| 其他摘要 | Neural crest is a multipotent, migratory cell population, which exists only in vertebrate embryos. These cells undergo migration along distinct pathways to various sites in the periphery, and then differentiate into various cell types, ranging from the peripheral nervous system to the craniofacial skeleton and pigment cells. The neural crest development is guided by a carefully orchestrated gene regulatory network, which can be divided into several modules, including: secreted signal molecules (BMP, Wnt, FGF, Delta), neural plate specifiers (Msx, Pax3/7, Zic1, Dlx3/5) and neural crest specifiers (Snail/Slug, AP-2, FoxD3, Twist, Id, cMyc, Sox9/10). In the first part, we mainly focused on the secreted signal pathways, which arise from different tissues, play distinct roles and, induce neural crest formation by integrations at multi-levels. The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In the second part, we described the cloning and expression patterns of the three Nkx6 family genes in Xenopus laevis. Like their mouse and chicken homologues, Xenopus Nkx6 family genes are mainly expressed in the CNS and anterior endodermal tissues during embryonic development. Nkx6.1 and Nkx6.2 share overlapping expression domains in the ventral neural tube at neurula stages and later in the ventral part of developing hindbrain and spinal cord. Nkx6.3 is detected in the non-neural ectoderm from cleavage to early neurula stages and in the caudal hindbrain and the mandibular arch at tail bud stages. In the endoderm, Nkx6.2 is expressed in the hypochord at tail bud stages. At tadpole stages, the three Nkx6 genes are differentially expressed in the anterior endoderm derivatives, including the pancreas, stomach, esophagus, and lung. Nkx6.3 is a recently reported member of Nkx6 family. Nkx6.3 shows distinct expression with the other two Nkx6 genes during early embryonic development in Xenopus laevis. In the third part, we reported the roles of Nkx6.3 in Xenopus by gain and loss of function studies. Both overexpression and knockdown of Nkx6.3 before gastrula stages led to gastrulation defects. Overepression of Nkx6.3 inhibited activin induced animal cap elongation, which could be rescued by co-injected dominant negative construct HDC. Further, we showed that Nkx6.3 regulated adhesion molecules expression by RT-PCR. These results suggest that Nkx6.3 plays roles in cell movements by regulating adhesion molecule expression levels. We also found that both gain and loss of function of Nkx6.3 inhibited the expression of neural crest marker Slug. Injection of Nkx6.3 mRNA at 32-cell stage led to inhibition or ectopic expression of Slug at different sites. In animal caps, overexpression of Nkx6.3 induced the expression of neural crest markers. These results indicate that Nkx6.3 is sufficient for neural crest induction. Further studies showed that Nkx6.3 up-regulated Wnt8 and Fgf8, but inhibited BMP4 in both animal caps and embryos. However, in the Nkx6.3 overexpressed embryos, the neural plate border specifiers, Msx1 and Pax3, as well as neural crest marker, Slug, were inhibited, indicating an existence of another regulating level of Nkx6.3 in the neural crest induction. Injection of Nkx6.3 at 32-cell stage inhibited Msx1, Pax3 and Slug in a cell autonomous manner while induced them in a cell unautonomous manner. In addition, we found that Nkx6.3 ectopicly induced Dlx5 while inhibited Dlx3, suggested that Dlx5 might be the direct target of Nkx6.3 at neural border specifier level. Thus, our data suggest that Nkx6.3 regulates neural crest induction at two levels: the positive contributions by regulating the secreted signaling molecules and the negative ones by inhibiting neural plate border specifiers. During the neural development of vertebrates, different types of neurons emerge along the dorso-ventral axis of the neural tube. These neurons are specified by different morphogens. A group of transcription factors are regulated by the gradient activities of these morphogens, and the cooperative regulation of these genes determines the fates of preneural cells. However, the way how these transcription factors interpret the gradient activities is not clear yet. In the fourth part of this thesis, we have tried to predict the potential regulatory sequences of these transcription factors by screening for conserved non-coding regions of these genes. In addition, we improved the transgenic method in Xenopus, by which we confirmed the regulatory region of Nkx6.2. The transcriptional regulatory regions of Dbx1, Nkx2.2 and Pax6 have been reported in mouse or Xenopus. Thus we got regulatory regions of two pairs of transcription factors which interact with each other during the neural tube pattern: Nkx6.2 and Dbx1, Nkx2.2 and Pax6. We showed that there were lots of cross talks between these four genes as well as Wnt signals by binding site predictions and TOP-flash assay. No binding site of Gli was predicted in the regulating regions of Nkx6.2 and Dbx1, indicating that these genes are not regulated by Shh signaling pathway directly. We also described the cloning and expression pattern of Dbx family genes, Dbx1 and Dbx2. These two genes are similarly expressed as two thin stripes flanking the midline in the neural plate and medially in the neural tube at the tailbud stages. Overexpression of Dbx2 inhibits N-tubulin, indicating a similar role of this gene in inhibiting neural differentiation as Dbx1. Dbx2 also inhibits expression of Nkx6.2 and Dbx1 but not Sox2, suggesting a patterning role of this gene in Xenopus neural differentiation. |
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