Table of contents

Sonic hedgehog and endoderm

The generation of the pancreas and small intestine from the embryonic gut depends on intercellular signalling between the endodermal and mesodermal cells of the gut. In particular, the differentiation of intestinal mesoderm into smooth muscle has been suggested to depend on signals from adjacent endodermal cells. One candidate mediator of endodermally derived signals in the embryonic hindgut is the secreted protein Sonic hedgehog (Shh). The Shh gene is expressed throughout the embryonic gut endoderm with the exception of the pancreatic bud endoderm, which instead expresses high levels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor 1/pancreatic and duodenal homeobox 1), an essential regulator of early pancreatic development. Does the differential expression of Shh in the embryonic gut tube control the differentiation of the surrounding mesoderm into specialised mesoderm derivatives of the small intestine and pancreas? To answer this question, the promoter of the Ipf1/Pdx1 gene was used to selectively express Shh in the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenic mice, the pancreatic mesoderm develops into smooth muscle and interstitial cells of Cajal, characteristic of the intestine, rather than into pancreatic mesenchyme and spleen. Also, pancreatic explants exposed to Shh undergo a similar program of intestinal differentiation. These results provide evidence that the differential expression of endodermally derived Shh controls the fate of adjacent mesoderm at different regions of the gut tube (Apelqvist, 1997).

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm; evidence is provided that the notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. Activin-B and FGF2 are identified as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes, including Pdx1 and insulin. Endoderm treatment with antibodies that block SHH activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting the early steps of chick pancreatic development (Hebrok, 1998).

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGFbeta signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh-/- and Shh-/-;Ihh+/- mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance (Hebrok, 2000).

The expression of Indian hedgehog mRNA and protein is upregulated dramatically as F9 teratocarcinoma cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies containing an outer visceral endoderm layer. The embryonic stem cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid; it also upregulates Indian hedgehog expression. Whereas little or no Indian hedgehog message is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells. In situ hybridization analysis for Indian hedgehog mRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mount in situ hybridization analysis of Days 7.5 and 8.5 embryos indicates that Indian hedgehog (Ihh) expression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full length Indian hedgehog cDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest that Indian hedgehog is involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis. An important area of future investigation is to determine if Ihh promotes the emergence of the primitive endoderm precursor cell and/or facilitates the terminal differentiation of both parietal and visceral endoderm cells (Becker, 1997).

The visceral yolk sac plays a critical role in normal embryogenesis, yet little is known about the specific molecules that regulate its development. Four winged-helix genes (HNF-3alpha, HNF-3beta, HNF-3gamma and HFH-4) are restricted to visceral endoderm. In the absence of HNF-3beta, visceral endoderm forms but the morphogenetic movements by which the embryo becomes enclosed within its yolk sac are disrupted and serum protein gene transcription is greatly reduced. Hedgehog and Bmp genes, which encode signaling molecules known to play multiple roles in embryonic development, are also differentially expressed in the closely apposed yolk sac mesoderm and endoderm layers. It is thought that Indian hedgehog signals from the visceral mesoderm to establish BMP2, BMP4 and BMP6 in the yolk sac mesoderm. All three BMPs may amplify their own transcription by an autoregulatory mechanism and participate in the differentiation of mesodermal cells. In an autocrine role, Indian hedgehog also signals to establish BMP6 in visceral mesoderm. Desert hedgehog may signal back from yolk sac mesoderm to induce BMP6 in visceral endoderm. These results suggest that similar mechanisms may be utilized to mediate inductive interactions in both extraembryonic and embryonic tissues (Farrington, 1997).

The mammalian lung, like many other organs, develops by branching morphogenesis of an epithelium. Development initiates with evagination of two ventral buds of foregut endoderm into the underlying splanchnic mesoderm. As the buds extend, they send out lateral branches at precise, invariant positions, establishing the primary airways and the lobes of each lung. Dichotomous branching leads to further extension of the airways. Grafting studies have demonstrated the importance of bronchial mesenchyme in inducing epithelial branching, but the significance of epithelial signaling has largely gone unstudied. The morphogen Sonic hedgehog (Shh) is widely expressed in the foregut endoderm and is specifically upregulated in the distal epithelium of the lung where branching is occurring. Ectopic expression of Shh disrupts branching and increases proliferation, suggesting that local Shh signaling regulates lung development. Shh is essential for development of the respiratory system. In Shh null mutants, the trachea and esophagus do not separate properly and the lungs form a rudimentary sac due to failure of branching and growth after formation of the primary lung buds. Interestingly, normal proximo-distal differentiation of the airway epithelium occurs, indicating that Shh is not needed for differentiation events. In addition, the transcription of several mesenchymally expressed downstream targets of Shh is abolished. These results highlight the importance of epithelially derived Shh in regulating branching morphogenesis of the lung (Pepicelli, 1998).

The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium, forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here is demonstrated the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non-smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm-derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons (Sukegawa, 2000).

The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. The function of Sonic hedgehog and Indian hedgehog genes have been assessed. Both are expressed in gut endoderm, whereas target genes are expressed in discrete layers in the mesenchyme. It has been unclear whether functional redundancy between the two genes would preclude a genetic analysis of the roles of Hedgehog signaling in the mouse gut. The mouse gut has both common and separate requirements for Sonic hedgehog and Indian hedgehog. Both Sonic hedgehog and Indian hedgehog mutant mice show reduced smooth muscle, gut malrotation and annular pancreas. Sonic hedgehog mutants display intestinal transformation of the stomach, duodenal stenosis (obstruction), abnormal innervation of the gut and imperforate anus. Indian hedgehog mutants show reduced epithelial stem cell proliferation and differentiation, together with features typical of HirschsprungÂ’s disease (aganglionic colon). These results show that Hedgehog signals are essential for organogenesis of the mammalian gastrointestinal tract and suggest that mutations in members of this signaling pathway may be involved in human gastrointestinal malformations (Ramalho-Santos, 2000).

Hedgehog ligands interact with receptor complexes containing Patched (PTC) and Smoothened (SMO) proteins to regulate many aspects of development. The mutation W535L (SmoM2) in human Smo is associated with basal cell skin cancers, causes constitutive, ligand-independent signaling through the Hedgehog pathway, and provides a powerful means to test effects of unregulated Hedgehog signaling. Expression of SmoM2 in Xenopus embryos leads to developmental anomalies that are consistent with known requirements for regulated Hedgehog signaling in the eye and pancreas. Additionally, it results in failure of midgut epithelial cytodifferentiation and of the intestine to lengthen and coil. The midgut mesenchyme shows increased cell numbers and attenuated expression of the differentiation marker smooth muscle actin. With the exception of the pancreas, differentiation of foregut and hindgut derivatives is unaffected. The intestinal epithelial abnormalities are reproduced in embryos or organ explants treated directly with active recombinant hedgehog protein. Ptc mRNA, a principal target of Hedgehog signaling, is maximally expressed at stages corresponding to the onset of the intestinal defects. In advanced embryos expressing SmoM2, Ptc expression is remarkably confined to the intestinal wall. Considered together, these findings suggest that the splanchnic mesoderm responds to endodermal Hedgehog signals by inhibiting the transition of midgut endoderm into intestinal epithelium and that attenuation of this feedback is required for normal development of the vertebrate intestine (Zhang, 2001).

These observations suggest that the response to Hh signaling in the developing gut is manifested in part in the same cells that produce Hh ligands, the gut endoderm. Early in gut development, it is inferred that the splanchnic mesoderm responds to Hh signals by actively inhibiting epithelial differentiation. Later, Hh signaling must be attenuated to allow proper midgut development, including elongation, coiling, radial intercalation of ventral endodermal cells, and ordered differentiation of both the epithelium and the surrounding mesenchyme. Constitutive signaling through SmoM2 is principally activated in the gut mesoderm, where morphologic correlates include patchy thickening and reduced expression of the differentiation marker smooth muscle actin. Although failure of the gut tube to elongate and coil is also likely related to mesenchymal defects, the major consequence is absence of midgut epithelial cytodifferentiation. Since effectors of Hh signaling localize to the gut mesoderm and not the endoderm, this is interpreted to represent a secondary effect reflecting a requirement for regulated attenuation of Hh activity in the differentiation of the intestinal epithelium. The decline in Hh mRNA levels after Stage 40 in Xenopus development is consistent with this model. However, the identity of the reverse signal is not known. BMP-4 is one important target of Hh signaling in the hindgut of chick embryos, and the Drosophila homolog decapentaplegic mediates signaling between splanchnic mesoderm and endoderm in flies. Factors of this class are hence candidate mediators of the isolated midgut phenotype observed in SmoM2- expressing Xenopus embryos. Treatment of Xenopus embryos with retinoic acid or injection of a truncated, dominant inhibitory FGF receptor results in intestinal developmental abnormalities partially resembling those seen with expression of SmoM2. In particular, FGF receptor signaling is required for synchronized differentiation of intestinal smooth muscle. Thus, a number of known signaling pathways appear to function in concert to effect intestinal organogenesis during vertebrate development (Zhang, 2001).

Recent studies have implicated the signaling factor Sonic hedgehog (Shh) as a negative regulator of pancreatic development, but as a positive regulator of pancreas function in amniotes. Here, using genetic analysis, it is shown that specification of the pancreas in the teleost embryo requires the activity of Hh proteins. Zebrafish embryos compromised in Hh signaling exhibit disruption in the expression of the pancreas-specifying homeobox gene pdx-1 and concomitantly show almost complete absence of the endocrine pancreas. Reciprocally, ubiquitous activation of the Hh pathway in wild-type embryos causes ectopic induction of endodermal pdx-1 expression and the differentiation of supernumerary endocrine cells. These results suggest that Hh proteins influence pancreas specification via inductive interactions from the axial midline rather than through their localized expression in the endodermal cells themselves (Roy, 2001b).

The genetic control of gut regionalization relies on a hierarchy of molecular events in which the Hox gene family of transcription factors is suspected to be key participant. The role of Hox genes in gut patterning has been examined using the Hoxa5-/- mice as a model. Hoxa5 is expressed in a dynamic fashion in the mesenchymal component of the developing gut. Its loss of function results in gastric enzymatic anomalies in Hoxa5-/- surviving mutants that are due to perturbed cell specification during stomach development. Histological, biochemical and molecular characterization of the mutant stomach phenotype may be compatible with a homeotic transformation of the gastric mucosa. As the loss of mesenchymal Hoxa5 function leads to gastric epithelial defects, Hoxa5 should exert its action by controlling molecules involved in mesenchymal-epithelial signaling. Indeed, in the absence of Hoxa5 function, there is alteration in the expression of genes encoding for signaling molecules such as Sonic hedgehog (Shh), Indian hedgehog (Ihh), transforming growth factor ß family members and fibroblast growth factor 10. These findings provide insight into the molecular controls of patterning events of the stomach, supporting the notion that Hoxa5 acts in regionalization and specification of the stomach by setting up the proper domains of expression of signaling molecules (Aubin, 2002).

Hoxa5 action in the establishment of Shh and Ihh gradients necessitates mesenchymally expressed intermediate(s). Bmps have been shown to be important regulators of glandular stomach development. Moreover in several species, a network exists between Hox, Bmp and Hh gut gene expression. For instance, ectopic Shh is able to induce Bmp4 expression in the chick hindgut and in the stomach. Although a complex situation prevails regarding the capacity of Shh to activate Bmp4 expression in foregut derivatives, it has been proposed that Hox genes influence the regionalized response to Shh. Even though the induction of Bmp4 by Shh in the stomach mesenchyme has not been directly addressed in the mouse, the change in the Bmp4 expression pattern observed in Hoxa5–/– stomachs is in agreement with this notion. It is also possible that Hoxa5 directly controls Bmp4 expression in the stomach. In the Drosophila midgut, the Ultrabithorax gene regulates at the transcriptional level the expression of the Bmp4 homolog decapentaplegic (Aubin, 2002 and references therein).

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. The morphogen Sonic hedgehog (Shh) is required for pancreatic development in zebrafish. Genetic mutants of Shh and its signaling pathway establish this dependence as specific to endocrine, but not exocrine, pancreas. Using cyclopamine to inhibit hedgehog signaling, it has been shown that transient Shh signaling is necessary during gastrulation for subsequent differentiation of endoderm into islet tissue. A second hedgehog-dependent activity occurring later in development was also identified and may be analogous to the known action of Shh in gut endoderm to direct localization of pancreatic development. The early action of Shh may be part of a more general process allowing neuroendocrine cells to originate in nonneuroectodermally derived tissues (diIorio, 2002).

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Evidence was found that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium, using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. Gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required (Kameda, 2003).

Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. The function of two hedgehog inhibitors, Hhip (a type I TM protein that attenuates hedgehog signalling by binding all three mammalian hedgehog proteins) and patched 1 (Ptch), during pancreas formation has been analyzed. Loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip–/–Ptch+/– embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages (Kawahira, 2003).

Sonic hedgehog (Shh) has been implicated as an important regulator of pharyngeal region development. Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh−/− mutants, the pharyngeal pouches and arches form by E9.5 and marker expression shows that initial patterning is normal. However, by E10.5-E11.0, the first arch has atrophied and the first pouch is missing. Although small, the second, third, and fourth arches and pouches are present. The expression patterns of Fgf8, Pax1, and Bmp4 suggest that pouch identity is abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers are expressed normally. These data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh−/− mutants suggest that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. These data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs (Moore-Scott, 2005).

Homeostasis of the vertebrate digestive tract requires interactions between an endodermal epithelium and mesenchymal cells derived from the splanchnic mesoderm. Signaling between these two tissue layers is also crucial for patterning and growth of the developing gut. From early developmental stages, sonic hedgehog (Shh) and indian hedgehog (Ihh) are secreted by the endoderm of the mammalian gut, indicative of a developmental role. Further, misregulated hedgehog (Hh) signaling is implicated in both congenital defects and cancers arising from the gastrointestinal tract. In the mouse, only limited gastrointestinal anomalies arise following removal of either Shh or Ihh. However, given the considerable overlap in their endodermal expression domains, a functional redundancy between these signals might mask a more extensive role for Hh signaling in development of the mammalian gut. To address this possibility, a conditional approach was adopted to remove both Shh and Ihh functions from early mouse gut endoderm. Analysis of compound mutants indicates that continuous Hh signaling is dispensable for regional patterning of the gut tube, but is essential for growth of the underlying mesenchyme. Additional in vitro analysis, together with genetic gain-of-function studies, further demonstrate that Hh proteins act as paracrine mitogens to promote the expansion of adjacent mesenchymal progenitors, including those of the smooth muscle compartment. Together, these studies provide new insights into tissue interactions underlying mammalian gastrointestinal organogenesis and disease (Mao, 2010).

Sonic Hedgehog and Ectodermal Patterning

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning, however, no molecular candidates have been identified. The feather tract is initiated by a continuous stripe of Shh, Fgf-4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, Shh and Fgf-4, which induce feather formation. Both Fgf-4 and Shh induce local expression of Bmp-4, while Bmp-4 suppresses local expression of both. Fgf-4 also induces Shh. Based on these findings, a model is proposed that involves (1) homogeneously distributed global activators that define the field; (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented (Jung, 1998).

The skin is responsible for forming a variety of epidermal structures that differ amongst vertebrates. In each case the specific structure (for example scale, feather or hair) arises from an epidermal placode as a result of epithelial-mesenchymal interactions with the underlying dermal mesenchyme. Expression of members of the Wnt, Hedgehog and bone morphogenetic protein families (Wnt10b, Sonic hedgehog [Shh] and Bmp2/Bmp4, respectively) in the epidermis correlates with the initiation of hair follicle formation. Further, their expression continues into either the epidermally derived hair matrix which forms the hair itself, or the dermal papilla, which is responsible for induction of the hair matrix. To address the role of Shh in the hair follicle, Shh null mutant mice were examined. Follicle development in the Shh mutant embryo is found to arrest after the initial epidermal-dermal interactions that lead to the formation of a dermal papilla anlage and ingrowth of the epidermis. Wnt10b, Bmp2 and Bmp4 continue to be expressed at this time, however. When grafted to nude mice (which lack T cells), Shh mutant skin give rise to large abnormal follicles each containing a small dermal papilla. Although these follicles show high rates of proliferation and some differentiation of hair matrix cells into hair-shaft-like material, no hair is formed. It is concluded that Shh signaling is not required for initiating hair follicle development. Shh signaling is essential, however, for controlling ingrowth and morphogenesis of the hair follicle (St-Jacques, 1998).

The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development is reported. Hair germs comprising epidermal placodes and associated dermal condensates are detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development is blocked in mutant skin. The expression of Gli1 and Ptc1 is reduced in Shh -/- dermal condensates and these condensates fail to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers are detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. These findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development (Chiang, 1999).

Spacing patterns are of fundamental importance in various repeated structures that develop at regular intervals such as feathers, teeth and insect ommatidia. The mouse tongue develops a regular papilla pattern and provides a good model to study pattern formation. The expression patterns of the signaling molecules, sonic hedgehog (Shh), bone morphogenetic proteins -2 and -4 (Bmp-2 and Bmp-4), and fibroblast growth factor-8 (Fgf-8) were studied in mouse embryos between E 10.5 and 15. All four genes are expressed uniformly in the tongue epithelium between E 10.5 and 11. At E 13, before morphologically detectable gustatory papillae initiation, Shh, Bmp-2 and Bmp-4 expression segregates into discrete spots, whereas, Fgf-8 is downregulated. At E 14, small eminences in the anterior part of the tongue are the first morphological indications of fungiform papillae, and they express Shh and Bmp-2, whereas, Bmp-4 is almost absent in the tongue. It is concluded that these conserved signaling molecules are associated with the initiation and early morphogenesis of the tongue papillae (Jung, 1999).

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal, sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog were overexpressed in the basal cells of mouse skin under the control of the human keratin 14 promoter. No overt epidermal morphogenesis phenotype was observed in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed among Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate (Adolphe, 2004).

Shh signaling induces proliferation of many cell types during development and disease, but how Gli transcription factors regulate these mitogenic responses remains unclear. By genetically altering levels of Gli activator and repressor functions in mice, it has been demonstrated that both Gli functions are involved in the transcriptional control of N-myc and Cyclin D2 during embryonic hair follicle development. The results also indicate that additional Gli-activator-dependent functions are required for robust mitogenic responses in regions of high Shh signaling. Through posttranscriptional mechanisms, including inhibition of GSK3-β activity, Shh signaling leads to spatially restricted accumulation of N-myc and coordinated cell cycle progression. Furthermore, a temporal shift in the regulation of GSK3-β activity occurs during embryonic hair follicle development, resulting in a synergy with β-catenin signaling to promote coordinated proliferation. These findings demonstrate that Shh signaling controls the rapid and patterned expansion of epithelial progenitors through convergent Gli-mediated regulation (Mill, 2005).

Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. This study discovered that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH; see Drosophila Hedgehog). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo (see Drosophila Smoothened), a gene required in SHH-receiving cells, it was found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg; see Drosophila Eip75b), to induce dermal adipogenesis. This study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages (Zhang, 2016).

Sonic Hedgehog and mammary gland development

Sonic Hedgehog (Shh) is a secreted morphogen that directs patterning and cellular differentiation through binding to its receptor Patched (Ptc). It is required for the development of skin-derived organs, such as hair, whiskers, and teeth. The mammary gland is a skin-derived organ that develops mainly during adult life in which Shh is expressed from puberty to lactation. The role of Shh in mammary gland morphogenesis and differentiation has been investigated by two transplantation approaches. Since Shh-null fetuses die at late embryogenesis, Shh-null mammary anlagen were transplanted into cleared fat pads and under the renal capsule of wild type host mice. Pregnancy-mediated functional differentiation of Shh-null mammary epithelium is indistinguishable from wild type transplants, while hair follicles derived from cotransplanted skin only develop in wild type transplants. Transplants of Ihh-null anlagen also develop normally. To assess the molecular consequences of Shh deletion in mammary tissue, mRNA levels of patched 1, a target gene of Hedgehog signaling, were compared in Shh-null and wild type mammary epithelial transplants. No reduction of Ptc1 transcripts was observed in Shh-null mammary tissues. These results demonstrate that neither Shh nor Ihh is required for mammary gland morphogenesis and functional differentiation, suggesting that the two members of the Hedgehog family may have redundant function in activating the Ptc1 signaling pathway during mammary gland development (Gallegao, 2002).

Hedgehog pathway and the preparation of the uterus for implantation

Genes encoding components of the hedgehog signaling pathway are dynamically expressed in the mouse uterus preparing for implantation. Indian hedgehog (Ihh), patched (Ptc), and Gli3 are expressed at low levels in the endometrial epithelium on day 1 of pregnancy. Transcription of Ihh increases dramatically in the luminal epithelium and glands from day 3, reaching very high levels on day 4. Over the same period, Ptc, Gli1, Gli2, and noggin are strongly upregulated in the underlying mesenchymal stroma. Transcription of Ihh in ovariectomized mice is induced by progesterone but not by estrogen. Lower induction of Ihh, Ptc, and Hoxa10 is seen in response to progesterone in the uteri of Pgr-/- mutant mice lacking progesterone nuclear steroid receptor. This finding suggests that the hormone may regulate Ihh through both nuclear receptor-dependent and -independent pathways. A method for culturing uterine explants in the absence of epithelium is described. Under these conditions, recombinant N-SHH protein promotes the proliferation of mesenchyme cells and the expression of noggin. It is proposed that IHH made by the epithelium normally functions as a paracrine growth factor for stromal cells during the early stages of pregnancy (Matsumoto, 2002).

Sonic Hedgehog and urogenital development

The prostate gland develops from the urogenital sinus by a testosterone-dependent process of ductal morphogenesis. Sonic hedgehog (Shh) is expressed in the urogenital sinus epithelium and the time course of expression coincides with the formation of the main prostatic ducts. Expression is most abundant in the lumen of the urogenital sinus and in the contiguous proximal duct segments. The initial upregulation of Shh expression in the male urogenital sinus depends on the presence of testosterone. The function of Shh was examined in the male urogenital sinus, which was transplanted under the renal capsule of an adult male host mouse. Blockade of Shh function by a neutralizing antibody interferes with Shh signaling and abrogates growth and ductal morphogenesis in the transplanted tissue. These observations show that testosterone-dependent Shh expression in the urogenital sinus is necessary for the initiation of prostate development (Podlasek, 1999).

Ductal budding in the developing prostate is a testosterone-dependent event that involves signaling between the urogenital sinus epithelium (UGE) and urogenital sinus mesenchyme (UGM). Ductal bud formation is associated with focused expression of Sonic hedgehog (Shh) in the epithelium of nascent prostate buds and in the growing tips of elongating prostate ducts. This pattern of localized Shh expression occurs in response to testosterone stimulation. The gene for the Shh receptor, Ptc1, is expressed in the UGM, as are the members of the Gli gene family of transcriptional regulators (Gli1, Gli2, and Gli3). Expression of Ptc1, Gli1, and Gli2 is localized primarily to mesenchyme surrounding prostate buds, whereas Gli3 is expressed diffusely throughout the UGM. A strong dependence of Gli1 (and Ptc1) expression on Shh signaling is demonstrated by induction of expression in both the intact urogenital sinus and the isolated UGM by exogenous SHH peptide. A similar dependence of Gli2 and Gli3 expression on Shh is not observed. Nonetheless, the chemical inhibitor of Shh signaling, cyclopamine, produced a graded inhibition of Gli gene expression (Gli1->Gli2->Gli3) in urogenital sinus explants that was paralleled by a severe inhibition of ductal budding. It is concluded that Shh activates mesenchymal Gli1 expression during prostate ductal bud formation. (Lamm, 2002).

External genital development in mammals begins with formation of paired genital swellings, which develop into the genital tubercle. Proximodistal outgrowth and axial patterning of the genital tubercle are coordinated to give rise to the penis or clitoris. The genital tubercle consists of lateral plate mesoderm, surface ectoderm, and endodermal urethral epithelium derived from the urogenital sinus. The molecular control of external genital development has been studied in the mouse embryo. Previous work has shown that the genital tubercle has polarizing activity, but the precise location of this activity within the tubercle is unknown. It was reasoned that if the tubercle itself is patterned by a specialized signaling region, then polarizing activity may be restricted to a subset of cells. Transplantation of urethral epithelium, but not genital mesenchyme, to chick limbs results in mirror-image duplication of the digits. Moreover, when grafted to chick limbs, the urethral plate orchestrates morphogenetic movements normally associated with external genital development. Signaling activity is therefore restricted to urethral plate cells. Before and during normal genital tubercle outgrowth, urethral plate epithelium expresses Sonic hedgehog. In mice with a targeted deletion of Shh, external genitalia are absent. Genital swellings are initiated, but outgrowth is not maintained. In the absence of Shh signaling, Fgf8, Bmp2, Bmp4, Fgf10, and Wnt5a are downregulated, and apoptosis is enhanced in the genitalia. These results identify the urethral epithelium as a signaling center of the genital tubercle, and demonstrate that Shh from the urethral epithelium is required for outgrowth, patterning, and cell survival in the developing external genitalia (Perriton, 2002).

While prostate gland development is dependent on androgens, other hormones including retinoids and estrogens can influence this process. Brief exposure to high-dose estrogen during the neonatal period in rats leads to permanent, lobe-specific aberrations in the prostate gland, a phenomenon referred to as developmental estrogenization. This response is mediated through alterations in steroid receptor expression; however, further downstream mechanisms remain unclear. Sonic hedgehog (Shh)-patched (ptc)-gli was investigated in the developing rat prostate gland, its role in branching morphogenesis, and the effects of neonatal estrogens on its expression and localization to determine whether a disturbance in this signaling pathway is involved in mediating the estrogenized phenotype. Shh is expressed in epithelial cells at the distal tips of elongating ducts in discreet, heterogeneous foci, while ptc and gli1–3 are expressed in the adjacent mesenchymal cells in the developing gland. The addition of Shh protein to cultured neonatal prostates reduces ductal growth and branching, decreases Fgf10 transcript, and increases Bmp4 expression in the adjacent mesenchyme. Shh-induced growth suppression is reversed by exogenous Fgf10, but not noggin, indicating that Fgf10 suppression is the proximate cause of the growth inhibition. A model is proposed to show how highly localized Shh expression along with regulation of downstream morphogens participates in dichotomous branching during prostate morphogenesis. Neonatal exposure to high-dose estradiol suppresses Shh, ptc, gli1, and gli3 expressions and concomitantly blocks ductal branching in the dorsal and lateral prostate lobes specifically. In contrast, ventral lobe branching and Shh-ptc-gli expression are minimally affected by estrogen exposure. Organ culture studies with lateral prostates confirms that estradiol suppresses Shh-ptc-gli expression directly at the prostatic level. Taken together, the present findings indicate that lobe-specific decreases in Shh-ptc-gli expression are involved in mediating estradiol-induced suppression of dorsal and lateral lobe ductal growth and branching during prostate morphogenesis (Pu, 2004).

The urogenital and reproductive organs, including the external genitalia, bladder and urethra, develop as anatomically aligned organs. Descriptive and experimental embryology suggest that the cloaca, and its derivative, the urogenital sinus, contribute to the formation of these organs. However, it is unknown how the primary tissue lineages in, and adjacent to, the cloaca give rise to the above organs, nor is bladder formation understood. While it is known that sonic hedgehog (Shh) is expressed by the cloacal epithelia, the developmental programs that regulate and coordinate the formation of the urogenital and reproductive organs have not been elucidated. This study reports that Shh mutant embryos display hypoplasia of external genitalia, internal urethra (pelvic urethra) and bladder. The importance of Shh signaling in the development of bladder and external genitalia was confirmed by analyzing a variety of mutant mouse lines with defective hedgehog signaling. By genetically labeling hedgehog-responding tissue lineages adjacent to the cloaca and urogenital sinus, the contribution of these tissues to the bladder and external genitalia is defined. Development of smooth muscle myosin-positive embryonic bladder mesenchyme requires Shh signaling, and the bladder mesenchyme and dorsal (upper) external genitalia derive from Shh-responsive peri-cloacal mesenchyme. Thus, the mesenchymal precursors for multiple urogenital structures derive from peri-cloacal mesenchyme and the coordination of urogenital organ formation from these precursors is orchestrated by Shh signals (Haraguchi, 2007).

Sonic Hedgehog and left-right asymmetries

Left-right (LR) asymmetry of the heart in vertebrates is regulated by early asymmetric signals in the embryo, including the secreted signal Sonic hedgehog (Shh), but less is known about LR asymmetries in visceral organs. Shh also specifies asymmetries in visceral precursors in the zebrafish (cardiac and visceral sidedness are independent of one another). The transcription factors fli-1 and Nkx-2.5 are expressed asymmetrically in the precardiac mesoderm and subsequently in the heart; an Eph receptor, rtk2, and an adhesion protein, DM-GRASP, mark early asymmetries in visceral endoderm. Misexpression on the right side of either shh mRNA, or a dominant negative form of protein kinase A, reverses the expression of these asymmetries in precursors of both the heart and the viscera. Reversals in the heart and gut are uncoordinated, suggesting that each organ interprets the signal independently. Misexpression of Bone Morphogenetic Protein (BMP4) on the right side reverses the heart, but visceral organs are unaffected, consistent with a function for BMPs locally in the heart field. Zebrafish mutants with midline defects show independent reversals of cardiac and visceral laterality. Thus, hh signals influence the development of multiple organ asymmetries in zebrafish and different organs appear to respond independently to a central cascade of midline signaling, which in the heart involves BMP4 (Schilling, 1999).

In chick embryos, the first signs of left-right asymmetry are detected in Hensen's node, essentially by left-sided Sonic Hedgehog (Shh) expression. After a gap of several hours, SHH induces polarized gene activities in the left paraxial mesoderm. During this time period, BMP4 signaling is necessary and sufficient to maintain Shh asymmetry within the node. SHH and BMP4 proteins negatively regulate each other's transcription, resulting in a strict complementarity between these two gene patterns on each side of the node. Noggin, present in the midline at this stage, limits BMP4 spreading. Moreover, BMP4 is downstream of Activin signals and controls Fgf8. Thus, early BMP4 signaling coordinates left and right pathways in Hensen's node (Monsoro-Burq, 2001).

Asymmetric expression of sonic hedgehog (Shh) in the left side of Hensen's node, a crucial step for specifying the left-right (LR) axis in the chick embryo, is established by the repression of Shh expression in the right side of the node. The transcriptional regulator that mediates this repression has not been identified. A novel chick Polycomblike 2 gene, chick Pcl2, has been isolated and characterized that encodes a transcription repressor and displays an asymmetric expression, downstream from Activin-ßB and Bmp4, in the right side of Hensen's node in the developing embryo. In vitro mapping studies define the transcription repression activity to the PHD finger domain of the chick Pcl2 protein. Repression of chick Pcl2 expression in the early embryo results in randomized heart looping direction, which is accompanied by the ectopic expression of Shh in the right side of the node and Shh downstream genes in the right lateral plate mesoderm (LPM), while overexpression of chick Pcl2 represses Shh expression in the node. The repression of Shh by chick Pcl2 is also supported by studies in which chick Pcl2 was overexpressed in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of chick Pcl2 in the developing mouse limb inhibits Shh expression in the ZPA. In vitro pull-down assays demonstrate a direct interaction of the chick Pcl2 PHD finger with EZH2, a component of the ESC/E(Z) repressive complex. Taken together with the fact that chick Pcl2 directly represses Shh promoter activity in vitro, these results demonstrate a crucial role for chick Pcl2 in regulating LR axis patterning in the chick by silencing Shh in the right side of the node (Wang, 2004).

Table of contents

hedgehog continued: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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