| 其他摘要 | Folate, a member of the vitamin B family, functions in a series of vital biological processes, including the synthesis of DNA and amino acid, cell cycle control, and one carbon unit cycle which is important for DNA and protein methylation. In most cases, different folate functions are mediated by different folate binding proteins. In mammals, three types of trans-membrane folate binding proteins have been identified termed Folbp1, RFC and HCP respectively. Secreted folate binding protein (sFBP), which has been reported in human, mouse and swine previously, is another type of folate binding protein. But their roles are poorly understood.
In this work, a new folate binding protein is identified in Xenopus laevis. We provide evidence that this protein is secreted and has the capability to bind folate. Therefore, it is named sFBP. sFBP mRNA expressed in the ventral area of neural plate and cement gland in stage 15 Xenopus embryos. Besides neural tube and cement gland, paraxial mesoderm cells begin to express sFBP after neural tube closue. Loss of function study shows that not only convergent extension but also neural tube closure and neural crest migration are blocked when sFBP morpholino is injected. In neural plate explant from sFBP morphants containing paraxial mesoderm and neural epithelial cells, the expression of adhesive molecules are abnormal. This may account for the failure of normal cell movement of the neural cells in these embryos. Knocking down sFBP also induce tremendous cell apoptosis in the neural plate region and absence of cement gland. At tail bud stage, the sFBP morphants exhibit shortened curve axis, small head and usually have no eyes. Unexpectedly, coinjection of folate
摘要
5
derivatives or methylation assistants such as S-Adenosylmethionine(SAM) or/and Leucine methyl transferase(LCMT) can not rescue sFBP morpholino phenotypes. These data imply that sFBP might work as a signal ligand in Xenopus neural development instead of its metabolism or epigenetic roles. Finally, we find that Islet-1 has similar expression pattern with sFBP. Islet-1 morphants are reminiscent of sFBP knocking down phenotypes. Co-injection of islet-1 mRNA can largely rescue the developmental abnormities caused by sFBP morpholino. Our data suggest that sFBP is required for neural cell survival and normal convergent extension and Islet-1 may works downstream of sFBP. Neural crest cells, the vertebrate specific pluripotent stem cells, are derived from the border between the neural plate and epidermis. During late gastrulation, morphogens such as Wnt, FGF and BMP in paraxial mesoderm and epidermis initiate the expression of a group of transcription factors (Pax3, Zic1, Msx1 Dlx3/5) and thereby determine the dorsal border region of the neural plate. Thereafter, these transcription factors activate the expression of the neural crest specification genes Slug and FoxD3 and accomplish the so-called neural crest induction process.
Nkx6.3 belongs to the Nkx transcription factor. Its expression is ubiquitous in blastula stage embryos and mainly in the neural crest region and endoderm at neural plate stage. In cultured mammalian cells, Nkx6.3 is detected in the nucleus and has a molecular weight of 32.5kDa. Interestingly，in Xenopus embryos, Nkx6.3 is also detected in the cell membrane area and has an additional 47.5kDa band in Western blot analysis. Loss of function of Nkx6.3 by specific morpholino injection blocks the expression of Wnt8, Fgf8, Pax3, Zic1 and Slug in the putative neural crest region. In animal cap assay, we provide evidence that Nkx6.3 can induce neural crest differentiation through enhancing expression level of Wnt8, Fgf8 and inhibiting BMP4 mRNA transcription in a nucleus translocation dependent manner. Eh1 the transcriptional inhibitory domain in Nkx6.3 is responsible for the Wnt activation whereas the EHL domain involves in the regulation of FGF and BMP signal. The induction of neural crest by Nkx6.3 can be inhibited by co-injection of Gsk3ß but not by the dominant negative FGF receptor XFD or FGFR4DN. Interestingly,
非洲爪蛙分泌型叶酸结合蛋白在早期胚胎神经发育中的功能及 Nkx6.3 影响原肠运动和神经嵴诱导调控中
的作用研究
6
microinjection of Nkx6.3 into one blastomere of 32cell embryo, Nkx6.3 can either induce ectopic or inhibit endogenous Slug expression depending on its injection site. The different response of Slug to Nkx6.3 may due to the different concentration of Fgf8 induced and different endogenous signaling context. In 4 cell embryo, Nkx6.3 gain of function inhibits the neural crest induction as proved by the inhibition of Pax3 and Slug mRNA expression. In these embryos, Nkx6.3 mRNA injection elevates the protein level of ß-catenin, Erk1/2 and also Smad1, but inhibits endogenous Dlx5 mRNA expression. The elevation of BMP (Smad1) signal and inhibition of Dlx5 might account for the inhibition of neural crest development by Nkx6.3 in Xenopus embryos. The contradictory effect of Nkx6.3 to neural crest induction between animal caps and whole embryos might due to the different regulation to BMP signal and Dlx5 expression in these two systems. Thus, our data suggest that Nkx6.3 is required for neural crest genesis and regulate neural crest induction through regulating the key signaling molecules, Wnt, FGF and BMP. It can also regulate the expression of Dlx5 at the neural-epidermis border and thus limit the range of neural crest induction. |
修改评论