RESUMO
Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição PAX2 , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX2/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Gastrulação/genética , Animais Geneticamente Modificados , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Embrião não Mamífero/metabolismo , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Desenvolvimento Embrionário/genética , Mesencéfalo/metabolismo , Mesencéfalo/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Somitos/metabolismo , Somitos/embriologia , Fatores de Crescimento de FibroblastosRESUMO
Meiotic recombination is critical for genetic exchange and generation of chiasmata that ensures faithful chromosome segregation during meiosis I. Meiotic recombination is initiated by DNA double-strand break (DSB) followed by multiple processes of DNA repair. The exact mechanisms for how recombinases localize to DSB remain elusive. Here, we show that C19orf57/4930432K21Rik/BRME1 is a player for meiotic recombination in mice. C19orf57/4930432K21Rik/BRME1 associates with single-stranded DNA (ssDNA) binding proteins, BRCA2 and MEILB2/HSF2BP, which are critical recruiters of recombinases onto DSB sites. Disruption of C19orf57/4930432K21Rik/BRME1 shows severe impact on DSB repair and male fertility. Remarkably, removal of ssDNA binding proteins from DSB sites is delayed, and reciprocally, the loading of RAD51 and DMC1 onto resected ssDNA is impaired in Brme1 knockout (KO) spermatocytes. We propose that C19orf57/4930432K21Rik/BRME1 modulates localization of recombinases to meiotic DSB sites through the interaction with the BRCA2-MEILB2/HSF2BP complex during meiotic recombination.