Developmental and molecular analysis of sperm production in Drosophila

果蝇精子产生的发育和分子分析

• 批准号: 1942113
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1942113
• 关键词: Developmental; molecular; analysis; sperm; production

This project links developmental biology and determination of cell fate, through transcriptional controls that interpret that cell fate decision and control differentiation to evolution and optimisation of reproductive function. Males from the Drosophila obscura group of flies make two types of sperm. Eusperm are long, and are capable of fertilising eggs, parasperm are short and protect eusperm from spermicide in the female reproductive tract. The mechanisms underlying both the specification and differential morphogenesis of eusperm vs parasperm is not known. Many testis-specifically expressed genes identified as single copy genes in other Drosophila species (eg melanogaster) are duplicated in D. pseudoobscura. Most genes required for spermatid differentiation are transcribed in spermatocytes, we hypothesise that the spermatocytes destined to differentiate into eusperm have a different transcript profile from those that differentiate into parasperm. This would include both differential expression of single copy genes, and expression of different paralogues of multicopy genes. We have already found differential presence of mRNA from one paralogous gene pair in spermatids. Two testis-specific transcriptional regulatory complexes have been identified in D. melanogaster. Several subunits of these complexes have duplicated in the obscura group, so we propose that there are distinct versions of the transcriptional complexes, one to promote the eusperm transcript profile, the other to promote the parasperm profile. To test this hypothesis, we are conducting RNAseq on isolated individual cysts or individual spermatocytes. This will identify whether there are two distinct populations of spermatocytes, and which transcripts are differentially expressed. You will analyse the RNAseq data sets and identify candidate genes for determining the differentiation into the two sperm morphs. You will apply bioinformatics tools to analyse evolution of both coding sequences and regulatory sequences of co-regulated genes. Q-RT-PCR on isolated cysts (early spermatocytes to spermatids) will validate and extend the analysis. You will experimentally test the roles of candidate genes you identified, in the broader context of understanding how two sperm morphs are generated. The methods applied will include primary culture of spermatogenic cysts; male fertility tests and sperm function assays; using CRISPR to knock out specific regulators and cytology and molecular analyses to determine the effects in mutant testes; generation of transgenic flies - a) expressing GFP-fusion proteins for cytology and ChIP; b) with reporter constructs to identify functional transcriptional elements; c) to over express specific genes; d) to employ clonal analysis to label progeny from single stem cells.

这个项目通过转录调控将发育生物学和细胞命运的决定联系起来，转录调控解释了细胞命运的决定和控制分化与生殖功能的进化和优化。雄性暗果蝇能产生两种类型的精子。胚乳是长的，能够使卵子受精，寄生虫是短的，可以保护胚乳免受雌性生殖道中杀精剂的伤害。胚乳和寄生胚乳之间的特殊和差异形态发生的机制尚不清楚。许多在其他果蝇物种(如黑腹果蝇)中被鉴定为单拷贝基因的睾丸特异表达基因在假黑腹果蝇中是复制的。大多数精细胞分化所需的基因都是在精母细胞中转录的，我们假设定向分化为真胚层的精母细胞与分化为寄生层的精母细胞具有不同的转录谱。这将包括单拷贝基因的差异表达，以及多拷贝基因的不同平行基因的表达。我们已经在精子细胞中发现了来自一对平行基因的不同存在的mRNA。两个睾丸特异的转录调控复合体已经在黑腹毛虫中被鉴定出来。这些复合体的几个亚基在暗色组中复制，所以我们认为存在不同版本的转录复合体，一个促进裸子植物转录谱，另一个促进被子植物转录谱。为了验证这一假设，我们对分离的单个包囊或单个精母细胞进行了RNAseq。这将确定是否存在两种不同的精母细胞群体，以及哪些转录本是差异表达的。您将分析RNAseq数据集，并确定决定两种精子形态分化的候选基因。你将应用生物信息学工具来分析共调控基因的编码序列和调控序列的进化。对分离的包囊(从早期精母细胞到精子细胞)的Q-RT-PCR将验证和扩展分析。你将在更广泛的背景下，通过实验测试你确定的候选基因的作用，了解两个精子形态是如何产生的。应用的方法将包括生精囊的原代培养；男性生育测试和精子功能分析；使用CRISPR敲除特定的调节因子以及细胞学和分子分析以确定对突变睾丸的影响；产生转基因果蝇-a)表达GFP融合蛋白用于细胞学和芯片；b)用报告构建物识别功能转录元件；c)过度表达特定基因；d)使用克隆分析来标记单个干细胞的后代。