Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata)

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.     

Hedgehog activity controls opening of the primary mouth

To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes.     

The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides

Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) are members of the Hedgehog (Hh) family of signaling molecules known to be involved in embryonic patterning and morphogenesis. The Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, we detect signaling peptides in producing cells, but we also find that ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, we show that induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.     

Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila

Hedgehog (Hh) signaling is important for development and homeostasis in vertebrates and invertebrates. Ligand-independent, deregulated Hh signaling caused by loss of negative regulators such as Patched causes excessive cell proliferation, leading to overgrowth in Drosophila and tumors in humans, including basal-cell carcinoma and medulloblastoma. We show that in Drosophila deregulated Hh signaling also promotes cell survival by increasing the resistance to apoptosis. Surprisingly, cells with deregulated Hh activity do not protect themselves from apoptosis; instead, they promote cell survival of neighboring wild-type cells. This non-cell autonomous effect is mediated by Hh-induced Notch signaling, which elevates the protein levels of Drosophila inhibitor of apoptosis protein-1 (Diap-1), conferring resistance to apoptosis. In summary, we demonstrate that deregulated Hh signaling not only promotes proliferation but also cell survival of neighboring cells. This non-cell autonomous control of apoptosis highlights an underappreciated function of deregulated Hh signaling, which may help to generate a supportive micro-environment for tumor development.     

Distinct Effects of the mesenchymal dysplasia Gene Variant of Murine Patched-1 Protein on Canonical and Non-canonical Hedgehog Signaling Pathways

Hedgehog (Hh) signaling requires regulation of the receptor Patched-1 (Ptch1), which, in turn, regulates Smoothened activity (canonical Hh signaling) as well as other non-canonical signaling pathways. The mutant Ptch1 allele mesenchymal dysplasia (mes), which truncates the Ptch1 C terminus, produces a limited spectrum of developmental defects in mice as well as deregulation of canonical Hh signaling in some, but not all, affected tissues. Paradoxically, mes suppresses canonical Hh signaling and binds to Hh ligands with an affinity similar to wild-type mouse Ptch1 (mPtch1). We characterized the distinct activities of the mes variant of mPtch1 mediating Hh signaling through both canonical and non-canonical pathways. We demonstrated that mPtch1 bound c-src in an Hh-regulated manner. Stimulation with Sonic Hedgehog (Shh) of primary mammary mesenchymal cells from wild-type and mes animals activated Erk1/2. Although Shh activated c-src in wild-type cells, c-src was constitutively activated in mes mesenchymal cells. Transient assays showed that wild-type mPtch1, mes, or mPtch1 lacking the C terminus repressed Hh signaling in Ptch1-deficient mouse embryo fibroblasts and that repression was reversed by Shh, revealing that the C terminus was dispensable for mPtch1-dependent regulation of canonical Hh signaling. In contrast to these transient assays, constitutively high levels of mGli1 but not mPtch1 were present in primary mammary mesenchymal cells from mes mice, whereas the expression of mPtch1 was similarly induced in both mes and wild-type cells. These data define a novel signal transduction pathway involving c-src that is activated by the Hh ligands and reveals the requirement for the C terminus of Ptch in regulation of canonical and non-canonical Hh signaling pathways.     

Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)^sq33-2 and Et(krt4:EGFP)^sqet3 that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.     

Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.     

Retinal ganglion cell-derived sonic hedgehog signaling is required for optic disc and stalk neuroepithelial cell development

The development of optic stalk neuroepithelial cells depends on Hedgehog (Hh) signaling, yet the source(s) of Hh protein in the optic stalk is unknown. We provide genetic evidence that sonic hedgehog (Shh) from retinal ganglion cells (RGCs) promotes the development of optic disc and stalk neuroepithelial cells. We demonstrate that RGCs express Shh soon after differentiation, and cells at the optic disc in close proximity to the Shh-expressing RGCs upregulate Hh target genes, which suggests they are responding to RGC-derived Shh signaling. Conditional ablation of Shh in RGCs caused a complete loss of optic disc astrocyte precursor cells, resulting in defective axon guidance in the retina, as well as conversion of the neuroepithelial cells in the optic stalk to pigmented cells. We further show that Shh signaling modulates the size of the Pax2(+) astrocyte precursor cell population at the optic disc in vitro. Together, these data provide a novel insight into the source of Hh that promotes neuroepithelial cell development in the mammalian optic disc and stalk.