, then they should be correcting for the sampling bias comparing larger objects to smaller objects in histological sections, e.g. by Abercrombie correction. If not, this is essential. Crucial for this calculation is the section thickness and the spacing between sections in relation to the size of the objects being studied. For example, if the section thickness is 15um and the object is 50um in depth, then each object is guaranteed to be counted twice if alternate sections are measured. Conversely, in the same set of sections a 10um object will be counted only once giving the impression that there are twice as many large objects as small objects when in fact, there would be equal numbers.

Also missing from this report is any mention of the development of taste bud primordia (placodes?) in vitro - a situation entirely devoid of innervation. This would seem highly relevant to this paper. Relevant references:

Mbiene, Maccallum and Mistretta, 1997.

Mistretta et al., 2003.

Hall, Bell and Finger, 2003.

Ozdener et al., 2006.

Reviewer #2:

Understanding the role of innervation in taste bud development is complicated by the normal occurrence of taste buds in the palate and different parts of the tongue, with taste cells maturing at different rates in each of these locations. Fan et al. exploited their genetic models to dissect the involvement of nerves in taste bud formation at different stages and in distinct locations.

Developmental defects in these mouse mutants include the absence of neurons that normally innervate the taste buds, and these unique neuronal knockouts therefore can be used to assess whether nerves are required in taste bud development. The authors showed that at early stages when nerves are just about to establish contacts with their peripheral targets, the circumvallate placode at the posterior of the tongue is unique in its nerve dependence for expression of certain early genes and for taste bud formation. Expression of some of the same genes during development of non-taste palatal placodes or lingual placodes giving rise to taste buds of the fungiform papillae in contrast remains largely unaffected. Nerves thus participate in taste bud formation at specific developmental windows that vary by location. This variation in nerve dependence for formation of distinct taste bud populations raises questions as to the choice of title, which understates the complexity of taste bud dependence on innervation.

It would be helpful if the authors could provide an overview describing taste bud development and maturation at each location and at distinct developmental stages. Using the Neurog2 knockout alone in palatal taste bud development, a previously unappreciated early involvement of nerves in taste bud maintenance was noted. The authors showed morphological defects in the number of K8+ cells and reduced proliferation and reduced expression of Sox2 associated with progenitor cells. However, sample numbers and quantification of these observations were not provided in the current manuscript. A revision of the manuscript is recommended before acceptance for publication. Please see below for specific questions and suggestions.

1) The Abstract and Title do not include any reference to the complexity of nerve involvement in formation of taste buds at different locations and developmental stages.

2) Does circumvallate atrophy occur in the Neurog2 single knockout, as was noted for the Neurog1/2 double KO?

3) Please report the number of mutant animals examined in Figure 1.

4) One of the most interesting aspects of the paper is mutant effects on progenitor cells, however, more samples are needed to quantify Sox2Sox2+ progenitor cells and proliferative status as indicated by Ki67+. Additional time points at E16.5 and E17.5 would also be informative.

Reviewer #3:

This focused and strong study answers an important biological question addressing the importance of innervation in initial development of taste buds and putting the regeneration of taste buds into an ontogeny-like framework. The authors provide a clear and straightforward answer in the form of a short report.

I have very few comments:

1) I think that the abstract slightly over-exaggerates the real conclusions: not all taste buds depend on the nerve presence in their development.

2) It should be very briefly discussed why different taste buds demonstrate differential preferences for the specific visceral sensory nerves during their development. Why the embryonic environment is permissive for some buds that are not innervated at all in case of a double Neurog1/2 KO? Can it be connected to the extraordinary high levels of Shh in developing rugi of the palate? This would fit the results described in the paper from Linda Barlow lab: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197660/

3) In Figure 2—figure supplement 1: In case of Neurog2 KO, Tuj1+ green fibers are very proximal to the CK8 staining and the location of forming taste bud. Are those nerves the remaining touch afferents? They stay in place in Neurog2 KO, although they do not support the development of fungiform papillae. It will be good if the authors clarify this in a figure legend for pedagogical purposes.

4) Can it be that nerve-associated cells (SCPs and Schwann cells) play a key role in signaling to the developing taste buds, and not the neurons, which support them (although I think this is unlikely)? Worth discussing in one or two phrases.

Overall, this is a good report that requires only some tuning of the text.

https://doi.org/10.7554/eLife.49226.016

Author response

Reviewer #1:

This is a nifty short report on genetic ablation of either somatosensory or visceral sensory neurons and the effects on development of taste buds. The work is generally clear and well-written and offers a modicum of new information on the neural dependency of the sensory end organs for taste.

A major obstacle to clarity in this work is the lack of a clear definition of how the authors use the term "Taste buds". As the MS describes, development in this system proceeds from non-innervated placodes through some intermediate stages culminating in a mature taste bud. The terminology used in this work seems inconsistent, sometimes referring to the intermediate stages as "taste anlage", but never clearly defining the term. Does a taste anlagen contain elongate, differentiated taste cells? When does a small collection of elongate taste cells become a "taste bud"?

The referee rightly points to the lack of clear-cut transition during taste bud formation. Indeed, we don’t know of any morphological criterion or global gene expression event that sharply demarcates the three canonical stages of taste bud formation: “placode”, “papilla” (whose mesenchymal core forms progressively), and “taste organ” (i.e. a papilla equipped with a mature taste bud). The expression of Shh or CK8 spans 3 and 2 of these phases, respectively, and their restriction to future taste bud cells is progressive. As for terminal differentiation markers (TRPM5, Pk2dL1 etc.), the onset of their expression is not precisely known. So, to the question “When does a small collection of elongate taste cells become a "taste bud"?” there is, in our opinion, no rigorous answer.

This said, to homogenize the nomenclature as requested, we now call any group of elongated CK8+ cells at E18.5 or later, “CK8+ cells clusters” whether they are mature taste buds, immature taste buds (that we formerly called “taste bud anlagen” — there was never mention of “taste anlagen” —, i.e. not yet functional or not containing the full complement of cells), or taste buds which are atrophic in the mutants, to any extent. This common term reflects the fact that all such formations are not distinguished by dichotomous morphological or gene expression criteria, but distributed on a gradient of sizes (and we quantify the sizes).

This leaves undefined the nature of CK8+ cells between E16.5 and 18.5, before they are clearly elongated or grouped in onion shaped structures, all the more so, since we show that some of these cells (on the dorsal aspect of the circumvallate papilla and on the rugae) never become taste cells.

Nevertheless, we have kept the word “taste bud” in the title of the paper and figures and in the Discussion section, because a large majority of CK8+ clusters have vanished in the mutants and the remaining ones are abnormally small, so that, despite the lack of definition of the exact moment where a collection of CK8+ cells qualifies as a “taste bud”, we feel justified in saying that “taste bud formation requires taste nerves” [see responses to criticisms of the title below].

For example, Figure 2 shows elongate differentiated taste cells in Neurog2 KO animals, but the caption suggests that taste buds do not form in this KO line. Since the bar graphs of this figure report on number of taste buds observed, it is essential that the authors define what structure was counted as a taste bud.

See explanation above. We have now replaced “taste buds” by “CK8+ cell clusters” in the legend and in the figure. And we counted all CK8+ cell clusters (down to single cells when that occurred in the mutants).

If in fact the authors are counting as taste buds these small collections of differentiated elongate cells, then they should be correcting for the sampling bias comparing larger objects to smaller objects in histological sections, e.g. by Abercrombie correction. If not, this is essential. Crucial for this calculation is the section thickness and the spacing between sections in relation to the size of the objects being studied. For example, if the section thickness is 15um and the object is 50um in depth, then each object is guaranteed to be counted twice if alternate sections are measured. Conversely, in the same set of sections a 10um object will be counted only once giving the impression that there are twice as many large objects as small objects when in fact, there would be equal numbers.

We do not think that the Abercrombie correction, which was devised to count cell nuclei in a volume of tissue, is appropriate for the comparison of very different objects between a wild type and mutant condition, particularly because of the indirect estimate of H (the height of the object perpendicular to the plane of section) and the “lost caps” effect We now make this reasoning explicit in the Material and methods section: in the following way:

“This raw number was used as an estimate of the number of CK8+ cell clusters in the palate, without multiplying by two and performing the Abercrombie correction. Indeed, all the clusters we counted in the wild type spanned the whole height of the epithelium and/or had a visible pore, de facto excluding objects that would be tangentially sectioned. This amounts to a systematic loss of “caps”, an important source of errors in the Abercrombie correction (Hedreen, 1998). Our method makes it next to impossible that clusters (with an average width of 28μm(E18.5) and 38μm (E20.5)) would be counted more than once on alternate 20μm sections. On the other hand, a few clusters were probably lost, if they were centered on one of the uncounted section. The accuracy of our method is verified by the fact that we found the same number of clusters at E20.5 and E18.5 despite the 36% increase in average diameter, and also that we found (not shown) the same number of clusters on alternate 30μm sections (as opposed to 20μm).”

In any case, this raw number is the only one relevant to the calculation of the total volume occupied by CK8+ cells, that we estimate through the proxy of the total surface occupied by CK8+ cells on the sections we count (one 20μm section out of two). The total surface occupied by taste cells is the total number of patches of CK8+ cells seen on sections, multiplied by the average size of the patches. If two patches on two sections happened to correspond to the same “CK8+ cell cluster”, it would be a realistic reflection of the fact that this cluster is big and occupies a large volume (which is what we evaluate, by a proxy). This is biologically the most important figure. If the number of CK8+ clusters was unchanged in the mutants, but their size decreased on average by 98%, the conclusion of our paper, i.e. that “taste bud formation depends on taste nerves” would remain unchanged.

Also missing from this report is any mention of the development of taste bud primordia (placodes?) in vitro - a situation entirely devoid of innervation. This would seem highly relevant to this paper. Relevant references:

Mbiene, Maccallum and Mistretta, 1997.

Mistretta et al., 2003.

Hall, Bell and Finger, 2003.

Ozdener et al., 2006.

We did cite the first paper, historically, to show that fungiform papillae develop in tongue explants. As requested, we have now added the following citation:, Maccallum and Mistretta, 1997 and Hall, Bell and Finger, 2003. And we added Mistretta et al., 2003 on the topic of the CV papilla, making explicit that we find, like her, that Shh is switched on in the CV placode, but leaving implicit the contradiction of her claim that CV papilla formation is nerve independent (on the basis of morphological evidence that we think is weak). On the other hand, it is not easy to see how the taste cell culture system described by Ozdener et al., 2006 informs about nerve dependency in vivo.

Reviewer #2:

[…] It would be helpful if the authors could provide an overview describing taste bud development and maturation at each location and at distinct developmental stages.

The circumvallate placode is unique in its dependency on nerves for subsequent papilla formation, but not for taste bud formation, which are just as dependent as in other papillae. To help conceptually disentangle the two issues, we now show, the absence of taste buds in the neonatal circumvallate organ (as expected since the organ itself does not form) (Figure 2—figure supplement 1).

Using the Neurog2 knockout alone in palatal taste bud development, a previously unappreciated early involvement of nerves in taste bud maintenance was noted. The authors showed morphological defects in the number of K8+ cells and reduced proliferation and reduced expression of Sox2 associated with progenitor cells. However, sample numbers and quantification of these observations were not provided in the current manuscript. A revision of the manuscript is recommended before acceptance for publication.

We do not document “maintenance” but appearance.

Please see below for specific questions and suggestions.

1) The Abstract and Title do not include any reference to the complexity of nerve involvement in formation of taste buds at different locations and developmental stages.

The “complexity” (which is rather an asynchrony) is in the development itself, not in the requirement for nerves in that development. The development of taste buds is not synchronous at every place (CV, soft palate, and tongue, and even different parts of the anterior tongue) and their demise at different stages in the absence of nerves simply reflects that.

The only true “complexity”, not encapsulated by the Title or the Abstract, is that the CV organ depends on nerves, not only for its resident taste buds but also for the papilla itself. In other words, it depends on the nerves even more than other taste organs. The title does not include this additional feature, but remains perfectly true, concerning taste buds.

We think that one virtue of our title (and of the paper), is precisely to simplify a field — which has become replete, over decades, with appearances of complexities and special cases, quantification of partial phenotypes, at different phases, in different locations, etc. — by encapsulating the fact that no complete, mature taste bud forms in the absence of nerves. This most important message would be jeopardized by the addition of what is basically a bonus, the idiosyncrasy of the circumvallate papilla (again, not of its taste buds).

2) Does circumvallate atrophy occur in the Neurog2 single knockout, as was noted for the Neurog1/2 double KO?

As requested, we now show in a new supplementary figure that the Neurog2 KO looks exactly like the double Neurog2/Neurog1 knock out. This is now stated in the text as: “A similar phenotype was obtained in single Neurog2 KO (Figure 1—figure supplement 3A,B), which lack a petrosal ganglion (Figure 1—figure supplement 3C).

3) Please report the number of mutant animals examined in Figure 1.

Two animals were used for each probe. This is now stated in the legend.

4) One of the most interesting aspects of the paper is mutant effects on progenitor cells, however, more samples are needed to quantify Sox2Sox2+ progenitor cells and proliferative status as indicated by Ki67+. Additional time points at E16.5 and E17.5 would also be informative.

As requested, we now counted Sox2+ cells and Ki67 cells in mutants and wild type ad E18.5, and add this data to Figure 4. On a pilot experiment (that we do not show), we could not see any difference at E16.5.

Reviewer #3:

This focused and strong study answers an important biological question addressing the importance of innervation in initial development of taste buds and putting the regeneration of taste buds into an ontogeny-like framework. The authors provide a clear and straightforward answer in the form of a short report.

I have very few comments:

1) I think that the abstract slightly over-exaggerates the real conclusions: not all taste buds depend on the nerve presence in their development.

It is not clear to us what are the taste buds that the referee has in mind that would not depend on nerves. Unless the referee means that our statement would be justified only if the disappearance of taste cells was by 100% as opposed to 96%? Also, we have no indication that the atrophic residual CK8+ cell clusters in the mutants are the same, from animal to animal. We believe that our short and clear abstract reflects the findings that we describe, without exaggeration, and that the field will benefit from that clear message [see response to referee #2]. Nevertheless, we softened the abstract as requested, it now reads “we show that the latter but not the former are absolutely required for the proper formation of their target organs, the taste buds”, leaving room for the formation of very rudimentary, atrophic organs.

2) It should be very briefly discussed why different taste buds demonstrate differential preferences for the specific visceral sensory nerves during their development.

Taste buds per se don’t demonstrate differential preferences (see response to referee#2). Only the circumvallate papilla shows a specific dependency that other papillae don’t.

Why the embryonic environment is permissive for some buds that are not innervated at all in case of a double Neurog1/2 KO? Can it be connected to the extraordinary high levels of Shh in developing rugi of the palate? This would fit the results described in the paper from Linda Barlow lab: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197660/

We do not see true “taste buds” (i.e. CK8+ clusters which would not be atrophic) in the mutants. The relationship of the atrophic residual taste buds (that we call now CK8+ cell clusters) to the ectopic ones in the Barlow paper is made explicit in the text. (And the rugae never contain taste buds: their CK8+ cells are not taste cell precursors, or possibly are taste cell precursors that never differentiate).

3) In Figure 2—figure supplement 1: In case of Neurog2 KO, Tuj1+ green fibers are very proximal to the CK8 staining and the location of forming taste bud. Are those nerves the remaining touch afferents? They stay in place in Neurog2 KO, although they do not support the development of fungiform papillae. It will be good if the authors clarify this in a figure legend for pedagogical purposes.

In the original main text we wrote: “In each single Neurog1 and Neurog2 knockout at E16.5, the epithelium of the tongue and of the soft palate retained nerve fascicles at regularly spaced locations (corresponding to the taste organs) from, presumably, visceral or somatic sensory fibers, respectively (which thus navigate to their target independently of each other)”. As requested, we have now added the following sentence in the legend of Figure 1—figure supplement 1: “Since Neurog2 KO have lost the geniculate, but keep the trigeminal ganglion, the residual innervation of taste organs in these mutants correspond to the somatic (touch and pain) fibers”.

4) Can it be that nerve-associated cells (SCPs and Schwann cells) play a key role in signaling to the developing taste buds, and not the neurons, which support them (although I think this is unlikely)? Worth discussing in one or two phrases.

As requested we now mention this possibility in the text as follows: “This specificity [of nerve fiber type] makes less likely the possibility, that we cannot exclude however, that the nerve associated cells (Schwann cell precursors and Schwann cells) have the inducing role, rather than the nerve fibers themselves.”

https://doi.org/10.7554/eLife.49226.017

Article and author information

Author details

  1. Di Fan

    1. Institut de Biologie de l’ENS (IBENS), Inserm, CNRS, École normale supérieure, PSL Research University, Paris, France
    2. School of Life Science, East China Normal University, Shanghai, China
    Contribution
    Conceptualization, Investigation, Writing—original draft
    Competing interests
    No competing interests declared
  2. Zoubida Chettouh

    Institut de Biologie de l’ENS (IBENS), Inserm, CNRS, École normale supérieure, PSL Research University, Paris, France
    Contribution
    Validation, Investigation, Visualization, Methodology, Writing—review and editing, Involved in every step of the production, acquisition and analysis of the data
    Competing interests
    No competing interests declared
  3. G Giacomo Consalez

    San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Milano, Italy
    Contribution
    Resources, Methodology, Writing—review and editing, Provided the main genetic tool for this study, the Neurog2 KO
    Competing interests
    No competing interests declared
  4. Jean-François Brunet

    Institut de Biologie de l’ENS (IBENS), Inserm, CNRS, École normale supérieure, PSL Research University, Paris, France
    Contribution
    Conceptualization, Supervision, Funding acquisition, Visualization, Writing—original draft, Project administration
    For correspondence
    jfbrunet@biologie.ens.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1985-6103

Funding

Agence Nationale de la Recherche (ANR-12-BSV4-0007-01)

  • Jean-François Brunet

Agence Nationale de la Recherche (ANR-10-LABX-54 MEMOLIFE)

  • Jean-François Brunet

Agence Nationale de la Recherche (ANR-11-IDEX-0001-02 PSL research University)

  • Jean-François Brunet

Fondation pour la Recherche Médicale (DEQ 2000326472)

  • Jean-François Brunet

Agence Nationale de la Recherche (ANR-17-CE16-00006-01)

  • Jean-François Brunet

China Scholarship Council

  • Di Fan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the Imaging Facility of IBENS, which is supported by grants from Fédération pour la Recherche sur le Cerveau, Région Ile-de-France DIM NeRF 2009 and 2011 and France-BioImaging. We wish to thank the animal facility of IBENS, C Goridis for helpful comments on the manuscript and all the members of the Brunet laboratory for discussions. This study was supported by the CNRS, the École Normale Supérieure, INSERM, ANR award 17-CE160006-01 (to J-FB), FRM award DEQ2000326472 (to J-FB), the ‘Investissements d’Avenir’ program of the French Government implemented by the ANR (referenced ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001–02 PSL Research University). D-F received a fellowship from the China Scholarship Council. GGC's lab was funded by the Italian Telethon Foundation grant GGP13146.

Ethics

Animal experimentation: All animal studies were done in accordance with the guidelines issued by the French Ministry of Agriculture and have been approved by the Direction Départementale des Services Vétérinaires de Paris.

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Jeremy Nathans, Johns Hopkins University School of Medicine, United States

Reviewer

  1. Igor Adameyko, Karolinska Institutet, Sweden

Publication history

  1. Received: June 11, 2019
  2. Accepted: September 23, 2019
  3. Accepted Manuscript published: October 1, 2019 (version 1)
  4. Version of Record published: October 9, 2019 (version 2)

Copyright

© 2019, Fan et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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