Sox gene function in Drosophila testis development

在人类中，发育中胚胎的性别是由Y染色体上编码的调节蛋白SRY的活性决定的。SRY的作用之一是启动早期性腺中的基因表达程序，指导细胞沿着雄性发育途径发展。虽然SRY的活性及其调控的靶基因的完整库仍然未知，但其靶基因之一是相关的调控蛋白SOX 9。几项研究表明，SOX 9单独能够指导哺乳动物早期性腺的雄性分化。在爬行动物中，Sox 9在睾丸的正确发育中发挥作用。最近，我们发现了一个与Sox9相关的果蝇基因（Sox100B），该基因在发育中的果蝇睾丸中特异表达。更值得注意的是，我们已经表明，果蝇基因的突变导致睾丸发育失败。因此，虽然决定性别的分子机制在多细胞生物中似乎完全不同，但似乎可能存在调节睾丸发育的基因的潜在保守性。由于指定一个睾丸是哺乳动物性别决定的关键事件，这是可能的，在苍蝇中的Sox100B活性的分析将导致深入了解人类性别决定和睾丸生物学的分子事件。我们将利用我们已经开发的几种方法，可以对苍蝇中的调节蛋白活性进行全局分析。使用一种称为DNA微阵列分析的技术，我们能够同时检查果蝇基因组中的所有基因。通过比较正常果蝇和Sox100B突变体，我们可以确定由于Sox100B缺失而下调或上调的基因组。我们已经进行的初步实验表明，这种方法是可行的，它可以识别果蝇基因与哺乳动物睾丸中表达的亲属，这表明我们可以识别人类基因，可能是Sox 9的目标。然而，与多细胞生物中的微阵列分析相关的问题之一是，它并不总是直接将基因表达的变化与特定调节剂的直接活性相关联。为了避免这一点，我们将使用我们最近开发的方法来确定在睾丸发育过程中Sox100B蛋白在果蝇基因组中的结合位置。通过用交联剂固定发育中的睾丸，我们可以将Sox100B“粘”在DNA上，同时它在特定基因的调节过程中是活跃的。我们可以分离出带有Sox100B的DNA，并使用含有整个果蝇基因组探针的微阵列来识别它来自果蝇基因组的何处。结合微阵列表达分析，这将明确确定一组基因，Sox100B是在发育中的睾丸调节。我们对果蝇睾丸中由于Sox100B缺失而导致的缺陷的分析，结合准确定义Sox100B靶基因的方法，将提供对Sox100B功能的非常详细的理解。这反过来可能有助于确定人类基因组中Sox 9的潜在靶点，Sox 9是涉及性别决定和男性不育的候选基因。在果蝇等模型系统中进行的此类研究对于帮助我们了解人类基因组是如何组织和表达的非常有价值。虽然苍蝇和人类看起来非常不同，但多年的分子和遗传分析表明，指导苍蝇关键发育过程的基因在哺乳动物中有类似的工作。由于苍蝇基因组要小得多，因此比人类或小鼠更容易通过实验处理，我们可以利用苍蝇来发现将从哺乳动物的集中分析中受益的基因和途径。我们提出的基于全基因组的方法为探索保守的调节蛋白提供了无与伦比的机会。

In humans, the sex of the developing embryo is determined by the activity of a regulatory protein encoded on the Y-chromosome, SRY. One of the roles of SRY is to initiate a program of gene expression in the early gonad that directs cells down the male pathway of development. While the activity of SRY and the complete repertoire of target genes it regulates in still unknown, one of its targets is the related regulatory protein SOX9. Several studies have shown that SOX9 alone is able to direct male differentiation of early gonads in mammals. In reptiles, Sox9 has a role to play in the correct development of the testis. Recently, we identified a Drosophila gene related to Sox9 (Sox100B) that shows specific expression in the developing fly testis. More remarkably, we have shown that mutations in the fly gene lead to a failure in testis development. Thus, while the molecular mechanisms that determine sex appear to be completely different in multicellar organisms, it appears that there may be underlying conservation of the genes regulating testis development. Since specifying a testis is the key event in mammalian sex determination, it is possible that an analysis of Sox100B activity in flies will lead to insights into the molecular events underlying human sex determination and testis biology in general. We will take advantage of several approaches we have developed that allow a global analysis of regulatory protein activity in the fly. Using a technique known as DNA microarray analysis, we are able to examine all of the genes in the fly genome simultaneously. By comparing normal flies with Sox100B mutants we can define the set of genes that are down or up regulated because of the loss of Sox100B. Preliminary experiments we have performed indicate that this approach is feasible and that it can identify fly genes with relatives that are expressed in the mammalian testis, suggesting we can identify human genes that may be targets of Sox9. However, one of the problems associated with microarray analysis in multicellular organisms is that it is not always straightforward to related changes in gene expression with the direct activity of a particular regulator. To circumvent this we will use a method we have recently developed to determine where the Sox100B protein is bound in the fly genome during testis development. By fixing developing testes with cross-linking agents, we can 'glue' Sox100B to the DNA while it is active during the regulation of specific genes. We can isolate the DNA with Sox100B attached and identify where in the fly genome it comes from using microarrays containing probes for the entire fly genome. Combined with the microarray expression analysis this will definitively identify a set of genes that Sox100B is regulating in the developing testis. Our analysis of defects in fly testes due to loss of Sox100B combined with the approaches to accurately define Sox100B target genes, will provide a very detailed understanding of the function of Sox100B. This in turn may help to identify potential targets of Sox9, candidate genes implicated in Sex determination and male infertility, in the human genome. Such studies in model systems such as Drosophila are very valuable in helping us understand how the human genome is organised and expressed. While it may appear that flies and humans are very different, years of molecular and genetic analysis have shown that genes which direct key developmental processes in the fly have relatives that perform similar jobs in mammals. Since the fly genome is considerably smaller, and therefore much easier to deal with experimentally than human or mouse, we can use the fly to discover genes and pathways that will benefit from a focused analysis in mammals. The genome-wide based approaches we propose offer an unparalleled opportunity to explore conserved a regulatory protein.