The coiled-coil domain containing protein Ccdc136b antagonizes maternal Wnt/ß-catenin activity during zebrafish dorsoventral axial patterning.

The coiled-coil domain containing protein CCDC136 is a putative tumor suppressor and significantly down-regulated in gastric and colorectal cancer tissues. However, little is known about its biological functions during vertebrate embryo development. Zebrafish has two CCDC136 orthologs, ccdc136a and ccdc136b, but only ccdc136b is highly expressed during early embryonic development. In this study, we demonstrate that ccdc136b is required for dorsal-ventral axial patterning in zebrafish embryos. ccdc136b morphants display strongly dorsalized phenotypes. Loss- and gain-of-function experiments in zebrafish embryos and mammalian cells show that Ccdc136b is a crucial negative regulator of the Wnt/ß-catenin signaling pathway, and plays a critical role in the establishment of the dorsal-ventral axis. We further find that Ccdc136b interacts with APC, promotes the binding affinity of APC with ß-catenin and then facilitates the turnover of ß-catenin. These results provide the first evidence that CCDC136 regulates zebrafish dorsal-ventral patterning by antagonizing Wnt/ß-catenin signal transduction and suggest a potential mechanism underlying its suppressive activity in carcinogenesis.

The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.

Molecular analysis and functions of p53R2 in zebrafish.

p53R2 is a newly identified small subunit of ribonucleotide reductase and plays a pivotal role in the supply of dNTPs for genomic DNA repair and mitochondrial DNA synthesis, but little is known about its functions in zebrafish. Herein, we obtained the cDNA of zebrafish p53R2 that shares 72.8% and 72.5% amino acid identities with human p53R2 and zebrafish R2, respectively. Residues crucial for enzymatic activity are highly conserved among p53R2 proteins from different species. p53R2 in zebrafish was maternally expressed, its transcripts were detected in developing embryos and all adult tissues examined. A 250-bp minimal promoter upstream of the translational initiation site was identified to drive basal expression of p53R2 in a p53-independent manner. Expression of p53R2 was induced by DNA-damaging reagents CPT or MMS, but suppressed by p53-knockdown in zebrafish embryos. Moreover, p53R2 was mainly distributed in the cytoplasm of cells under normal condition and upon DNA damage. Furthermore, overexpression of p53R2 attenuated apoptosis of embryonic cells caused by CPT or MMS treatment and protected developing embryos from death. Therefore, functions of p53R2 in zebrafish are closely associated with its activity in DNA repair and synthesis.