Maternal Vsxl Plavs An Essential Role In Regulating Prechordal Mesendoderm And Forebrain Formation In Zebrafish

The vertebrate brain development includes intricate moleculear, cellular and morphogenetic programs which proceed in a given precise spatiotemporal pattern. The tremendous complexity of the vertebrate brain makes it a challenging task to elucidate how this structure forms during embryonic development. The construction of brain is evolutionarily conserved among vertebrates, as well as the development of the central nervous system. It has been firmly established that the vertebrate forebrain arises from the anterior neuroectoderm under the induction of underlying prechordal mesendoderm (PME) during gastrulation. Therefore, the establishment of PME in a desired shape and size and the proper spatial relationship between PME and neuroectoderm during early embryogenesis are critical for normal forebrain development. However, it remains largely unclear how the PME and forebrain are generated in a desired form and size at a correct position during early embryogenesis. Recently, it has been observed that in maternal Vsxl knockdown zebrafish embryos the axial mesoderm domain is shorter than that in the wild type control at bud stage, implying that maternal Vsxl is involved in regulating anterior-posterior patterning. Here we show that maternal Vsxl plays a pivotal role in controlling PME and forebrain formation in a desired shape and size at a correct position during early embryogenesis in zebrafish.1. Knocking down maternal vsxl resulted in impaired PME formation and progression associated with a deficient and posteriorized forebrain. These results indicate that maternal Vsxl plays a pivotal role in regulating PME and forebrain formation and position.2. Supplying a deficiency of Dkkl, which antagonizes the caudalizing Wnt signaling to induce forebrain formation, effectively rescued the forebrain phenotype but not the position in vsxl morphants, indicating that maternal Vsxl could not directly regulate the fate specification of the forebrain progenitors, but rather regulates PME formation and positioning to control the forebrain formation and positioning during early embryogenesis.3. Changes of cell shape analysis demonstrated that, at60%epiboly stage, directional elongation of the cells at the leading edge of the mesendoderm, close to the border between epiblast and hypoblast, was inhibited in maternal Vsxl knockdown embryos, thereby, the presumptive PME cells could not migrate to the animal pole. These results demonstrated that maternal Vsxl regulates PME formation by promoting the directional elongation and migration of presumptive PME cells.4. Loss-and gain-of-function experiments in combination with whole-mount in situ hybridization and real-time quantitative RT-PCR analysis revealed that maternal Vsxl is essential for repressing ntl ectopic expression in more animal region at early gastrula stages.5. Chromatin immunoprecipitation assay in combination with core consensus sequence mutation analysis further revealed that maternal Vsxl can directly repress ntl transcription by binding to the proximal promoter at a specific site.6. Simultaneous inhibition of ntl function successfully suppressed the defects of both PME and forebrain formation in maternal Vsxl knockdown embryos. On the contrary, misexpression of ntl would result in head truncation in zebrafish. These results indicate that ntl misexpression resulted in the aberrant formation and positioning of PME and forebrain in maternal Vsxl knockdown embryos.Taken together, our study revealed a maternal factor controlled mechanism in establishing normal PME and forebrain at a correct position during early embryogenesis in zebrafish:1) maternal Vsxl protein functions as a direct transcriptional repressor of ntl in the animal half at early gastrula stages;2) removing the repression will result in ntl ectopic expression more animally and severely disturb PME and forebrain formation by inhibiting the directional elongation and migration of presumptive PME cells. These results provide an insight into how the intricate molecular, cellular and morphogenetic processes of forebrain formation are initially regulated.