Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

NC在脊椎动物的头、脸和颚的发育中起着至关重要的作用，它提供了颅面骨骼、周围神经系统和其他颅组织的主体。正常颅面发育依赖于NC细胞及其衍生物的适当诱导、迁移和分化。这些步骤的任何缺陷，无论是由于NC本身的内在缺陷，还是NC细胞无法与邻近组织正常相互作用，都可能导致出生缺陷：所有先天性畸形中有三分之一本质上是颅面畸形。在这个项目中，我们使用一个关键的NC控制因子TFAP2作为分子工具来鉴定编码NC迁移和分化到颅面组织所需蛋白质的靶基因。构成本研究重点的三个基因要么本身是新的（PCNS和Inka），要么以前没有显示在NC （MyoX）中起作用，并且具有极好的潜力，可以为颅面发育提供新的见解。特别是，我们与Inka的研究结果有望在通过小gtpase和PAK4调节细胞骨架和通过调节细胞-细胞信号传导调节基因表达之间建立新的联系。在过去的两年里，我们完成并发表了PCNS和MyoX的研究，现在我们将重点放在Inka及其在细胞和胚胎中的交互伙伴上。

The NC plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations are craniofacial in nature. In this project we have used a key NC control factor, TFAP2, as a molecular tool to identify target genes encoding proteins that are required for NC migration and differentiation into craniofacial tissue. The three genes that comprise the focus of this research are either themselves novel (PCNS and Inka) or have not previously been shown to function in NC (MyoX), and have an excellent potential to yield new insights into craniofacial development. In particular, our findings with Inka promise to lead to a new link between cytoskeletal regulation through small GTPases and PAK4 and regulation of gene expression through modulation of cell-cell signaling. In the previous two years we completed and published our studies on PCNS and MyoX, and now focus on focus on Inka and its interaction partners in cells and embryos. Using yeast two-hybrid screening we discovered that Inka interacts with p21-activated kinase 4 (PAK4). This kinase is part of a family of proteins that mediate cell-cell signaling through the small GTPase molecules Rac, Rho and Cdc42. We have found that stable expression of mouse Inca in NIH3T3 cells results in inhibition of PAK4 phosphorylation. PAK4 has been reported to bind to actin filaments and regulate their dynamic remodeling through LIM kinase 1 and cofilin. If Inka controls PAK4 activity, this would predict an effect on the microfilament network in cells. We have tested and confirmed this hypothesis by examining phalloidin staining of a stable Inca expressing NIH3T3 line and showing that these cells have enhanced stress fiber and focal adhesion networks, and also migrate more rapidly than controls. Rescue/reversion experiments showed that these functions depend on the kinase activity of PAK4. Several lines of evidence link the regulation of microfilaments and microtubules, suggesting the possibility that Inka might also affect microtubule dynamics, either indirectly by altering microfilaments or by a more direct mechanism. This was also investigated in the Inca expressing cell lines, and also in Xenopus embryos. We discovered that the level of alpha-tubulin acetylation is inhibited by Inka. This also appears to be mediated via PAK4, but via a mechanism that is independent of the kinase activity. The inhibition appears to be exerted at the level of acetylation, rather than the deacetylation step that has been characterized by other laboratories. Interestingly, Inka also inhibits the acetylation of histones, suggesting a possible role in the control of chromatin architecture. Such a molecular link between the regulation of gene expression and cytoskeletal dynamics would be represent a novel mechanism for coordinating cell behavior and differentiation.