Molecular Regulation of Pronephric Development

前肾发育的分子调控

• 批准号: 9630621
• 负责人: Peter Vize
• 金额: $ 33.9万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630621&HistoricalAwards=false
• 关键词: Molecular; Regulation; Pronephric; Development

9630621 Vize This proposal aims to investigate the genetic interactions required for pronephric specification and patterning. Xenopus orthologs of a number of genes previously demonstrated to play essential, but poorly defined, roles in development of the mammalian adult kidney have been isolated. These genes include frog orthologs of the Wilms' tumor suppressor gene WT1, pax2, wnt4, c-met, HGF, N-myc and lim~1. A novel Xenopus pax gene which is expressed at high levels very early in kidney development has also been identified. Vertebrates form at least two different forms of kidney during their life span, normally an embryonic kidney of low or moderate complexity, and an adult kidney of much greater complexity. Once the more complex kidney is functional, the embryonic kidney either degenerates or becomes integrated into the reproductive system. The pronephros is a simple embryonic kidney used by the embryos and larvae of the lower vertebrates to excrete wastes and control water balance. The structure of one entire pronephros resembles a much larger version of a single nephron from an adult kidney. A pair of these large nephrons sustain the embryo until the adult kidney is functional, which in amphibians is around the time of metamorphosis. In comparison, the human adult kidney requires around one million nephrons to perform all of its required functions. The pronephros has a number of advantages as an experimental model for studying organogenesis of the kidney. The simple organization and large size of the organ make it easy to visualize, and as it undergoes a stereotypical pattern of morphogenesis, subtle changes in its development are easy to detect. Molecular markers are available for each of the different components of the pronephros. The pronephros is fully formed in under three days in a Xenopusembryo, so the effects of experimental interventions can be rapidly analyzed. This early development also means that this organ is induced and patterned in the period of develo pment that is easily manipulated in Xenopus by either microdissection or microinjection. In this proposal the mechanism by which ectopic Xenopus WT1 disrupts kidney development will be determined by analyzing the effect of this gene product on cell proliferation, apoptosis, and the expression of other pronephric genes. The second part of the proposal will examine the role of each of the genes listed above in pronephric development. All of these experiments will be performed by microinjecting either in vitro synthesized mRNA encoding the gene product, or expression plasmids into early blastulae, that will then be allowed to develop to tadpole stages. The effects of aberrant gene expression will be analyzed by examining the effect on the expression of other pronephric genes, and by studying the structure of the differentiated pronephros with immunohistochemistry and confocal microscopy. These experiments will use a simple model system to determine how these genes interact to pattern and form a nephron. The results will have direct relevance not only to organogenesis of other types of kidney, but also to kidney disease, as a number of these genes are involved in the development of Wilms' tumor, a pediatric tumor of the urogenital system.

9630621 VIZE该提案旨在研究倾斜规范和模式所需的遗传相互作用。许多先前证明在哺乳动物成年肾脏发育中起着必不可少但定义较差的作用的基因的爪蟾直系同源物已被分离出来。这些基因包括Wilms肿瘤抑制基因WT1，PAX2，WNT4，C-MET，HGF，N-MYC和LIM 〜1的青蛙直系同源物。 也已经确定了一种新型的Xenopus Pax基因，在肾脏发育中很早就表达。 脊椎动物在其寿命中至少形成了两种不同形式的肾脏，通常是低复杂或中度复杂性的胚胎肾脏，成年肾脏具有更大的复杂性。一旦更复杂的肾脏起作用，胚胎肾脏就会退化或整合到生殖系统中。 pronephros是一种简单的胚胎肾脏，由下脊椎动物的胚胎和幼虫用来排泄废物并控制水平衡。整个pronephros的结构类似于成年肾脏中的单个肾单位的更大版本。一对这些大肾肾脏维持胚胎，直到成年肾功能性，在两栖动物中，大约在变态时代。相比之下，人类的成年肾脏需要大约一百万个肾脏才能执行其所有必需的功能。 pronephros具有许多优势，是研究肾脏器官发生的实验模型。简单的组织和大尺寸的器官使其易于可视化，并且由于经历了形态发生的刻板印象，因此其发育的细微变化很容易检测到。分子标记可用于俯卧的每个不同成分。在异种菜单中，在三天内完全形成了pronephros，因此可以迅速分析实验干预措施的影响。这种早期发展还意味着，该器官是在脱水阶段诱导和图案化的，该时期很容易通过显微注射或显微注射在Xenopus中操纵。 在此提案中，将通过分析该基因产物对细胞增殖，凋亡和其他促脑基因的表达的影响来确定异位爪诺泊WT1破坏肾脏发育的机制。该提案的第二部分将研究上面列出的每个基因在俯卧开发中的作用。所有这些实验将通过对编码基因产物或表达质粒的体外合成mRNA进行显微注射来进行，或者将其表达为早期的囊肿，然后将其发展到tadpole阶段。通过检查对其他促脑基因表达的影响，并研究具有免疫组织化学和共聚焦显微镜的分化性促脑细胞的结构，可以分析异常基因表达的影响。 这些实验将使用简单的模型系统来确定这些基因如何相互作用并形成肾单位。结果将不仅与其他类型的肾脏的器官发生有关，而且与肾脏疾病有关，因为这些基因的许多基因参与了Wilms肿瘤的发展，Wilms肿瘤是泌尿生殖系统的儿科肿瘤。