The Role Of Repression In Regulating The Spatiotemproal Expression Patterns Of Flh And Ntl

Gastrulation is a key developmental event of body plan establishment, floating head (flh) and no tail (ntl) are crucial developmental regulatory genes required for notochord formation and body plan patterning in fish. Homeobox gene flh specifically expresses in the dorsal organizer and prospective notochord and is required for promoting axial mesoderm development and repressing paraxial mesoderm formation. T-box gene ntl expresses initially in all the prospective mesodermal cells and then restricted in axial mesodermal cells, it plays important roles in mesoderm specification, convergent extension and axial mesoderm differentiation. However, the regulatory mechanism, especially the repressive regulatory mechanism, of the spatial and temporal expression pattern of flh and ntl remains unclear. Therefore, we investigated the role of repressive regulation in determining the spatiotemporal expression patterns of flh and ntl during early embryogenesis. The main results and conclusions are as follows:1. Loss-and gain-of-function studies revealed that vsxl plays a pivotal role in establishing convergent extension movement pattern and body plan, vsxl inhibits flh ectopic expression in a cell-autonomous manner and preserves the expression of spt and papc, genes required in regulating paraxial mesodermal cell specification and convergence, in the ventrolateral mesoderm.2. Promoter binding sites and expression analysis demonstrated that Boz directly represses vsxl transcription by binding to the tandem double core (C)TAAT(C) site in vsxl proximal promoter and preserves flh expression in the organizer.3. Knock-down and over-expression assays showed that Wnt8a and Bmp2b signaling pathways might be required for preserving vsxl function in the ventrolateral mesoderm by repressing boz ectopic expression outside the organizer.4. These results revealed a global mesoderm and convergent extension patterning mechanism in zebrafish:In the ventrolateral region vsxl directly inhibits flh ectopic expression in a cell autonomous manner and preserves the expression of spt in the paraxial mesoderm. In the dorsal region Boz directly represses vsxl transcription and preserves flh expression in the organizer. So, vsxl and boz directly depict the spatially complementary expression pattern of spt and flh in a cell autonomous manner, and establish the model of mesodermal cell convergent extension movements.5. To understand the biological significance of maternal-specific methylation of ntl, we measured the expression difference of ntl between gynogenetic haploids and inbreeding diploids by real-time quantitative RT-PCR and whole-mount in situ hybridization. Our results revealed that the expression level of ntl in haploids is significantly lower than that in diploids, which resulted in the failure of early mesoderm specification and convergent extension and the lack of the anterior prospective notochord in the gynogenetic haploids.6. By knocking down Kaiso, a methyl-CpG dependent binding protein and a global repressor of methylated genes, the expression level of methylated maternal ntl allele was significantly elevated in the gynogenetic haploids at sphere stage but was not elevated at early gastrula stage when the maternal ntl allele was completely demethylated. These results demonstrated that methylation modification at the ntl promoter represses maternal ntl allele transcription during early embryogenesis.7. We further directly examined the transcriptional repression effect of methylation modification at ntl promoter by analyzing the reporter gene GFP expression under the control of methylated or unmethylated ntl promoter. GFP is actively expressed in the embryos injected with unmethylated ntl promoter driven GFP sensor, but is not detected in the embryos injected with methylated ntl promoter driven GFP sensor, confirming the conclusion that methylation modification at the promoter represses maternal ntl allele expression.8. The results showed in5-7prove that methylation modification at ntl promoter plays a crucial role in regulating the timing of maternal ntl allele activation. Parent-specific methylation during gametogenesis results in the methylated maternal ntl allele is transcriptional inactive during early embryogenesis. Thus, the expression of unmethylated paternal allele is essential for initiating early mesodermal cell specification and convergent extension, and gynogenetic reproduction is prevented.These results and conclusions provide insight into the global regulation mechanisms of vertebrate body patterning and the evolutionary function of parent-specific methylation during vertebrate evolution.