Hedgehog signaling in pancreas development

Hedgehog proteins are secreted molecules that bind to their cell surface receptors to elicit concentration dependent responses essential for numerous tissue patterning and cell differentiation events during embryogenesis. However, during early stages of pancreas organogenesis, hedgehog signaling has been shown to inhibit tissue morphogenesis and cell differentiation. By contrast, recent cell culture studies indicate that an active hedgehog pathway might be required for maintenance of adult endocrine cell functions. This review describes our current understanding of the requirement of hedgehog signaling during pancreas morphogenesis and cell differentiation and discusses how individual hedgehog genes might act at various stages to ensure proper pancreas development and organ function.     

Hedgehog signaling in skeletal development

Hedgehog signaling coordinates a variety of patterning processes during early embryonic development. Drosophila hedgehog and its vertebrate orthologs, Sonic hedgehog, Indian hedgehog, and Desert hedgehog, share a generally conserved signal transduction cascade. However, the particular mechanisms by which the lipid-modified molecules specify embryonic tissues differ substantially. Vertebrate skeletal patterning is one of the most intensively studied biological processes. During skeletogenesis, Sonic and Indian hedgehog provide positional information and initiate or maintain cellular differentiation programs regulating the formation of cartilage and bone. They either signal directly to adjacent cells or form tightly regulated gradients that act over long distances to pattern the axial and appendicular skeleton and regulate crucial steps during endochondral ossification. As a consequence, malfunction of the hedgehog signaling network can cause severe skeletal disorders and tumors.     

Hedgehog signaling in gastrointestinal development and disease

The development of the gastrointestinal (GI) tract and its associated parenchymal organs depends on Hedgehog signals from the endoderm to the surrounding mesoderm. During development, Hedgehog signaling is essential for patterning the GI tract along anterior-posterior (A-P), dorsal-ventral (D-V), and radial axes, as well as in maintenance of stem cells. Our knowledge about these roles for Hedgehog signaling is derived from studies of developmental defects that result from disrupted or activated Hedgehog signaling in model organisms including mouse, chick, and frog. These studies provide evidence for distinct roles of specific Hedgehog ligands in GI development. Studies in model organisms have also elucidated how Hedgehog signaling may function in development and function of the GI tract in humans. Several diseases and congenital syndromes are known to result from genetic defects in Hedgehog signaling components, and this pathway may ultimately prove to be an important target for future diagnostic and therapeutic tools.     

Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling

The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

Sequential roles of Hedgehog and Wnt signaling in osteoblast development

Signals that govern development of the osteoblast lineage are not well understood. Indian hedgehog (Ihh), a member of the hedgehog (Hh) family of proteins, is essential for osteogenesis in the endochondral skeleton during embryogenesis. The canonical pathway of Wnt signaling has been implicated by studies of Lrp5, a co-receptor for Wnt proteins, in postnatal bone mass homeostasis. In the present study we demonstrate that beta-catenin, a central player in the canonical Wnt pathway, is indispensable for osteoblast differentiation in the mouse embryo. Moreover, we present evidence that Wnt signaling functions downstream of Ihh in development of the osteoblast lineage. Finally Wnt7b is identified as a potential endogenous ligand regulating osteogenesis. These data support a model that integrates Hh and Wnt signaling in the regulation of osteoblast development.     

The adventures of Sonic Hedgehog in development and repair. I. Hedgehog signaling in gastrointestinal development and disease

Hedgehog (Hh) proteins are members of a family of secreted signaling factors that orchestrate the development of many organs and tissues including those of the gastrointestinal (GI) tract. The requirement for Hh activity is not limited to early development but underlies the homeostasis of a number of tissues, and abnormal activity of the Hh pathway is associated with several GI malignancies. Understanding the roles and mechanisms of action of Hh signaling both in development and postnatally should thus give novel insights into potential treatments for these diseases. Here we focus on the Hh signaling pathway and its role in GI tract development and maintenance and consider the diseases resulting from aberrant Hh activity.     

Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.     

BMP and Hedgehog signaling during the development of scleral ossicles

Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has largely focused on endochondral bones while intramembranous bones, such as the scleral ossicles within the avian eye, have received less attention. Our previous research directly demonstrated the involvement of sonic hedgehog and suggested the involvement of bmp2 and 4 during the development of scleral ossicles. The bones of the sclerotic ring are induced by overlying conjunctival papillae at HH 35 and 36. Here, we examine the spatial and temporal expression patterns of ptc1, ihh, bmp2, bmp4 and bmp7. We show that the cells of conjunctival papillae express ptc1, ihh and bmp2 at these stages; coincident with shh expression previously described. Interestingly, both ihh and ptc1 are also expressed in the mesenchyme underlying the papillae unlike shh and bmp2. Bmp4 and bmp7 are not expressed in these regions at any stages examined. Furthermore, using Noggin soaked beads implanted adjacent to papillae, we provide direct evidence that the BMP family of genes are important factors in the development of scleral ossicles. Localized inhibition of BMPs in this way causes a reduced expression of ihh in the surrounding tissue demonstrating that the BMP and Hedgehog pathways interact. Our data also demonstrates that the sclerotic ring has an intrinsic ability to compensate for missing elements. The scleral ossicle system provides a unique opportunity to investigate the epithelial-mesenchymal induction of intramembranous bones of the vertebrate skull.     

Notch signaling maintains Leydig progenitor cells in the mouse testis

During testis development, fetal Leydig cells increase their population from a pool of progenitor cells rather than from proliferation of a differentiated cell population. However, the mechanism that regulates Leydig stem cell self-renewal and differentiation is unknown. Here, we show that blocking Notch signaling, by inhibiting gamma-secretase activity or deleting the downstream target gene Hairy/Enhancer-of-split 1, results in an increase in Leydig cells in the testis. By contrast, constitutively active Notch signaling in gonadal somatic progenitor cells causes a dramatic Leydig cell loss, associated with an increase in undifferentiated mesenchymal cells. These results indicate that active Notch signaling restricts fetal Leydig cell differentiation by promoting a progenitor cell fate. Germ cell loss and abnormal testis cord formation were observed in both gain- and loss-of-function gonads, suggesting that regulation of the Leydig/interstitial cell population is important for male germ cell survival and testis cord formation.     

Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Shifting paradigms in Hedgehog signaling

Hedgehog (Hh) signaling proteins regulate multiple developmental and adult homeostatic processes. A defining feature of Hh signaling is that relatively small changes in the concentration of Hh ligand elicit dramatically different cellular responses. As a result, the processing, release and trafficking of Hh ligands must be tightly regulated to ensure proper signaling. In addition, sensitive and specific intracellular signaling cascades are needed to interpret subtle differences in the level of Hh signal to execute an appropriate response. A detailed understanding of the mechanisms that regulate these responses is critical to shaping our view of this key regulatory system.     

Hedgehog signaling: a smoothened conformational switch

An intracellular conformational switch in the serpentine transmembrane protein Smoothened appears to underlie Hedgehog pathway activation. The switch is gated by electrostatic interactions that are regulated by multiple phosphorylations, potentially endowing a dose-dependent response.