Naked1 Antagonizes Wnt Signaling by Preventing Nuclear Accumulation of β-Catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Nodal-Dependent Mesendoderm Specification Requires the Combinatorial Activities of FoxH1 and Eomesodermin

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA–binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words

Characterization of immune-matched hematopoietic transplantation in zebrafish

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.Stem Cell and Regenerative Biolog

The zebrafish dorsal axis is apparent at the four-cell stage

10.1038/nature04184Nature43870701030-1035NATU