Pattern formation in the Drosophila eye disc

描述（由申请人提供）：果蝇眼是一个极好的模型系统，用于研究参与人眼发育以及其他发育和疾病背景的信号通路。一个遗传镶嵌屏幕已被用来确定新的基因所需的正常模式的感光细胞分化的视盘。该提案将研究表皮生长因子受体（EGFR）信号通路的正常活性所需的几个这样的基因，该信号通路控制视网膜祖细胞的增殖，存活和分化，并且在许多人类癌症中被错误调节。一个这样的基因，mago nashi，编码外显子连接复合物（EJC）的一个组成部分，它沉积在所有剪接的mRNA上。缺乏Mago nashi或其他EJC亚基的突变体显示编码丝裂原活化蛋白激酶（MAPK）的mRNA水平的特异性和显著降低，丝裂原活化蛋白激酶是EGFR和其他受体酪氨酸激酶信号传导途径的重要下游组分。该建议的第一个目的是确定EJC是否是正常mapk前mRNA剪接所需的，或者调节成熟mRNA的稳定性。这两种机制都代表了这种知之甚少的复合物的一种新的作用模式，并将有助于我们理解MAPK表达的调控。感光细胞分化所需的两个额外的基因编码内体蛋白，近视和Vps 4;该提案的第二个目的将寻求了解EGFR和其他受体的信号转导中的内吞作用。在近视突变细胞中，EGFR蛋白积聚但不能发出信号。将确定近视增强EGFR信号传导的机制。还将研究内吞途径内形成的EGFR切割片段在转录调节中的可能作用。Vps 4中的突变在蛋白质分选成多泡体以进行降解的最后步骤中起作用，将用作确定在该分选步骤期间受体活性如何改变的工具。第三个目标将寻求克隆和表征在X染色体筛选中发现的两个新基因，这两个基因可能影响通过EGFR途径的信号传导。ayn和cassandra已被定位在染色体的小区域，并将通过对候选基因进行测序来确定。将使用生物化学方法来确定它们在每个信号通路中的作用。最后，影响CSN 1b（COP 9信号体的一个亚基）的突变显示EGFR靶基因argos的表达显著增加。这种变化是由转录因子泛素化的改变介导的假说将被研究。总之，该提案将揭示对视网膜发育至关重要的保守信号通路中的新分子机制。公共卫生相关性：视网膜发育受一组保守的信号通路调节。果蝇遗传学提供了一个强大的，快速和经济的方法来确定新的组件所需的这一过程，并了解其分子机制。我们建议表征表皮生长因子受体信号传导所需的新基因，表皮生长因子受体对感光细胞分化至关重要，其失调是癌症的主要原因。进化保护表明，我们的发现将适用于包括人类在内的高等生物，并可能导致诊断或治疗影响眼睛或其他器官发育的遗传疾病的工具，或新的癌症疗法。

DESCRIPTION (provided by applicant): The Drosophila eye is an excellent model system in which to study signaling pathways that are involved in the development of the human eye as well as in other developmental and disease contexts. A genetic mosaic screen has been used to identify novel genes required for the normal pattern of photoreceptor differentiation in the eye disc. This proposal will investigate several such genes that are required for normal activity of the Epidermal growth factor receptor (EGFR) signaling pathway, which controls the proliferation, survival and differentiation of retinal progenitor cells and is misregulated in many human cancers. One such gene, mago nashi, encodes a component of the exon junction complex (EJC), which is deposited onto all spliced mRNAs. Mutants lacking Mago nashi or other EJC subunits show a specific and dramatic reduction in the levels of the mRNA encoding Mitogen-activated protein kinase (MAPK), an essential downstream component of the EGFR and other receptor tyrosine kinase signaling pathways. The first aim of this proposal is to determine whether the EJC is required for normal mapk pre-mRNA splicing, or regulates the stability of the mature mRNA. Either mechanism would represent a novel mode of action for this poorly understood complex, and would contribute to our understanding of the regulation of MAPK expression. Two additional genes required for photoreceptor differentiation encode endosomal proteins, Myopic and Vps4; the second aim of this proposal will seek to understand the role of endocytosis in signaling by the EGFR and other receptors. In myopic mutant cells, EGFR protein accumulates but is unable to signal. The mechanism by which Myopic enhances EGFR signaling will be determined. The possible role in transcriptional regulation of a cleavage fragment of the EGFR formed within the endocytic pathway will also be investigated. The mutation in Vps4, which acts at the final step of protein sorting into multivesicular bodies for degradation, will be used as a tool to determine how receptor activity is altered during this sorting step. The third aim will seek to clone and characterize two novel genes found in a screen of the X chromosome that are likely to affect signaling through the EGFR pathway. ayn and cassandra have been mapped to small chromosomal regions and will be identified by sequencing candidate genes. Biochemical methods will be used to determine their roles within each signaling pathway. Finally, mutations affecting CSN1b, a subunit of the COP9 signalosome, show a dramatic increase in the expression of the EGFR target gene argos. The hypothesis that this change is mediated by alterations in transcription factor ubiquitination will be investigated. Taken together, the proposal will reveal new molecular mechanisms in a conserved signaling pathway that is critical for retinal development. PUBLIC HEALTH RELEVANCE: Retinal development is regulated by a conserved set of signaling pathways. Drosophila genetics offers a powerful, rapid and economical method to identify novel components required for this process and to understand their molecular mechanisms. We propose to characterize new genes required for signaling by the Epidermal growth factor receptor, which is essential for photoreceptor differentiation and the misregulation of which is a major cause of cancer. Evolutionary conservation suggests that our findings will be applicable to higher organisms, including humans, and may lead to tools for diagnosis or treatment of genetic disorders affecting the development of the eye or other organs, or to new cancer therapies.