Wnt signalling: a moving picture emerges from van gogh

Recent studies on vertebrate homologues of the van gogh/strabismus (vang/stbm) gene, a key player in planar cell polarity signalling in Drosophila, show that vang/stbm is involved in patterning and morphogenesis during vertebrate gastrulation where it modulates two distinct Wnt signals.     

The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.     

Non-canonical Wnt signalling and regulation of gastrulation movements

Members of the Wnt family have been implicated in a variety of developmental processes including axis formation, patterning of the central nervous system and tissue morphogenesis. Recent studies have shown that a Wnt signalling pathway similar to that involved in the establishment of planar cell polarity in Drosophila regulates convergent extension movements during zebrafish and Xenopus gastrulation. This finding provides a good starting point to dissect the complex cell biology and genetic regulation of vertebrate gastrulation movements.     

An essential and highly conserved role for Zic3 in left-right patterning, gastrulation and convergent extension morphogenesis

Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting of left-right (L-R) patterning defects, midline abnormalities, and cardiac malformations. Similarly, loss of function of Zic3 in mouse results in abnormal L-R patterning and cardiac development. However, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting the hypothesis that Zic3 is necessary for proper convergent extension (C-E) morphogenesis. To further investigate the role of Zic3 in early embryonic development, we utilized two model systems, Xenopus laevis and zebrafish, and performed loss of function analysis using antisense morpholino-mediated gene knockdown. Both Xenopus and zebrafish demonstrated significant impairment of C-E in Zic3 morphants. L-R patterning was also disrupted, indicating that the role of Zic3 in L-R axis development is conserved across species. Correlation of L-R patterning and C-E defects in Xenopus suggests that early C-E defects may underlie L-R patterning defects at later stages, since Zic3 morphants with moderate to severe C-E defects exhibited an increase in laterality defects. Taken together, these results demonstrate a functional conservation of Zic3 in L-R patterning and uncover a previously unrecognized role for Zic3 in C-E morphogenesis during early vertebrate development.     

Intra-endodermal interactions are required for pancreatic beta cell induction

The cellular origin of signals that regulate pancreatic beta cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic beta cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in beta cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic beta cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate beta cell specification, and uncover complex patterning and signaling interactions within the endoderm.     

Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin

Wnt11 plays a central role in tissue morphogenesis during vertebrate gastrulation, but the molecular and cellular mechanisms by which Wnt11 exerts its effects remain poorly understood. Here, we show that Wnt11 functions during zebrafish gastrulation by regulating the cohesion of mesodermal and endodermal (mesendodermal) progenitor cells. Importantly, we demonstrate that Wnt11 activity in this process is mediated by the GTPase Rab5, a key regulator of early endocytosis, as blocking Rab5c activity in wild-type embryos phenocopies slb/wnt11 mutants, and enhancing Rab5c activity in slb/wnt11 mutant embryos rescues the mutant phenotype. In addition, we find that Wnt11 and Rab5c control the endocytosis of E-cadherin and are required in mesendodermal cells for E-cadherin-mediated cell cohesion. Together, our results suggest that Wnt11 controls tissue morphogenesis by modulating E-cadherin-mediated cell cohesion through Rab5c, a novel mechanism of Wnt signaling in gastrulation.     

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.     

Essential roles of G{alpha}12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements

Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.     

Gastrulation in zebrafish: what mutants teach us.

A major approach to the study of development is to compare the phenotypes of normal and mutant individuals for a given genetic locus. Understanding the development of a complex metazoan therefore requires examination of many mutants. Relatively few organisms are being studied this way, and zebrafish is currently the best example of a vertebrate for which large-scale mutagenesis screens have successfully been carried out. The number of genes mutated in zebrafish that have been cloned expands rapidly, bringing new insights into a number of developmental pathways operating in vertebrates. Here, we discuss work on zebrafish mutants affecting gastrulation and patterning of the early embryo. Gastrulation is orchestrated by the dorsal organizer, which forms in a region where maternally derived beta-catenin signaling is active. Mutation in the zygotic homeobox gene bozozok disrupts the organizer genetic program and leads to severe axial deficiencies, indicating that this gene is a functional target of beta-catenin signaling. Once established, the organizer releases inhibitors of ventralizing signals, such as BMPs, and promotes dorsoanterior fates within all germ layers. In zebrafish, several mutations affecting dorsal-ventral (D/V) patterning inactivate genes functioning in the BMP pathway, stressing the central role of this pathway in the gastrula embryo. Cells derived from the organizer differentiate into several axial structures, such as notochord and prechordal mesoderm, which are thought to induce various fates in adjacent tissues, such as the floor plate, after the completion of gastrulation. Studies with mutants in nodal-related genes, in one-eyed pinhead, which is required for nodal signaling, and in the Notch pathway reveal that midline cell fate specification is, in fact, initiated during gastrulation. Furthermore, the organizer coordinates morphogenetic movements, and zebrafish mutants in T-box mesoderm-specific genes help clarify the mechanism of convergence movements required for the formation of axial and paraxial mesoderm.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Analysis of maternal-zygotic ugdh mutants reveals divergent roles for HSPGs in vertebrate embryogenesis and provides new insight into the initiation of left–right asymmetry

Growth factors and morphogens regulate embryonic patterning, cell fate specification, cell migration, and morphogenesis. The activity and behavior of these signaling molecules are regulated in the extracellular space through interactions with proteoglycans (Bernfield et al., 1999, Perrimon and Bernfield, 2000, Lander and Selleck, 2000 and Selleck, 2000). Proteoglycans are high molecular-weight proteins consisting of a core protein with covalently linked glycosaminoglycan (GAG) side chains, which are thought to mediate ligand interaction. Drosophila mutant embryos deficient for UDP-glucose dehydrogenase activity (Ugdh, required for GAG synthesis) exhibit abnormal Fgf, Wnt and TGFß signaling and die during gastrulation, indicating a broad and critical role for proteoglycans during early embryonic development (Lin et al., 1999 and Lin and Perrimon, 2000) (Hacker et al., 1997). Mouse Ugdh mutants also die at gastrulation, however, only Fgf signaling appears disrupted ( Garcia-Garcia and Anderson, 2003). These findings suggested a possible divergence in the requirement for proteoglycans during Drosophila and mouse embryogenesis, and that mammals may have evolved alternative means of regulating Wnt and TGFß activity. To further examine the function of proteoglycans in vertebrate development, we have characterized zebrafish mutants devoid of both maternal and zygotic Ugdh/Jekyll activity (MZjekyll). We demonstrate that MZjekyll mutant embryos display abnormal Fgf, Shh, and Wnt signaling activities, with concomitant defects in central nervous system patterning, cardiac ventricular fate specification and axial morphogenesis. Furthermore, we uncover a novel role for proteoglycans in left–right pattern formation. Our findings resolve longstanding questions into the evolutionary conservation of Ugdh function and provide new mechanistic insights into the initiation of left–right asymmetry.     

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.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.     

Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.