A Maternal Transcription Factor Controls The Paraxial Mesoderm Patterning In Zebrafish

Axial-paraxial mesoderm patterning is a special dorsal-ventral (DV) patterning event of vertebrates, However, the identified DV patterning mechanisms could not explain the initiation of axial-paraxial mesoderm patterning. In zebrafish, spt cell-autonomously regulates paraxial mesoderm specification and flh represses spt expression to promote axial mesoderm fate, but the expression domains of spt and flh initially overlap in the entire margin zone of the embryo. Defining flh and spt territories is therefore a premise of axial-paraxial patterning. Our revious studies have shown that a transcriptional factor Vsxl directly represses axial mesoderm gene flh transcription to preserve paraxial mesoderm gene spt expression in zebrafish. The present investigations further revealed the function and molecular mechanisn of Vsxl in regulating axial-paraxial mesoderm patterning, as well as the roles of different Vsxl domains. The main results and conclusions are as follows:1. Whole mount in situ immunohistochemistry analysis showed that maternal vsxl mRNA directs Vsxl translation in the ventrolaternal region during cleavage.2. By injection of vsxl tbMO, which can blocking the translation of vsxl mRNA, and vsxl sbMO, which can interfere with the splicing of newly synthesized zygotic vsxl mRNA but leave the maternal vsx1mRNA intact, we determine that maternal Vsxl acts as a ventralizing factor essential for paraxial mesoderm domain patterning at blastula stage.3. Maternal Vsxl promotes paraxial mesoderm specification in the ventrolateral margin via direct transcriptional represson of flh to preserve spt expression.4. Maternal Vsxl regulate the maintenance of axial-paraxial mesoderm patterning. This function is independent of Wnt8and Bmp2b signaling pathways. It seems that there are two parallel mechanisms co-regulating the maintenance of axial-paraxial mesederm patterning.5. We created a transcriptional fusion construct En-Vsxl by replacing the N-terminal125amino acids of Vsxl with Engrailed repressing domain and a transcriptional activator fusion construct VP16-Vsxl with the VP16activating domain. By analyzing the phenotypes and flh expression of the En-vsxl or VP16-vsxl mRNA injected embryos, we demonstrated that the function of N-terminal region of Vsxl is similar to the Engrailed repressor domain and Vsxl acts as a transcriptional repressor.6. The sole binding site for mediating Vsxl repressing flh at the proximal promoter of flh was identified by analyzing the mutation of consensus sequences at the binding site detected by CHIP.7. The sequence characteristics of Vsxl-specific binding site at Vsxl target genes was determined by comparing the up-and down-stream sequences of potential binding sites of flh proximal promoters. Mutation analysis revealed that, except the consensus sequences TAATTG, at last a CCC motif is essential for Vsxl-DNA reciprocal recognition and binding, and the CVC domain might be involved in Vsx1-DNA binding.These results provide an evidence for the first time that materal Vsxl controls paraxial mesoderm patterning via direct transcriptional repression in vertebrates. Therefore, zygotic signaling gradients generated after DV axis formation is unlikely the principal regulatory component of primary axial-paraxial patterning. The sequence characteristics of Vxsl binding site and the function CVC domain will be usful for revealing the roles of paired-like homeobox genes.