Co-localization of patched and activated sonic hedgehog to lysosomes in neurons

We demonstrate co-localization of the Patched 1 (Ptc1) receptor and its ligand sonic hedgehog (Shh) in lysosomes suggests an intracellular sorting mechanism for this receptor and its ligand. Treatment of murine brain primary cultures and a human teratoma cell line with the N-terminal activated form of Shh (ShhNT), a Ptc1-Shh complex was observed in lysosomes. Consistent with this interaction, Western immunoblot analysis revealed intracellular localization of native Ptc1 and ShhNT. Examination of the topological model of the Ptc1 receptor revealed a number of Yxxphi lysosomal targeting sequences consistent with our observations for Ptc1 sorting.     

Erb and c-Kit receptors have distinctive patterns of expression in adult and developing taste papillae and taste buds.

Twenty four different protein tyrosine kinases (PTKs) were amplified from a taste-enriched cDNA library using PCR. The expression of four protein tyrosine kinase receptors (EGFR, ErbB2, ErbB3, and c-kit) was examined in adult and developing rat taste papillae. All four of these receptors were expressed in overlapping populations of differentiated taste cells within adult taste buds. Taste bud basal cells were ErbB2(+) but did not express the other Erb receptors. During prenatal development, the Erb receptors were expressed extensively in the basal cells around developing papillae, and ErbB2 and c-kit immunoreactive neuronal fibers were seen in close association with taste papillae. In early postnatal stages, ErbB2(+) and c-kit(+) neuronal fibers were often seen entering the taste papillae epithelium, where new taste buds form, and by postnatal day 2 (P2), individual ErbB2(+) and c-kit(+) cells were seen in this region as well. Between P3 and P8, c-kit was highly expressed at the bottom of foliate papillae trenches. The extensive expression of the Erb and c-kit receptors in adult taste buds and in and around developing papillae suggests that these receptors may play a role in the prenatal and postnatal development of gustatory papillae and taste buds.     

Brain-derived neurotrophic factor mRNA is expressed in the developing taste bud-bearing tongue papillae of rat

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are expressed in many areas of the nervous system and its target tissues. Using in situ hybridization we have investigated the possible presence of NGF mRNA and BDNF mRNA in the developing fungiform and circumvallate papillae of the rat tongue. BDNF mRNA is present in the epithelium of the developing fungiform papillae in E15, E16, and E17 rat embryos with peak concentration at E16. It starts to diminish after E17 and is almost absent at E21. There is a specific temporospatial change in the expression of BDNF mRNA in developing circumvallate papillae. It is expressed in the epithelium of the superior and posterior surfaces of the papillae at E15, E16, and E17. Already at E17 the BDNF mRNA labeling has started to decrease in the superior epithelium. At E19 and E21, BDNF mRNA is exclusively present in the epithelium of the inner and outer walls of the trench, surrounding the papilla at the posterior and lateral surfaces where the taste buds are located later in life. BDNF mRNA was also detected in the developing palatal taste buds. NGF mRNA was below detection level in the developing papillae. The highly localized expression of BDNF mRNA in areas where taste buds are to be formed suggests that BDNF may be one crucial factor in the formation of the epithelial innervation prior to taste bud formation. It might also participate in the formation and/or maintenance of the papillary and/or taste bud innervation apparatus. We conclude that the neurotrophin BDNF is expressed in early development of taste bud-bearing papillae in the rat tongue in a temporally and spatially controlled manner, presumably to act as a target-derived chemoat tractant for the early nerve fibers. © 1995 Wiley-Liss, Inc.     

Timing of Sonic hedgehog and Gli1 expression segregates midbrain dopamine neurons

The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using genetic inducible fate mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages' contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the embryonic day 12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains.     

Expression of GDNF and GFR alpha 1 in mouse taste bud cells

GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.     

Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs.

Sensory ganglia that innervate taste buds and gustatory papillae (geniculate and petrosal) are reduced in volume by about 40% in mice with a targeted deletion of the gene for brain-derived neurotrophic factor (BDNF). In contrast, the trigeminal ganglion, which innervates papillae but not taste buds on the anterior tongue, is reduced by only about 18%. These specific alterations in ganglia that innervate taste organs make possible a test for roles of lingual innervation in the development of appropriate number, morphology, and spatial pattern of fungiform and circumvallate papillae and associated taste buds. We studied tongues of BDNF null mutant and wild-type littermates and made quantitative analyses of all fungiform papillae on the anterior tongue, the single circumvallate papilla on the posterior tongue, and all taste buds in both papilla types. Fungiform papillae and taste buds were reduced in number by about 60% and were substantially smaller in diameter in mutant mice 15-25 days postnatal. Remaining fungiform papillae were selectively concentrated in the tongue tip region. The circumvallate papilla was reduced in diameter and length by about 40%, and papilla morphology was disrupted. Taste bud number in the circumvallate was reduced by about 70% in mutant tongues, and the remaining taste buds were smaller than those on wild-type tongues. Our results demonstrate a selective dependence of taste organs on a full complement of appropriate innervation for normal growth and morphogenesis. Effects on papillae are not random but are more pronounced in specific lingual regions. Although the geniculate and petrosal ganglia sustain at least half of their normal complement of cell number in BDNF -/- mice, remaining ganglion cells do not substitute for lost neurons to rescue taste organs at control numbers. Whereas gustatory ganglia and the taste papillae initially form independently, our results suggest interdependence in later development because ganglia derive BDNF support from target organs and papillae require sensory innervation for morphogenesis.     

Development of fungiform papillae,taste buds,and their innervation in the hamster

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5–11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These “eminences” selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13–14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14–15 (birth = E15–16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth. © Wiley-Liss, Inc.     

Persistence of taste buds in denervated fungiform papillae

Taste buds in hamster fungiform papillae persist in an atrophic state for as long as 330 days after chorda tympani denervation or 50 days after combined chorda tympani-lingual nerve resection. Although taste bud structure depends on innervation, there is no absolute neural requirement for taste bud survival.     

The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord

Netrin-1 is an attractive axon guidance molecule required for the proper navigation of commissural axons to the floor plate in the spinal cord. A new study now shows that sonic hedgehog (Shh) from midline structures collaborates with netrin-1 to guide commissural axons. This new role for the morphogen Shh raises the possibility that principles similar to those used to establish positional information in embryonic patterning are also employed during axon navigation.     

TRPM8 protein localization in trigeminal ganglion and taste papillae

TRPM8 is a TRP family cation channel which can be activated by cold stimuli or l-menthol. However, TRPM8 protein localization of nerve terminals in sensory organs remains unknown. Here we generated an antibody against TRPM8 and analyzed TRPM8 protein localization in trigeminal ganglia (TG) and in sensory nerve fibers in the tongue. TRPM8 immunoreactivity was detected in a subset of neurons with a small diameter in TG and in nerve fibers in the tongue. TRPM8-immunoreactive nerve fibers were rich in fungiform papillae, but sparse in foliate and circumvallate papillae. The TRPM8-immunoreactive nerve fibers reached the outer epithelial layer in each papilla, while no TRPM8-immunoreactive nerve fibers penetrated into taste buds. Double labeling analysis revealed that TRPM8 immunoreactivity was co-expressed with a part of TRPV1 or CGRP-immunoreactive neurons in TG. However, TRPM8 immunoreactivity was not observed in TRPV1- or CGRP-positive nerve fibers in fungiform, foliate, and circumvallate papillae. These results suggest that TRPM8 protein is present in sensory lingual nerve fibers mainly projected from TG and might work as cold and l-menthol receptors on tongue.     

Expression of connexin 31 in the developing mouse cochlea

Connexin 31 (Cx31) mutations cause an autosomal dominant form of high-frequency hearing loss. The immunohistochemical localization of Cx31 in mouse cochlea was studied at different ages between 0 and 60 days after birth (DAB). Cx31-like immunoreactivity was detected in fibrocytes of spiral ligament and spiral limbus at 12 DAB, gradually enhanced with the increase of age and reached the adult pattern on 60 DAB. Immunoreactivity decreased gradually from the basal to apical turn in all developmental stages. The mRNA of Cx31 was also identified by RT-PCR. The distribution of Cx31 and connexin 26 were obviously different in the developing mouse cochlea. The expression and distribution of Cx31 in the development may explain the progressive hearing loss in human Cx31 mutations.     

Caffeine induces sonic hedgehog gene expression in cultured astrocytes and neurons

Caffeine affects early in vivo murine brain development by accelerating the evagination of the primitive neuroepithelium into telencephalic vesicles. In this model, caffeine induces the expression of the regulatory subunit α of protein kinase A (PKA RI α) and of Sonic hedgehog (Shh). The understanding of the molecular mechanisms linking caffeine and neural gene expression would benefit from a reproducible in vitro model. Accordingly, the present study aimed to determine whether caffeine modulated the expression of these genes in primary neuronal and astroglial cultures derived from developing murine neocortex. Using real-time PCR, the results showed that caffeine induced robust overexpression of Shh mRNA in both cell types without significantly modifying PKA RI α gene expression.     

Expression of connexin 30 in the developing mouse cochlea

Mutations in the GJB6 gene encoding connexin 30 (Cx30) can cause dominant forms of nonsyndromic deafness. By studying immunohistochemical localization of Cx30 in the mouse cochlea at different ages from 0 to 30 days after birth, we found that the expression of Cx30 is nearly the same as that of Cx26. These findings suggest that as well as Cx26, Cx30 may also contribute to the generation and maturation of endocochlear potential.     

Reduced number of taste papillae in patients with eating disorders

Taste affects dietary behavior and in turn taste response and food preferences are altered in eating disorders. Fungiform papillae on the tongue are the first line of the gustatory apparatus to provide information about taste. Aim of this study is determination of their number in patients with eating disorders. Twenty-seven female adolescents with eating disorders and 16 age-matched healthy female controls were examined. Tongues were stained with blue food coloring and the number of fungiform papillae was quantified using digital photography and image processing. Patients with restrictive type eating disorders showed a more distinct reduction (p < 0.001) of fungiform papillae than patients with vomiting and/or binge eating (p < 0.05), compared with those of healthy control subjects. Causes may be an initially disturbed development of fungiform papillae or secondary to changes in eating behavior which may be mutually causative.