Table of contents

Hedgehogs, mesodermal induction and differentiation, hematopoiesis, chondrogenesis and bone growth

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. vascular endothelial growth factor (vegf), synthesized by somites, acts downstream of the notochordal sonic hedgehog signal and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells (Lawson, 2002).

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh-/- mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, an examination was made of the ability of ES cells deficient for Ihh or smoothened to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh-/- lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, the phenotypes of embryo yolk sacs deficient for Ihh or Smo were examined. Whereas Ihh-/- yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo-/- yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh-/- yolk sacs. This study supports a role for hedgehog signaling in yolk sac angiogenesis (Byrd, 2002).

During gastrulation in the mouse, mesoderm is induced and patterned by secreted signaling molecules, giving rise first to primitive erythroblasts and vascular endothelial cells. Development of these lineages requires a signal(s) secreted from the adjacent primitive endoderm. Indian hedgehog (Ihh) is a primitive endoderm-secreted signal that alone is sufficient to induce formation of hematopoietic and endothelial cells. Strikingly, as seen with primitive endoderm, Ihh can respecify prospective neural ectoderm (anterior epiblast) along hematopoietic and endothelial (posterior) lineages. Downstream targets of the hedgehog signaling pathway (the genes encoding patched, smoothened and Gli1) are upregulated in anterior epiblasts cultured in the presence of Ihh protein, as is Bmp4, which may mediate the effects of Ihh. Blocking Ihh function in primitive endoderm inhibits activation of hematopoiesis and vasculogenesis in the adjacent epiblast, suggesting that Ihh is an endogenous signal that plays a key role in the development of the earliest hemato-vascular system. It is possible that these are the earliest functions for a hedgehog protein in post-implantation development in the mouse embryo (Dyer, 2001).

An extra-embryonic signal regulated by the transcriptional coactivator Smad2 confers molecular asymmetry to the epiblast by specifying the anterior pole and by restricting the site of primitive streak formation. It is proposed that one function of Ihh is to help antagonize the Smad2 pathway in the posterior and/or extra-embryonic region of the embryo. Such a role would provide at least a partial explanation for the ability of primitive endoderm to reprogram the anterior epiblast in recombinant explants. A role for the anterior visceral endoderm (AVE) in inhibiting activation of hematopoietic and vascular development in the anterior epiblast appears very unlikely. If the AVE antagonized formation of these cell types, then genes such as epsilon-globin, Flk1 and Runx1/Cbfa2 should be activated upon removal of VE from anterior epiblasts. However, no activation of these or any other genes investigated in these studies was detected in anterior epiblasts stripped of VE. Either anterior or posterior VE can activate hematopoiesis in anterior epiblasts, suggesting that the signal(s) is(are) not strongly regionalized in an anteroposterior direction (Dyer, 2001).

Anti-hedgehog activity such as that produced by Hip in the anterior epiblast or a mouse homolog of Dispatched in the AVE could in principle result in local inhibition of Ihh, restricting its inductive activity and, thus, the formation of hematopoietic mesoderm to the posterior epiblast. Hedgehog signaling stimulates cellular proliferation in a variety of systems. Treatment of anterior epiblasts with IHH-N, the biologically active N-terminal portion of IHH, clearly promotes a proliferative response. However, proliferation per se is not sufficient to activate hematopoiesis and vasculogenesis in IHH-N-treated ectoderm. This conclusion is based on the following observations: (1) epsilon-globin-lacZ transgenic anterior epiblasts co-cultured on fibroblast monolayers or treated with epidermal growth factor shows significant cell proliferation but does not form beta-galactosidase-positive erythroblasts, and (2) incubation of anterior epiblasts with the hedgehog blocking antibody 5E1 inhibits formation of erythroid cells but not cell proliferation. The antibody blocking experiments suggest that visceral endoderm provides a signal(s) in addition to Ihh that can stimulate cell proliferation (Dyer, 2001).

The axial structures, consisting of the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. The role of the axial structures in the mediolateral patterning of the somite has been investigated by using a newly identified murine homeobox gene, Nkx-3.1 (homologous to Drosophila bagpipe), as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. The expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. By exposing explants to either COS cells transfected with a Shh expression construct or to recombinant Shh, it is shown that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures. Shh can induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites, but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression are antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. The expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial structures, but Shh, by itself, is not capable of inducing or maintaining it. It is suggested that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites (Kos, 1998).

In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are expressed in these inducing tissues during times of active cell fate specification, including members of the Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic hedgehog (expressed in the notochord and floor plate of the nerve cord), the functions of these signaling molecules with respect to dorsoventral somite patterning have not been determined. The role of Wnt-1 (expressed in the dorsal neural tube), a candidate dorsalizing factor, has been investigated in the regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. In response to Shh expression in dorsal somitic tissues, a marked diminution of BMP-4 expression is observed. This finding is consistent with the notion that Shh influences myotome formation through the elimination of BMP-4, which is a known repressor of MyoD transcription. Since MyoD expression is not significantly affected in response to Wnt signaling, it is concluded that Wnt-mediated up-regulation of BMP-4 message is not sufficient to down-regulate MyoD expression. Taken together, these results support a model for somite patterning where sclerotome formation is controlled by the antagonistic activities of Shh and Wnt signaling pathways. Shh is clearly required to suppress dorsal cell fates and promote ventral cell fates (Capdevila, 1998).

In vitro somite myogenesis is regulated by neural tube and notochord factors including Wnt, Sonic hedgehog (Shh), and basic fibroblast growth factor (bFGF) together with transforming growth factor-beta1 (TGF-beta1). Insulin and insulin-like growth factors I and II (IGF-I and -II) also promote myogenesis in explant cultures containing single somites or somite-sized pieces of segmental plate mesoderm from 2-day (stage 10-14) chicken embryos. The combination of insulin/IGFs with bFGF plus TGF-beta1 promotes even higher levels of myogenesis. Shh promotes myogenesis in this in vitro system, and Shh interacts synergistically with insulin/IGFs to promote high levels of myogenesis. RT-PCR analysis has detected insulin, IGF-II, insulin receptor, and IGF receptor mRNAs in both the neural tube and the somites, whereas IGF-I transcripts are detected in entire embryos but not in the neural tube or somites. Treatment of somite-neural tube cocultures with anti-insulin, anti-IGF-II, anti-insulin receptor, or anti-IGF receptor blocking antibodies causes a significant decrease in myogenesis. These results are consistent with the hypothesis that systemic IGF-I as well as insulin and IGF-II secreted by the neural tube act as additional early myogenic signals during embryogenesis. Further studies indicate that insulin, IGFs, bFGF, and Shh also stimulate somite cell proliferation and influence apoptosis (Pirskanen, 2000).

The Fgf4 gene encodes an important signaling molecule that is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, overlapping but distinct enhancer elements have been identified in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. The hypothesis that Fgf4 is a target of myogenic bHLH factors has been investigated in this study. By mutational analysis it has been shown that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. Expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner (Fraidenraich, 2000).

In vertebrates, somite differentiation is mediated in part by Sonic Hedgehog (Shh), secreted by the notochord and the floor plate. However, Shh-null mice display close to normal expression of molecular markers for dermomytome, myotome, and sclerotome, indicating that Shh might not be required for their initial induction. This paper addresses the capacity of Shh to regulate in vivo the expression of the somite differentiation markers Pax-1, MyoD, and Pax-3 after separation of paraxial mesoderm from axial structures. Pax-1, which is lost under these experimental conditions, is rescued by Shh. In contrast, Shh maintains, but cannot induce MyoD expression, while Pax-3 expression is independent of the presence of axial structures or Shh. Shh is a potent mitogen for somitic cells, supporting the idea that it may serve to expand subpopulations of cells within the somite (Marcelle, 1999).

Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. The role of Shh signaling has been examined in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. Shh is required for Myf5 expression in the specification of dorsal somite epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, transgenic analysis with lacZ reporter genes was used to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification (Gustafsson, 2002).

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. The generation of slow and fast muscle has been studied in zebrafish embryos. Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh, either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. It is suggested that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm (Blagden, 1997).

Previous work has indicated that signals from the floor plate and notochord promote chondrogenesis of the somitic mesoderm. These tissues, acting through the secreted signaling molecule Sonic hedgehog (Shh), appear to be critical for the formation of the sclerotome. Later steps in the differentiation of sclerotome into cartilage may be independent of the influence of these axial tissues. Although the signals involved in these later steps have not yet been pinpointed, there is substantial evidence that the analogous stages of limb bud chondrogenesis require bone morphogenetic protein (BMP) signaling. Presomitic mesoderm (psm) cultured in the presence of Shh will differentiate into cartilage: the later stages of this differentiation process specifically depend on BMP signaling. Shh not only acts in collaboration with BMPs to induce cartilage, but it changes the competence of target cells to respond to BMPs. In the absence of Shh, BMP administration induces lateral plate gene expression in cultured psm. After exposure to Shh, BMP signaling no longer induces expression of lateral plate markers but now induces robust chondrogenesis in cultured psm. Shh signals are required only transiently for somitic chondrogenesis in vitro, and act to provide a window of competence during which time BMP signals can induce chondrogenic differentiation. These findings suggest that chondrogenesis of somitic tissues can be divided into two separate phases: Shh-mediated generation of precursor cells, which are competent to initiate chondrogenesis in response to BMP signaling, and later exposure to BMPs, which act to trigger chondrogenic differentiation (Murtaugh, 1999).

Shh acts early in the development of the axial skeleton, to induce a prochondrogenic response to later BMP signaling. Somitic expression of the transcription factor Nkx3.2, homolog of Drosophila bagpipe, is initiated by Shh and sustained by BMP signals. Misexpression of Nkx3.2 in somitic tissue confers a prochondrogenic response to BMP signals. The transcriptional repressor activity of Nkx3.2 is essential for this factor to promote chondrogenesis. Conversely, a 'reverse function' mutant of Nkx3.2 that has been converted into a transcriptional activator inhibits axial chondrogenesis in vivo. It is concluded that Nkx3.2 is a critical mediator of the actions of Shh during axial cartilage formation, acting to inhibit expression of factors that interfere with the prochondrogenic effects of BMPs (Murtaugh, 2001).

Zebrafish have three distinct embryonic muscle fiber types: muscle pioneer slow muscle fibers, non-pioneer slow muscle fibers and fast muscle fibers. Slow muscle fibers develop from adaxial cells, which are adjacent to the notochord in the segmental plate. These cells are the first to express myogenic transcription factors such as MyoD and the first to differentiate into muscle fibers. Adaxial cells begin to elongate while adjacent to the notochord and then migrate radially to the surface of the somite, forming a superficial monolayer of embryonic slow twitch muscle fibers. A small subset of these cells express Engrailed proteins and are known as muscle pioneer slow muscle cells. Muscle pioneers span the somite from its medial to its lateral surface at the future position of the horizontal myoseptum, which establishes a separation between the dorsal and ventral myotome. The position of slow muscle precursors adjacent to the notochord suggests that notochord signaling may play a role in their development. In support of this, mutants with disrupted notochord development have a loss of muscle pioneers, and muscle pioneer development can be rescued by transplanting wild-type notochord cells into mutant embryos. Non-pioneer slow muscle cells are also dependent on notochord signaling. After adaxial cell migration to the surface of the myotome, fast muscle fibers develop from cells that are initially lateral to adaxial cells in the segmental plate. Their development does not depend on notochord signaling (Barresi, 2000).

Hedgehog proteins mediate many of the inductive interactions that determine cell fate during embryonic development. Hedgehog signaling has been shown to regulate slow muscle fiber type development. Mutations in the zebrafish slow-muscle-omitted (smu) gene disrupt many developmental processes involving Hedgehog signaling. smu-/- embryos have a 99% reduction in the number of slow muscle fibers and a complete loss of Engrailed-expressing muscle pioneers. In addition, mutant embryos have partial cyclopia, and defects in jaw cartilage, circulation and fin growth. The smu-/- phenotype is phenocopied by treatment of wild-type embryos with forskolin, which inhibits the response of cells to Hedgehog signaling by indirect activation of cAMP-dependent protein kinase (PKA). Overexpression of Sonic hedgehog (Shh) or dominant negative PKA (dnPKA) in wild-type embryos causes all somitic cells to develop into slow muscle fibers. Overexpression of Shh does not rescue slow muscle fiber development in smu-/- embryos, whereas overexpression of dnPKA does. Cell transplantation experiments confirm that smu function is required cell-autonomously within the muscle precursors: wild-type muscle cells rescue slow muscle fiber development in smu-/- embryos, whereas mutant muscle cells cannot develop into slow muscle fibers in wild-type embryos. Slow muscle fiber development in smu mutant embryos is also rescued by expression of rat Smoothened. Therefore, Hedgehog signaling through Slow-muscle-omitted is necessary for slow muscle fiber type development. It is proposed that smu encodes a vital component in the Hedgehog response pathway (Barresi, 2000).

Bone morphogenetic proteins (BMPs) have been implicated in regulating multiple stages of bone development. Constitutive activation of the BMP receptor-IA blocks chondrocyte differentiation in a similar manner as does the misexpression of Indian hedgehog. The role of BMPs has been analzyed; BMPs are possible mediators of Indian hedgehog signaling; Noggin misexpression was used to gain insight into additional roles for BMPs during cartilage differentiation. Comparative analysis of BMP and Ihh expression domains shows that the borders of Indian hedgehog expression in the chondrocytes are reflected in changes of the expression level of several BMP genes in the adjacent perichondrium. Misexpression of Indian hedgehog appears to directly upregulate BMP2 and BMP4 expression, independent of the differentiation state of the flanking chondrocytes. In contrast, changes in BMP5 and BMP7 expression in the perichondrium correspond to altered differentiation states of the flanking chondrocytes. In addition, Noggin and Chordin, which are both expressed in the developing cartilage elements, also change their expression pattern after Ihh misexpression. Finally, retroviral misexpression of Noggin, a potent antagonist of BMP signaling, has been used to gain insight into additional roles of BMP signaling during cartilage differentiation. BMP signaling is found to be necessary for the growth and differentiation of the cartilage elements. In addition, this analysis reveals that the members of the BMP/Noggin signaling pathway are linked in a complex autoregulatory network (Pathi, 1999).

Is the development of embryonic muscle fiber type regulated by competing influences between Hedgehog and TGF-beta signals? Ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. It is proposed that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells (Du, 1997).

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs reveals an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is closely related to Bf2, with only one amino acid difference within the winged helix domain; the protein is also closely related to two other mouse forkhead proteins, Hfh2 and Fkh2. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system, Mf2 is transcribed in restricted regions of the midbrain and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing Sonic hedgehog (Shh). In explant cultures of presomitic mesoderm, Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH (Wu, 1998).

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. The long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product of Shh. Pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. These results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members (Fan, 1995).

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth. Activating mutations in Fgfr3cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, the expression of an activated FGFR3 was targeted to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. FGFR3 inhibits endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulates the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the Hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium (Naski, 1998).

Sonic hedgehog (Shh), produced by the notochord and floor plate, is proposed to function as an inductive and trophic signal that controls somite and neural tube patterning and differentiation. To investigate Shh functions during somite myogenesis in the mouse embryo, an analysis was carried out of the expression of the myogenic determination genes, Myf5 and MyoD, and other regulatory genes in somites of Shh null embryos and in explants of presomitic mesoderm from wild-type and Myf5 null embryos. Shh has an essential inductive function in the early activation of the myogenic determination genes, Myf5 and MyoD, in the epaxial somite cells that give rise to the progenitors of the deep back muscles. Shh is not required for the activation of Myf5 and MyoD at any of the other sites of myogenesis in the mouse embryo, including the hypaxial dermomyotomal cells that give rise to the abdominal and body wall muscles, or the myogenic progenitor cells that form the limb and head muscles. Shh also functions in somites to establish and maintain the medio-lateral boundaries of epaxial and hypaxial gene expression. Myf5, and not MyoD, is the target of Shh signaling in the epaxial dermomyotome, since MyoD activation by recombinant Shh protein in presomitic mesoderm explants is defective in Myf5 null embryos. In further support of the inductive function of Shh in epaxial myogenesis, Shh has been shown to be not essential for the survival or the proliferation of epaxial myogenic progenitors. However, Shh is required specifically for the survival of sclerotomal cells in the ventral somite as well as for the survival of ventral and dorsal neural tube cells. It is concluded, therefore, that Shh has multiple functions in the somite, including inductive functions in the activation of Myf5, leading to the determination of epaxial dermomyotomal cells to myogenesis, as well as trophic functions in the maintenance of cell survival in the sclerotome and adjacent neural tube. These findings, therefore, indicate that other Shh-independent signals function to induce myogenesis at other sites in the embryo. In vitro explant studies have shown that different Wnts produced by surface ectoderm and dorsal neural tube can differentially induce Myf5 and MyoD, and therefore are candidates for Shh-independent signals that regulate epaxial and hypaxial myogenesis (Borycki, 1999).

The developmental signals that govern cell specification and differentiation in vertebrate somites are well understood. However, little is known about the downstream signaling pathways involved. A combination of Shh protein and Wnt1 or Wnt3a-expressing fibroblasts is sufficient to activate skeletal muscle-specific gene expression in somite explants. The molecular mechanisms by which the Wnt-mediated signal acts on myogenic precursor cells has been examined. Chick frizzled 1 (Fz1), beta-catenin and Lef1 are expressed during somitogenesis. Lef1 and beta-catenin transcripts become restricted to the developing myotome. Furthermore, beta-catenin is expressed prior to the time at which MyoD transcripts can be detected. Expression of beta-catenin mRNA is regulated by positive and negative signals derived from neural tube, notochord and lateral plate mesoderm. These signals include Bmp4, Shh and Wnt1/Wnt3a itself. In somite explants, Fz1, beta-catenin and Lef1 are expressed prior to activation of myogenesis in response to Shh and Wnt signals. Thus, these data show that a combination of Shh and Wnt1 upregulates expression of Wnt pathway components in developing somites prior to myogenesis (Schmidt, 2000).

Indian hedgehog (Ihh) and Parathyroid Hormone-related Protein (PTHrP) play a critical role in the morphogenesis of the vertebrate skeleton. Targeted deletion of Ihh results in short-limbed dwarfism, with decreased chondrocyte proliferation and extensive hypertrophy, features shared by mutants in PTHrP and its receptor. PTHrP is expressed in the periarticular perichondrium, and the mRNA for its receptor is expressed in a region that includes proliferating cells and extends into the prehypertrophic zone. Null mutations in PTHrP (PTHrP -/-) result in decreased numbers of mitotically active chondrocytes, whereas overexpression of PTHrP increases this pool, establishing a role for PTHrP in determining the size of the population of proliferating chondrocytes. Activation of Ihh signaling upregulates PTHrP at the articular surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null explants, suggesting that Ihh acts through PTHrP. To investigate the relationship between these factors during development of the appendicular skeleton, mice were produced with various combinations of an Ihh null mutation (Ihh -/- ), a PTHrP null mutation, and a constitutively active PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen II promoter (PTHrPR*). PTHrPR* rescues PTHrP -/- embryos, demonstrating that this construct can completely compensate for PTHrP signaling. At 18.5 of development, limb skeletons of Ihh;PTHrP compound mutants were identical to Ihh single mutants, suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in chondrocytes of Ihh -/- mice prevents premature chondrocyte hypertrophy but does not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation. These experiments demonstrate that the molecular mechanism that prevents chondrocyte hypertrophy is distinct from that which drives proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent chondrocyte hypertrophy and maintain a normal domain of cells competent to undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte proliferation in a pathway that can not be rescued by PTHrP signaling. This identifies Ihh as a coordinator of skeletal growth and morphogenesis, and refines the role of PTHrP in mediating a subset of Ihh's actions (Karp, 2000).

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. BMP and Ihh/PTHrP signaling were found to interact to coordinate chondrocyte proliferation and differentiation. To analyze the relationship between the two signaling pathways, an organ culture system was used for limb explants of mouse and chick embryos. Chondrocyte differentiation was manipulated by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice does not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, can not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. The role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes was further investigated and three functions of BMP signaling in this process were identified: (1) maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel; (2) a role for BMP signaling in modulating the expression of Ihh has been identified. Third, the application of Noggin to mouse limb explants results in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway (Minina, 2001).

These results suggest the following model for the regulation of chondrogenesis by the Ihh/PTHrP and BMP signaling pathways. Ihh produced by prehypertrophic chondrocytes promotes proliferation of the adjacent chondrocytes and, in addition, induces the expression of several Bmp genes in the perichondrium and in the proliferating chondrocytes. Ihh, furthermore, induces the expression of PTHrP in the periarticular region. PTHrP, in turn, negatively regulates the onset of hypertrophic differentiation. The range of PTHrP activity determines the distance from the joint region at which chondrocytes initiate the hypertrophic differentiation program and thereby the size of the domain of chondrocytes that are competent to proliferate. Ihh and BMP signaling together regulate the level of chondrocyte proliferation thereby pushing some cells out of the PTHrP signaling range. These cells are then released from the block of hypertrophic differentiation and activate the expression of Ihh, which, as discussed above, might be directly or indirectly regulated by BMP signaling. Since Ihh signaling regulates the expression of both Bmp genes and PTHrP, it tightly controls its own activation (Minina, 2001).

The development of endochondral bones requires the coordination of signals from several cell types within the cartilage rudiment. A signaling cascade involving Indian hedgehog (Ihh) and parathyroid hormone related peptide (PTHrP) has been described in which hypertrophic differentiation is limited by a signal secreted from chondrocytes as they become committed to hypertrophy. In this negative-feedback loop, Ihh inhibits hypertrophic differentiation by regulating the expression of Pthrp, which in turn acts directly on chondrocytes in the growth plate that express the PTH/PTHrP receptor. PTHrP also acts downstream of transforming growth factor ß (TGFß) in a common signaling cascade to regulate hypertrophic differentiation in embryonic mouse metatarsal organ cultures. Since members of the TGFß superfamily have been shown to mediate the effects of Hedgehog in several developmental systems, a model is proposed where TGFß acts downstream of Ihh and upstream of PTHrP in a cascade of signals that regulate hypertrophic differentiation in the growth plate. This report tests the hypothesis that TGFß signaling is required for the effects of Hedgehog on hypertrophic differentiation and expression of Pthrp. Sonic hedgehog (Shh), a functional substitute for Ihh, stimulates expression of Tgfß2 and Tgfß3 mRNA in the perichondrium of embryonic mouse metatarsal bones grown in organ cultures and that TGFß signaling in the perichondrium is required for inhibition of differentiation and regulation of Pthrp expression by Shh. The effects of Shh are specifically dependent on TGFß2, as cultures from Tgfß3-null embryos respond to Shh but cultures from Tgfß2-null embryos do not. Taken together, these data suggest that TGFß2 acts as a signal relay between Ihh and PTHrP in the regulation of cartilage hypertrophic differentiation (Alvarez, 2002).

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 has been assumed to occur independently of Shh. This study shows that formation of limb muscles is severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect becomes apparent relatively late and initial stages of hypaxial muscle development are unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicate 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. It is proposed 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 indicates that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. These data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis (Kruger, 2001).

Developmental programs that govern the embryonic diversification of distinct kinds of muscles in vertebrates remain obscure. For instance, the most widely recognized attribute of early diversity among skeletal myoblasts is their ability to differentiate exclusively into fibers with slow or fast contractile properties. Little is known about the developmental basis and genetic regulation of this seminal event in vertebrate myogenesis. In the zebrafish, the u-boot gene acts as a myogenic switch that regulates the choice of myoblasts to adopt slow versus fast fiber developmental pathways. Hedgehog activity singles out a subpopulation of somitic cells (the adaxial cells) and directs them to mature into slow muscle fibers. This Hh-dependent fiber-type specification operates through the activation of a myogenic switch that selectively propels naïve myoblasts to the slow muscle differentiation pathway. In u-boot mutant embryos, slow muscle precursors abort their developmental program, fail to activate expression of the homeobox gene prox1 and transfate into muscle cells with fast fiber properties. Using oligonucleotide-mediated translational inhibition, the role of prox1 in this program was investigated. It functions in the terminal step of the u-boot controlled slow fiber developmental pathway in the regulation of slow myofibril assembly. These findings provide new insight into the genetic control of slow versus fast fiber specification and differentiation and indicate that dedicated developmental pathways exist in vertebrates for the elaboration of distinct elements of embryonic muscle pattern (Roy, 2001a).

Indian hedgehog (Ihh), one of the three mammalian hedgehog proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, the Cre-loxP approach was used to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihhn/Ihhn), chondrocyte differentiation proceeds normally. However, like Ihhn/Ihhn mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, it has been demonstrated that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, results from the present study establishes Ihh as a key mitogen in the endochondral skeleton (Long, 2001).

Transient Shh signals from the notochord and floor plate confer a competence in somitic tissue for subsequent BMP signals to induce chondrogenesis. It has therefore been proposed that Shh induces a factor(s) that renders somitic cells competent to chondrify in response to subsequent BMP signals. Forced expression of Nkx3.2 (Drosophila homolog: Bagpipe), a transcriptional repressor induced by Shh, is able to confer chondrogenic competence in somites. Administration of Shh or forced Nkx3.2 expression induces the expression of the transcription factor Sox9 in the somitic tissue. Forced expression of Sox9 can, in turn, induce robust chondrogenesis in somitic mesoderm, provided that BMP signals are present. In the presence of BMP signals, Sox9 and Nkx3.2 induce each other's expression. Thus, Nkx3.2 may promote axial chondrogenesis by derepressing the expression of Sox9 in somitic mesoderm. Furthermore, forced expression of either Sox9 or Nkx3.2 not only activates expression of cartilage-specific genes in somitic mesoderm, but also promotes the proliferation and survival of the induced chondrocytes in the presence of BMP signals. However, unlike Nkx3.2, Sox9 is able to induce de novo cartilage formation in non-cartilage-forming tissues. These findings suggest that Shh and BMP signals work in sequence to establish a positive regulatory loop between Sox9 and Nkx3.2, and that Sox9 can subsequently initiate the chondrocyte differentiation program in a variety of cellular environments (Zeng, 2002).

In the zebrafish embryo, the differentiation of distinct muscle fiber types has been shown to require the activity of members of the Hedgehog (Hh) family of secreted proteins. Evidence from other systems suggests that Hh behaves as a morphogen, inducing cell fates in a concentration-dependent manner. Exactly how Hh signaling contributes to the generation of the correct pattern of cells within the zebrafish myotome, however, has remained obscure. Four distinct myotomal cell identities in the zebrafish embryo are distinguished on the basis of their position, morphology, and gene expression patterns. Using morpholino oligonucleotides (MOs) to diminish the activities of the Hh pathway components Patched (Ptc), Fused (Fu), and Suppressor of Fused [Su(fu)], and the teratogen cyclopamine to inhibit the Hh transducer Smoothened (Smo), it has been shown that the appropriate differentiation of each cell type depends upon the levels and range of Hh signaling within the myotome. In addition, by transiently modulating Hh activity by using cyclopamine and a heat-inducible transgene, it has been demonstrated that the competence of myotomal cells to respond to Hh changes with time. Finally, it has been show that the Gli1 and Gli2 transcription factors mediate most of the response of myotomal cells to Hh. It is concluded that Hh signaling acts in a dosage-dependent manner to specify cell fate in the zebrafish myotome. Allocation of the correct number of cells to a specific fate depends upon the range of Hh activity. The timing of exposure to Hh determines the response of cells to the signal (Wolff, 2003).

The fact that lowest levels of Hh signaling direct cells to follow the slow-twitch differentiation pathway raises the issue of how higher levels of Hh activity can induce a specific type of fast-twitch fiber. Such elevated levels might be expected to convert presumptive fast myoblasts to the slow lineage, as is the case when Shh is ectopically expressed throughout the myotome. it is proposed that the correct specification of each cell type is a function not simply of the levels of Hh to which they are exposed, but also of the time at which they receive the signal and of their competence to respond to it. In this view, exposure of myotomal precursors to Hh is initially restricted to the adaxial cells that lie initially in the immediate vicinity of the embryonic shield that secretes the signal. As these cells respond to Hh, they upregulate Ptc, which sequesters the ligand, thereby limiting the extent of its influence to a relatively narrow domain close to the midline. In addition, this early exposure seems to be essential for the singling out of MP precursors from amongst the slow myoblasts that respond to the highest levels of signaling, while the remainder become fated to form the SSFs. It is only after the outward movement of the superficial slow fiber cells that a subset of presumptive fast myoblasts becomes exposed to the Hh signal; by this time, they are irreversibly committed to the fast lineage. In support for such a possibility, it has been found that the specification of the medial fast fibers requires Hh activity significantly later than do the slow-lineage fibers. Moreover, whereas early misexpression of Shh can restore MPs in sonic you (syu: zebrafish sonic hedgehog) mutants or induce supernumerary MPs in wild-type embryos, later misexpression results exclusively in the specification of Eng+ fast fibers (Wolff, 2003).

Sonic hedgehog (Shh) signaling is essential for sclerotome development in the mouse. Gli2 and Gli3 are thought to be the primary transcriptional mediators of Shh signaling; however, their roles in Shh induction of sclerotomal genes have not been investigated. Using a combination of mutant analysis and in vitro explant assays, it has been demonstrated that Gli2 and Gli3 are required for Shh-dependent sclerotome induction. Gli2–/–Gli3–/– embryos exhibit a severe loss of sclerotomal gene expression, and somitic mesoderm from these embryos cannot activate sclerotomal genes in response to exogenous Shh. One copy of either Gli2 or Gli3 is required to mediate Shh induction of sclerotomal markers Pax1 and Pax9 in vivo and in vitro. Although Gli2 is generally considered an activator and Gli3 a repressor, the results also reveal a repressor function for Gli2 and an activator function for Gli3 in the developing somite. To further dissect the function of each Gli, adenovirus was used to overexpress Gli1, Gli2 and Gli3 in presomitic mesoderm explants. Each Gli preferentially activates a distinct set of Shh target genes, suggesting that the functions of Shh in patterning, growth and negative feedback are divided preferentially between different Gli proteins in the somite (Buttitta, 2003).

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals, was genetically manipulated. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smon/n cells indicates that Smon/n cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, the bone collar was analyzed in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. It is suggested that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation (Long, 2004).

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, myogenesis has been examined in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. A model is proposed for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk (Grimaldi, 2004).

It is suggested that Hh signalling from ventral midline acts on medial somitic cells to promote XMyf5 expression and early slow myogenesis. These cells rapidly differentiate, express XMyoD and move to the superficial somite surface where they elongate anteroposteriorly to make superficial slow fibres. Simultaneously, most somitic cells differentiate into fast fibres, also elongating anteroposteriorly to form the bulk of somitic muscle. Undifferentiated cells form a dermomyotome. At later stages, a second population of slow muscle fibres is generated from dermomyotome, probably at dorsomedial and ventrolateral lips, independent of Hh signalling. In anterior somites, despite early notochord-dependent XMyf5 expression, a block on slow muscle formation prevents appearance of the first wave of slow fibres. Fast fibre formation is abundant, and precocious compared with zebrafish. However, some cells remain undifferentiated to form the superficial dermomyotome. Dorsal and ventral dermomyotomal lips continue to express XMyf5 and XMyoD, reflecting their continued role as myogenic centres. Slow fibre formation is initiated from dermomyotome independently of Hh signalling. Extra fast fibres probably also arise from dermomyotome at all anteroposterior levels. At even later stages Hh signalling is again required for XMyoD expression, somite growth and third wave slow fibre formation at dermomyotomal lips throughout the axis (Grimaldi, 2004).

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, endodermally derived sonic hedgehog is shown to be both necessary and sufficient for vascular tube formation in avian embryos. Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation (Vokes, 2004).

In zebrafish, skeletal muscle precursors can adopt at least three distinct fates: fast, non-pioneer slow, or pioneer slow muscle fibers. Slow muscle fibers develop from adaxial cells and depend on Hedgehog signaling. This study analyzed when precursors become committed to their fates and the step(s) along their differentiation pathway affected by Hedgehog. Unexpectedly, embryos deficient in Hedgehog signaling still contain postmitotic adaxial cells that differentiate into fast muscle fibers instead of slow. By the onset of gastrulation, slow and fast muscle precursors are already spatially segregated but uncommitted to their fates until much later, in the segmental plate when slow precursors become independent of Hedgehog. In contrast, pioneer and non-pioneer slow muscle precursors share a common lineage from the onset of gastrulation. These results demonstrate that slow muscle precursors form independently of Hedgehog signaling and further provide direct evidence for a multipotent muscle precursor population whose commitment to the slow fate depends on Hedgehog at a late stage of development when postmitotic adaxial cells differentiate into slow muscle fibers (Hirsinger, 2004).

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 it is demonstrated that beta-catenin, a central player in the canonical Wnt pathway, is indispensable for osteoblast differentiation in the mouse embryo. Moreover, evidence is presented 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 (Hu, 2005).

Studies with embryonic explants and embryonic stem cells have suggested a role for Hedgehog (Hh) signaling in hematopoiesis. However, targeted deletion of Hh pathway components in the mouse has so far failed to provide in vivo evidence. Zebrafish embryos mutant in the Hh pathway or treated with the Hh signaling inhibitor cyclopamine display defects in adult hematopoietic stem cell (HSC) formation but not in primitive hematopoiesis. Hh is required in the trunk at three consecutive stages during vascular development: for the medial migration of endothelial progenitors of the dorsal aorta (DA), for arterial gene expression, and for the formation of intersomitic vessel sprouts. Interference with Hh signaling during the first two stages also interferes with HSC formation. Furthermore, HSC and DA formation also share Vegf and Notch requirements, which further distinguishes them from primitive hematopoiesis and underlines their close relationship during vertebrate development (Gering, 2005).

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in both foregut endoderm and cardiogenic mesoderm and is required for earliest stages of heart development. isl1 is also required upstream of Shh. In isl1 null mice, Sonic hedgehog (Shh) is downregulated in foregut endoderm. Shh signals through the unique activating receptor smoothened (Smo). To investigate the role of hedgehog signaling in the isl1 domain, smo utilizing isl1-cre was ablated. Isl1-cre;smo mutants exhibit cardiovascular defects similar to those observed in Shh null mice, defining a spatial requirement for hedgehog signaling within isl1 expression domains for aortic arch and outflow tract formation. Semaphorin signaling through neuropilin receptors npn1 and npn2 is required for aortic arch and outflow tract formation. Expression of npn2 is downregulated in isl1-cre;smo mutants, suggesting an isl1/Shh/npn pathway required to affect morphogenesis at the anterior pole of the heart (Lin, 2007).

Hematopoietic stem cells (HSCs) are first detected in the floor of the embryonic dorsal aorta (DA), this study investigated the signals that induce the HSC program there. While continued Hedgehog (Hh) signaling from the overlying midline structures maintains the arterial program characteristic of the DA roof, a ventral Bmp4 signal induces the blood stem cell program in the DA floor. This patterning of the DA by Hh and Bmp is the mirror image of that in the neural tube, with Hh favoring dorsal rather than ventral cell types, and Bmp favoring ventral rather than dorsal. With the majority of current data supporting a model whereby HSCs derive from arterial endothelium, these data identify the signal driving this conversion. These findings are important for the study of the production of HSCs from embryonic stem cells and establish a paradigm for the development of adult stem cells (Wilkinson, 2009).

Shh promotes direct interactions between epidermal cells and osteoblast progenitors to shape regenerated zebrafish bone

Zebrafish innately regenerate amputated fins by mechanisms that expand and precisely position injury-induced progenitor cells to re-form tissue of the original size and pattern. For example, cell signaling networks direct osteoblast progenitors (pObs) to rebuild thin cylindrical bony rays with a stereotypical branched morphology. Hedgehog/Smoothened (Hh/Smo) signaling has been variably proposed to stimulate overall fin regenerative outgrowth or promote ray branching. Using a photoconvertible patched2 (see Drosophila Patched) reporter, active Hh/Smo output to a narrow distal regenerate zone comprising pObs and adjacent motile basal epidermal cells was identified. This Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which nevertheless functions atypically to support bone maturation. Using BMS-833923, a uniquely effective Smo inhibitor, and high-resolution imaging, it was shown that Shha/Smo is functionally dedicated to ray branching during fin regeneration. Hh/Smo activation enables transiently divided clusters of Shha-expressing epidermis to escort pObs into similarly split groups. This co-movement likely depends on epidermal cellular protrusions that directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. Progressively separated pObs pools then continue regenerating independently to collectively re-form a now branched skeletal structure (Armstrong, 2017).

Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. This ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh-/- chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh-/- skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh-/- cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh-/- cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is the finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh-/- cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage - comprising osteoblast precursors and endothelial cells - as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh-/- embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling (Colnot, 2005).

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development, including proliferation and maturation of chondrocytes, osteoblast development and cartilage vascularization. Although it is known that Gli transcription factors are key effectors of hedgehog signaling, it has not been established which Gli protein mediates Ihh activity in skeletal development. This study shows that removal of Gli3 in Ihh-null mouse embryos restores normal proliferation and maturation of chondrocytes, but only partially rescues the defects in osteoblast development and cartilage vascularization. Remarkably, in both Ihh-/- and Ihh-/-; Gli3-/- embryos, vascularization promotes osteoblast development in perichondrial progenitor cells. These results not only establish Gli3 as a critical effector for Ihh activity in the developing skeleton, but also identify an osteogenic role for a vasculature-derived signal, which integrates with Ihh and Wnt signals to determine the osteoblast versus chondrocyte fate in the mesenchymal progenitors (Hilton, 2005).

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, tests were performed to see whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells. Rather, it is due to instructive signaling from ventral tissues. Furthermore, Hedgehog protein(s) was identifed as an HSC inducing signal (Peeters, 2009).

Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation

During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs, was analyzed. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh–/– mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh–/–;Gli3–/– mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3–/– mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. The results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression (Koziel, 2005).

Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression

Hedgehog (Hh) signaling is required for osteoblast differentiation from mesenchymal progenitors during endochondral bone formation. However, the role of Hh signaling in differentiated osteoblasts during adult bone homeostasis remains to be elucidated. The postnatal bone Hh signaling activity is progressively reduced as osteoblasts mature. Upregulating Hh signaling selectively in mature osteoblasts leads to increased bone formation and excessive bone resorption. As a consequence, these mutant mice showed severe osteopenia. Conversely, inhibition of Hh signaling in mature osteoblasts resulted in increased bone mass and protection from bone loss in older mice. Cellular and molecular studies showed that Hh signaling indirectly induces osteoclast differentiation by upregulating osteoblast expression of PTHrP, which promotes RANKL expression via PKA and its target transcription factor CREB. These results demonstrate that Hh signaling in mature osteoblasts regulates both bone formation and resorption and that inhibition of Hh signaling reduces bone loss in aged mice (Mak, 2008).

Hedgehog and heart development

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. This study shows that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, it was found that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells (Thomas, 2008).

Sonic hedgehog and caudal development

The progressive generation of murine embryonic trunk structures relies on the proper patterning of the caudal epiblast involving the integration of several signalling pathways. The function of retinoic acid (RA) signalling during this process was investigated. In addition to posterior mesendoderm, primitive streak and node cells transiently express the RA-synthesizing enzyme Raldh2 prior to the headfold stage. RA-responsive cells (detected by the RA-activated RARE-lacZ transgene) are additionally found in the epiblast layer. Analysis of RA-deficient Raldh2-/- mutants reveals early caudal patterning defects, with an expansion of primitive streak and mesodermal markers at the expense of markers of the prospective neuroepithelium. As a result, many genes involved in neurogenesis and/or patterning of the embryonic spinal cord are affected in their expression. RA signalling is required at late gastrulation stages for mesodermal and neural progenitors to respond to the Shh signal. Whole-embryo culture experiments indicate that the proper response of cells to Shh requires two RA-dependent mechanisms: (1) a balanced antagonism between Fgf and RA signals, and (2) a RA-mediated repression of Gli2 expression. Thus, an interplay between RA, Fgf and Shh signalling is likely to be an important mechanism underpinning the tight regulation of caudal embryonic development (Ribes, 2009).

Table of contents

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

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