Mechanisms of Embryonic Patterning and Lineage Specification

0318768 EtkinsI.在非洲爪蛙的智力优势研究中，定位的母体RNA和蛋白质在早期胚胎发育中发挥着重要作用。在大规模植物定位的母体RNA筛选中，克隆了编码FATVG的基因。Fatvg通过Metro(早期)和晚期途径定位，并与卵裂期胚胎的种质有关。它与一组哺乳动物蛋白有关，包括脂肪细胞分化相关蛋白(ADRP)和TIP47，它们具有不同的功能，包括脂体运输、信号通路调节和各种细胞类型内的分子运输。用反义寡核苷酸对FATVG功能丧失的分析表明，FATVG在背侧/腹侧模式和生殖细胞谱系的指定中具有双重作用。数据还表明，这种双重效应是通过母体背靠背和生殖细胞决定因素在脂肪耗尽的胚胎中无法正确分离而产生的。这表明，决定生殖细胞谱系和背轴/腹轴的决定因素可能利用共同的机制将其产物分离到胚胎中，或者它们的功能通过共同的途径进行调节。很可能，参与背部和生殖细胞决定因素运输的机制依赖于携带这些因素的小泡的运动。这与观察到的FATVG蛋白包被位于植物皮层的囊泡状结构是一致的。这代表了一种新的、新的机制，涉及到调节生殖细胞规格和背腹模式。根据FATVG相关蛋白在哺乳动物细胞分子运输中的潜在功能，推测FATVG通过调节分子运输，在启动胚系指定和背腹模式的分子通路中发挥关键作用。这一假说将通过实现以下特定目标来验证：a.分析FATVG蛋白与休眠因子和生殖细胞决定因素在细胞结构上的共存以及在FATVG缺失的胚胎中这一点是如何改变的；b.确定含有Fatvg的囊泡、细胞骨架和分子马达之间的关系；c.确定Fatvg通过哪些相互作用的蛋白质发挥作用。ii.拟议研究产生的更广泛的影响。FATVG通过其在分子运输中的作用在模式形成和细胞谱系指定中的作用是独特的，并将阐明一种整合这两个过程的新机制。这将是对理解早期发育机制的重要贡献，并将对发育生物学、细胞生物学和胚胎学领域产生广泛影响。除了潜在的科学进步，拟议的项目还将涉及本科生(实验室暑期学生)、研究生和博士后科学家的培训。这是我们作为科学家工作的一个重要方面，也是这一领域的工作者如何与社会联系的一个重要方面。

0318768EtkinsI. Intellectual merit of the proposed studyIn the frog, Xenopus laevis, localized maternal RNAs and proteins play important roles in early embryogenesis. In a large scale screen for vegetally localized maternal RNAs the cDNA encoding fatvg was cloned. Fatvg is localized through both the METRO (Early) and late pathways and is associated with the germ plasm in cleavage stage embryos. It is related to a group of mammalian proteins that include adipocyte differentiation related protein (ADRP) and TIP47 that have diverse functions including transport of lipid bodies, regulation of signaling pathways, and molecular trafficking within a variety of cell types. A loss of function analysis of fatvg using antisense oligodeoxynucleotides demonstrated that fatvg plays a dual role in dorsal/ventral patterning and in the specification of the germ cell lineage. The data also show that this dual effect is through the inability of maternal dorsalizing and germ cell determinants to segregate properly in fatvg?depleted embryos. This indicates that both the determinants specifying the germ cell lineage and the dorsal/ventral axis may utilize a common mechanism in segregating their products in the embryo or that their functions are regulated through a common pathway. It is likely, that the mechanism involved in the transport of dorsal and germ cell determinants relies on the movement of vesicles that carry these factors. This is consistent with the observations that fatvg protein is detected coating vesicle-like structures located at the vegetal cortex. This represents a new and novel mechanism involved in regulating germ cell specification and dorsal?ventral patterning. Based on the potential function of fatvg related proteins in molecular trafficking in mammalian cells it is hypothesized that fatvg is a key player in initiating the molecular pathways responsible for germ line specification and dorsal ventral patterning through its role regulating molecular trafficking. This hypothesis will be tested by carrying out the following specific aims: A. To analyze the co localization of fatvg protein with dorsalizing factors and germ cell determinants on cellular structures and how this is altered in fatvg depleted embryos; B. To determine the relationship between fatvg containing vesicles, the cytoskeleton and molecular motors; C. To identify the interacting proteins through which Fatvg functions.II. Broader Impact resulting from the proposed study.The role of fatvg in both patterning and cell lineage specification through its role in molecular trafficking is unique and will illistrates a novel mechanism that integrates both processes. This would be an important contribution to the understanding of early developmental mechanisms and would have a broad impact on the fields of developmental biology, cell biology, and embryology. In addition to the potential scientific advance, the proposed project will involve the training of undergraduates (summer students in the laboratory), graduate students, and postdoctoral scientists. This is an important aspect of our work as scientists and how workers in this field relate to society.