Wnt signaling is required for early development of zebrafish swimbladder

BACKGROUND: Wnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter. SIGNIFICANCE/CONCLUSION: We first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.     

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.     

Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm

Formation of mesoderm from the pluripotent epiblast depends upon canonical Wnt/beta-catenin signaling, although a precise molecular basis for this requirement has not been established. To develop a robust model of this developmental transition, we examined the role of Wnt signaling during the analogous stage of embryonic stem cell differentiation. We show that the kinetics of Wnt ligand expression and pathway activity in vitro mirror those found in vivo. Furthermore, inhibition of this endogenous Wnt signaling abrogates the functional competence of differentiating ES cells, reflected by their failure to generate Flk1(+) mesodermal precursors and subsequent mature mesodermal lineages. Microarray analysis at various times during early differentiation reveal that mesoderm- and endoderm-associated genes fail to be induced in the absence of Wnt signaling, indicating a lack of germ layer induction that normally occurs during gastrulation in vivo. The earliest genes displaying Wnt-dependent expression, however, were those expressed in vivo in the primitive streak. Using an inducible form of stabilized beta-catenin, we find that Wnt activity, although required, does not autonomously promote primitive streak-associated gene expression in vitro. Our results suggest that Wnt signaling functions in this model system to regulate the thresholds or stability of responses to other effector pathways and demonstrate that differentiating ES cells represent a useful model system for defining complex regulatory interactions underlying primary germ layer induction.     

Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth

Transforming growth factor-beta (TGF-beta) plays an essential role in growth and patterning of the mammary gland, and alterations in its signaling have been shown to illicit biphasic effects on tumor progression and metastasis. We demonstrate in mice that TGF-beta (Tgfbeta) regulates the expression of a non-canonical signaling member of the wingless-related protein family, Wnt5a. Loss of Wnt5a expression has been associated with poor prognosis in breast cancer patients; however, data are lacking with regard to a functional role for Wnt5a in mammary gland development. We show that Wnt5a is capable of inhibiting ductal extension and lateral branching in the mammary gland. Furthermore, Wnt5a(-/-) mammary tissue exhibits an accelerated developmental capacity compared with wild-type tissue, marked by larger terminal end buds, rapid ductal elongation, increased lateral branching and increased proliferation. Additionally, dominant-negative interference of TGF-beta signaling impacts not only the expression of Wnt5a, but also the phosphorylation of discoidin domain receptor 1 (Ddr1), a receptor for collagen and downstream target of Wnt5a implicated in cell adhesion/migration. Lastly, we show that Wnt5a is required for TGF-beta-mediated inhibition of ductal extension in vivo and branching in culture. This study is the first to show a requirement for Wnt5a in normal mammary development and its functional connection to TGF-beta.     

Fgf8 is required for anterior heart field development

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8neo) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Wnt4 is required for proper male as well as female sexual development

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 -/- gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development.     

Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus

Epithelial-mesenchymal interactions play a crucial role in the correct patterning of the mammalian female reproductive tract (FRT). Three members of the Wnt family of growth factors are expressed at high levels in the developing FRT in the mouse embryo. The expression of Wnt genes is maintained in the adult FRT, although levels fluctuate during estrous. Wnt4 is required for Müllerian duct initiation, whereas Wnt7a is required for subsequent differentiation. In this study, we show that Wnt5a is required for posterior growth of the FRT. We further demonstrate that the mutant FRT has the potential to form the posterior compartments of the FRT using grafting techniques. Postnatally, Wnt5a plays a crucial role in the generation of uterine glands and is required for cellular and molecular responses to exogenous estrogens. Finally, we show that Wnt5a participates in a regulatory loop with other FRT patterning genes including Wnt7a, Hoxa10 and Hoxa11. Data presented provide a mechanistic basis for how uterine stroma mediates both developmental and estrogen-mediated changes in the epithelium and demonstrates that Wnt5a is a key component in this process. The similarities of the Wnt5a and Wnt7a mutant FRT phenotypes to those described for the Hoxa11 and Hoxa13 mutant FRT phenotypes reveal a mechanism whereby Wnt and Hox genes cooperate to pattern the FRT along the anteroposterior axis.     

Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse

We present here an analysis of cardiovascular and pharyngeal arch development in mouse embryos hypomorphic for Fgf8. Previously, we have described the generation of Fgf8 compound heterozygous (Fgf8(neo/-)) embryos. Although early analysis demonstrated that some of these embryos have abnormal left-right (LR) axis specification and cardiac looping reversals, the number and type of cardiac defects present at term suggested an additional role for Fgf8 in cardiovascular development. Most Fgf8(neo/-) mutant embryos survive to term with abnormal cardiovascular patterning, including outflow tract, arch artery and intracardiac defects. In addition, these mutants have hypoplastic pharyngeal arches, small or absent thymus and abnormal craniofacial development. Neural crest cells (NCCs) populate the pharyngeal arches and contribute to many structures of the face, neck and cardiovascular system, suggesting that Fgf8 may be required for NCC development. Fgf8 is expressed within the developing pharyngeal arch ectoderm and endoderm during NCC migration through the arches. Analysis of NCC development in Fgf8(neo/-) mutant embryos demonstrates that NCCs are specified and migrate, but undergo cell death in areas both adjacent and distal to where Fgf8 is normally expressed. This study defines the cardiovascular defects present in Fgf8 mutants and supports a role for Fgf8 in development of all the pharyngeal arches and in NCC survival.     

Kruppel is a gap gene in the intermediate germband insect Oncopeltus fasciatus and is required for development of both blastoderm and germband-derived segments

Segmentation in long germband insects such as Drosophila occurs essentially simultaneously across the entire body. A cascade of segmentation genes patterns the embryo along its anterior-posterior axis via subdivision of the blastoderm. This is in contrast to short and intermediate germband modes of segmentation where the anterior segments are formed during the blastoderm stage and the remaining posterior segments arise at later stages from a posterior growth zone. The biphasic character of segment generation in short and intermediate germ insects implies that different formative mechanisms may be operating in blastoderm-derived and germband-derived segments. In Drosophila, the gap gene Krüppel is required for proper formation of the central portion of the embryo. This domain of Krüppel activity in Drosophila corresponds to a region that in short and intermediate germband insects spans both blastoderm and germband-derived segments. We have cloned the Krüppel homolog from the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae), an intermediate germband insect. We find that Oncopeltus Krüppel is expressed in a gap-like domain in the thorax during the blastoderm and germband stages of embryogenesis. In order to investigate the function of Krüppel in Oncopeltus segmentation, we generated knockdown phenotypes using RNAi. Loss of Krüppel activity in Oncopeltus results in a large gap phenotype, with loss of the mesothoracic through fourth abdominal segments. Additionally, we find that Krüppel is required to suppress both anterior and posterior Hox gene expression in the central portion of the germband. Our results show that Krüppel is required for both blastoderm-derived and germband-derived segments and indicate that Krüppel function is largely conserved in Oncopeltus and Drosophila despite their divergent embryogenesis.     

Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Here, we report that mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling.     

Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.