REGULATION OF CELL AND ORGAN SIZE IN VIVO

动物最终达到的大小既取决于细胞总数，也取决于单个细胞的大小。细胞总数由细胞增殖和细胞死亡决定。近年来，我们对这两个过程的监管的理解取得了实质性进展。相比之下，调节单个细胞大小的机制还没有被很好地理解。这项提案中详细介绍的实验旨在了解体内调节细胞大小的机制。利用FLP-重组酶在果蝇眼中诱导有丝分裂重组的遗传筛选被用来鉴定导致细胞大小增加的突变。到目前为止分离出的突变映射到六个不同的基因位点。其中一个基因座对应于Gigas基因，其他基因已经确定了该基因的特征，并导致了巨大的多倍体细胞。Gigas基因是TSC2基因的果蝇同源物，TSC2基因在大约70%的结节性硬化症患者中发生突变。在屏幕上识别的基因座之一，Rocky的表型与Gigas极其相似，并映射到TSC1基因的位置，该基因在30%的结节性硬化症患者中发生了突变。另一种突变是在不改变倍性的情况下增加细胞大小，它映射到PTEN基因的附近，该基因在许多不同的人类癌症中发生突变。另一个扩展的，似乎是以非自治的方式行动。另外两个还没有被绘制出来。该提案的具体目标1描述了完成一项全面的基因筛查，以确定导致细胞大小增加的突变。突变将被分类为互补组，并将绘制基因图谱。具体目标2描述了巨型、落基型、狼型和扩张型的详细表型分析。实验将解决这些突变增加细胞大小的确切机制。在每种情况下，突变对倍性、生长和周期调节的影响将被确定，该基因的作用将在已知的细胞大小调节的背景下进行研究。具体目标3描述了描述剩余突变和那些将在筛查接近完成时被识别的突变的表型的一般策略。还描述了一种克隆基因的方法。对筛查中发现的突变的分析将有助于我们理解体内细胞大小的调节。

The eventual size reached by an animal is determined both by the total number of cells as well as the size of individual cells. Total cell number is determined by cell proliferation and cell death. Recent years have witnessed substantial progress our understanding of the regulation of these two processes. In contrast, the mechanisms that regulate the size of individual cells are not well understood. The experiments detailed in this proposal are aimed at understanding the mechanisms that regulate cell size in vivo. A genetic screen using FLP-recombinase induced mitotic recombination in the Drosophila eye was used to identify mutations that resulted in increased cell size. The mutations isolated to date map to six different genetic loci. One of the loci corresponds to the gigas gene which has been characterized by others and results in large polyploid cells. The gigas gene is the Drosophila homologue of the TSC2 gene which is mutated in approximately 70 percent of patients with tuberous sclerosis. One of the loci identified in the screen, rocky has a phenotype that is extremely similar to that of gigas and maps to the location of the TSC1 gene which is mutated in 30 percent of patients with tuberous sclerosis. Another mutation, which increases cell size without altering ploidy, maps to the vicinity of PTEN gene which is mutated in a number of different human cancers. Another, expanded, appears to act in a non-autonomous manner. Two others have not yet been mapped. Specific Aim 1 of this proposal describes the completion of a comprehensive genetic screen to identify mutations that lead to an increase in cell size. Mutations will be sorted into complementation groups and the genes will be mapped. Specific Aim 2 describes a detailed phenotypic analysis of gigas, rocky, wolf and expanded. Experiments will address the precise mechanism by which these mutations increase cell size. In each case the effect of the mutation on ploidy, growth and cycle regulation will be determined and the role of the gene will be studied in the context of known regulators of cell size. Specific Aim 3 describes a general strategy for characterizing the phenotype of the remaining mutations and those that will be identified as the screen moves to completion. An approach to cloning the genes is also described. Analysis of the mutations identified in the screen will contribute to our understanding of cell size regulation in vivo.