Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog

An early step in the development of vertebrae, ribs, muscle, and dermis is the differentiation of the somitic mesoderm into dermomyotome dorsally and sclerotome ventrally. To analyze this process, we have developed an in vitro assay for somitic mesoderm differentiation. We show that sclerotomal markers can be induced by a diffusible factor secreted by notochord and floor plate and that heterologous cells expressing Sonic hedgehog (shh/vhh-1) mimic this effect. In contrast, expression of dermomyotomal markers can be caused by a contact-dependent signal from surface ectoderm and a diffusible signal from dorsal neural tube. Our results extend previous studies by suggesting that dorsoventral patterning of somites involves the coordinate action of multiple dorsalizing and ventralizing signals and that a diffusible form of Shh/Vhh-1 mediates sclerotome induction.     

Postnatal development under conditions of simulated weightlessness and space flight

The axial structures, 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. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, 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. We found that 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. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could 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 Nkr-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial structures but that Shh by itself is not capable of inducing or maintaining it. We suggest 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.     

Wnt signaling from the dorsal neural tube is required for the formation of the medial dermomyotome

Signals originating from tissues surrounding somites are involved in mediolateral and dorsoventral patterning of somites and in the differentiation of the myotome. Wnt-1 and Wnt-3a, which encode members of the Wnt family of cystein-rich secreted signaling molecules, are coexpressed at the dorsal midline of the developing neural tube, an area adjacent to the dorsomedial portion of the somite. Several lines of evidence indicate that Wnt-1 and Wnt-3a have the ability to induce the development of the medial and dorsal portion of somites, as well as to induce myogenesis. To address whether these Wnt signalings are really essential for the development of somites during normal embryogenesis, we investigated the development of somites in mouse embryos lacking both Wnt-1 and Wnt-3a. Here we demonstrate that the medial compartment of the dermomyotome is not formed and the expression of a lateral dermomyotome marker gene, Sim-1, is expanded more medially in the absence of these Wnt signalings. In addition, the expression of a myogenic gene, Myf-5, is decreased at 9.5 days post coitum whereas the level of expression of a number of myogenic genes in the later stage appeared normal. These results indicate that Wnt-1 and Wnt-3a signalings actually regulate the formation of the medial compartment of the dermomyotome and the early part of myogenesis.     

Sonic hedgehog promotes somitic chondrogenesis by altering the cellular response to BMP signaling

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. We show here that presomitic mesoderm (psm) cultured in the presence of Shh will differentiate into cartilage, and that the later stages of this differentiation process specifically depend on BMP signaling. We find that Shh not only acts in collaboration with BMPs to induce cartilage, but that 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. Our 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.     

Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show that Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.     

The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction

The steroidal alkaloid cyclopamine produces cyclopia and holoprosencephaly when administered to gastrulation-stage amniote embryos. Cyclopamine-induced malformations in chick embryos are associated with interruption of Sonic hedgehog (Shh)-mediated dorsoventral patterning of the neural tube and somites. Cell types normally induced in the ventral neural tube by Shh are either absent or appear aberrantly at the ventral midline after cyclopamine treatment, while dorsal cell types normally repressed by Shh appear ventrally. Somites in cyclopamine-treated embryos show Pax7 expression throughout, indicating failure of sclerotome induction. Cyclopamine at concentrations of 20-100 nM blocks the response of neural plate explants to recombinant Shh-N in a dose-dependent manner. Similar concentrations have no effect on the post-translational modification of Shh by cholesterol in transfected COS-1 cells. Comparison of the effects of cyclopamine to those of the holoprosencephaly-inducing cholesterol synthesis inhibitor AY-9944 shows that cyclopamine does not induce malformations by interfering with cholesterol metabolism. Although AY-9944 does not interrupt Shh signaling in ovo, it blocks the response to Shh-N in explants cultured without an exogenous cholesterol source. As predicted by current models of the regulation of cholesterol metabolism, the response to Shh-N in AY-9944-treated explants is restored by providing exogenous cholesterol. However, exogenous cholesterol does not restore Shh signaling in cyclopamine-treated explants. These findings suggest that cyclopamine-induced teratogenesis is due to a more direct antagonism of Shh signal transduction.     

The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail

The zebrafish locus one-eyed pinhead (oep) is essential for the formation of anterior axial mesoderm, endoderm and ventral neuroectoderm. At the beginning of gastrulation anterior axial mesoderm cells form the prechordal plate and express goosecoid (gsc) in wild-type embryos. In oep mutants the prechordal plate does not form and gsc expression is not maintained. Exposure to lithium, a dorsalizing agent, leads to the ectopic induction and maintenance of gsc expression in wild-type embryos. Lithium treatment of oep mutants still leads to ectopic gsc induction but not maintenance, suggesting that oep acts downstream of inducers of dorsal mesoderm. In genetic mosaics, wild-type cells are capable of forming anterior axial mesoderm in oep embryos, suggesting that oep is required in prospective anterior axial mesoderm cells before gastrulation. The oep gene is also essential for endoderm formation and the early development of ventral neuroectoderm, including the floor plate. The loss of endoderm is already manifest during gastrulation by the absence of axial-expressing cells in the hypoblast of oep mutants. These findings suggest that oep is also required in lateral and ventral regions of the gastrula margin. The sonic hedgehog (shh).gene is expressed in the notochord of oep animals. Therefore, the impaired floor plate development in oep mutants is not caused by the absence of the floor plate inducer shh. This suggests that oep is required downstream or in parallel to shh signaling. The ventral region of the forebrain is also absent in oep mutants, leading to severe cyclopia. In contrast, anterior-posterior brain patterning appears largely unaffected, suggesting that underlying prechordal plate is not required for anterior-posterior pattern formation but might be involved in dorsoventral brain patterning. To test if oep has a wider, partially redundant role, we constructed double mutants with two other zebrafish loci essential for patterning during gastrulation. Double mutants with floating head, the zebrafish Xnot homologue, display enhanced floor plate and adaxial muscle phenotypes. Double mutants with no tail (ntl), the zebrafish homologue of the mouse Brachyury locus, display severe defects in midline and mesoderm formation including absence of most of the somitic mesoderm. These results reveal a redundant function of oep and ntl in mesoderm formation. Our data suggest that both oep and ntl act in the blastoderm margin to specify mesendodermal cell fates.     

Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that 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. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.     

Evidence for a frizzled-mediated wnt pathway required for zebrafish dorsal mesoderm formation

We have used zebrafish as a model system for the study of vertebrate dorsoventral patterning. We isolated a maternally expressed and dorsal organizer localized member of the frizzled family of wnt receptors. Wild-type and dominant, loss-of-function molecules in misexpression studies demonstrate frizzled function is necessary and sufficient for dorsal mesoderm specification. frizzled activity is antagonized by the action of GSK-3, and we show GSK-3 is also required for zebrafish dorsal mesoderm formation. frizzled cooperatively interacts with the maternally encoded zebrafish wnt8 protein in dorsal mesodermal fate determination. This frizzled -mediated wnt pathway for dorsal mesoderm specification provides the first evidence for the requirement of a wnt-like signal in vertebrate axis determination.     

Formation and specification of ventral neuroblasts is controlled by vnd in Drosophila neurogenesis

During Drosophila neural development, neuroblasts delaminate from the neuroectoderm of each hemisegment in a stereotypic orthogonal array of five rows and three columns (ventral, intermediate, and dorsal). Prevailing evidence indicates that the individual neuroblast fate is determined by the domain-specific expression of genes along the dorsoventral and anteroposterior axis. Here, we analyze the role of Vnd, a NK-2 homeodomain protein, expressed initially in the ventral neuroectoderm adjacent to the ventral midline, in the dorsoventral patterning of the neuroectoderm and the neuroblasts. We show that in vnd null mutants most ventral neuroblasts do not form and the few that form do not develop ventral fates, but instead develop intermediate-like fates. Furthermore, we demonstrate that Vnd influences the gene expression patterns in the ventral proneural clusters and neuroectoderm, and that its action in neuroblast formation includes, but is not exclusive to the activation of proneural AS-C genes. Through the use of GAL4/UAS gene-expression system we show that ectopic Vnd expression can promote ventral-like fates in intermediate and dorsal neuroblasts and can suppress certain normal characteristics of the intermediate and dorsal neuroectoderm. Our results are discussed in the context of the current evidence in dorsoventral patterning in the Drosophila neuroectoderm.     

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.     

Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro

The Wnt family of secreted signaling proteins are implicated in regulating morphogenesis and tissue patterning in a wide variety of organ systems. Several Wnt genes are expressed in the developing limbs and head, implying roles in skeletal development. To explore these functions, we have used retroviral gene transfer to express Wnt-1 ectopically in the limb buds and craniofacial region of chick embryos. Infection of wing buds at stage 17 and tissues in the head at stage 10 resulted in skeletal abnormalities whose most consistent defects suggested a localized failure of cartilage formation. To test this hypothesis, we infected micromass cultures of prechondrogenic mesenchyme in vitro and found that expression of Wnt-1 caused a severe block in chondrogenesis. Wnt-7a, a gene endogenously expressed in the limb and facial ectoderm, had a similar inhibitory effect. Further analysis of this phenomenon in vitro showed that Wnt-1 and Wnt-7a had mitogenic effects only in early prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema occurred normally, and that the block to differentiation was at the late-blastema/early-chondroblast stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence skeletal morphology by localized inhibition of chondrogenesis.     

Eph signaling is required for segmentation and differentiation of the somites

Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. Here we show a role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. We have examined the role of these proteins by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. We suggest that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites.     

Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus

The marginal zone is a ring of tissue that gives rise to a characteristic dorsoventral pattern of mesoderm in amphibian embryos. Bmp-4 is thought to play an important role in specifying ventral mesodermal fate. Here we show (1) that different doses of Bmp-4 are sufficient to pattern four distinct mesodermal cell types and to pattern gene expression in the early gastrula marginal zone into three domains, (2) that there is a graded requirement for a Bmp signal in mesodermal patterning, and (3) that Bmp-4 has long-range activity which can become graded in the marginal zone by the antagonizing action of noggin. The results argue that Bmp-4 acts as a morphogen in dorsoventral patterning of mesoderm.     

Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the msh homeobox gene. Here we describe a new Drosophila homeobox gene, intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. Vertebrate genes closely related to vnd (Nkx2.1 and Nkx2.2), ind (Gsh1 and Gsh2), and msh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved.     

The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos

Segmentation of the vertebrate embryo begins when the paraxial mesoderm is subdivided into somites, through a process that remains poorly understood. To study this process, we have characterized X-Delta-2, which encodes the second Xenopus homolog of Drosophila Delta. Strikingly, X-Delta-2 is expressed within the presomitic mesoderm in a set of stripes that corresponds to prospective somitic boundaries, suggesting that Notch signaling within this region establishes a segmental prepattern prior to somitogenesis. To test this idea, we introduced antimorphic forms of X-Delta-2 and Xenopus Suppressor of Hairless (X-Su(H)) into embryos, and assayed the effects of these antimorphs on somite formation. In embryos expressing these antimorphs, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment properly. Both antimorphs also disrupted the segmental expression of X-Delta-2 and Hairy2A, a basic helix-loop-helix (bHLH) gene, within the presomitic mesoderm. These observations suggest that X-Delta-2, via X-Notch-1, plays a role in segmentation, by mediating cell-cell interactions that underlie the formation of a segmental prepattern prior to somitogenesis.     

BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos

Bone morphogenetic protein 1 (BMP1) is a metalloproteinase closely related to Drosophila Tolloid (Tld). Tld regulates dorsoventral patterning in early Drosophila embryos by enhancing the activity of Dpp, a member of the TGF-beta family most closely related to BMP2 and BMP4. In Xenopus BMP4 appears to play an essential role in dorsoventral patterning, promoting the development of ventral fates during gastrula stages. To determine if BMP1 has a role in regulating the activity of BMP4, we have isolated cDNAs for Xenopus BMP1 and a novel closely related gene that we have called xolloid (xld). Whereas xbmp1 is uniformly expressed at all stages tested, the initial uniform expression of xld becomes localized to two posterior ectodermal patches flanking the neural plate and later to the inner ectoderm of the developing tailbud. xld is also expressed in dorsal regions of the brain during tailbud stages and is especially abundant in the ventricular layer of the dorsal hindbrain caudal to the otic vesicle. Overexpression of either gene inhibits the development of dorsoanterior structures in whole embryos and ventralizes activin-induced dorsal mesoderm in animal caps. Since ventralization of activin-induced animal caps can be blocked by coinjecting a dominant-inhibitory receptor for BMP2 and BMP4, we suggest a role for BMP1 and Xld in regulating the ventralizing activity of these molecules.     

A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.