Table of contents

DPP homologs and dorsoventral patterning: Echinoderms, Ascidians and Cephalochordates

To examine whether a BMP signaling pathway functions in specification of cell fates in sea urchin embryos, sea urchin BMP2/4 has been cloned, its expression in time and space has been analyzed in developing embryos, and the developmental consequences of changing its concentration has been assayed through mRNA injection experiments. These studies show that BMP4 mRNAs accumulate transiently during blastula stages, beginning around the 200-cell stage, 14 hours postfertilization. Soon after the hatching blastula stage, BMP2/4 transcripts can be detected in presumptive ectoderm, where they are enriched on the oral side. Injection of BMP2/4 mRNA at the one-cell stage causes a dose-dependent suppression of commitment of cells to vegetal fates and ectoderm differentiates almost exclusively as a squamous epithelial tissue. In contrast, NOGGIN, an antagonist of BMP2/4, enhances differentiation of endoderm, a vegetal tissue, and promotes differentiation of cells characteristic of the ciliated band, which contains neurogenic ectoderm. These findings support a model in which the balance of BMP2/4 signals produced by animal cell progeny and opposing vegetalizing signals sent during cleavage stages regulate the position of the ectoderm/ endoderm boundary. In addition, BMP2/4 levels influence the decision within ectoderm between epidermal and nonepidermal differentiation (Angerer, 2000).

BMP4 and NOGGIN misexpression cause reciprocal shifts in the allocation of cell fates along the animal-vegetal axis. BMP4 compresses the endodermal field and the ectoderm/endoderm border shifts vegetally, as does the primary mesenchyme cell (PMC) ring, whereas NOGGIN has the opposite effects. This argues that precisely controlled levels of BMP signaling are required for correct patterning along the animal-vegetal axis of sea urchin embryos, as is the case for the DV axes of both Drosophila and Xenopus embryos. In the latter systems, BMP4 is thought to behave as a morphogen, and that the fates cells adopt are sensitive to BMP4 concentration. In Xenopus embryos, although the most thoroughly studied effects of BMP signaling are on dorsal-ventral patterning of mesoderm, and epidermal versus neural specification of ectoderm, BMP4 also affects endoderm differentiation, since either CHORDIN or NOGGIN can induce expression of an endodermal marker in animal caps. In normal sea urchin embryos, the ectoderm/endoderm boundary is not conditionally specified until late blastula stages and not determined until late gastrula stage. This decision depends on cell-cell signaling, since the boundary does not conform to lineage boundaries and can regulate after removal of the archenteron. BMP2/4 expression occurs in progeny of animal blastomeres at an appropriate time to participate in ectoderm/endoderm boundary decisions (Angerer, 2000).

The ascidian tadpole larva is thought to be close to a prototype of the ancestral chordate. The vertebrate body plan is established by a series of inductive cellular interactions, whereas ascidians show a highly determinate mode of development. Recent studies however, suggest some roles of cell-cell interaction during ascidian embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, HrBMPb, an ascidian homolog of the vertebrate bone morphogenetic protein (BMP) gene, was isolated from Halocynthia roretzi. The amino acid sequence of HrBMPb closely resembles that of vertebrate BMP-2 and BMP-4 and of Drosophila Decapentaplegic (DPP). In addition to the sequence similarity, HrBMPb overexpression induced the ventralization of Xenopus embryos, suggesting functional conservation. The zygotic expression of HrBMPb is first detected at gastrulation. HrBMPb expression is maintained in some cells at the lateral edges of the neural plate, through gastrulation to neurulation, although expression in the presumptive muscle cells is downregulated. HrBMPb was not expressed in the presumptive epidermis during gastrulation. When HrBMPb mRNA is injected into fertilized Halocynthia eggs, cells that normally give rise to the neural tissue differentiate into epidermis, causing a loss of anterior neural tissue in the larva. HrBMPb might function synergistically with HrBMPa, an ascidian homologue of BMPs-5 to 8 (belonging to the 60A subclass of BMPs, a different subclass than that occupied by BMP-2, BMP-4 and Dpp). However, HrBMPb overexpression does not affect differentiation of the notochord and muscle cells (Miya, 1997).

Whereas Xenopus BMP-4 is expressed during gastrulation in the marginal zone, except for the organizer region, as well as in the entire ectoderm except for the future neural plate, the ascidian HrBMPb begins to be expressed in three pairs of cells, A9.15, A8.16 and B7.5. B7.5 gives rise to muscle and trunk ventral cells. However, HrBMPb expression in the cells is quickly downregulated after B7.5. In contrast, A8.15 and A8.16 are located at the edges of the future neural plate. A8.15 is destined to give rise to spinal cord and A8.16 to spinal cord and muscle. HrBMPb expression is retained in the descendent cells that give rise to spinal cord, but not in those that give rise to muscle cells during gastrulation and neurulation. HrBMPb is not expressed in the presumptive epidermis during gastrulation, in contrast to Xenopus Bmp-4. HrBMPb expression in some epidermal cells becomes evident after gastrulation is completed. In tailbud stage embryos, HrBMPb is expressed in the anterior ectoderm, which forms palps, and in the anterior neuroectoderm. Although these neural tissues are not formed in embryos injected with HrBMPb mRNA, neural fate seems to be specified. In vertebrate embryos, Bmp-2 and Bmp-4 are expressed in anterior neural tissues including the eye, otic vesicle and olfactory placode. Thus, though BMPs act as neural inhibitors during gastrulation and neurulation, they seem to have roles in the later differentiation of these anterior neural tissues. Thus HrBMPb functions as a neural inhibitor and as an epidermal inducer but not as a ventralizing agent or mesodermal inducer as is found in vertebrates (Miya, 1997).

AmphiBMP2/4 of Amphioxus, a cephalochordate, appears to be a single gene closely related to vertebrate BMP2 and BMP4. In amphioxus embryos, the expression patterns of AmphiBMP2/4 suggest patterning roles in the ectodermal dorsoventral axis (comparable to dorsoventral axis establishment in the ectoderm by Drosophila Decapentaplegic and vertebrate BMP4). In addition, AmphiBMP2/4 may be involved in somite evagination, tail bud growth, pharyngeal differentiation (resulting in club-shaped gland morphogenesis), hindgut regionalization, differentiation of olfactory epithelium, patterning of the anterior central nervous system, and establishment of the heart primordium. One difference between the developmental role of amphioxus AmphiBMP2/4 and vertebrate BMP4 is that the former does not appear to be involved in the initial establishment of the dorsoventral polarity of the mesoderm (Panopoulou, 1998).

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. Early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate these genes control formation of the oral-aboral axis. Overexpression of nodal converts the whole ectoderm into oral ectoderm and induces ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal is blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm are specified. Injection of nodal mRNA into Nodal-deficient embryos induces an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes (Duboc, 2004).

The following model is proposed to explain specification of the oral-aboral axis: specification of the oral-aboral axis relies on inductive interactions that involve at least three different signals and that rely on formation of a signaling center in the presumptive oral ectoderm. First, between fertilization and sixth cleavage, an initial labile oral-aboral asymmetry combined with signaling from vegetal cells causes the restricted expression of nodal and specification of the oral ectoderm. Activation of the Nodal signaling pathway in the oral ectoderm would in turn lead to the production of a signal(s) that would diffuse outside the oral ectoderm and cooperate with signals from vegetal cells to specify aboral fates and pattern the embryo along the oral-aboral axis. A good candidate for an aboral-inducing signal is BMP2/4, because it is expressed in the oral ectoderm, it is downstream of nodal, and it has been shown to promote aboral fates. However, it is also possible that BMP2/4 acts locally and indirectly by inducing a yet unidentified signal that would act as a long-range relay to induce aboral fates (Duboc, 2004).

DPP homologs and patterning in fish

Experiments with Drosophila indicate that hedgehog-related genes require the downstream activities of dpp-related signalling molecules for patterning functions related to hedgehog activity. But to date, there has been no example of an epistatic relation between HH-related members and dpp-related members during ventral neural tube formation in vertebrates. Dynamo, a new zebrafish DVR (dpp-vg-related) protein detected in the posterior neural plate from late gastrula on, becomes restricted to the ventral region of the trunk neural tube, exclusive of the most ventral cells (the floor plate and adjacent cells). Analysis of dynamo expression in zebrafish axial mutants such as cyclops, and floating head indicates that dynamo expression in the ventral region of the central nervous system (CNS) is induced by axial mesoderm and maintained by notochord, but is independent of a differentiated floor plate. Ectopic Sonic hedgehog expression can up-regulate dynamo expression in the posterior neural tube providing evidence that cells of the posterior neural tube are competent to respond to shh signaling and that the close relationship between DVR members and hedgehog-related genes might also apply to vertebrate CNS development (Bruneau, 1997).

Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homolog and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded, while ventral structures such as blood and nephros are missing. The swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. zBMP2 has no maternal role in mesoderm induction. This analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo (Kishimoto, 1997).

The patterning activity of the Spemann organizer in early amphibian embryos has been characterized by a number of organizer-specific secreted proteins including Chordin, Noggin, and Follistatin, which all share the same inductive properties. They can neuralize ectoderm and dorsalize ventral mesoderm by blocking the ventralizing signals Bmp2 and Bmp4. In the zebrafish, null mutations in the chordin gene, named chordino, lead to a severe reduction of organizer activity, indicating that Chordino is an essential, but not the only, inductive signal generated by the zebrafish organizer. A second gene required for zebrafish organizer function is mercedes, but the molecular nature of its product is not known as yet. To investigate whether and how Follistatin and Noggin are involved in dorsoventral (D-V) patterning of the zebrafish embryo, their zebrafish homologs were isolated and characterized. Overexpression studies demonstrate that both proteins have the same dorsalizing properties as their Xenopus homologs. However, unlike the Xenopus genes, zebrafish follistatin and noggin are not expressed in the organizer region, nor are they linked to the mercedes mutation. Expression of both genes starts at midgastrula stages. While no patterned noggin expression is detectable by in situ hybridization during gastrulation stages, later expression is confined to presumptive cartilage cells in the branchial arches and the neurocranium and to proximal regions of the pectoral fin buds. follistatin transcripts in gastrulating embryos are confined to anterior paraxial regions, which give rise to head mesoderm and the first five somites. The dorsolateral extent of this expression domain is regulated by Bmp2b, Chordino, and Follistatin itself. In addition, transient expression is observed in a subset of cells in the posterior notochord anlage. Later, follistatin is expressed in brain, eyes, and somites. Comparison of the spatiotemporal expression pattern of follistatin and noggin with those of bmp2b and bmp4, as well as overexpression studies suggest that Noggin and Follistatin may function as Bmp antagonists in later processes of zebrafish development, including late phases of D-V patterning, to refine the early pattern set up by the interaction of Chordino and Bmp2/4. It thus appears that many, but not all, aspects of early dorsoventral patterning are shared among different vertebrate species (Bauer, 1999).

The dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin activity in the early patterning of organisms other than Xenopus. The contribution of Noggin to the embryonic development of the zebrafish has been studied. While single-copy noggin genes have been characterized in several vertebrate species, the zebrafish genome harbors three noggin homologs. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish (Furthauer, 1999).

Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double loss-of-function mutations in BMP inhibitors have less dramatic effects. Combining mutations in the zebrafish genes bozozok, coding for a homeodomain transcription factor, and chordino (din) causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk (Gonzalez, 2000).

If boz and din function redundantly in limiting BMP signaling, then the severe morphological defects in boz;din mutants should be correlated with greatly increased bmp expression compared with either single mutant. During early gastrulation, bmp4 is normally expressed ventrolaterally and in a discrete dorsal domain. The dorsal domain was absent in boz and boz;din mutants. Consistent with the above hypothesis, the ventro-lateral bmp4 expression domain is more dorsally expanded in boz;din mutants compared with either single mutant. Similarly, the ventrolateral marker eve1 is more expanded in boz;din mutants than in either single mutant. Later, during segmentation, the tailbud bmp4 expression domain is almost normal in boz, somewhat expanded in din, but dramatically expanded in boz;din embryos. Together, these morphological and gene expression analyses indicate that most cells in boz;din mutants reside in the prospective tailbud region within a greatly expanded bmp4 expression domain. Furthermore, boz and din function synergistically in negative regulation of bmp4 expression (Gonzalez, 2000).

The findings described here show that several distinct effects of high BMP activity cause the head/trunk deficiency in boz;din double mutants. Within the ectoderm, high levels of BMP activity transform the neuroectoderm into nonneural ectoderm. These data suggest that almost all of the mesodermal precursors in boz;din mutants experience very high levels of BMP activity and, instead of contributing to head and trunk, are misallocated to the tailbud, express ventro-posterior markers, and form a tail. This idea is in accord with fate-mapping studies showing that ventral cells in the zebrafish gastrula migrate to the tailbud and contribute to the tail, and this behavior is expanded dorsally in mutants with elevated BMP activity. Therefore, it is proposed that the high levels of BMP signaling in boz;din embryos specify posterior structures at the expense of anterior structures. This proposal is consistent with the phenotype of BMP signaling mouse mutants, in which the major defect arises in posterior structures, such as the tail and allantois. In summary, boz and din represent the major overlapping pathways that are absolutely essential to limit BMP activity dorso laterally and allow head and trunk formation in the vertebrate embryo (Gonzalez, 2000).

A bone morphogenetic protein (BMP) signaling pathway is implicated in dorsoventral patterning in Xenopus. Three genes in the zebrafish -- swirl, snailhouse, and somitabun -- function as critical components within a BMP pathway to pattern ventral regions of the embryo. The dorsalized mutant phenotypes of these genes can be rescued by overexpression of bmp4, bmp2b, an activated BMP type I receptor, and the downstream functioning Smad1 gene. Consistent with a function as a BMP ligand, swirl functions cell nonautonomously to specify ventral cell fates. Chromosomal mapping of swirl and cDNA sequence analysis demonstrate that swirl is a mutation in the zebrafish bmp2b gene. Interestingly, this analysis suggests that the previously described nonneural/neural ectodermal interaction specifying the neural crest occurs through a patterning function of swirl/bmp2b during gastrulation. A loss in neural crest progenitors is observed in swirl/bmp2b mutant embryos, while somitabun mutants display an opposite, dramatic expansion of the prospective neural crest. In snailhouse, somitabun, and swirl mutant embryos, examination of dorsally and ventrally restricted markers during gastrulation reveals a successive reduction and reciprocal expansion in nonneural and neural ectoderm, respectively, with swirl/bmp2b mutants exhibiting almost no nonneural ectoderm. Based on the alterations in tissue-specific gene expression, a model is proposed whereby swirl/bmp2b acts as a morphogen to specify different cell types along the dorsoventral axis (V. H. Nguyen, 1998).

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through ß-catenin signaling. The molecular mechanism by which boz contributes to the establishment of the organizer has been investigated. The homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. This Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide (CHX). Two high-affinity binding sites for Boz have been identified within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. It is proposed that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin) modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms (Leung, 2003).

The effects of signal perturbation on expression domains of molecular markers for the mesoderm and ectoderm have been analysed across the dorso-ventral axis in zebrafish embryos. Injection of RNA encoding bone morphogenetic protein-4 (BMP-4) ventralizes the embryo, expanding the intermediate mesoderm and non-neural ectoderm at the expense of the dorso-anterior mesoderm and neural plate. A dose-dependent response is observed both morphologically and in expression of gta3, MyoD and pax2. Conversely, increases in dorso-anterior mesoderm and neurectoderm are generated by injection of RNA encoding either a dominant-negative BMP receptor (delta BMPR) or noggin, as demonstrated by goosecoid and pax2 expression. Ventral BMP-4 expression is also inhibited. Thus, patterning of both the mesoderm and the ectoderm during gastrulation appears to depend, directly or indirectly, on the level of BMP activity. Consistent with their locations prior to formation of the neural tube, elevated BMP-4 increases the number of dorsal spinal cord neurons whilst sonic hedgehog and islet1 expression in the ventral spinal cord are reduced. However, the ectopic neurons are not positioned more ventrally, implicating a prepattern in the dorsal neural tube that is independent of the ventral central nervous system (Neave, 1997).

The expression pattern of zebrafish bone morphogenetic protein-4 (BMP4) has been examined as a start to evaluating signals which might participate in the fashioning of organ systems. The predicted sequence of the mature zebrafish protein is more than 75% identical to that of other vertebrates and 66% identical to Drosophila Decapentaplegic (Dpp). As in other species, BMP4 is expressed ventrally during gastrulation, but the zebrafish is unusual in having an additional dorsal domain of expression. Expression is first detected at the margin over the circumference of the zebrafish embryo, at about 30% epiboly stage. The level increases rapidly thereafter. At the onset of gastrulation, expression is strong and restricted mostly to the ventral margin, diminishing towards the dorsal side of the embryo. Co-hybridization with zebrafish Otx2, which is expressed in the dorsal ectoderm, confirms that at the dorsal side of the embryo BMP4 is confined to the hypoblast. By the tailbud stage, dorsal expression is restricted to a diffuse domain at the tailbud and a horseshoe-shaped domain flanking the most anterior portion of the neural tube. Subsequent BMP4 expression is especially prominent in sensory organs, fin buds, and in the gut, kidney, and heart. At all these sites, it becomes particularly enriched in regions of inductive demarcations. For example, expression initially extends through the entire heart tube but then becomes limited to the boundaries between cardiac chambers (sinus venosus-atrial junction, atrio-ventricular junction, and aortic root) prior to cushion formation. In early pectoral fin development, BMP4 is at first expressed uniformly but then becomes restricted to the mesenchyme subjacent to the apical ectodermal ridge. This suggests that among its roles in development, BMP4 serves as a signal in primordial outgrowth and also as a signal demarcating the borders within organs or structures where subspecializations occur. However, the expression of BMP4 in the dorsal margin of the zebrafish gastrula does not correspond to known sites of BMP4 expression in other embryos (Chin, 1997).

Nodal-related genes belong to a separate branch of the transforming growth factor-beta superfamily than that made up of Dpp homologs. Nodal-related 1 (ndr1) and nodal-related 2 (ndr2) genes in zebrafish encode members of the nodal subgroup of the transforming growth factor-beta superfamily. The expression patterns and functional characteristics of these factors are reported, implicating them in the establishment of dorsal-ventral polarity and left-right asymmetry. Ndr1 is expressed maternally, and ndr1 and ndr2 are expressed during blastula stage in the blastoderm margin. During gastrulation, ndr expression subdivides the shield into two domains: a small group of noninvoluting cells, the dorsal forerunner cells, express ndr1, while ndr2 RNA is found in the hypoblast layer of the shield and later in notochord, prechordal plate, and overlying anterior neurectoderm. During somitogenesis, ndr2 is expressed asymmetrically in the lateral plate as are nodal-related genes of other organisms, and in a small domain in the left diencephalon, providing the first observation of asymmetric gene expression in the embryonic forebrain. RNA injections into Xenopus animal caps show that Ndr1 acts as a mesoderm inducer, whereas Ndr2 is an efficient neural but very inefficient mesoderm inducer. It is suggested that Ndr1 has a role in mesoderm induction, while Ndr2 is involved in subsequent specification and patterning of the nervous system and establishment of laterality (Rebagliati,1998).

The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mutations mercedes(tm305), ogon(m60), and short tail(b180) produce ventralized phenotypes, including expanded bmp2b/4 expression domains. The three mutations are allelic and the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo(m60) and ogo(b180) mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo(tm305) retains partial function. Aspects of the ogo(m60) and ogo(tm305) mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl(tc300), a null mutation in bmp2b, is epistatic to ogo(m60) mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. Zebrafish noggin homologs have been ruled out as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity (Miller-Bertoglio, 1999).

Bone morphogenetic proteins (Bmps) are signaling molecules that have been implicated in a variety of inductive processes. Zebrafish Bmp7 is disrupted in snailhouse (snh) mutants. The allele snhst1 is a translocation deleting the snhst1 gene, while snhty68 displays a Val to Gly exhange in a conserved motif of the Bmp7 prodomain. The snhty68 mutation is temperature-sensitive, leading to severalfold reduced activity of mutant Bmp7 at 28°C and non-detectable activity at 33°C. This prodomain lesion affects secretion and/or stability of secreted mature Bmp7 after processing has occurred. Both snhty68 and snhst1 mutant zebrafish embryos are strongly dorsalized, indicating that bmp7 is required for the specification of ventral cell fates during early dorsoventral patterning. At higher temperature, the phenotype of snhty68 mutant embryos is identical to that caused by an amorphic bmp2b mutation and similar to that caused by the smad5 mutation somitabun. mRNA injection studies and double mutant analyses indicate that Bmp2b and Bmp7 closely cooperate and that Bmp2b/Bmp7 signaling is transduced by Smad5 and antagonized by Chordino (Dick, 2000).

The mutant Bmp7 allele snhty68 with its Val to Gly exchange in the prodomain allows some insights into the importance of the prodomain for the biological activity of the mature protein. Bmp precursor proteins are supposed to be processed intracellularly, after dimer formation and before secretion. For snhty68 mutant Bmp7, normal levels of the precursor and the mature protein were found in Xenopus oocyte lysates, indicating that stability and processing of the precursor are not affected by the mutation. However, dramatically reduced amounts of both the prodomain peptide and the mature protein are found in the conditioned medium, which could be due either to impaired secretion or to reduced stability of the processing products after they have been secreted. The fact that intracellular levels of mutant mature protein are unaltered might argue against a defect in the secretion process itself, although additional experiments will be necessary to clarify this point. A similar effect of prodomains on the stability of secreted mature protein takes place for two other TGFbeta-family proteins: mouse Bmp4 and Nodal. These data provide genetic evidence for an essential role of the prodomain on the secretion and/or turnover of mature TGFbeta polypeptides (Dick, 2000).

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. This report demonstrates genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, it was determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. The original snailhouse allele is a hypomorphic mutation and a snailhouse/bmp7 null mutant has been identified. The snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo. snailhouse maps to LG11 and is syntenic to bmp7 in the mouse (Schmid, 2000).

Both in situ hybridization and RT-PCR fail to detect any maternally deposited bmp7 RNA, consistent with the lack of a maternal-effect phenotype of homozygous snh females. bmp7 expression first becomes detectable uniformly throughout the blastoderm shortly after the activation of the zygotic genome. At 30% epiboly, transcripts start to be excluded from the presumptive dorsalmost region of the embryo and this restriction of bmp7 expression to ventrolateral regions becomes more pronounced at the beginning of gastrulation. At 60% epiboly, bmp7 is expressed in the ventral half of the gastrula, as well as in the yolk syncytial layer and weakly in the prechordal plate. The expression of bmp7 in the ventral blastula and gastrula stage embryo supports a role for this factor in the early specification of ventral cell fates. At late gastrulation stages, expression delineates the boundary of the anterior neural plate, while weak expression is observed throughout the non-neural ectoderm. Moreover, transcripts are detected in the ventral marginal zone (Schmid, 2000).

It was shown in Xenopus and chick that Spemann's organizer activity is regulated through the negative action of Anti-dorsalizing morphogenetic protein (ADMP). Admp is a growth factor that is most closely related to human bone morphogenetic protein-3. Characterization and functional properties of admp in zebrafish is reported. admp expression profile is consistent with a role in the organizer, including the tail organizer. admp function was studied through overexpression experiments, with the use of a dominant-negative form (TR-ADMP) and of an antisense morpholino-modified oligonucleotide. The ADMP pathway causes the restriction of anterior and axial fates and ADMP, BMP2b, and BMP7 pathways have distinct actions but cooperate in establishing proper dorso-ventral regionalization. This is shown by partial rescue of the dorsalized mutant snailhouse and of the ventralized mutant chordino, upon admp and tr-admp RNA injection, respectively. Moreover, ADMP and BMP7 probably form heterodimers as shown by the ability of TR-ADMP and BMP7 to antagonize each other. A MYC-tagged ADMP is secreted and can be detected in the extracellular space, suggesting that ADMP can act at a distance. Simultaneous local inhibition of bmp function at the blastoderm margin and impairment of ADMP secretion leads to the induction of secondary head structures, confirming that the two pathways cooperatively regulate organizer formation and activity (Willot, 2002).

Interactions between mutations in antagonistic BMP pathway signaling components were analyzed to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, it was found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, a mutual suppression is observed of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, it was found that the absence of Ogon or Chordin antagonism does not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly, a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype is observed when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead an apparent decrease is observed in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly ortholog of chordin, short gastrulation, these paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity (Wagner, 2002).

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. By measuring movements of cell populations in vivo, the presence of three domains of convergent extension movements have been revealed in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, these domains are demonstrated to be specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicate that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses reveals that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. High Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension (Myers, 2002).

Overall, this work strongly advocates the idea that the Bmp activity gradient plays an instructive role in establishing distinct morphogenetic domains of convergence and extension movements along the dorsoventral axis of zebrafish gastrulae. Furthermore, it provides the basis for an intriguing hypothesis, that by regulating activities of signaling pathway(s) required for specific morphogenetic movements, like Wnt11/Wnt5a, possibly in parallel to cell fate specification, the Bmp activity gradient coordinates cell fate specification with the morphogenetic process of convergent extension. Recent work investigating the role of FGF signaling in vertebrate gastrulation and neurulation supports this hypothesis by also highlighting the extraordinary interconnectedness of patterning and morphogenesis (Myers, 2002).

The maternal effect dorsalization of zebrafish embryos from sbndtc24 heterozygous mothers is caused by a dominant negative mutation in Smad5 (Drosophila homolog: Mad), a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbndtc24 mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it has remained open to what extent Smad5 itself is required for dorsoventral patterning. The identification is reported of novel smad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. m169 mutant embryos were rescued by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169-/- females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169-/- embryos) are even more strongly dorsalized than bmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169-/- embryos display reduced bmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry (Kramer, 2002).

The BMP signaling pathway plays a key role during dorsoventral pattern formation of vertebrate embryos. In zebrafish, all cloned mutants affecting this process are deficient in members of the BMP pathway. In a search for factors differentially expressed in swirl/bmp2b mutants compared with wild type, zebrafish Sizzled (a member of the secreted Frizzled-related protein family and putative Wnt inhibitor) was isolated. The knockdown of sizzled using antisense morpholino phenocopies the ventralized mutant ogon (also known as mercedes or short tail). By sequencing and rescue experiments, it has been demonstrated that ogon encodes sizzled. Correlating with its role in dorsoventral patterning, overexpression of sizzled results in strongly dorsalized phenotypes. Similarly related to its role in dorsoventral patterning, sizzled expression has been localized to the ventral side during gastrulation and is restricted to the posterior end during segmentation stages. The expanded expression domain of sizzled in both ogon and chordino mutants, together with its downregulation in swirl, suggests a BMP2b-dependent negative autoregulation of sizzled. Indicating a novel role for a secreted Frizzled-related protein, the Wnt signaling pathway is shown to be required for dorsoventral pattern formation in zebrafish (Martyn, 2003).

The establishment of dorsoventral (DV) patterning in vertebrate embryos depends on the morphogenic activity of a group of Tgfß superfamily members, the bone morphogenetic proteins (Bmps) (which specify ventral cell fates), and on their interaction with their dorsally secreted cognate inhibitors chordin and noggin. In the zebrafish, genetic analysis has revealed that Bmp2b and Bmp7, as well as their antagonist chordin, are required for proper DV patterning. The expression of Bmp genes is initially activated in the whole blastula. Well before the beginning of gastrulation, Bmp gene expression progressively disappears from the dorsal side to become restricted to the ventral part of the embryo. This early restriction of Bmp gene expression, which occurs independently of noggin and chordin, is an essential step in the establishment of DV patterning. The progressive ventral restriction of Bmp gene transcripts is coincident with the spreading of Fgf activity from the dorsal side of the embryo, suggesting that Fgf signalling is implicated in dorsal downregulation of Bmp gene expression. In accordance with this, activation of the Fgf/Ras/Mapk-signalling pathway inhibits ventral Bmp gene expression, thereby causing a dorsalisation of the embryo. Conversely, inhibition of Fgf signalling causes Bmp gene expression to expand dorsally, leading to an expansion of ventral cell fates. In accordance with an important role of Fgf signalling in the DV patterning of the zebrafish, loss of Fgf8 function enhances the ventralisation of chordin-deficient embryos. These results thereby demonstrate that pre-gastrula stage Fgf-signalling is essential to delimit the expression domain of the genes encoding the functional morphogen of the dorsoventral axis of the early zebrafish embryo (Fürthauer, 2004).

Studies in fish and amphibia have shown that graded Bmp signalling activity regulates dorsal-to-ventral (DV) patterning of the gastrula embryo. In the ectoderm, it is thought that high levels of Bmp activity promote epidermal development ventrally, whereas secreted Bmp antagonists emanating from the organiser induce neural tissue dorsally. However, in zebrafish embryos, the domain of cells destined to contribute to the spinal cord extends all the way to the ventral side of the gastrula, a long way from the organiser. In vegetal (trunk and tail) regions of the zebrafish gastrula, neural specification is initiated at all DV positions of the ectoderm in a manner that is unaffected by levels of Bmp activity and independent of organiser-derived signals. Instead, Fgf activity is required to induce vegetal prospective neural markers and can do so without suppressing Bmp activity. Bmp signalling is shown to occur within the vegetal prospective neural domain and Bmp activity promotes the adoption of caudal fate by this tissue (Kudoh, 2004).

To explore the epistatic relationships between the Fgf and Bmp pathways, the consequences of locally activating or suppressing Fgf signalling were examined. fgf3-expressing ectodermal cells transplanted into animal pole regions of host embryos induce sox3 non-autonomously in surrounding host cells. This induction still occurs if the donor cells are from embryos co-expressing a truncated Fgf receptor, suggesting that the Fgf signal from the donor cells acts directly on the host. When both donor and host cells are overexpressing bmp2b, fgf3-expressing cells still induce sox3 and suppress expression of the epidermal marker gene, foxi1, suggesting that exogenous Bmp activity does not block induction of prospective neural marker genes by Fgf (Kudoh, 2004).

Next, Fgf signalling was locally suppressed by transplanting truncated Fgf receptor (XFD) expressing donor cells to various positions in the prospective neural ectoderm of host embryos. sox3 expression was suppressed in XFD-expressing cells transplanted to dorsal, lateral or ventral vegetal ectoderm, and foxi1 was ectopically induced in transplants targeted to ventral-vegetal ectoderm. These results suggest that ventral vegetal ectoderm needs to receive Fgf to express sox3, otherwise it expresses the prospective epidermal marker, foxi1 (Kudoh, 2004).

To directly assess if Fgf signals are essential for vegetal ectoderm to form neural tissue, the eventual fate was traced of XFD-expressing donor cells transplanted to wild-type hosts. In these experiments, labelled wild-type cells were co-transplanted with XFD-expressing cells to the same locations in the vegetal ectoderm of unlabelled host embryos at the end of blastula stage. When the donor cells were transplanted to the dorsal side, wild-type cells primarily contributed to the hindbrain, whereas the XFD-expressing cells localised more anteriorly, mainly in the midbrain. However, when transplanted to ventral vegetal ectoderm, wild-type donor cells contributed to spinal cord and muscle whereas XFD-expressing cells were excluded from the CNS and found in tissues such as the epidermis and fin. These results suggest that Fgf signalling is required for vegetal ectoderm to contribute to caudal neural tissue. They also suggest that the consequences of suppression of Fgf signalling in cells in dorsal and ventral domains of the vegetal ectoderm are different: dorsally, cells with compromised Fgf signalling frequently move into anterior neural tissue; ventrally, cells move into the prospective epidermis and are excluded from neural tissue. These results are consistent with analyses of embryos in which XFD is expressed ubiquitously and which show loss of posterior neural structures and anteriorisation of remaining CNS tissue on the dorsal side of the embryo (Kudoh, 2004).

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, tbx6 was identified as a posterior mesodermal gene activated by both of these signaling pathways. A genomic fragment was isolated from the tbx6 gene that recapitulates the endogenous tbx6 expression, and this was used to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. The tbx6 promoter was found to utilize distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, overexpression of either signaling pathway was found to activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. A model is presented in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures (Szeto, 2004).

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, morpholino (MO)-based knockdown studies of Tsg1 were performed in zebrafish. Loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhance dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppresses the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). These results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning (Little, 2004).

Bone morphogenetic protein (Bmp) signaling is crucial for the formation and patterning of zebrafish ventral and posterior mesoderm. Mutants defective in the Bmp pathway have expanded trunk muscle, abnormal tails and severely impaired development of ventral mesodermal derivatives such as vasculature, blood and pronephros. Since Bmps continue to be expressed in the ventral and posterior mesoderm after gastrulation, it is likely that Bmp signaling continues to play an important developmental role during outgrowth of the posterior body. However, because Bmp signaling plays an essential role during the gastrula stages, it has not been possible with mutants or standard disruption techniques to determine the later functions of the Bmp pathway. To study the role of Bmp signaling in the ventral and posterior mesoderm during trunk and tail outgrowth, a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion was generated. The data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. It is concluded that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds (Pyati, 2005).

Cranial sensory neurons largely derive from neurogenic placodes (epibranchial and dorsolateral), which are ectodermal thickenings that form the sensory ganglia associated with cranial nerves, but the molecular mechanisms of placodal development are unclear. This study shows that the pharyngeal endoderm induces epibranchial neurogenesis in zebrafish, and that BMP signaling plays a crucial role in this process. Using a her5:egfp transgenic line to follow endodermal movements in living embryos (the her5 promoter drives egfp expression in the pharynx), it has been shown that contact between pharyngeal pouches and the surface ectoderm coincides with the onset of neurogenesis in epibranchial placodes. By genetic ablation and reintroduction of endoderm by cell transplantation, it has been shown that these contacts promote neurogenesis. Using a genetic interference approach bmp2b and bmp5 were identified as crucial components of the endodermal signals that induce epibranchial neurogenesis. Dorsolateral placodes (trigeminal, auditory, vestibular, lateral line) develop independently of the endoderm and BMP signaling, suggesting that these two sets of placodes are under separate genetic control. These results show that the endoderm regulates the differentiation of cranial sensory ganglia, which coordinates the cranial nerves with the segments that they innervate (Holzschuh, 2005).

Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. Via bead implantations, this study shows that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca2+/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. It is proposed that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula (von der Hardt, 2007).

Formation of the vertebrate embryo is known to depend on the activity of organizing centers. The dorsal Spemann organizer is the source of growth factor antagonists that participate in the creation of signaling gradients. In various species, the existence of head, trunk and trunk-tail inducers has been proposed to explain the formation of different parts of the embryo along the anteroposterior (A/P) axis. In zebrafish, two organizing centers have been described, the dorsal and tail organizers, located at the dorsal and ventral gastrula margins, respectively. This study reports that organizer functions are executed not only by the dorsal and ventral margins, but also by all parts of the blastula-gastrula margin. The position of different marginal territories along the dorsoventral axis defines the A/P nature of the structures they are able to organize. At the molecular level, this organizing activity results from the simultaneous activation of BMP and Nodal signaling pathways. Furthermore, the A/P character of the organized structures is not defined by absolute levels but instead by the ratio of BMP and Nodal activities. Rather than resulting from the activity of discrete centers, organization of the zebrafish embryo depends on the activity of the entire margin acting as a continuous and global organizer that is established by a gradual ventral-to-dorsal modulation of the ratio of marginal BMP to Nodal activity (Fauny, 2009).

A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. It has been shown that Notch signaling inhibits the projection neuron fate. This study shows that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. Crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. These results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/ Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. These results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS (Quillien, 2011).

During ectodermal patterning the neural crest and preplacodal ectoderm are specified in adjacent domains at the neural plate border. BMP signalling is required for specification of both tissues, but how it is spatially and temporally regulated to achieve this is not understood. This study shows that at the beginning of neurulation in zebrafish, the ventral-to-dorsal gradient of BMP activity evolves into two distinct domains at the neural plate border: one coinciding with the neural crest and the other abutting the epidermis. In between is a region devoid of BMP activity, which is specified as the preplacodal ectoderm. The ligands required for these domains of BMP activity have been identified. The BMP-interacting protein Crossveinless 2 is expressed in the BMP activity domains and is under the control of BMP signalling. Crossveinless 2 functions at this time in a positive-feedback loop to locally enhance BMP activity and is required for neural crest fate. It was further demonstrated that the Distal-less transcription factors Dlx3b and Dlx4b, which are expressed in the preplacodal ectoderm, are required for the expression of a cell-autonomous BMP inhibitor, Bambi-b, which can explain the specific absence of BMP activity in the preplacodal ectoderm. It is proposed that high BMP activity observed ventrally as generated by bmp2b/4/7a ligand expression and reinforced by Cvl2. BMP activity is inhibited dorsally by expression of the diffusible antagonists Chordin and Noggin. Prospective preplacodal ectoderm and neural crest are formed at intermediate levels of BMP signalling. Taken together, these data define a BMP regulatory network that controls cell fate decisions at the neural plate borde (Reichert, 2012).

Construction of a vertebrate embryo from two opposing morphogen gradients

Development of vertebrate embryos involves tightly regulated molecular and cellular processes that progressively instruct proliferating embryonic cells about their identity and behavior. Whereas numerous gene activities have been found to be essential during early embryogenesis, little is known about the minimal conditions and factors that would be sufficient to instruct pluripotent cells to organize the embryo. This study shows that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, in vivo or in vitro, uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo (Zu, 2014).

Bmp15 is an oocyte-produced signal required for maintenance of the adult female sexual phenotype in zebrafish

Although the zebrafish is a major model organism, how they determine sex (see Drosophila sex determination) is not well understood. In domesticated zebrafish, sex determination appears to be polygenic, being influenced by multiple genetic factors that may vary from strain to strain, and additionally can be influenced by environmental factors. However, the requirement of germ cells for female sex determination is well documented: animals that lack germ cells, or oocytes in particular, develop exclusively as males. Recently, it has been determined that oocytes are also required throughout the adult life of the animal to maintain the differentiated female state. How oocytes control sex differentiation and maintenance of the sexual phenotype is unknown. This study generated targeted mutations in genes for two oocyte produced signaling molecules, Bmp15 and Gdf9 (see Drosophila Dpp) and found a novel role for Bmp15 in maintaining adult female sex differentiation in zebrafish. Females deficient in Bmp15 begin development normally but switch sex during the mid- to late- juvenile stage, and become fertile males. Additionally, by generating mutations in the aromatase cyp19a1a (see Drosophila Cyp311a1), it was shown that estrogen production is necessary for female development and that the function of Bmp15 in female sex maintenance is likely linked to the regulation of estrogen biosynthesis via promoting the development of estrogen-producing granulosa cells in the oocyte follicle (Dranow, 2016).

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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