Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish

The vertebrate body plan arises during gastrulation, when morphogenetic movements form the ectoderm, mesoderm, and endoderm. In zebrafish, mesoderm and endoderm derive from the marginal region of the late blastula, and cells located nearer the animal pole form the ectoderm [1]. Analysis in mouse, Xenopus, and zebrafish has demonstrated that Nodal-related proteins, a subclass of the TGF-beta superfamily, are essential for mesendoderm development [2], but previous mutational studies have not established whether Nodal-related signals control fate specification, morphogenetic movements, or survival of mesendodermal precursors. Here, we report that Nodal-related signals are required to allocate marginal cells to mesendodermal fates in the zebrafish embryo. In double mutants for the zebrafish nodal-related genes squint (sqt) and cyclops (cyc) [3] [4] [5], dorsal marginal cells adopt neural fates, whereas in wild-type embryos, cells at this position form endoderm and axial mesoderm. Involution movements characteristic of developing mesendoderm are also blocked in the absence of Nodal signaling. Because it has been proposed [6] that inhibition of Nodal-related signals promotes the development of anterior neural fates, we also examined anteroposterior organization of the neural tube in sqt;cyc mutants. Anterior trunk spinal cord is absent in sqt;cyc mutants, despite the presence of more anterior and posterior neural fates. These results demonstrate that nodal-related genes are required for the allocation of dorsal marginal cells to mesendodermal fates and for anteroposterior patterning of the neural tube.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development

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. As 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, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our 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. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.     

Nodal signaling patterns the organizer

Spemann's organizer plays an essential role in patterning the vertebrate embryo. During gastrulation, organizer cells involute and form the prechordal plate anteriorly and the notochord more posteriorly. The fate mapping and gene expression analyses in zebrafish presented in this study reveal that this anteroposterior polarity is already initiated in the organizer before gastrulation. Prechordal plate progenitors reside close to the blastoderm margin and express the homeobox gene goosecoid, whereas notochord precursors are located further from the margin and express the homeobox gene floating head. The nodal-related genes cyclops and squint are expressed at the blastoderm margin and are required for prechordal plate and notochord formation. We show that differential activation of the Nodal signaling pathway is essential in establishing anteroposterior pattern in the organizer. First, overexpression of cyclops and squint at different doses leads to the induction of floating head at low doses and the induction of both goosecoid and floating head at higher doses. Second, decreasing Nodal signaling using different concentrations of the antagonist Antivin inhibits goosecoid expression at low doses and blocks expression of both goosecoid and floating head at higher doses. Third, attenuation of Nodal signaling in zygotic mutants for the EGF-CFC gene one-eyed pinhead, an essential cofactor for Nodal signaling, leads to the loss of goosecoid expression and expansion of floating head expression in the organizer. Concomitantly, cells normally fated to become prechordal plate are transformed into notochord progenitors. Finally, activation of Nodal signaling at different times suggests that prechordal plate specification requires sustained Nodal signaling, whereas transient signaling is sufficient for notochord development. Together, these results indicate that differential Nodal signaling patterns the organizer before gastrulation, with the highest level of activity required for anterior fates and lower activity essential for posterior fates.     

A role for MKP3 in axial patterning of the zebrafish embryo

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Sequential antagonism of early and late Wnt-signaling by zebrafish colgate promotes dorsal and anterior fates

The establishment of the vertebrate body plan involves patterning of the ectoderm, mesoderm, and endoderm along the dorsoventral and antero-posterior axes. Interactions among numerous signaling molecules from several multigene families, including Wnts, have been implicated in regulating these processes. Here we provide evidence that the zebrafish colgate(b382) (col) mutation results in increased Wnt signaling that leads to defects in dorsal and anterior development. col mutants display early defects in dorsoventral patterning manifested by a decrease in the expression of dorsal shield-specific markers and ectopic expression of ventrolaterally expressed genes during gastrulation. In addition to these early patterning defects, col mutants display a striking regional posteriorization within the neuroectoderm, resulting in a reduction in anterior fates and an expansion of posterior fates within the forebrain and midbrain-hindbrain regions. We are able to correlate these phenotypes to the overactivation of Wnt signaling in col mutants. The early dorsal and anterior patterning phenotypes of the col mutant embryos are selectively rescued by inactivation of Wnt8 function by morpholino translational interference. In contrast, the regionalized neuroectoderm posterioriorization phenotype is selectively rescued by morpholino-mediated inactivation of Wnt8b. These results suggest that col-mediated antagonism of early and late Wnt-signaling activity during gastrulation is normally required sequentially for both early dorsoventral patterning and the specification and patterning of regional fates within the anterior neuroectoderm.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

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, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that 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) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

nemo-like kinase is an essential co-activator of Wnt signaling during early zebrafish development

Wnt/beta-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of beta-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. We show that overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate.     

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.     

Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation

During gastrulation, diffusible "organizer" signals, including members of the TGFbeta Nodal subfamily, pattern dorsal mesoderm and the embryonic axes. Simultaneously, negative regulators of these signals, including the Nodal inhibitor Lefty, an atypical TGFbeta factor, are induced by Nodal. This suggests that Lefty-dependent modulation of organizer signaling might regulate dorsal mesoderm patterning and axial morphogenesis. Here, Xenopus Lefty (Xlefty) function was blocked by injection of anti-Xlefty morpholino oligonucleotides (MO). Xlefty-deficient embryos underwent exogastrulation, an aberrant morphogenetic process not predicted from deregulation of the Nodal pathway alone. In the absence of Xlefty, both Nodal- (Xnr2, gsc, cer, Xbra) and Wnt-responsive (gsc, Xnr3) organizer gene expression expanded away from the dorsal blastopore lip. Conversely, coexpression of Xlefty with Nodal or Wnt reduced the ectopic expression of Nodal- (Xbra) and Wnt-responsive (Xnr3) genes in a dose-dependent manner. Furthermore, Xlefty expression in the ectodermal animal pole inhibited endogenous Nodal- and Wnt-responsive gene expression in distant mesoderm cells, indicating that Xlefty inhibition can spread from its source. We hypothesize that Xlefty negatively regulates the spatial extent of Nodal- and Wnt-responsive gene expression in the organizer and that this Xlefty-dependent inhibition is essential for normal organizer patterning and gastrulation.     

Dissecting Wnt/beta-catenin signaling during gastrulation using RNA interference in mouse embryos

Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/beta-catenin signaling, we have used genome-wide expression profiling of conditional beta-catenin mutant embryos. Known Wnt/beta-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets.     

The cnidarian and the canon: the role of Wnt/beta-catenin signaling in the evolution of metazoan embryos

In a recent publication, Wikramanayake and colleagues have implicated the canonical Wnt/beta-catenin signaling pathway as a mediator of axial polarity and germ-layer specification in embryos of the cnidarian Nematostella. In this anthozoan, beta-catenin is localized in nuclei of blastomeres in one region of the 16- to 32-cell embryo whose descendants subsequently form the entoderm of the embryo. They claim that the pattern of nuclear localization is significant for two reasons: (1) when nuclear localization of beta-catenin was inhibited, gastrulation does not occur, and (2) when localization of beta-catenin took place in all cells of the pregastrula embryo, the number of entodermal cells increases. Since the Wnt/beta-catenin signaling pathway also plays a role in establishing axial polarity and specifying endoderm and mesoderm in a number of bilaterians, Wikramanayake et al. imply that this developmental mechanism is an evolutionary inheritance from a radially symmetrical ancestor. Some of the gaps in the current evidence, which must be filled to evaluate their interpretation, are discussed.