Identifying a transcriptional program that regulates compensatory proliferation

描述(申请人提供)：所有动物在发育和衰老过程中都会受到损害，许多生物体都进化出了应对和修复这种损害的机制。一种这样的机制是补偿性增殖，即由于损伤而死亡的细胞将有丝分裂地向周围细胞发出信号，以促进增殖并取代丢失的细胞。这一过程被大量用于发育期间受损的前体细胞群体和成年后的干细胞。然而，许多有丝分裂后的细胞似乎无法重新进入细胞周期并对损伤做出反应而增殖，这阻碍了成年组织的再生；这种无法再生的原因尚不清楚。在果蝇的眼睛前体组织中，有一群有丝分裂后的未分化细胞能够进行补偿增殖。这些细胞如何能够克服负的细胞周期调节并重新进入细胞周期目前尚不清楚。阐明与此相关的基因 这一过程对于理解如何阻止或逆转许多与衰老相关的表型很重要。例如，随着年龄的增长，组织的再生能力会下降，从而导致损伤的累积。如果在这些组织中能够刺激代偿性增殖所需的基因，就可能诱导再生。此外，损伤往往导致炎症和过度增殖，这有助于疾病的发展。因此，阻断参与代偿增殖的基因可能会阻止多余组织的积累和疾病的进展。这项建议旨在确定果蝇有丝分裂后眼睛前体种群中参与补偿性增殖的基因，假设这一过程需要一个独特的转录程序，因为已经显示了转录调控的要求。我将通过使用荧光激活的细胞分类来表征这些细胞中的转录图谱来验证这一假设 (FACS)分离代偿性增殖群体，RNA-SEQ鉴定转录组。在这些细胞中高表达的基因，而不是在非增殖细胞中表达的基因，很可能参与了代偿性增殖。除了提供补偿增殖过程中可能需要的候选基因外，这些RNA-SEQ实验的结果还将为补偿增殖提供一个假想的分子网络。第二种互补方法将利用RNAi来识别补偿增殖所需的转录因子。我们将通过分别下调所有果蝇转录因子的表达来确定这些必要的基因，其中大多数转录因子具有脊椎动物的同源物；发育中的眼睛受到损伤时，补偿增殖所需的基因将由于存在的细胞数量的减少而产生粗糙的小成年眼睛。来自rna-seq实验的任何其他候选者也将使用该测试进行测试。将进行进一步的遗传和分子分析，以表征多个途径之间的相互作用。这些实验的结果将提供对补偿性增殖的全面看法，并将提供可能参与脊椎动物再生和疾病的候选基因。

DESCRIPTION (provided by applicant): All animals undergo damage as they develop and age, and many organisms have evolved mechanisms to respond to and repair this damage. One such mechanism is compensatory proliferation, in which cells that are dying due to damage will mitogenically signal surrounding cells to increase proliferation and replace lost cells. This process is heavily utilized in damaged precursor cell populations during development and stem cells during adult life. However, many post-mitotic cells appear unable to re-enter the cell cycle and proliferate in response to damage, which hinders regeneration of adult tissues; the reasons for this inability are not known. In the eye precursor tissue of the fruit fly Drosophila melanogaster, there is a population of post-mitotic, undifferentiated cells that are able to undergo compensatory proliferation. How these cells are able to overcome negative cell cycle regulation and re-enter the cell cycle is currently unknown. Elucidating the genes involved in this process is important for understanding how to stop and perhaps reverse many of the phenotypes associated with aging. For example, tissues often accumulate damage with age as their regenerative abilities decrease. If the genes required for compensatory proliferation can be stimulated in these tissues, regeneration may be induced. Furthermore, damage often leads to inflammation and hyperproliferation, which contribute to disease development. Therefore, blocking genes involved in compensatory proliferation may prevent the accumulation of excess tissue and disease progression. This proposal aims to identify the genes involved in compensatory proliferation in the Drosophila post-mitotic eye precursor population, with the hypothesis that a unique transcriptional program is necessary for this process since a requirement for transcriptional regulation has already been shown. I will test this hypothesis by characterizing the transcriptional profile in these cells using fluorescence-activated cell sorting (FACS) to isolate the compensatory proliferating population and RNA-seq to identify the transcriptome. Genes that are highly expressed in these cells and not in their non-proliferating counterparts are likely to be involved in compensatory proliferation. In addition to providing candidate genes that may be required during compensatory proliferation, the results from these RNA-seq experiments will provide a hypothetical molecular network for compensatory proliferation. The second complementary approach will utilize RNAi to identify transcription factors required for compensatory proliferation. We will identify these necessary genes by individually knocking down expression of all Drosophila transcription factors, the majority of which have vertebrate homologs; genes that are required for compensatory proliferation in developing eyes subjected to damage will produce rough, small adult eyes due to a decrease in the number of cells present. Any additional candidates from the RNA-seq experiments will also be tested using this assay. Further genetic and molecular analysis will be performed to characterize the interactions between multiple pathways. The results from these experiments will provide a comprehensive view of compensatory proliferation and will provide candidate genes that may be involved in regeneration and disease in vertebrates.