Sonic hedgehog signaling from the urethral epithelium controls external genital development

External genital development 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. We have investigated the molecular control of external genital development 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. We 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 (Shh). 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.     

Apaf1 apoptotic function critically limits Sonic hedgehog signaling during craniofacial development

Apaf1 is an evolutionarily conserved component of the apoptosome. In mammals, the apoptosome assembles when cytochrome c is released from mitochondria, binding Apaf1 in an ATP-dependent manner and activating caspase 9 to execute apoptosis. Here we identify and characterize a novel mouse mutant, yautja, and find it results from a leucine-to-proline substitution in the winged-helix domain of Apaf1. We show that this allele of Apaf1 is unique, as the yautja mutant Apaf1 protein is stable, yet does not possess apoptotic function in cell culture or in vivo assays. Mutant embryos die perinatally with defects in craniofacial and nervous system development, as well as reduced levels of apoptosis. We further investigated the defects in craniofacial development in the yautja mutation and found altered Sonic hedgehog (Shh) signaling between the prechordal plate and the frontonasal ectoderm, leading to increased mesenchymal proliferation in the face and delayed or absent ossification of the skull base. Taken together, our data highlight the time-sensitive link between Shh signaling and the regulation of apoptosis function in craniofacial development to sculpt the face. We propose that decreased apoptosis in the developing nervous system allows Shh-producing cells to persist and direct a lateral outgrowth of the upper jaw, resulting in the craniofacial defects we see. Finally, the novel yautja Apaf1 allele offers the first in vivo understanding of a stable Apaf1 protein that lacks a function, which should make a useful tool with which to explore the regulation of programmed cell death in mammals.     

Sonic hedgehog expression during early tooth development in Suncus murinus

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.     

Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.     

Vitronectin regulates Sonic hedgehog activity during cerebellum development through CREB phosphorylation

During development of the cerebellum, Sonic hedgehog (SHH) is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. We have previously demonstrated that SHH interacts with vitronectin in the differentiation of spinal motor neurons. Here, we analysed whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development. Laminins and their integrin receptor subunit alpha6 accumulate in the outer most external germinal layer where proliferation of granule cell precursors is maximal. Consistent with this expression pattern, laminin significantly increases SHH-induced proliferation in primary cultures of cerebellar granule cells. Vitronectin and its integrin receptor subunits alpha(v) are expressed in the inner part of the external germinal layer where granule cell precursors exit the cell cycle and commence differentiation. In cultures, vitronectin is able to overcome SHH-induced proliferation, thus allowing granule cell differentiation. Our studies indicate that the pathway in granule cell precursors responsible for the conversion of a proliferative SHH-mediated response to a differentiation signal depends on CREB. Vitronectin stimulates phosphorylation of cyclic-AMP responsive element-binding protein (CREB), and over-expression of CREB is sufficient to induce granule cell differentiation in the presence of SHH. Taken together, these data suggest that granule neuron differentiation is regulated by the vitronectin-induced phosphorylation of CREB, a critical event that terminates SHH-mediated proliferation and permits the differentiation program to proceed in these cells.     

Sonic hedgehog is a survival factor for hypaxial muscles during mouse development

Sonic hedgehog (Shh) has been proposed to function as an inductive and trophic signal that controls development of epaxial musculature in vertebrate embryos. In contrast, development of hypaxial muscles was assumed to occur independently of Shh. We here show that formation of limb muscles was severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect became apparent relatively late and initial stages of hypaxial muscle development were unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicated that Shh is required for the maintenance of the expression of myogenic regulatory factors (MRFs) and, consecutively, for the formation of differentiated limb muscle myotubes. We propose that Shh acts as a survival and proliferation factor for myogenic precursor cells during hypaxial muscle development. Detection of a reduced but significant level of Myf5 expression in the epaxial compartment of somites of Shh homozygous mutant embryos at E9.5 indicated that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. Our data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis.     

Sonic hedgehog signalling in T-cell development and activation

The production of mature functional T cells in the thymus requires signals from the thymic epithelium. Here, we review recent experiments showing that one way in which the epithelium controls the production of mature T cells is by the secretion of sonic hedgehog (SHH). We consider the increasing evidence that SHH-induced signalling is not only important for the differentiation and proliferation of early thymocyte progenitors, but also for modulating T-cell receptor signalling during repertoire selection, with implications for positive selection, CD4 versus CD8 lineage commitment, and clonal deletion of autoreactive cells. We also review the influence of hedgehog signalling in peripheral T-cell activation.     

Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.     

Molecular mechanisms of external genitalia development

External genitalia development occurs through a combination of hormone independent, hormone dependent, and endocrine pathways. Perturbation of these pathways can lead to abnormal external genitalia development. We review human and animal mechanisms of normal and abnormal external genitalia development, and we evaluate abnormal mechanisms that lead to hypospadias. We also discuss recent laboratory findings that further our understanding of animal models of hypospadias.     

Sonic hedgehog signaling in forebrain development and its interactions with pathways that modify its effects

During the development of the nervous system and other organs in the embryo, a limited set of master signaling pathways are used repeatedly for induction, patterning and growth. Among these, the Sonic hedgehog (Shh) pathway is crucial for the development of many structures in the brain. How the context-specific interplay between these various signaling pathways produces distinct temporal and spatial outcomes is not clear. Resolving this problem is a major goal in the study of cell and organ development. Here, we focus on signaling events during dorso-ventral patterning of the embryonic forebrain in vertebrates. In particular, we discuss the role of the Shh pathway in this process and on its interactions with the FGF, retinoic acid and Nodal pathways and other information cascades that modify its effects.     

Sonic hedgehog signaling in kidney fibrosis: a master communicator

The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog (Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease (CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelial- mesenchymal communication (EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients.     

Sonic hedgehog is required early in pancreatic islet development

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. We demonstrate that 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, we show 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.     

Some distal limb structures develop in mice lacking Sonic hedgehog signaling

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.     

The adventures of sonic hedgehog in development and repair. II. Sonic hedgehog and liver development, inflammation, and cancer

Hedgehog (Hh) signaling modulates tissue remodeling by controlling the fate of Hh-responsive cells. Healthy adult livers exhibit little Hh activity. However, cells involved in adult liver repair, including myofibroblasts and progenitors, are capable of producing and responding to Hh ligands. During adult liver injury, Hh ligand production increases and populations of Hh-responsive cells expand. This process is accompanied by fibrosis. Ligand production and Hh-responsive cells diminish as fibrosis resolves and normal hepatic architecture is restored, but Hh signaling persists in hepatocellular carcinomas. These findings suggest that the Hh pathway mediates remodeling responses that are triggered by adult liver damage.     

Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.