Developmental and molecular analysis of sperm production in Drosophila

该项目将发育生物学和细胞命运的决定联系起来，通过转录控制来解释细胞命运决定和控制分化，以进化和优化生殖功能。果蝇中的雄性产生两种精子。精胚很长，能够使卵子受精；超胚很短，可以保护精子不受雌性生殖道中杀精剂的伤害。原种与异胚乳的形态发育差异机制尚不清楚。许多睾丸特异性表达的基因在其他果蝇物种（如黑腹果蝇）中被鉴定为单拷贝基因，在伪眼果蝇中被复制。精子分化所需的大多数基因在精母细胞中转录，我们假设注定分化为真精子的精母细胞与分化为异精子的精母细胞具有不同的转录谱。这既包括单拷贝基因的差异表达，也包括多拷贝基因的不同同源物的表达。我们已经在精子中发现了同源基因对mRNA的差异存在。两种睾丸特异性转录调控复合物已在黑腹蝶中被鉴定出来。这些复合物的几个亚基在暗室群中有重复，因此我们提出存在不同版本的转录复合物，一个促进真种转录谱，另一个促进准种转录谱。为了验证这一假设，我们正在对分离的单个囊肿或单个精母细胞进行RNAseq。这将确定是否存在两个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数据集并确定确定分化为两种精子形态的候选基因。您将应用生物信息学工具来分析编码序列和共调节基因的调节序列的进化。对分离的包囊（早期精子细胞到精子细胞）进行Q-RT-PCR将验证并扩展分析。在了解两种精子形态是如何产生的更广泛的背景下，你将通过实验测试你确定的候选基因的作用。应用的方法包括原代培养生精囊肿；男性生育能力测试和精子功能分析；使用CRISPR敲除特定的调节因子，并用细胞学和分子分析来确定突变睾丸的影响；产生转基因果蝇- a)表达gfp融合蛋白用于细胞学和ChIP；B)使用报告结构来识别功能性转录元件；C)过表达特定基因；D)利用克隆分析来标记单个干细胞的后代。

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.