Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

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.     

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.     

Involvement of Hex in the initiation of feather morphogenesis

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.     

Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon

Wnt signaling is involved in numerous processes during vertebrate CNS development. In this study, we used conditional Cre/loxP system in mouse to ablate or activate beta-catenin in the telencephalon in two time windows: before and after the onset of neurogenesis. We show that beta-catenin mediated Wnt signals are required to maintain the molecular identity of the pallium. Inactivation of beta-catenin in the telencephalon before neurogenesis results in downregulated expression of dorsal markers Emx1, Emx2 and Ngn2, and in ectopic up-regulation of ventral markers Gsh2, Mash1 and Dlx2 in the pallium. In contrast, ablation of ss-catenin after the onset of cortical neurogenesis (E11.5) does not result in a dorso-ventral fate shift. In addition, activation of canonical Wnt signaling in the subpallium leads to a repression of ventral telencephalic cell identities as shown by the down-regulation of subpallial markers Dlx2, Nkx2.1, Gsh2, Olig2 and Mash1. This was accompanied with an expansion of dorsal identities ventrally as shown by the expanded expression domains of pallial markers Pax6 and Ngn2. Thus, our data suggest that canonical Wnt signals are involved in maintaining the identity of the pallium by controlling expression of dorsal markers and by suppressing ventral programs from being activated in pallial progenitor cells.     

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.     

Regulation of hhex expression in the yolk syncytial layer, the potential Nieuwkoop center homolog in zebrafish

The Nieuwkoop center is the earliest signaling center during dorsal-ventral pattern formation in amphibian embryos and has been implied to function in induction of the Spemann-Mangold organizer. In zebrafish, Nieuwkoop-center-like activity resides in the dorsal yolk syncytial layer (YSL) at the interface of the vegetal yolk cell and the blastoderm. hex homologs are expressed in the anterior endomesoderm in frogs (Xhex), the anterior visceral endoderm in mice, and the dorsal YSL in zebrafish (hhex). Here, we investigate the control of hhex expression in the YSL. We demonstrate that bozozok (boz) is absolutely required for early hhex expression, while overexpression of boz causes ectopic hhex expression. Activation of Wnt/beta-catenin signaling by LiCl induces hhex expression in wild-type YSL but not in boz mutant embryos, revealing that boz activity is required downstream of Wnt/beta-catenin signaling for hhex expression. Further, we show that the boz-mediated induction of hhex is independent of the Boz-mediated repression of bmp2b. Our data reveal that repressive effects of both Vega1 and Vega2 may be responsible for the exclusion of hhex expression from the ventral and lateral parts of the YSL. In summary, zebrafish hhex appears to be activated by Wnt/beta-catenin in the dorsal YSL, where Boz acts in a permissive way to limit repression of hhex by Vega1 and Vega2.     

Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain beta-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium (Ca(2+)) and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, we first tested the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs. We report that Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, we then tested the hypothesis that CamKII participates in axis formation in the early embryo. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos revealed elevated activity on the prospective ventral side, which was suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants were consistent with this prediction, revealing that CamKII contributes to a ventral cell fate.     

Ventral vs. dorsal chick dermal progenitor specification

The dorsal and the ventral trunk integuments of the chick differ in their dermal cell lineage (originating from the somatic and somatopleural mesoderm respectively) and in the distribution of their feather fields. The dorsal macropattern has a large spinal pteryla surrounded by semi-apteria, whereas the ventral skin has a true medial apterium surrounded by the ventral pterylae. Comparison of the results of heterotopic transplantations of distal somatopleure in place of somatic mesoderm (Mauger 1972) or in place of proximal somatopleure (our data), leads to two conclusions. These are that the fate of the midventral apterium is not committed at day 2 of incubation and that the signals from the environment which specify the ventral and dorsal featherforming dermal progenitors are different. Effectively, Shh, but not Wnt -1 signalling can induce the formation of feather forming dermis from the embryonic somatopleure. Shh is not able, however, to trigger the formation of a feather forming dermis from the extra embryonic somatopleure. This brief report constitutes the first attempt, by comparing old and new preliminary results, to understand whether dermal progenitors at different sites are specified by different signalling pathways.     

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.     

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.     

A role for maternal beta-catenin in early mesoderm induction in Xenopus

Mesoderm formation results from an inducing process that requires maternal and zygotic FGF/MAPK and TGFbeta activities, while maternal activation of the Wnt/beta-catenin pathway determines the anterior-dorsal axis. Here, we show a new role of Wnt/beta-catenin signaling in mesoderm induction. We find that maternal beta-catenin signaling is not only active dorsally but also all around the equatorial region, coinciding with the prospective mesoderm. Maternal beta-catenin function is required both for expression of dorsal genes and for activation of MAPK and the mesodermal markers Xbra and eomesodermin. beta-catenin acts in a non- cell-autonomous manner upstream of zygotic FGF and nodal signals. The Wnt/beta-catenin activity in the equatorial region of the early embryo is the first example of a maternally provided mesoderm inducer restricted to the prospective mesoderm.