Zebrafish ΔNp63 Is a Direct Target of Bmp Signaling and Encodes a Transcriptional Repressor Blocking Neural Specification in the Ventral Ectoderm

Abstract

Bone morphogenetic proteins (Bmps) promote ventral specification in both the mesoderm and the ectoderm of vertebrate embryos. Here we identify zebrafish DeltaNp63, encoding an isoform of the p53-related protein p63, as an ectoderm-specific direct transcriptional target of Bmp signaling. DeltaNp63 itself acts as a transcriptional repressor required for ventral specification in the ectoderm of gastrulating embryos. Loss of DeltaNp63 function leads to reduced nonneural ectoderm followed by defects in epidermal development during skin and fin bud formation. In contrast, forced DeltaNp63 expression blocks neural development and promotes nonneural development, even in the absence of Bmp signaling. Together, DeltaNp63 fulfills the criteria to be the neural repressor postulated by the "neural default model."

Perp is required for tissue-specific cell survival during zebrafish development

“…Efficiency of used p53 MO was confirmed by parallel coinjections with mdm2 MO, leading to a rescue of the mdm2 morphant phenotype, as described elsewhere. 20 Efficiency of DNp63 and TAp73 MOs was confirmed by the skin and craniofacial phenotypes of injected sibling embryos at later stages of development 23,26 Pro-and antiapoptotic roles of zebrafish Perp M Nowak et al is not affected (data not shown). However, TUNEL stainings revealed a significant increase in the number of dying cells in the notochord, the pectoral fins, and the skin of perp morphant embryos (Figure 7a-f).…”

“…28 During zebrafish embryogenesis, only DNp63 and TAp73 transcripts could be detected, while the other isoforms appear to be absent or expressed at much lower levels. 23,24,27,26 However, both DNp63 and TAp73 morphant embryos, although displaying specific developmental defects, 23,27,26 show an unaltered perp expression pattern, including coexpressing tissues like skin and pronephric ducts (Figure 5e; and data not shown). Also, there was no indication that loss of any of the p53/63/73 family members affects the intensity of perp expression.…”

“…At 48 hpf, p53 shows prominent expression in the brain, eyes, and gut 22 (Figure 5a). p63 is expressed in the skin 23,24 (Figure 5b), overlapping with the epidermal expression of perp, but not in the other perp expression domains. However, even within the skin, the expression patterns of perp and p63 differ.…”

“…At 24 hpf, strong expression is detected in the notochord, eye vesicles, lenses, neural tissues of the brain (ventral diencephalon and hindbrain), otic vesicles, and skin (Figure 4g-k). In the skin, perp is expressed in both epidermal cell layers, the basal layer characterized by the coexpression of the putative keratinocyte stem cell factor p63, 23,24 and the outer keratinocyte layer (Figure 4h), while the EVL cells covering the embryo during earlier stages of development have already been shed. 25 At 36 hpf, perp shows expression in the skin, nasal pits, gut, liver, and pronephric ducts (Figure 4i, m).…”

“…DNp63 MO and TAp73 MO were used as described. 23,26 Immunoblotting Embryos were injected with 75 pg perp mRNA together with either perp MO or 4 mm-perp control MO, grown until shield stage (early gastrula, 6 hpf), and embryonic protein extracts were generated as described. 71 Protein samples were separated via SDS-PAGE on 12% acrylamid/bisacrylamid gels, and blotted on Hybond P membranes (Amersham).…”