Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.     

Distinct Wnt signaling pathways have opposing roles in appendage regeneration

In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/beta-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, we show that Wnt/beta-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/beta-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/beta-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/beta-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/beta-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/beta-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration.     

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.     

Reiterated Wnt signaling during zebrafish neural crest development

While Wnt/beta-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.     

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.     

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.     

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.     

<Emphasis Type="Italic">Wnt</Emphasis> gene loss in flatworms

Wnt genes encode secreted glycoproteins that act in cell–cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/β-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5–6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/β-catenin) and non-canonical (planar cell polarity and Wnt/Ca²⁺) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.     

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.     

Wnt5a及其信号通路与血管新生的研究进展

Wnt5a是Wnt蛋白家族中的成员之一,在细胞成熟、胚胎发育等过程中发挥着重要作用。研究表明Wnt5a的表达调控及其信号通路与血管新生密切相关,并且在血管新生性相关疾病中发挥了重要作用。本文从Wnt5a与其相关信号转导通路对血管新生的影响以及分子机制等方面进行阐述和展望,旨在为以Wnt5a为靶点进行血管新生性疾病的防治提供理论依据。