Sox gene function in Drosophila testis development

在人类中，发育中的胚胎的性别由Y染色体上编码的调节蛋白SRY的活性决定。SRY的作用之一是在性腺早期启动一个基因表达程序，引导细胞沿着男性发育的道路前进。虽然SRY的活性及其调控的靶基因的完整谱系尚不清楚，但其靶标之一是相关的调控蛋白SOX9。多项研究表明，SOX9单独能够引导哺乳动物早期性腺的雄性分化。在爬行动物中，Sox9在睾丸的正确发育中扮演着重要角色。最近，我们发现了一个与Sox9(Sox100B)相关的果蝇基因，该基因在果蝇发育过程中的睾丸中有特异表达。更值得注意的是，我们已经证明苍蝇基因的突变会导致睾丸发育失败。因此，尽管决定性别的分子机制在多细胞生物体中似乎完全不同，但似乎控制睾丸发育的基因可能存在潜在的保守性。由于确定睾丸是哺乳动物性别决定的关键事件，因此对果蝇中Sox100B活性的分析可能有助于深入了解人类性别决定的分子事件和总体上的睾丸生物学。我们将利用我们开发的几种方法，这些方法可以对苍蝇中的调节蛋白活动进行全球分析。使用一种名为DNA微阵列分析的技术，我们能够同时检查苍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集。我们进行的初步实验表明，这种方法是可行的，它可以识别哺乳动物睾丸中表达的苍蝇基因和近亲基因，这表明我们可以识别可能是Sox9靶标的人类基因。然而，与多细胞生物体中的微阵列分析相关的问题之一是，它并不总是直接地与特定调控因子的直接活性相关的基因表达变化。为了绕过这一点，我们将使用我们最近开发的一种方法来确定Sox100B蛋白在睾丸发育过程中与苍蝇基因组的结合位置。通过用交联剂固定发育中的睾丸，我们可以在Sox100B在调节特定基因的过程中活跃的时候将其‘粘合’到DNA上。我们可以分离连接了Sox100B的DNA，并使用包含整个苍蝇基因组的探针的微阵列来识别它在苍蝇基因组中的位置。结合微阵列表达分析，这将最终确定Sox100B在发育中的睾丸中调节的一组基因。我们对Sox100B缺失导致的果蝇睾丸缺陷的分析，结合精确定义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在人类基因组中确定Sox9的潜在靶点，Sox9是与性别决定和男性不育有关的候选基因。在果蝇等模型系统中进行的这种研究，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。尽管苍蝇和人类可能看起来非常不同，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与哺乳动物的亲缘关系相似。由于苍蝇的基因组要小得多，因此在实验上比人类或老鼠更容易处理，我们可以利用苍蝇来发现基因和途径，这些基因和途径将从哺乳动物的集中分析中受益。我们提出的基于全基因组的方法为探索保守的调控蛋白提供了一个无与伦比的机会。

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.