Table of contents

DPP homologs and dorsoventral patterning: Frogs - part 2

The work of the Spemann organizer of the amphibian embryo can be subdivided into two discrete activities: trunk organizer and head organizer. Several factors, secreted from the organizer and involved in trunk organization, are thought to act by repressing Bmp signaling. With the exception of the secreted factor cerberus (a divergent member of the TGFbeta superfamily), little is known about head-organizer inducers. Co-expression of a dominant-negative Bmp receptor with inhibitors of the Wnt-signaling pathway in Xenopus leads to the induction of complete secondary axes, including a head. This induction does not require expression of the siamois marker of Nieuwkoop center signaling, suggesting that cells are directly shifted to head-organizer fate. Cerberus is a potent inhibitor of Wnt signaling. These results indicate that head-organizer activity results from the simultaneous repression of Bmp and Wnt signaling and suggest a mechanism for region-specific induction by the organizer (Glinka, 1997).

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. The Cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signaling by BMP and Wnt. In order for the head territory to form, it is proposed that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions (Piccolo, 1999).

The Xenopus homeobox gene twin is involved in the Wnt-mediated induction of Spemann's organizer. Additionally, several lines of evidence indicate that bone morphogenetic proteins (BMPs) play a role in repressing the formation of the organizer by antagonizing the expression of genes involved in organizer establishment. In order to determine at what level BMPs exert their effect, the activity of different genes expressed within the organizer region were measured. BMP signaling can antagonize the induction of the dorsal-specific gene goosecoid but is unable to affect Wnt signaling at the level of twin. These results suggest that the antagonistic activities of BMPs in organizer formation occur postzygotically, independent of twin regulation, and that Wnt-like dorsal determinant signaling pathways do not crosstalk with BMPs (Laurent, 1999).

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins have been isolated from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones are designated as zbmp-2 and zbmp-4. In gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, is also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, synthetic mRNA was injected into zebrafish embryos. Overexpression of this gene eliminates dorsal structures, including the notochord at both the morphological and molecular level. In contrast, expression of the ventral marker gene eve1 is expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts (Nikaido, 1997).

The heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. Indirect evidence is provided that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. It is proposed that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development (Nishimatsu, 1998).

The Xenopus laevis BMP-4 gene shows an evolutionarily conserved structure containing two coding exons and a leader exon. The transcripts that are detected after zygotic activation of the gene in ventral mesoderm of late blastula stage embryos either contain the leader exon or begin within the first intron. Luciferase reporter/promoter studies reveal multiple elements being required for the activation and for the spatial control of transcription. These elements are located within the upstream region and within the second intron; they interact with the most proximal basal promoter and are indispensable for transcriptional activation. The auto-activatory capacity of BMP-4 is mediated by several enhancer elements that are responsive not only to BMP-4 but also to BMP-2 signaling. Thus, BMP-2 might function as a natural activator of the BMP-4 gene in the early embryo. Since reporter activity obtained with distinct BMP-2/4 responsive promoter deletion mutants is simultaneously inhibited by the dominant negative BMP receptor as well as by chordin, it is suggested that down-regulation of the BMP-4 gene by chordin results from an interference with the auto-regulatory loop at the level of protein-protein interactions (Metz, 1998).

Bone morphogenetic protein 4 (BMP4), the vertebrate homolog of Dpp, controls the fundamental choice between neural and epidermal fates in the vertebrate ectoderm, under the control of antagonists secreted by the organizer region of the mesoderm. BMP4 can act as a morphogen, evoking distinct responses in Xenopus ectodermal cells at high and low concentrations, in a pattern consistent with the positions of the corresponding cell types in the embryo. Moreover, this complex cellular response to extracellular BMP4 concentration does not require subsequent cell-cell communication and is thus direct, as required of a classical morphogen. The same series of cell types--epidermis, cement gland and neural tissue--can be produced by progressively inhibiting endogenous BMP signaling with specific antagonists, including the organizer factor noggin. BMP4 suppresses the neural marker NCAM; high doses of BMP antagonists induce NCAM, and lower doses of antigonist induce cement gland. Expression of increasing doses of the signal transduction molecule Smad1 (Drosophila homolog Mothers against Dpp) accurately reproduces the response to BMP4 protein. Since Smads have been shown to act in the nucleus, this finding implies a direct translation of extracellular morphogen concentration into transcription factor activity. It is proposed that a graded distribution of BMP activity controls the specification of several cell types in the gastrula ectoderm, and that this extracellular gradient acts by establishing an intracellular and then nuclear gradient of Smad activity (Wilson, 1997).

Recent work suggests that signaling molecules such as activin are capable of acting at long range to establish a morphogen gradient in the amphibian embryo: responding cells activate different genes at distinct threshold levels of activin. Other signaling molecules, like BMP-4 and Xnr-2, also exert concentration-dependent effects, but these factors appear to diffuse less freely. This raises the question of whether gradients of these inducing factors are indeed established, and if so, how they are generated. BMP-4 is shown to elicit graded responses in gastrula-stage embryos. Marked clones of cells expressing BMP-4 were generated in the marginal zone of Xenopus. BMP-4 regulated Myf-5 transcripts are absent from BMP-4 expressing cells, and from cells three to five cells away, showing that BMP-4 is able to exert its influence over a range of 3-5 cell diameters. In contrast, the effects of noggin protein can be detected over at least a quarter of the embryo. An effective BMP-4 gradient is established not by diffusion of BMP-4 protein but by the long-range effects of two BMP-4 inhibitors, noggin and chordin. This provides a novel mechanism for the establishment of a morphogen gradient in vertebrate embryos (Jones, 1998).

Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. A distantly related member of the Xenopus Smad family, Smad8, has been identified that lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. An intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Smad8 can block BMP-4-mediated induction of ventral mesoderm-specific gene expression in ectodermal explants. However, overexpression of Smad8 within dorsal cells causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-beta family ligands (Nakayama, 1998b).

Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signaling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation-mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription. A Xenopus Smad (Smad6) has been identified that is 52% identical to mammalian Smad6, an inhibitory Smad. The spatial pattern of expression of Smad6 changes dynamically during embryogenesis and is similar to that of BMP-4 at the tailbud stage. Overexpression of Smad6 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to dorsalization of mesoderm and neuralization of ectoderm. Xenopus Smad6 completely blocks the activity of exogenous BMP-4, and, unlike human Smad6, partially blocks the activity of activin, in a mesoderm induction assay. Smad6 protein accumulates at the membrane in some cells but is partially or completely restricted to nuclei of most overexpressing cells. Thus Smad6 functions as an intracellular antagonist of activin and BMP-4 signaling. The finding that Smad6 protein is partially or completely restricted to the nuclei of most overexpressing cells suggests that it may employ a novel or additional mechanism of action to antagonize TGF-beta family signaling other than that reported for other inhibitory Smads (Nakayama, 1998b).

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated by the activity of an activin/BVg1-like protein and Bone Morphogenetic Proteins (BMP), respectively. Interestingly, these two TGF-beta subfamilies are found in overlapping regions during mesoderm patterning. This raises the question of how the presumptive mesodermal cells recognize the multiple TGF-beta signals and differentially interpret this information to assign a particular cell fate. The well characterized model of Xenopus mesoderm induction was exploited to determine the intracellular interactions between BMP-2/4 and activin/BVg1 signaling cascades. Using a constitutively active BMP-2/4 receptor that transduces BMP-2/4 signals in a ligand-independent fashion, it has been demonstrated that signals provided by activin/BVg1 and BMP modulate each other's activity; this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, it has been determined that the specificity of BMP-2/4 and activin/BVg1 signaling is mediated by Smad1 and Smad2, respectively. These Smads should be considered as the mediators of the intracellular antagonism between BMP-2/4 and activin/BVg1, possibly signaling through sequestration of a limited pool of Smad4. Consistent with such a mechanism, Smad4 interacts functionally with both Smad1 and -2 to potentiate their signaling activities; a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling. An activin/BVg1-dependent transcriptional complex contains both Smad2 and Smad4 and thereby provides a physical basis for the functional involvement of both Smads in TGF-beta-dependent transcriptional regulation. Thus, Smad4 plays a central role in synergistically activating activin/BVg1 and BMP-dependent transcription, and functions as an intracellular sensor for TGF-beta-related signals (Candia, 1997).

Comparative studies on a dpp related gene (bone morphogenetic protein-4 [BMP-4]) in Xenopus and mouse, point to a conserved role in specifying posteroventral mesoderm during gastrulation. Analysis of other polypeptide signaling molecules during gastrulation suggests that their interaction in the generation of the overall body plan has also been conserved during vertebrate evolution (Hogan, 1994).

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. Different doses of Bmp-4 are sufficient to pattern four distinct mesodermal cell types (notochord, muscle pronephros and blood) and to pattern gene expression in the early gastrula marginal zone into three domains. There is a graded requirement for a Bmp signal in mesodermal patterning; Bmp-4 has long-range activity which can become graded in the marginal zone by the antagonizing action of noggin. This suggests that Bmp-4 activity rather than protein distribution may be graded, with noggin and chordin being responsible for the graded activity of Bmp-4. The expression of the homeobox genes Xvent-1 and Xvent-2 are nuclear targets of Bmp-4 and potential mediators of Bmp signaling. It is unlikely that Bmp-4 signaling is sufficient to specify all mesoderm. For example, the secondary axis induced by dominant-negative Bmp receptor injections are incomplete, they do not form heads suggesting that no head mesoderm is present. Other factors for head mesoderm formation will be required, and these factors, probably eminating from the Nieuwkoop center, may not be equivalent to the absence of Bmp-4 signaling (Dosch, 1997).

BMP-4 induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation: the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4mRNA reduces Goosecoid (the vertebrate homolog of Drosophila Goosecoid) and forkhead-1(the vertebrate homolog of Drosophila forkhead) transcription in whole embryos and in activin-treated animal-pole cell explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of Goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits the mesodermal response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin (a protein that binds to and inactivates BMP-4, the vertebrate homolog of Decapentaplegic) protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm (Re'em-Kalma, 1995).

Ectopic expression of the ventralizing morphogen BMP-4 in the dorsal lip (Spemann organizer) of Xenopus embryos blocks transcription of dorsal-lip-specific early response genes. The molecular mechanism underlying the BMP-4-induced inhibition of the fork head gene XFD-1' (pintallavis) was examined. The promoter of XFD-1' contains a BMP-triggered inhibitory element (BIE) that prevents gene activation at the ventral/vegetal side of the embryo in vivo. BMP-4-induced inhibition is not direct but indirect, and is mediated by Xvent homeobox proteins. Xvent proteins have no known Drosophila homolog(s). Micro-injections of Xvent-1 RNA and XFD-1' promoter deletion mutants demonstrate that Xvent-1 mimics the effect of BMP-4 signaling, not only by suppression of the XFD-1' gene, but also by utilizing the BIE. Suppression can be reverted using a dominant-negative Xvent-1 mutant. The repressor domain localizes to the N-terminal region of the protein. Gel-shift and footprint analyses prove that Xvent-1 binds to the BIE. PCR-based target-site selection for the Xvent-1 homeodomain confirms distinct motifs within the BIE as preferential binding sites. Thus, biological and molecular data suggest that Xvent-1 acts as a direct repressor for XFD-1' transcription and mediates BMP-4-induced inhibition (Friedle, 1998).

BMP-4 is believed to play a central role in the patterning of the mesoderm by providing a strong ventral signal. As part of this ventral patterning signal, BMP-4 has to activate a number of transcription factors to fulfill this role. Among the transcription factors regulated by BMP-4 are the Xvent and the GATA genes. A novel homeobox gene has been isolated termed Xvex-1, which represents a new class of homeobox genes. Transcription of Xvex-1 initiates soon after the midblastula transition. Xvex-1 transcripts undergo spatial restriction from the onset of gastrulation to the ventral marginal zone, and the transcripts will remain in this localization, including at the tailbud stage in the proctodeum. Expression of Xvex-1 during gastrula stages requires normal BMP-4 activity as evidenced from the injection of BMP-4, Smad1, Smad5 and Smad6 mRNA and antisense BMP-4 RNA. Xvex-1 overexpression ventralizes the Xenopus embryo in a dose dependent manner. Partial loss of Xvex-1 activity induced by antisense RNA injection results in the dorsalization of embryos and the induction of secondary axis formation. Xvex-1 can rescue the effects of overexpressing the dominant negative BMP receptor. These results place Xvex-1 downstream of BMP-4 during gastrulation and suggest that Xvex-1 represents a novel homeobox family in Xenopus which is part of the ventral signaling pathway (Shapira, 1999).

Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. This study makes three comparisons. (1) A systematic comparison was made of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT-), ß-catenin-depleted (ß-catenin-) and double depleted (VegT-/ß-catenin-) embryos and early zygotic genes were placed into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. (2) The roles of the Wnt and VegT pathways in producing dorsal signals were compared. Explant co-culture experiments showed that the Wnt pathway does not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. (3) The extent to which inhibiting Smad 1 phosphorylation in one area of VegT-, or ß-catenin- embryos would rescue organizer and axis formation was measured. BMP inhibition with cm-BMP7 mRNA has no rescuing effects on VegT- embryos, while cm-BMP7 and noggin mRNA causes a complete rescue of the trunk, but not of the anterior pattern in ß-catenin- embryos. One likely missing component required for normal anterior patterning could be later BMP signaling, which would remain inhibited by the over-expression of cm-BMP7 or noggin mRNAs. Also, early organizer elements still missing in these embryos include the dorsoventral waves of Xnr1, 2 and 4 expression, the expression of siamois, Xnr3 and the correct level of expression of Xhex and Xlim1. Many studies have implicated all of these in aspects of neural, head, heart and anterior endoderm specification. The challenge is to work out the hierarchy of the regulatory networks. One simple possibility is that the early high dorsal level of Xnr expression is needed for the high level of expression of siamois, Xnr3, Xhex and Xlim1. This view is supported by the fact that a dose response of Xnr1, 2 and 4 mRNAs injected into VegT- embryos restores increasing amounts of head formation. However, the specific roles played by individual Xnrs have yet to be examined by loss-of-function analysis (Xanthos, 2002).

Transplanted Spemann's organizer induces dorsal embryonic cell fates such as the nervous system and somites, but in normal development, elimination of individual organizer signals (such as the bone morphogenetic protein [BMP] antagonists) has surprisingly modest effects on these tissues. Thus, the role of BMP antagonists may be limited to fine tuning the size of the dorsal domain. However, at least five BMP antagonists are specifically expressed in the organizer, and all can mimic aspects of organizer function, suggesting overlapping functions. The function of three BMP antagonists, chordin, noggin, and follistatin, has been depleted in Xenopus tropicalis. This results in catastrophic failure of dorsal development and expansion of ventral and posterior fates. It is concluded that BMP antagonists are required for formation of the neural plate and dorsal mesoderm. In addition, the results show that neural specification requires the continuous activity of BMP antagonists from blastula through gastrula stages (Khokha, 2005).

Table of contents

decapentaplegic: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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