Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.     

Analysis of<Emphasis Type="Italic"> Fz10</Emphasis> expression in mouse embryos

Wnt signaling molecules regulate the development of multiple organs in vertebrate embryos. We have isolated cDNA clones for frizzled10 (Fz10), which encodes a putative Wnt receptor, to further characterize the mechanisms of Wnt signaling in mouse embryos. Interestingly, Fz10 is expressed in the same regions as Wnt7a in the neural tube, limb buds, and Müllerian duct.Edited by R.P. Elinson     

A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling

Background

Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/β-catenin signalling, and also interacts with those Wnt ligands that act on β-catenin-independent Wnt pathways.

Findings

Here we show that Xenopus Fz4-v1 can activate and inhibit the β-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/β-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced β-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin.

Conclusions

For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators.

     

Wnt3‐frizzled 1 chimera as a model to study canonical Wnt signaling

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low‐density lipoprotein receptor‐related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF‐luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF‐luciferase activation. DKK‐1 protein blocks upregulation of the TCF‐luciferase reporter by the Wnt3–Fz1 chimera, suggesting involvement of LRP in Wnt3–Fz1 signaling. Expression of a Wnt3–Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3–Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt–Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases. J. Cell. Biochem. 109: 876–884, 2010. © 2009 Wiley‐Liss, Inc.     

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.     

Phosphatidylinositol 4‐kinase IIα function at endosomes is regulated by the ubiquitin ligase Itch

Phosphatidylinositol (PI) 4‐phosphate (PI(4)P) and its metabolizing enzymes serve important functions in cell signalling and membrane traffic. PI 4‐kinase type IIα (PI4KIIα) regulates Wnt signalling, endosomal sorting of signalling receptors, and promotes adaptor protein recruitment to endosomes and the trans‐Golgi network. Here we identify the E3 ubiquitin ligase Itch as binding partner and regulator of PI4KIIα function. Itch directly associates with and ubiquitinates PI4KIIα, and both proteins colocalize on endosomes containing Wnt‐activated frizzled 4 (Fz4) receptor. Depletion of PI4KIIα or Itch regulates Wnt signalling with corresponding changes in Fz4 internalization and degradative sorting. These findings unravel a new molecular link between phosphoinositide‐regulated endosomal membrane traffic, ubiquitin and the modulation of Wnt signalling.     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/β-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise β-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3], [4], [5] and [6], the effect of other Dkks on the Wnt/β-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/β-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/β-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

Wnt7a Regulates Multiple Steps of Neurogenesis

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a^−/− dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin–cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin–neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.     

Normal forebrain development may require continual Wnt antagonism until mid-somitogenesis in zebrafish

During normal forebrain development in vertebrates, rostral neural tissue must be protected from Wnt signals via the actions of locally expressed Wnt antagonistic factors. In zebrafish zygotic oep (Zoep) mutants, forebrain structure is severely disrupted with reduced expression of the Wnt antagonists secreted frizzled related protein1 and dickkopf1. To analyze the temporal effects of Wnt antagonism on forebrain development, we generated transgenic zebrafish that overexpressed the dominant negative form of frizzled8a (DNfz8a) in wild-type and Zoep mutants under the control of a heat-inducible promoter. This model allowed for assessment of the dynamics of Wnt antagonistic signaling during forebrain development. Our results demonstrated that overexpression of DNfz8a in Zoep embryos between 7 and 16 hpf increased putative forebrain region demarcated by anf and distal-less2 expressions. These results suggest that normal forebrain development requires continual Wnt antagonism from the early gastrula to the mid-somitogenesis stage.     

Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration

We have investigated the requirement for the FGF and Wnt/beta-catenin pathways for Xenopus tadpole tail regeneration. Pathways were modified either by treatment with small molecules or by induction of transgene expression with heat shocks. Regeneration is inhibited by treatment with the FGF inhibitor SU5402, or by activation of a dominant negative FGF receptor, or by activation of expression of the Wnt inhibitor Dkk1. Agents promoting Wnt activity: the small molecule BIO, or a constitutively active form of beta-catenin, led to an increased growth rate. Combination of a Wnt activator with FGF inhibitor suppressed regeneration, while combination of a Wnt inhibitor with a FGF activator allowed regeneration. This suggests that the Wnt activity lies upstream of the FGF activity.Expression of both Wnt and FGF components was inhibited by activation of noggin, suggesting that BMP signalling lies upstream of both Wnt and FGF.The results show that the molecular mechanism of Xenopus tadpole tail regeneration is surprisingly similar to that of the Xenopus limb bud and the zebrafish caudal fin, despite the difference of anatomy.     

Metalloprotease ADAM9 cleaves ephrin-B ligands and differentially regulates Wnt and mTOR signaling downstream of Akt kinase in colorectal cancer cells

Ephrin-B signaling has been implicated in many normal and pathological processes, including neural crest development and tumor metastasis. We showed previously that proteolysis of ephrin-B ligands by the disintegrin metalloprotease ADAM13 is necessary for canonical Wnt signal activation and neural crest induction in Xenopus, but it was unclear if these mechanisms are conserved in mammals. Here, we report that mammalian ADAM9 cleaves ephrin-B1 and ephrin-B2 and can substitute for Xenopus ADAM13 to induce the neural crest. We found that ADAM9 expression is elevated in human colorectal cancer (CRC) tissues and that knockdown (KD) of ADAM9 inhibits the migration and invasion of SW620 and HCT116 CRC cells by reducing the activity of Akt kinase, which is antagonized by ephrin-Bs. Akt is a signaling node that activates multiple downstream pathways, including the Wnt and mTOR pathways, both of which can promote CRC cell migration/invasion. Surprisingly, we also found that KD of ADAM9 downregulates Wnt signaling but has negligible effects on mTOR signaling in SW620 cells; in contrast, mTOR activity is suppressed while Wnt signaling remains unaffected by ADAM9 KD in HCT116 cells. These results suggest that mammalian ADAM9 cleaves ephrin-Bs to derepress Akt and promote CRC migration and invasion; however, the signaling pathways downstream of Akt are differentially regulated by ADAM9 in different CRC cell lines, reflecting the heterogeneity of CRC cells in responding to manipulations of upstream Akt regulators.     

XsFRP5 modulates endodermal organogenesis in Xenopus laevis

Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated β-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posterior stomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventral pancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.     

A role for Wnt/planar cell polarity signaling during lens fiber cell differentiation?

Wnt signaling through frizzled (Fz) receptors plays key roles in just about every developmental system that has been studied. Several Wnt-Fz signaling pathways have been identified including the Wnt/planar cell polarity (PCP) pathway. PCP signaling is crucial for many developmental processes that require major cytoskeletal rearrangements. Downstream of Fz, PCP signaling is thought to involve the GTPases, Rho, Rac and Cdc42 and regulation of the JNK cascade. Here we report on the localization of these GTPases and JNK in the lens and assess their involvement in the cytoskeletal reorganisation that is a key element of FGF-induced lens fiber cell differentiation.     

Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.