Rapid structural remodeling of peripheral taste neurons is independent of taste cell turnover.

Taste bud cells are constantly replaced in taste buds as old cells die and new cells migrate into the bud. The perception of taste relies on new taste bud cells integrating with existing neural circuitry, yet how these new cells connect with a taste ganglion neuron is unknown. Do taste ganglion neurons remodel to accommodate taste bud cell renewal? If so, how much of the structure of taste axons is fixed and how much remodels? Here, we measured the motility and branching of individual taste arbors (the portion of the axon innervating taste buds) in mice over time with two-photon in vivo microscopy. Terminal branches of taste arbors continuously and rapidly remodel within the taste bud. This remodeling is faster than predicted by taste bud cell renewal, with terminal branches added and lost concurrently. Surprisingly, blocking entry of new taste bud cells with chemotherapeutic agents revealed that remodeling of the terminal branches on taste arbors does not rely on the renewal of taste bud cells. Although terminal branch remodeling was fast and intrinsically controlled, no new arbors were added to taste buds, and few were lost over 100 days. Taste ganglion neurons maintain a stable number of arbors that are each capable of high-speed remodeling. We propose that terminal branch plasticity permits arbors to locate new taste bud cells, while stability of arbor number supports constancy in the degree of connectivity and function for each neuron over time.

Taste arbor structural variability analyzed across taste regions.

Taste ganglion neurons are functionally and molecularly diverse, but until recently morphological diversity was completely unexplored. Specifically, taste arbors (the portion of the neuron within the taste bud) vary in structure, but the reason for this variability is unclear. Here, we analyzed structural variability in taste arbors to determine which factors determine their morphological diversity. To characterize arbor morphology and its relationship to taste bud cells capable of transducing taste stimuli (taste-transducing cell) number and type, we utilized sparse cell genetic labeling of taste ganglion neurons in combination with whole-mount immunohistochemistry. Reconstruction of 151 taste arbors revealed variation in arbor size, complexity, and symmetry. Overall, taste arbors exist on a continuum of complexity, cannot be categorized into discrete morphological groups, and do not have stereotyped endings. Arbor size/complexity was not related to the size of the taste bud in which it was located or the type of taste-transducing cell contacted (membranes within 180 nm). Instead, arbors could be broadly categorized into three groups: large asymmetrical arbors contacting many taste-transducing cells, small symmetrical arbors contacting one or two taste-transducing cells, and unbranched arbors. Neurons with multiple arbors had arbors in more than one of these categories, indicating that this variability is not an intrinsic feature of neuron type. Instead, we speculate that arbor structure is determined primarily by nerve fiber remodeling in response to cell turnover and that large asymmetrical arbors represent a particularly plastic state.

Variable Branching Characteristics of Peripheral Taste Neurons Indicates Differential Convergence.

Taste neurons are functionally and molecularly diverse, but their morphologic diversity remains completely unexplored. Using sparse cell genetic labeling, we provide the first reconstructions of peripheral taste neurons. The branching characteristics across 96 taste neurons show surprising diversity in their complexities. Individual neurons had 1-17 separate arbors entering between one and seven taste buds, 18 of these neurons also innervated non-taste epithelia. Axon branching characteristics are similar in gustatory neurons from male and female mice. Cluster analysis separated the neurons into four groups according to branch complexity. The primary difference between clusters was the amount of the nerve fiber within the taste bud available to contact taste-transducing cells. Consistently, we found that the maximum number of taste-transducing cells capable of providing convergent input onto individual gustatory neurons varied with a range of 1-22 taste-transducing cells. Differences in branching characteristics across neurons indicate that some neurons likely receive input from a larger number of taste-transducing cells than other neurons (differential convergence). By dividing neurons into two groups based on the type of taste-transducing cell most contacted, we found that neurons contacting primarily sour transducing cells were more heavily branched than those contacting primarily sweet/bitter/umami transducing cells. This suggests that neuron morphologies may differ across functional taste quality. However, the considerable remaining variability within each group also suggests differential convergence within each functional taste quality. Each possibility has functional implications for the system.SIGNIFICANCE STATEMENT Taste neurons are considered relay cells, communicating information from taste-transducing cells to the brain, without variation in morphology. By reconstructing peripheral taste neuron morphologies for the first time, we found that some peripheral gustatory neurons are simply branched, and can receive input from only a few taste-transducing cells. Other taste neurons are heavily branched, contacting many more taste-transducing cells than simply branched neurons. Based on the type of taste-transducing cell contacted, branching characteristics are predicted to differ across (and within) quality types (sweet/bitter/umami vs sour). Therefore, functional differences between neurons likely depends on the number of taste-transducing cells providing input and not just the type of cell providing input.

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Variation in taste ganglion neuron morphology: insights into taste function and plasticity.

Chemical information from food is transduced by cells in the taste bud (taste-transducing cells) and carried to the brain by peripheral taste ganglion neurons. These neurons are thought to act simply as cables without any transformation of the signal or circuitry between the taste-transducing cells and the neurons. However, these neurons vary in structure, particularly in the extent of their peripheral axon branching. Such structural differences would be expected to underlie differences in the number of taste-transducing cells providing convergent information to these neurons. However, axon branching may vary over time and morphological differences between neurons might also reflect neuron plasticity. Because taste-transducing cells die and are replaced, the morphology of neurons may change as they form connections with new cells within the taste bud. Modern genetic approaches may permit investigations of the complex relationship among gustatory neuron morphology, circuitry, and function. This review discusses potential relationships among peripheral taste neuron morphology, function, and plasticity to help advance our understanding of taste system function and dysfunction.

TrkB expression and dependence divides gustatory neurons into three subpopulations.

BACKGROUND: During development, gustatory (taste) neurons likely undergo numerous changes in morphology and expression prior to differentiation into maturity, but little is known this process or the factors that regulate it. Neuron differentiation is likely regulated by a combination of transcription and growth factors. Embryonically, most geniculate neuron development is regulated by the growth factor brain derived neurotrophic factor (BDNF). Postnatally, however, BDNF expression becomes restricted to subpopulations of taste receptor cells with specific functions. We hypothesized that during development, the receptor for BDNF, tropomyosin kinase B receptor (TrkB), may also become developmentally restricted to a subset of taste neurons and could be one factor that is differentially expressed across taste neuron subsets. METHODS: We used transgenic mouse models to label both geniculate neurons innervating the oral cavity (Phox2b+), which are primarily taste, from those projecting to the outer ear (auricular neurons) to label TrkB expressing neurons (TrkBGFP). We also compared neuron number, taste bud number, and taste receptor cell types in wild-type animals and conditional TrkB knockouts. RESULTS: Between E15.5-E17.5, TrkB receptor expression becomes restricted to half of the Phox2b + neurons. This TrkB downregulation was specific to oral cavity projecting neurons, since TrkB expression remained constant throughout development in the auricular geniculate neurons (Phox2b-). Conditional TrkB removal from oral sensory neurons (Phox2b+) reduced this population to 92% of control levels, indicating that only 8% of these neurons do not depend on TrkB for survival during development. The remaining neurons failed to innervate any remaining taste buds, 14% of which remained despite the complete loss of innervation. Finally, some types of taste receptor cells (Car4+) were more dependent on innervation than others (PLCß2+). CONCLUSIONS: Together, these findings indicate that TrkB expression and dependence divides gustatory neurons into three subpopulations: 1) neurons that always express TrkB and are TrkB-dependent during development (50%), 2) neurons dependent on TrkB during development but that downregulate TrkB expression between E15.5 and E17.5 (41%), and 3) neurons that never express or depend on TrkB (9%). These TrkB-independent neurons are likely non-gustatory, as they do not innervate taste buds.

Insulin-Like Growth Factors Are Expressed in the Taste System, but Do Not Maintain Adult Taste Buds.

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1rlox/lox). While K14-Cre::Igf1rlox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1rlox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C ß2- (PLCß2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1rlox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1rlox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.

Postnatal reduction of BDNF regulates the developmental remodeling of taste bud innervation.

The refinement of innervation is a common developmental mechanism that serves to increase the specificity of connections following initial innervation. In the peripheral gustatory system, the extent to which innervation is refined and how refinement might be regulated is unclear. The initial innervation of taste buds is controlled by brain-derived neurotrophic factor (BDNF). Following initial innervation, taste receptor cells are added and become newly innervated. The connections between the taste receptor cells and nerve fibers are likely to be specific in order to retain peripheral coding mechanisms. Here, we explored the possibility that the down-regulation of BDNF regulates the refinement of taste bud innervation during postnatal development. An analysis of BDNF expression in Bdnf(lacZ/+) mice and real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that BDNF was down-regulated between postnatal day (P) 5 and P10. This reduction in BDNF expression was due to a loss of precursor/progenitor cells that express BDNF, while the expression of BDNF in the subpopulations of taste receptor cells did not change. Gustatory innervation, which was identified by P2X3 immunohistochemistry, was lost around the perimeter where most progenitor/precursor cells are located. In addition, the density of innervation in the taste bud was reduced between P5 and P10, because taste buds increase in size without increasing innervation. This reduction of innervation density was blocked by the overexpression of BDNF in the precursor/progenitor population of taste bud cells. Together these findings indicate that the process of BDNF restriction to a subpopulation of taste receptor cells between P5 and P10, results in a refinement of gustatory innervation. We speculate that this refinement results in an increased specificity of connections between neurons and taste receptor cells during development.

Taste Bud-Derived BDNF Is Required to Maintain Normal Amounts of Innervation to Adult Taste Buds.

Gustatory neurons transmit chemical information from taste receptor cells, which reside in taste buds in the oral cavity, to the brain. As adult taste receptor cells are renewed at a constant rate, nerve fibers must reconnect with new taste receptor cells as they arise. Therefore, the maintenance of gustatory innervation to the taste bud is an active process. Understanding how this process is regulated is a fundamental concern of gustatory system biology. We speculated that because brain-derived neurotrophic factor (BDNF) is required for taste bud innervation during development, it might function to maintain innervation during adulthood. If so, taste buds should lose innervation when Bdnf is deleted in adult mice. To test this idea, we first removed Bdnf from all cells in adulthood using transgenic mice with inducible CreERT2 under the control of the Ubiquitin promoter. When Bdnf was removed, approximately one-half of the innervation to taste buds was lost, and taste buds became smaller because of the loss of taste bud cells. Individual taste buds varied in the amount of innervation each lost, and those that lost the most innervation also lost the most taste bud cells. We then tested the idea that that the taste bud was the source of this BDNF by reducing Bdnf levels specifically in the lingual epithelium and taste buds. Taste buds were confirmed as the source of BDNF regulating innervation. We conclude that BDNF expressed in taste receptor cells is required to maintain normal levels of innervation in adulthood.

The neurotrophin receptor p75 regulates gustatory axon branching and promotes innervation of the tongue during development.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) regulate the survival of gustatory neurons, axon growth and branching, and innervation of taste buds during development. These actions are largely, but not completely, mediated through the tyrosine kinase receptor, TrkB. Here, we investigated the role of p75, the other major receptor for BDNF and NT4, in the development of the taste system. RESULTS: We found that p75-/-mice showed delayed axon outgrowth and reduced branching of gustatory axons at embryonic day (E)13.5. From E14.5 to E18.5, gustatory neurons innervated fewer papillae and completely failed to innervate the mid-region of the tongue in p75-/-mice. These early effects of the p75 mutation on gustatory axons preceded the loss of geniculate ganglion neurons starting at E14.5 and also contributed to a loss of taste buds at and after birth. Because knockouts for the TrkB receptor (TrkB-/-) do not lose as many taste buds as hybrid knockouts for its two ligands (BDNF and NT4), we asked if p75 maintains those additional taste buds in the absence of TrkB. It does not; hybrid TrkB-/-/p75-/-mice had more taste buds than TrkB-/-mice; these additional taste buds were not due to an increase in neurons or innervation. CONCLUSIONS: p75 regulates gustatory neuron axon branching and tongue innervation patterns during taste system development. This function is likely accomplished independently of BDNF, NT4, and TrkB. In addition, p75 does not support the remaining neurons or taste buds in TrkB-/-mice.

BDNF and NT4 play interchangeable roles in gustatory development.

A limited number of growth factors are capable of regulating numerous developmental processes, but how they accomplish this is unclear. The gustatory system is ideal for examining this issue because the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) have different developmental roles although both of them activate the same receptors, TrkB and p75. Here we first investigated whether the different roles of BDNF and NT4 are due to their differences in temporal and spatial expression patterns. Then, we asked whether or not these two neurotrophins exert their unique roles on the gustatory system by regulating different sets of downstream genes. By using Bdnf(Nt4/Nt4) mice, in which the coding region for BDNF is replaced with NT4, we examined whether the different functions of BDNF and NT4 are interchangeable during taste development. Our results demonstrated that NT4 could mediate most of the unique roles of BDNF during taste development. Specifically, caspase-3-mediated cell death, which was increased in the geniculate ganglion in Bdnf(-/-) mice, was rescued in Bdnf(Nt4/Nt4) mice. In BDNF knockout mice, tongue innervation was disrupted, and gustatory axons failed to reach their targets. However, disrupted innervation was rescued and target innervation is normal when NT4 replaced BDNF. Genome wide expression analyses revealed that BDNF and NT4 mutant mice exhibited different gene expression profiles in the gustatory (geniculate) ganglion. Compared to wild type, the expression of differentiation-, apoptosis- and axon guidance-related genes was changed in BDNF mutant mice, which is consistent with their different roles during taste development. However, replacement of BDNF by NT4 rescued these gene expression changes. These findings indicate that the functions of BDNF and NT4 in taste development are interchangeable. Spatial and temporal differences in BDNF and NT4 expression can regulate differential gene expression in vivo and determine their specific roles during development.

Taste neurons consist of both a large TrkB-receptor-dependent and a small TrkB-receptor-independent subpopulation.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) are two neurotrophins that play distinct roles in geniculate (taste) neuron survival, target innervation, and taste bud formation. These two neurotrophins both activate the tropomyosin-related kinase B (TrkB) receptor and the pan-neurotrophin receptor p75. Although the roles of these neurotrophins have been well studied, the degree to which BDNF and NT-4 act via TrkB to regulate taste development in vivo remains unclear. In this study, we compared taste development in TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice to determine if these deficits were similar. If so, this would indicate that the functions of both BDNF and NT-4 can be accounted for by TrkB-signaling. We found that TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice lose a similar number of geniculate neurons by E13.5, which indicates that both BDNF and NT-4 act primarily via TrkB to regulate geniculate neuron survival. Surprisingly, the few geniculate neurons that remain in TrkB(-/-) mice are more successful at innervating the tongue and taste buds compared with those neurons that remain in Bdnf(-/-)/Ntf4(-/-) mice. The remaining neurons in TrkB(-/-) mice support a significant number of taste buds. In addition, these remaining neurons do not express the TrkB receptor, which indicates that either BDNF or NT-4 must act via additional receptors to influence tongue innervation and/or targeting.

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor.

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.

Developmental expression of Bdnf, Ntf4/5, and TrkB in the mouse peripheral taste system.

Brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4), and their TrkB receptor regulate taste system development. To determine where and when gustatory neurons come in contact with these important factors, temporospatial expression patterns of Bdnf, Ntf4/5, and TrkB in the peripheral taste system were examined using RT-PCR. In the lingual epithelium, Ntf4/5 mRNA expression was higher than that of Bdnf at embryonic day 12.5 (E12.5), and the expression of both factors decreased afterwards. However, Ntf4/5 expression decreased at an earlier age than Bdnf. Bdnf and Ntf4/5 are expressed in equal amounts at E12.5 in geniculate ganglion, but Bdnf expression increased from E14.5 to birth, whereas Ntf4/5 expression decreased. These findings indicate that NT4 functions at early embryonic stages and is derived from different sources than Bdnf. TrkB expression in the geniculate ganglion is robust throughout development and is not a limiting factor for neurotrophin function in this system.

Lingual and palatal gustatory afferents each depend on both BDNF and NT-4, but the dependence is greater for lingual than palatal afferents.

Neurons of the geniculate ganglion innervate taste buds located in two spatially distinct targets, the tongue and palate. About 50% of these neurons die in Bdnf(-/-) mice and Ntf4/5(-/-) mice. Bdnf(-/-)/Ntf4/5(-/-) double mutants lose 90-95% of geniculate ganglion neurons. To determine whether different subpopulations are differentially influenced by neurotrophins, we quantified neurons from two ganglion subpopulations separately and remaining taste buds at birth within each target field in wild-type, Bdnf(-/-), Ntf4/5(-/-), and Bdnf(-/-)/Ntf4/5(-/-) mice. In wild-type mice the same number of neurons innervated the anterior tongue and soft palate and each target contained the same number of taste buds. Compared to wild-type mice, Bdnf(-/-) mice showed a 50% reduction in geniculate neurons innervating the tongue and a 28% loss in neurons innervating the soft palate. Ntf4/5(-/-) mice lost 58% of the neurons innervating the tongue and 41% of the neurons innervating the soft palate. Taste bud loss was not as profound in the NT-4 null mice compared to BDNF-null mice. Tongues of Bdnf(-/-)/Ntf4/5(-/-) mice were innervated by 0 to 4 gustatory neurons and contained 3 to 16 taste buds at birth, indicating that some taste buds remain even when all innervation is lost. Thus, gustatory neurons are equally dependent on BDNF and NT-4 expression for survival, regardless of what peripheral target they innervate. However, taste buds are more sensitive to BDNF than NT-4 removal.

BDNF is required for the survival of differentiated geniculate ganglion neurons.

In mice lacking functional brain-derived neurotrophic factor (BDNF), the number of geniculate ganglion neurons, which innervate taste buds, is reduced by one-half. Here, we determined how and when BDNF regulates the number of neurons in the developing geniculate ganglion. The loss of geniculate neurons begins at embryonic day 13.5 (E13.5) and continues until E18.5 in BDNF-null mice. Neuronal loss in BDNF-null mice was prevented by the removal of the pro-apoptotic gene Bax. Thus, BDNF regulates embryonic geniculate neuronal number by preventing cell death rather than promoting cell proliferation. The number of neurofilament positive neurons expressing activated caspase-3 increased on E13.5 in bdnf(-/-) mice, compared to wild-type mice, demonstrating that differentiated neurons were dying. The axons of geniculate neurons approach their target cells, the fungiform papillae, beginning on E13.5, at which time we found robust BDNF(LacZ) expression in these targets. Altogether, our findings establish that BDNF produced in peripheral target cells regulates the survival of early geniculate neurons by inhibiting cell death of differentiated neurons on E13.5 of development. Thus, BDNF acts as a classic target-derived growth factor in the developing taste system.

Fate mapping of mammalian embryonic taste bud progenitors.

Mammalian taste buds have properties of both epithelial and neuronal cells, and are thus developmentally intriguing. Taste buds differentiate at birth within epithelial appendages, termed taste papillae, which arise at mid-gestation as epithelial thickenings or placodes. However, the embryonic relationship between placodes, papillae and adult taste buds has not been defined. Here, using an inducible Cre-lox fate mapping approach with the ShhcreER(T2) mouse line, we demonstrate that Shh-expressing embryonic taste placodes are taste bud progenitors, which give rise to at least two different adult taste cell types, but do not contribute to taste papillae. Strikingly, placodally descendant taste cells disappear early in adult life. As placodally derived taste cells are lost, we used Wnt1Cre mice to show that the neural crest does not supply cells to taste buds, either embryonically or postnatally, thus ruling out a mesenchymal contribution to taste buds. Finally, using Bdnf null mice, which lose neurons that innervate taste buds, we demonstrate that Shh-expressing taste bud progenitors are specified and produce differentiated taste cells normally, in the absence of gustatory nerve contact. This resolution of a direct relationship between embryonic taste placodes with adult taste buds, which is independent of mesenchymal contribution and nerve contact, allows us to better define the early development of this important sensory system. These studies further suggest that mammalian taste bud development is very distinct from that of other epithelial appendages.

Epithelial-derived brain-derived neurotrophic factor is required for gustatory neuron targeting during a critical developmental period.

Brain-derived neurotrophic factor (BDNF) is expressed in epithelial targets of gustatory neurons (i.e., fungiform papillae) before their innervation, and BDNF overexpression in nontaste regions of the tongue misdirects gustatory axons to these sites, suggesting that BDNF is necessary for gustatory axons to locate and innervate their correct targets during development. To test this hypothesis, we examined the targeting of taste neurons in BDNF-null mice (bdnf(-/-)). Analysis of bdnf(-/-) mice using a combination of DiI labeling and electron microscopy revealed that taste regions were not innervated by gustatory axons. Instead, branching was increased and many nontaste regions were innervated. The increased branching by gustatory axons in these animals was facilitated by neurotrophin 4 (NT4), because branching was virtually eliminated in bdnf(-/-)/nt4(-/-) mice. No abnormalities in gustatory innervation patterns and targeting were observed in nt4(-/-) mice. Conditional removal of BDNF selectively in epithelial cells disrupted targeting at the tongue tip, where gene recombination removed bdnf by embryonic day 13.5 (E13.5). However, innervation patterns were normal in the midregion and caudal portions of the tongue, where gene recombination did not occur until E14.5. These findings demonstrate that BDNF derived from gustatory epithelia is required for gustatory axons to correctly locate and innervate fungiform papillae. In addition, they show that BDNF-mediated targeting is restricted to a critical period of development, on or before E13.5.

Exuberant neuronal convergence onto reduced taste bud targets with preservation of neural specificity in mice overexpressing neurotrophin in the tongue epithelium.

A mouse fungiform taste bud is innervated by only four to five geniculate ganglion neurons; their peripheral fibers do not branch to other buds. We examined whether the degree or specificity of this exclusive innervation pattern is influenced by brain-derived neurotrophic factor (BDNF), a prominent lingual neurotrophin implicated in taste receptoneural development. Labeled ganglion cells were counted after injecting single buds with different color markers in BDNF-lingual-overexpressing (OE) mice. To evaluate the end-organs, taste buds and a class of putative taste receptor cells were counted from progeny of BDNF-OE mice crossbred with green fluorescent protein (GFP) (gustducin) transgenic mice. Fungiform bud numbers in BDNF-OE mice are 35%, yet geniculate neuron numbers are 195%, of wild-type mice. Neurons labeled by single-bud injections in BDNF-OE animals were increased fourfold versus controls. Injecting three buds, each with different color markers, resulted in predominantly single-labeled ganglion cells, a discrete innervation pattern similar to controls. Thus, hyper-innervation of BDNF-OE buds involves many neurons innervating single buds, not increased fiber branching. Therefore, both wild-type and BDNF-OE mice exhibit, in fungiform buds, the same, "discrete" receptoneural pattern, this despite dramatic neurotrophin overexpression-related decreases in bud numbers and increases in innervation density. Hyperinnervation did not affect GFP positive cell numbers; proportions of GFP cells in BDNF-OE buds were the same as in wild-type mice. Total numbers of ganglion cells innervating buds in transgenic mice are similar to controls; the density of taste input to the brain appears maintained despite dramatically reduced receptor organs and increased ganglion cells.

Factors that regulate embryonic gustatory development.

Numerous molecular factors orchestrate the development of the peripheral taste system. The unique anatomy/function of the taste system makes this system ideal for understanding the mechanisms by which these factors function; yet the taste system is underutilized for this role. This review focuses on some of the many factors that are known to regulate gustatory development, and discusses a few topics where more work is needed. Some attention is given to factors that regulate epibranchial placode formation, since gustatory neurons are thought to be primarily derived from this region. Epibranchial placodes appear to arise from a pan-placodal region and a number of regulatory factors control the differentiation of individual placodes. Gustatory neuron differentiation is regulated by a series of transcription factors and perhaps bone morphongenic proteins (BMP). As neurons differentiate, they also proliferate such that their numbers exceed those in the adult, and this is followed by developmental death. Some of these cell-cycling events are regulated by neurotrophins. After gustatory neurons become post-mitotic, axon outgrowth occurs. Axons are guided by multiple chemoattractive and chemorepulsive factors, including semaphorins, to the tongue epithelium. Brain derived neurotrophic factor (BDNF), functions as a targeting factor in the final stages of axon guidance and is required for gustatory axons to find and innervate taste epithelium. Numerous factors are involved in the development of gustatory papillae including Sox-2, Sonic hedge hog and Wnt-beta-catenin signaling. It is likely that just as many factors regulate taste bud differentiation; however, these factors have not yet been identified. Studies examining the molecular factors that regulate terminal field formation in the nucleus of the solitary tract are also lacking. However, it is possible that some of the factors that regulate geniculate ganglion development, outgrowth, guidance and targeting of peripheral axons may have the same functions in the gustatory CNS.