Table of contents

Regulation of Hedgehog in axial patterning and head development

The functional interaction between Hedgehog and Wingless is conserved in vertebrates. In chick embryos, a combination of signals from the neural tube and the floor plate/notochord complex synergistically induce the expression of myogenic bHLH genes and myogenic differentiation markers in unspecified somites. Sonic hedgehog (Shh), the vertebrate homolog of hedgehog, is expressed in the floor plate/notochord. A subset of Wnt family members (Wnt-1, Wnt-3, and Wnt-4), is expressed in dorsal regions of the neural tube, mimicking the muscle inducing activity of these tissues (Munsterberg, 1995). The Wnt family in vertebrates is homologous to wingless in the fly.

The signaling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co-expressed with the gene axial (HNF3ß in mammals), a transcription regulator of the winged helix family. Misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial. Expression of axial or rat HNF3ß in HeLa cells results in activation of co-transfected sonic hedgehog promoter-CAT fusion genes. This effect is mediated by two Axial (HNF3beta) recognition sequences. A retinoic acid response element (RARE) was identified in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro. This confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. These results suggest that both Axial (HNF3beta) and retinoic acid receptors are direct regulators of the sonic hedgehog gene (Chang, 1997).

In the vertebrate embryo, the lateral compartment of the somite gives rise to muscles of the limb and body wall and is patterned in response to lateral-plate-derived BMP4. Activation of the myogenic program distinctive to the medial somite, i.e. relatively immediate development of the epaxial muscle lineage, requires neutralization of this lateral signal. The properties of molecules likely to play a role in opposing lateral somite specification by BMP4 were examined. It is proposed that the BMP4 antagonist Noggin plays an important role in promoting medial somite patterning in vivo. Noggin expression in the somite is under the control of a neural-tube-derived factor, whose effect can be mimicked experimentally by Wnt1. Wnt1 is appropriately expressed in the neural tube. It is shown that Sonic Hedgehog, expressed in both the notochord and neural tube is able to activate ectopic expression of Noggin resulting in the blocking of BMP4 specification of the lateral somite. These results are consistent with a model in which Noggin activation in the medial somite lies downstream of the SHH and Wnt pathways signaling from the notochord and neural tube (Hirsinger, 1997).

Notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilagedifferentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of Pax family (Pax1/Pax9, Drosophila homolog: Pox meso) transcription factors in the ventrolateral domain, and Msx family transcription factors in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. Shh acts antagonistically to Bmp4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibit the differentiation of the spinous process (Watanabe, 1998).

In spite of the fact that vertebrae are formed by a single cell type, cartilage, their development involves different molecular pathways according to the vertebral region considered. The ventrolateral part of the vertebra (i.e. vertebral body and neural arches) develops from the ventral sclerotomal cells that express the transcription factor Pax1 before the onset of chondrogenesis. Previous work has shown that chondrogenesis of the ventrolateral part of the vertebra takes place under the influence of the notochord: a supernumerary notochord grafted dorsomedially to the somite extends the Pax1-expressing somitic domain dorsally, and subsequently its differentiation into cartilage takes place to the point that the development of the dorsal somitic derivatives (i.e. the dermomyotome) can be totally suppressed. The most dorsal part of the vertebra that closes the vertebral arch differentiates from mesenchymal cells of somitic origin. This occurs between two ectodermal layers: the superficial ectoderm and the roof plate. Thus, the unilateral graft of quail somites into chick embryos results in the formation of chimeric vertebrae with a hemivertebral body and hemispinous process, and a neural arch made up of donor cells on the operated side and host cells on the intact side. The limit between the host's and donor's territories corresponds strikingly to the sagittal plane of the embryo. Therefore, somitic cells with a chondrogenic fate must migrate medially in order to surround the neural tube and form the vertebral body ventrally and the spinous process dorsally. The cells that migrate dorsally from E3 onward fail to express Pax1 but start to express Msx1 and Msx2 as they become positioned between the superficial ectoderm and the roof plate, which produces BMP4. Moreover, the lateral graft of a roof plate or of cells producing BMP4 induces ectopic expression of Msx genes in the host somitic mesenchyme. Such an induction, however, can occur only if the inducer (e.g. the roof plate) is placed in close proximity to the superficial ectoderm. This supports the contention that bone formation in the subcutaneous site, where the spinous process is formed, is under the control of BMP4, and that Msx genes are involved in the pathway leading to chondrogenesis. This view was confirmed by the fact that overexpression of BMP4 (or of the closely related compound BMP2) dorsal to the neural tube results in the expansion of the Msx1- and Msx2-positive mesenchymal territory and subsequently in the enlargement of the spinous process. Duality in vertebral chondrogenesis was further underlined by the opposite effect of BMPs on the development of the ventrolateral part of the vertebra. Chondrogenesis was strongly inhibited by the graft of BMP2/4-producing cells in a ventrolateral position, with respect to the neural tube (Watanabe, 1998 and references).

These observations raised the question of the nature of the factor of notochord/floor plate origin that is responsible for chondrogenesis in the ventrolateral domain of the vertebra. The most obvious candidate was the protein SHH. Lateral grafts of SHH-producing cells do indeed enhance Pax1 expression in sclerotomal cells and induce the over-development of cartilage laterally at the level of the neural arches. The positive influence of SHH protein on Pax1 expression by somitic cells has already been demonstrated by in vitro experiments and in vivo by the use of retroviral vectors controlling Shh gene expression. This paper demonstrates that enhancement of the number of Pax1-expressing cells by SHH is followed in vivo by the increase in size of the ventrolateral part of the vertebral cartilage. In contrast, dorso-medial grafts of notochord and of SHH-QT6 cells inhibit the expression of the Bmp4 gene in dorsal ectoderm, dorsal mesenchyme and roof plate. Since Msx gene expression has been shown to be controlled by BMP signaling in several induction systems, it is probable that, under the experimental conditions described here, the inhibition of Bmp4 expression is primarily responsible for that of Msx1 and Msx2 and for the failure of chondrogenesis in the dorsal part of the vertebra. This leads to the identification of two molecular pathways in bone development. They concern cartilage and bone formation in 'deep' and 'subectodermal' positions, respectively. Ectoderm has previously been shown to reduce or inhibit chondrogenesis in somitic explant cultures. Such an inhibition is proposed to be relieved by the local production of BMP4 by the dorsal ectoderm and neural tube, thus allowing the formation of superficial bony structures from mesodermal (or mesectodermal) mesenchyme to take place. The deep vertebral cartilage that develops at a distance from the ectoderm and surrounds the notochord and the ventrolateral part of the neural tube requires SHH signaling to differentiate from the sclerotome (Watanabe, 1998 and references).

The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, the mechanism controlling its spatial expression has been studied in the zebrafish. An enhancer screening strategy in zebrafish embryos was employed, based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in introns 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo reveals that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in introns 1 and 2, respectively. These regions mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three 40 bp regions each essential for activity. These regions do not contain binding sites for HNF3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos (Muller, 1999).

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGFbeta family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in the specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Cyc signaling can act directly on shh expression in neural tissue. Modulation of the Cyc signaling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. These data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube (Muller, 2000).

The data best fit a model in which Cyc signaling is required for the establishment of shh expression in the neuroectoderm. This mode of regulation also operates presumably on the paralogous gene twhh. Hh signaling, as assessed by ectopic induction of floor plate marker genes, such as netrin1 and axial (HNF3 beta), is not impaired in cyc mutant embryos or in embryos injected with FAST-1 SID. A two-step model is proposed to explain the lack of floor plate and ventral brain identity in cyc mutant embryos. The first step requires the action of Cyc signalling to establish shh (twhh) expression in the ventral neural tube. Once turned on in the neural tube, local Hh signals lead to activation of the downstream Hh target genes netrin1, pax2.1 or axial, a step that can occur in the absence of Cyc signaling. These data, however, do not rule out that these genes can also be activated directly by Cyc signaling in an Hh-independent manner. The data imply an involvement of FAST-1 in the regulation of shh. In agreement with this, schmalspur, which has a ventral neural phenotype very similar to cyc mutants, encodes Fast-1/FoxH1, a zebrafish homolog of FAST-1. It remains to be established whether Smad2/FAST-1 interacts directly with the shh enhancer. Several homologies to the binding sites of FAST-1 (FAST binding elements, FBE are present in the -2430/-563 region. In one model, Shh is envisaged to activate HNF3beta in the neural tube, which would then turn on shh expression. HNF3beta is also a target of activins and may be responsive to Cyc/Nodal signals (Muller, 2000).

The secreted protein sonic hedgehog is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified that direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. The activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2-/-embryos, it is absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon (Epstein, 2000).

Significant differences have been identified between Drosophila and mammals in the use of pathways that mediate ventral midline induction and downstream signaling properties. For instance in Drosophila, sim is expressed in cells fated to make up the ventral midline and is required for their formation. Sim functions by regulating a number of midline-specific genes including spitz, a secreted TGFalpha-like molecule that operates in a graded distribution in the ectoderm to establish distinct cell fates. This contrasts with floor plate induction within the spinal cord of higher vertebrates, which appears to be independent of Sim function and reliant on graded Shh signaling for the specification of distinct neuronal fates. hedgehog is expressed in the Drosophila neurectoderm, however it is localized to transverse stripes and does not play a role in signaling from the ventral midline. Although, the genes involved in ventral midline induction differ between the two organisms, the employment of a patterning strategy that relies on the graded response to a factor secreted from the ventral midline is a feature common to both. Given the many similarities between ventral midline cells of the CNS in Drosophila and mouse, it is rather intriguing that a role for Sim2 in ventral midline determination has re-emerged in vertebrates through its ability to regulate Shh expression in the ventral diencephalon. Whether this points to a conserved role for Sim2 or an example of convergent evolution remains to be determined (Epstein, 2000).

The Rel/NF-kappaB gene family encodes a large group of transcriptional activators involved in myriad differentiation events, including embryonic development. Xrel3, a Xenopus Rel/NF-kappaB-related gene, is expressed in the forebrain, dorsal aspect of the mid- and hind-brain, the otocysts and notochord of neurula and larval stage embryos. Overexpression of Xrel3 causes formation of embryonic tumors. Xrel3-induced tumors and animal caps from embryos injected with Xrel3 RNA express Otx2, Shh and Gli1. Heterodimerization of a C-terminally deleted mutant of Xrel3 with wild-type Xrel3 inhibits in vitro binding of wild-type Xrel3 to Rel/NF-kappaB consensus DNA sequences. This dominant interference mutant disrupts Shh, Gli1 and Otx2 mRNA patterning and inhibits anterior development when expressed in the dorsal side of zygotes: anterior development is rescued by co-injecting wild-type Xrel3 mRNA. In chick development, Rel activates Shh signaling, which is required for normal limb formation -- Shh, Gli1 and Otx2 encode important neural patterning elements in vertebrates. The activation of these genes in tumors by Xrel3 overexpression and the inhibition of their expression and head development by a dominant interference mutant of Xrel3 indicates that Rel/NF-kappaB is required for activation of these genes and for anterior neural patterning in Xenopus (Lake, 2001).

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. Development of the mandibular arch of Prx1-/-Prx2-/- mutants was investigated to obtain insight into the molecular basis of the lower jaw abnormalities. In mutant embryos a local decrease in proliferation of mandibular arch mesenchyme is observed in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression is strongly reduced, indicative of a function for Prx genes in indirect regulation of Shh. Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopy the lower jaw defects of Prx1-/-Prx2-/- mutants. In addition, this treatment leads to loss of the mandibular incisors. A model is presented that describes how loss of Shh expression in Prx1-/-Prx2-/- mutants leads to abnormal morphogenesis of the mandibular arch. According to this model, Prx1 and Prx2 expression in mandibular mesenchyme is required to stimulate the expression of Shh in the medial domain of the oral epithelium via an as yet unknown intermediate. The Shh protein then promotes cell proliferation in part of the underlying mesenchyme, which is required for correct morphogenesis. In the Prx1-/-Prx2-/- mutant, reduction of Shh expression leads to reduction of mesenchymal proliferation in the medial-oral region. This lack of proliferation causes a malformation of the mandibular processes such that the oral region develops in a more medial position, and the aboral region in a more lateral position. Consequently, the oral expression domains of Dlx2, Alx3 and Fgf8 shift medially, while the aboral domains of Dlx2 and Alx3 shift laterally. Fgf8 expression in the medial region subsequently induces the formation of the medial incisor (ten Berge, 2001).

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. Msx1-deficient mice have been used as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. Msx1 expression is restricted to the anterior of the first upper molar site in the palatal mesenchyme and Msx1 is required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicate that the cleft palate seen in Msx1 mutants results from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1–/– palatal mesenchyme rescues the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate is a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicate that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme, which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. These findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations (Zhang, 2002).

The role of Notch signalling was analyzed during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway it was found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. It is proposed that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favors floor plate development at the expense of the notochord, preferentially before mid gastrula. Evidence is presented that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. Notch signalling is shown to require Presenilin to modulate this switch (López, 2003).

Much of the skeleton and connective tissue of the vertebrate head is derived from cranial neural crest. During development, cranial neural crest cells migrate from the dorsal neural tube to populate the forming face and pharyngeal arches. Fgf8 and Shh, signaling molecules known to be important for craniofacial development, are expressed in distinct domains in the developing face. Specifically, in chick embryos these molecules are expressed in adjacent but non-overlapping patterns in the epithelium covering crest-derived mesenchyme that will give rise to the skeletal projections of the upper and lower beaks. It has been suggested that these molecules play important roles in patterning the developing face. The ability of FGF8 and SHH signaling, singly and in combination, to regulate cranial skeletogenesis, has been examined both in vitro and in vivo. SHH and FGF8 were found to have strong synergistic effects on chondrogenesis in vitro and are sufficient to promote outgrowth and chondrogenesis in vivo, suggesting a very specific role for these molecules in producing the elongated beak structures during chick facial development (Abzhanov, 2004).

Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these interactions, the Sonic hedgehog (SHH) pathway was activated in the brain. Beginning 72 hours after activation of the SHH pathway, growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, it was observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered. Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to hypoplasia of the skeleton, and this phenotype was reproduced when the FGF pathway was blocked. Thus, the brain establishes multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as a result of disease can alter these centers and thereby generate morphological variation (Hu, 2009).

Hedgehog and vascularization

The first vasculature of the developing vertebrate embryo forms by assembly of endothelial cells into simple tubes from clusters of mesodermal angioblasts. Maturation of this vasculature involves remodeling, pruning and investment with mural cells. Hedgehog proteins are part of the instructive endodermal signal that triggers the assembly of the first primitive vessels in the mesoderm. A combination of genetic and in vitro culture methods was used to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. Bmps, in particular Bmp4, are crucial for vascular tube formation, Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1 (Drosophila homolog: Biniou), and the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. In the allantois. genetic disruption of hedgehog signaling (Smo-/-) has no effect on Foxf1 expression, and neither Bmp4 expression nor vasculogenesis are disturbed. By contrast, targeted inactivation of Foxf1 leads to loss of allantoic Bmp4 and vasculature. In vitro, the avascular Foxf1-/- phenotype can be rescued by exogenous Bmp4, and vasculogenesis in wild-type tissue can be blocked by the Bmp antagonist noggin. Hedgehogs are required for activation of Foxf1, Bmp4 expression and vasculogenesis in the yolk sac. However, vasculogenesis in Smo-/- yolk sacs can be rescued by exogenous Bmp4, consistent with the notion that the role of hedgehog signaling in primary vascular tube formation is as an activator of Bmp4, via Foxf1 (Astorga, 2006).

Choroid plexuses (ChPs) are vascularized secretory organs involved in the regulation of brain homeostasis, and function as the blood-cerebrospinal fluid (CSF) barrier. Despite their crucial roles, there is limited understanding of the regulatory mechanism driving ChP development. Sonic hedgehog (Shh), a secreted signal crucial for embryonic development and cancer, is strongly expressed in the differentiated hindbrain ChP epithelium (hChPe). However, a distinct epithelial domain in the hChP that does not express Shh, but displays Shh signaling, has been identified. This distinct Shh target field that adjoins a germinal zone, the lower rhombic lip (LRL), functions as a progenitor domain by contributing directly to the hChPe. By conditional Shh mutant analysis, it was shown that Shh signaling regulates hChPe progenitor proliferation and hChPe expansion through late embryonic development, starting around E12.5. Whereas previous studies show that direct contribution to the hChPe by the LRL ceases around E14, these findings reveal a novel tissue-autonomous role for Shh production and signaling in driving the continual growth and expansion of the hindbrain choroid plexus throughout development (X. Huang, 2009).

Hedgehog and neural crest

To define the timing of neural crest formation, the fate of presumptive neural crest cells was challenged by grafting notochords, Sonic Hedgehog- (Shh) or Noggin-secreting cells at different stages of neurulation in chick embryos. Notochords or Shh-secreting cells are able to prevent neural crest formation at open neural plate levels, as assayed by DiI-labeling and expression of the transcription factor Slug, suggesting that neural crest cells are not committed to their fate at this time. In contrast, the BMP signaling antagonist, Noggin, does not repress neural crest formation at the open neural plate stage, but does so if injected into the lumen of the closing neural tube. The period of Noggin sensitivity corresponds to the time when BMPs are expressed in the dorsal neural tube but are down-regulated in the non-neural ectoderm. To confirm the timing of neural crest formation, either Shh or Noggin were added to neural folds at defined times in culture. Shh inhibits neural crest production at early stages (0-5 hours in culture), whereas Noggin exerts an effect on neural crest production only later (5-10 hours in culture). These results suggest three phases of neurulation that relate to neural crest formation: (1) an initial BMP-independent phase that can be prevented by Shh-mediated signals from the notochord; (2) an intermediate BMP-dependent phase around the time of neural tube closure, when BMP-4 is expressed in the dorsal neural tube, and (3) a later pre-migratory phase, which is refractory to exogenous Shh and Noggin (Selleck, 1998).

Hedgehog signal transduction is directly required in zebrafish neural crest-derived dorsal root ganglia (DRG) precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursor (Ungos, 2003).

Neural crest cells that ultimately populate the DRG migrate ventrally on the medial pathway along with sympathetic and pigment cell precursors. However, at a point adjacent to the notochord, sensory precursors stop and return dorsally to the position of the DRG where they begin to express ngn1. The timing of onset of ngn1 expression suggests that Hh signals emanating from the notochord and/or neural tube may be involved in initiation of ngn1 expression in DRG precursors. Ngns are known to be sufficient for conferring neuronal identity on uncommitted precursors. Furthermore, Ngns are thought to reinforce the neuronal program by inhibiting genes necessary for gliogenesis. This difference in migration behavior between sensory precursors and autonomic and pigment cell precursors further suggests that DRG precursors are already predisposed to respond to Hh signals early in their migration. Rather than biasing neural crest cells toward a sensory fate, Hh signaling may be influencing DRG precursors to adopt a neuronal cell fate by promoting ngn1 expression (Ungos, 2003).

Neural crest cells that form the vertebrate head skeleton migrate and interact with surrounding tissues to shape the skull, and defects in these processes underlie many human craniofacial syndromes. Signals at the midline play a crucial role in the development of the anterior neurocranium, which forms the ventral braincase and palate; this study examines the role of Hedgehog (Hh) signaling in this process. Using sox10:egfp transgenics to follow neural crest cell movements in the living embryo, and vital dye labeling to generate a fate map, it was shown that distinct populations of neural crest form the two main cartilage elements of the larval anterior neurocranium: the paired trabeculae and the midline ethmoid. By analyzing zebrafish mutants that disrupt sonic hedgehog (shh) expression, it was demonstrated that shh is required to specify the movements of progenitors of these elements at the midline, and to induce them to form cartilage. Treatments with cyclopamine, to block Hh signaling at different stages, suggest that although requirements in morphogenesis occur during neural crest migration beneath the brain, requirements in chondrogenesis occur later, at the time that cells form separate trabecular and ethmoid condensations. Cell transplantations indicate that these also reflect different sources of Shh, one from the ventral neural tube that controls trabecular morphogenesis and one from the oral ectoderm that promotes chondrogenesis. These results suggest a novel role for Shh in the movements of neural crest cells at the midline, as well as in their differentiation into cartilage, and help to explain why both skeletal fusions and palatal clefting are associated with the loss of Hh signaling in holoprosencephalic humans (Wada, 2005).

Hedgehog and hypothalamus development

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. This study reports on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. The downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. Forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2+-proliferative progenitor state. These data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain (Manning, 2007).

Hedgehog and pituitary development

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. The role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland has been investigated using loss- and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke's pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. Oral ectoderm/Rathke's pouch-specific 5' regulatory sequences (Pitx1HS) from the bicoid related pituitary homeobox gene (Pitx1) were used to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling. It was found that SHH is required for proliferation of the pituitary gland. In addition, evidence is provided that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke's pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, the data suggest that Hedgehog signaling, together with FGF8/10 signaling that arises from the dorsally located infundibulum, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development (Treier, 2001).

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, four cDNAs were cloned encoding different pituitary hormones [ prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh)] and their expression patterns were analyzed relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, no pituitary-specific invagination equivalent to Rathke's pouch formation appears to take place. Rather, pituitary cells move inward, together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Loss- and gain-of-function experiments has shown that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes (Herzog, 2003).

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog, FGF, and BMP families. To better understand when and where Hh signaling influences pituitary development, the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants), has been examined. While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, the data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. The Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. Zebrafish Hh pathway mutants were used to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. These results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland (Sbrogna, 2003).

Facial abnormalities in human SHH mutants have implicated the Hedgehog (Hh) pathway in craniofacial development, but early defects in mouse Shh mutants have precluded the experimental analysis of this phenotype. Hh-responsiveness has been removed specifically in neural crest cells (NCCs), the multipotent cell type that gives rise to much of the skeleton and connective tissue of the head. Hh-responsiveness was removed from the entire neural crest lineage by crossing mice harboring Wnt1-Cre with those that are conditionally null for Smoothened (Smo), an obligatory and cell-autonomous component of Hh signal transduction in responding tissue. In these mutants, many of the NCC-derived skeletal and nonskeletal components are missing, but the NCC-derived neuronal cell types are unaffected. Although the initial formation of branchial arches (BAs) is normal, expression of several Fox genes, specific targets of Hh signaling in cranial NCCs, is lost in the mutant. The spatially restricted expression of Fox genes suggests that they may play an important role in BA patterning. Removing Hh signaling in NCCs also leads to increased apoptosis and decreased cell proliferation in the BAs, which results in facial truncation that is evident by embryonic day 11.5 (E11.5). Together, these results demonstrate that Hh signaling in NCCs is essential for normal patterning and growth of the face. Further, this analysis of Shh-Fox gene regulatory interactions leads to the proposal that Fox genes mediate the action of Shh in facial development (Jeong, 2004).

These data indicate that Hh signaling regulates ectomesenchymal expression of five Fox genes, Foxc2, Foxd1, Foxd2, Foxf1, and Foxf2. Although several of these have been reported to be induced by Shh in somites, foregut, or tissue culture, little attention has been given to their expression in facial primordia. Consequently, prior to this work, Foxc2 was the only one of these members that had been shown to be transcribed downstream of Hh signaling in the ectomesenchyme. Based on these findings, it is proposed that the Fox genes are the major mediators of the function of Hh signaling in craniofacial morphogenesis. Further support for this model comes from the mutant phenotype of Foxc2. The head skeleton of Foxc2 mutants exhibits defects that overlap those of Wnt1-Cre;Smon/c mutants, suggesting that the loss of Foxc2 expression can account for at least part of the phenotype of Wnt1-Cre;Smon/c embryos. In particular, the absence of the palate components (palatal process of the maxilla and palatine) and the middle ear ossicles (incus and stapes) correlates with the expression of Foxc2 in the MXA and second BAs. Foxf2 mutants also have a cleft palate, although this is likely to be secondary to the influence of Foxf2 on tongue morphogenesis. In contrast, no craniofacial abnormalities were reported in the mutants of either Foxd1 or Foxd2. This lack of an overt phenotype could be due to a functional redundancy between these or other Fox family members that obscures their importance. Unfortunately, the early lethality caused by mutation of Foxf1 precludes an assessment of its role in facial development (Jeong, 2004).

Although the transcription of Foxc2, Foxd1, Foxd2, Foxf1, and Foxf2 are clearly all under the positive regulation by Hh signaling in facial primordia, the Fox genes are dissimilar from one another in their normal expression patterns. Furthermore, in the ectomesenchyme of Wnt1-Cre;R26SmoM2 embryos, the distribution and level of each Fox gene transcripts are spatially regulated despite the uniform activation of the Hh pathway; in the MNA, Foxc2 and Foxd1 are expressed ubiquitously except at the midline, whereas Foxf1 is excluded from the lateral ends. Foxd2 and Foxf2 are both expressed along the entire mediolateral axis, but Foxd2 has an increasing, and Foxf2 a decreasing, gradient of intensity from medial to lateral. These observations suggest that Fox genes may be at the regulatory intersection between a Hh pathway input and that of another signaling activity present in a mediolateral gradient in the MNA. For example, if a hypothetical signaling molecule forms an increasing concentration gradient from medial to lateral, then induction of Foxc2, Foxd1, and Foxd2, and repression of Foxf1 and Foxf2 at different thresholds could result in the Fox gene expression patterns described above (Jeong, 2004).

How do Fox genes participate in craniofacial development? First, they may be functionally redundant permissive factors that serve common needs of cells such as survival or proliferation. In this case, a certain amount of Fox protein may be required in order for a cranial neural crest cell to participate in facial morphogenesis, but the exact combination of Fox proteins may not be important. Further, when more than one Fox gene is expressed in the same cell, inactivating one of these may or may not produce abnormalities, depending on its expression level and the sensitivity of the particular cell to the overall Fox gene dosage. Alternatively, certain combinations of Fox genes may have instructive information specifying distinct cell fates. When combined together, the unique expression patterns of each Fox gene make an intriguing map of 'Fox codes' in the developing face. How these domains defined by different Fox codes correlate with facial structures is not clear, because a fate map of facial development is not yet available. However, if one assumes that the relative positions of the facial element precursor domains at E10.5 are the same as those of the facial elements in the newborns, this leads to some interesting predictions. For example, the mesenchyme around the first pharyngeal cleft is expected to make the skeleton associated with the otic capsule, such as stapes, malleus, gonial bone, and tympanic ring. This mesenchyme expresses Foxc2 + Foxd1 + Foxd2. On the other hand, the tongue arises at the midline of the MNAs, where Foxf1, but none of these three Fox genes, is expressed. The domain anterior to the tongue, where the lower incisors form, has still another Fox code, Foxd1 + Foxd2 + Foxf1 + Foxf2. All these facial structures are lost in Wnt1-Cre;Smon/c embryos, consistent with all the Fox codes being lost. The absence of craniofacial defects in Foxd1 or Foxd2 mutants could be explained by some degree of tolerance in the Fox codes, which would allow more than one combination to encode the same element. The molars and the body of the dentary apparently develop outside of the Fox gene expression domains. Accordingly, they are present in Wnt1-Cre;Smon/c heads, suggesting that they are specified by other mechanisms. Similar correlations can be found for the MXA-derived elements, but not for the FNP-derived ones. The FNP derivatives (nasal bone, nasal cartilage, premaxilla, and upper incisor) suffer relatively mild defects in Wnt1-Cre;Smon/c embryos, where none of them are completely lost. Furthermore, no defects in these structures are observed in Foxc2–/– embryos. Therefore, it is speculated that unlike the first and second BAs, specification of individual skeletal elements in the FNP is independent of Hh signaling or Fox gene expression in the ectomesenchyme, though FNP growth is dependent on Hh signaling. Clearly, distinguishing between the two models for Fox gene function will require additional loss-of-function, gain-of-function, and gene swapping experiments (Jeong, 2004).

Cardiac outflow tract (OFT) septation is crucial to the formation of the aortic and pulmonary arteries. Defects in the formation of the OFT can result in serious congenital heart defects. Two cell populations, the anterior heart field (AHF) and cardiac neural crest cells (CNCCs), are crucial for OFT development and septation. In this study, a series of tissue-specific genetic manipulations were used to define the crucial role of the Hedgehog pathway in these two fields of cells during OFT development. These data indicate that endodermally-produced Shh ligand is crucial for several distinct processes, all of which are required for normal OFT septation. (1) Shh is required for CNCCs to survive and populate the OFT cushions; (2) Shh mediates signaling to myocardial cells derived from the AHF to complete septation after cushion formation; (3) endodermal Shh signaling is required in an autocrine manner for the survival of the pharyngeal endoderm, which probably produces a secondary signal required for AHF survival and for OFT lengthening. Disruption of any of these steps can result in a single OFT phenotype (Goddeeris, 2007).

Table of contents

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

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