Alk8 is required for neural crest cell formation and development of pharyngeal arch cartilages.

The type I TGFbeta family member receptor alk8 acts in bone morphogenetic protein (BMP) signaling pathways to establish dorsoventral patterning in the early zebrafish embryo. Here, we present evidence that alk8 is required for neural crest cell (NCC) formation and that alk8 signaling gradients direct the proper patterning of premigratory NCCs. We extend our previous functional studies of alk8 to demonstrate that ectopic expression of constitutively active and dominant negative Alk8, consistently results in more medially or laterally positioned premigratory NCCs, respectively. We also demonstrate that patterning defects in premigratory NCCs, induced by alk8 misexpression, correlate with subsequent defects in NCC-derived pharyngeal arch cartilages. Furthermore, an anteroposterior effect is revealed, where overexpression of Alk8 more severely affects anterior arch cartilages and decreased Alk8 activity more severely affects posterior arch cartilage formation. Ectopic expression studies of alk8 are supported by analyses of zygotic and maternal-zygotic laf/alk8 mutants and of several BMP pathway mutants. Pharyngeal mesodermal and endodermal defects in laf/alk8 mutants suggest additional roles for alk8 in patterning of these tissues. Our results provide insight into alk8-mediated BMP signaling gradients and the establishment of premigratory NCC mediolateral positioning, and extend the model for BMP patterning of the neural crest to include that of NCC-derived pharyngeal arch cartilages.     

A direct requirement for Hedgehog signaling for normal specification of all ventral progenitor domains in the presumptive mammalian spinal cord

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, we examined the behavior of Smo null mutant neural progenitor cells in the developing vertebrate spinal cord, and we show that direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), we observe the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities.     

Zebrafish ift57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect hedgehog signaling

Cilia formation requires intraflagellar transport (IFT) proteins. Recent studies indicate that mammalian Hedgehog (Hh) signaling requires cilia. It is unclear, however, if the requirement for cilia and IFT proteins in Hh signaling represents a general rule for all vertebrates. Here we examine zebrafish ift57, ift88, and ift172 mutants and morphants for defects in Hh signaling. Although ift57 and ift88 mutants and morphants contained residual maternal protein, the cilia were disrupted. In contrast to previous genetic studies in mouse, mutations in zebrafish IFT genes did not affect the expression of Hh target genes in the neural tube and forebrain and had no quantitative effect on Hh target gene expression. Zebrafish IFT mutants also exhibited no dramatic changes in the craniofacial skeleton, somite formation, or motor neuron patterning. Thus, our data indicate the requirement for cilia in the Hh signal transduction pathway may not represent a universal mechanism in vertebrates. Developmental Dynamics 238:1744–1759, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia

The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.     

Developmentally regulated expression of Shh and Ihh in the developing mouse cranial base: comparison with Sox9 expression

The cranial base, located between the cranial vault and the facial bones, plays an important role in integrated craniofacial development and growth. Transgenic Shh and Sox9-deficient mice show similar defects in cranial base patterning. Therefore, in order to examine potential interactions of Shh, Ihh, another member of the Hh family, and Sox9 during cranial base development and growth, we investigated their cellular mRNA expression domains in the embryonic (E) and early postnatal (PN) cranial base from E10 to PN5 using sectional radioactive 35-S in situ hybridization. Of the Hhs, Shh was observed in the foregut epithelium and the notochord, while Sox9 showed broad expression in the loose mesenchyme of the cranial base area during E10-E11. Subsequently, from E12 onward, all genes were observed in the developing cranial base, and after birth the genes were prominently colocalized in the prehypertrophic chondrocytes of the synchondroses. Collectively, these data suggest that Hh-Sox9 auto- and paracrine signaling interactions may provide a critical mechanism for regulating the patterning of the cranial base as well as for its development and growth.     

Hedgehog serves as a mitogen and survival factor during embryonic stem cell neurogenesis

Hedgehog (Hh) signaling is involved in a wide range of important biological activities. Within the vertebrate central nervous system, Sonic Hedgehog (Shh) can act as a morphogen or mitogen that regulates the patterning, proliferation, and survival of neural stem cells (NSCs). However, its role in embryonic stem cell (ESC) neurogenesis has not been explored in detail. We have previously shown that Hh signaling is required for ESC neurogenesis. In order to elucidate the underlying mechanism, we utilized the Sox1-GFP ESC line, which has a green fluorescent protein (GFP) reporter under the control of the Sox1 gene promoter, providing an easy means of detecting NSCs in live cell culture. We show here that ESC differentiation in adherent culture follows the ESC--> primitive ectoderm --> neurectoderm transitions observed in vivo. Selective death of the Sox1-GFP-negative cells contributes to the enrichment of Sox1-GFP-positive NSCs. Interestingly, Shh is expressed exclusively by the NSCs themselves and elicits distinct downstream gene expression in Sox1-GFP-positive and -negative cells. Suppression of Hh signaling by antagonist treatment leads to different responses from these two populations as well: increased apoptosis in Sox1-GFP-positive NSCs and decreased proliferation in Sox1-GFP-negative primitive ectoderm cells. Hedgehog agonist treatment, in contrast, inhibits apoptosis and promotes proliferation of Sox1-GFP-positive NSCs. These results suggest that Hh acts as a mitogen and survival factor during early ESC neurogenesis, and evidence is presented to support a novel autocrine mechanism for Hh-mediated effects on NSC survival and proliferation.     

Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein,Hip1, modulates Fgf signaling during branching morphogenesis of the lung

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generated Hip1 mutants in the mouse. Loss of Hip1 function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated in Hip1 mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching.     

Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition

We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity.     

Hedgehog signaling regulates size of the dorsal aortae and density of the plexus during avian vascular development

Signaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved. Developmental Dynamics 240:1354–1364, 2011. © 2011 Wiley‐Liss, Inc.     

The seven-transmembrane receptor smoothened cell-autonomously induces multiple ventral cell types

Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the developing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2) mimics concentration-dependent actions of Shh in the developing neural tube, including activation of ventral marker genes (HNF3beta, patched, Nkx2.2, netrin-1), suppression of dorsal markers (Pax-3, Gli-3, Ephrin A5) and induction of ventral neurons (dopaminergic, serotonergic) and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+, CHX10+). Furthermore, Smo-M2's patterning activities were cell autonomous, occurring exclusively in cells expressing Smo-M2. These findings suggest that Smo is a key signaling component in the Hh receptor and that Shh patterns the vertebrate nervous system as a morphogen, rather than through secondary relay signals.     

Hedgehog signaling regulates proliferation of prostate cancer cells via stathmin1

Hedgehog (Hh) signaling is an essential pathway in embryonic development of prostate. Hh also plays roles in the proliferation of progenitor cells and cancer cells of adult prostate. However, how Hh signaling contributes to carcinogenesis of prostate is poorly understood. Stathmin1 is a microtubule-regulating protein that plays an important role in the assembly and disassembly of the mitotic spindle. Stathmin1 is expressed in normal developing mouse prostate and in prostate cancer. The expression pattern of stathmin1 is similar to that of Shh in prostate development and cancer, suggesting a connection between these two proteins. In this study, we examined the relationship between stathmin1 and Hh signaling. Here, we show that stathmin1 expression is regulated by Hh signaling in prostate cancer cells. Cyclopamine, a specific inhibitor of Hh signaling, reduced the expression of stathmin1 in prostate cancer cells. However, the Shh peptide induced stathmin1 expression. Overexpression of Gli1 further confirmed the relationship. Co-expression of stathmin1 and Patched 1, a receptor for Hh signaling was observed in prostate cancer tissues. Cyclopamine and stathmin1 siRNA both decreased proliferation of prostate cancer cells but did not produce an additive effect, suggesting a common pathway. These results suggest that Hh signaling regulates proliferation of prostate cancer cells by controlling stathmin1 expression.     

Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles

We investigated the roles of bare morphogenetic protein (BMP), sonic hedgehog (SHH) and fibroblast growth factor (FGF)-expressing signaling centers in regulating the patterned outgrowth of the telencephalic and optic vesicles. Implantation of BMP4 beads in the anterior neuropore of stage 10 chicken embryos repressed FGF8 and SHH expression. Similarly, loss of SHH expression in Shh mutant mice leads to increased BMP signaling and loss of Fgf8 expression in the prosencephalon. Increased BMP signaling and loss of FGF and SHH expression was correlated with decreased proliferation, increased cell death, and hypoplasia of the telencephalic and optic vesicles. However, decreased BMP signaling, through ectopic expression of Noggin, a BMP-binding protein, also caused decreased proliferation and hypoplasia of the telencephalic and optic vesicles, but with maintenance of Fgf8 and Shh expression, and no detectable increase in cell death. These results suggest that optimal growth requires a balance of BMP, FGF8 and SHH signaling. We suggest that the juxtaposition of Fgf8, Bmp4 and Shh expression domains generate patterning centers that coordinate the growth of the telencephalic and optic vesicles, similar to how Fgf8, Bmp4 and Shh regulate growth of the limb bud. Furthermore, these patterning centers regulate regional specification within the forebrain and eye, as exemplified by the regulation of Emx2 expression by different levels of BMP signaling.In summary, we present evidence that there is cross-regulation between BMP-, FGF- and SHH-expressing signaling centers in the prosencephalon which regulate morphogenesis of, and regional specification within, the telencephalic and optic vesicles.     

Zebrafish acvr2a and acvr2b exhibit distinct roles in craniofacial development.

To examine the roles of activin type II receptor signaling in craniofacial development, full-length zebrafish acvr2a and acvr2b clones were isolated. Although ubiquitously expressed as maternal mRNAs and in early embryogenesis, by 24 hr postfertilization (hpf), acvr2a and acvr2b exhibit restricted expression in neural, hindbrain, and neural crest cells (NCCs). A morpholino-based targeted protein depletion approach was used to reveal discrete functions for each acvr2 gene product. The acvr2a morphants exhibited defects in the development of most cranial NCC-derived cartilage, bone, and pharyngeal tooth structures, whereas acvr2b morphant defects were largely restricted to posterior arch structures and included the absence and/or aberrant migration of posterior NCC streams, defects in NCC-derived posterior arch cartilages, and dysmorphic pharyngeal tooth development. These studies revealed previously uncharacterized roles for acvr2a and acvr2b in hindbrain and NCC patterning, in NCC derived pharyngeal arch cartilage and joint formation, and in tooth development.     

The role of morphogens in T-cell development

The Hedgehog (Hh) and Wnt family proteins, and the bone morphogenetic proteins (BMPs) 2 and 4, act as morphogens during vertebrate embryogenesis and organogenesis by regulating patterning and cell fate. They have recently been found to have a role in regulating cell fate and determination in self-renewing tissues in adults, such as the immune system and haematopoietic system. This Review presents studies on the role of Sonic Hh (Shh), Wnts and BMP2/4 in the regulation of thymocyte development. Shh and BMP2/4 act as negative regulators of thymocyte development. By contrast, Wnt signalling, through beta-catenin, has a positive role in the control of T-cell development, such that an absence or reduction in the Wnt signal leads to a reduction in cell number and cell proliferation rate and differentiation to the CD4+CD8+ double-positive stage.     

Tissue- and stage-specific modulation of Wingless signaling by the segment polarity gene lines

Wnt signaling controls a variety of developmental programs but the mechanisms by which the same signal leads to distinct outputs remain unclear. To address this question, we identified stage-specific modulators of Wingless (Wg) signaling in the Drosophila embryonic epidermis. We show that lines (lin) is essential for Wg-dependent patterning in dorsal epidermis. lin encodes a novel protein that acts cell-autonomously, downstream or in parallel to Armadillo (Arm) and upstream of Wg-dependent target genes. Lin can accumulate in nuclei of cells signaled by Wg, suggesting that signaling promotes entry of Lin into the nucleus, where it cooperates with Arm and Pangolin. Thus, a stage-specific modulator is used to mediate Wg signaling activity in dorsal patterning. Hedgehog (Hh) controls half of the parasegmental pattern dorsally and antagonizes Wg function to do so. Lin can accumulate in the cytoplasm of cells signaled by Hh, suggesting that Hh antagonizes Wg function by prohibiting Lin from entering the nucleus.