Developmental and molecular analysis of sperm production in Drosophila

该项目将发育生物学和细胞命运的确定联系起来，通过转录控制来解释细胞命运的决定，并控制分化到进化和生殖功能的优化。果蝇的雄性产生两种类型的精子。真精子长，能够使卵受精，副精子短，保护真精子不受雌性生殖道中杀精剂的伤害。真精子与准精子的特化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果蝇中被复制。伪暗精子细胞分化所需的基因大多在精母细胞中转录，我们假设，精母细胞注定要分化成真精子有不同的转录谱从那些分化成parasperm。这将包括单拷贝基因的差异表达和多拷贝基因的不同旁系同源物的表达。我们已经在精子细胞中发现了来自一对旁系同源基因的mRNA的差异。在D.黑腹菌这些复合物的几个亚基在暗组中重复，因此我们提出转录复合物有不同的版本，一个促进真精子转录谱，另一个促进旁精子谱。为了验证这一假设，我们正在对分离的单个包囊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，以及哪些转录本差异表达。您将分析RNAseq数据集，并识别用于确定两种精子形态分化的候选基因。您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（早期精母细胞到精子细胞）进行Q-RT-PCR将验证和扩展分析。您将通过实验测试您确定的候选基因的作用，在更广泛的背景下了解两种精子变体是如何产生的。所应用的方法将包括生精囊肿的原代培养;雄性生育力测试和精子功能测定;使用CRISPR敲除特定调节剂以及细胞学和分子分析以确定突变睾丸中的作用;产生转基因果蝇- a）表达GFP融合蛋白用于细胞学和ChIP; B）具有报告构建体以鉴定功能性转录元件; c）过表达特定基因; d）采用克隆分析来标记来自单个干细胞的后代。

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.