Genetic variation and function of body axis determinants in midges and other flies

项目总结 我们的目标是发现密切相关的物种如何采用不相关的轴决定因素来指定 前胚胎。动物发育的许多方面在不同物种之间是保守的，就像人类一样 和苍蝇，但一些关键的调节器在进化过程中变化非常迅速，原因不明。 解释发育基因网络中这种出人意料的可塑性将有助于我们理解基础科学 包括人类在内的所有动物的发育健壮性和先天性疾病。比较多个 亲缘关系密切的物种是理解基因网络中可塑性原因的有力途径。这 这种方法在脊椎动物模型生物中很难实现，但很容易实现 在昆虫身上。苍蝇(双翅目)特别适合，因为它们包括黑腹果蝇，一种 在发育生物学中领先的模式生物，以及因为其他双翅目物种可以在 实验室环境对功能研究是适用的。我们对多种双翅目模式生物的研究 在之前的资助期间，确定了这些密切相关的物种使用广泛的前额 确定胚胎极性和前特异性基因表达的决定因素(ADS)，很可能是通过 形成具有类似形态原活性的长程转录因子梯度。这一发现使我们能够 来研究为什么关键的发育调节因子在进化过程中会高度不稳定。 虽然果蝇的AD是一种经典的形态发生模型，但目前尚不清楚其发病机制。 行为是可以概括的，也不知道向双曲面依赖的图案形成的进化转变是如何 已实现。这两个问题中的第一个问题将通过识别和表征染色质和 白斑潜蝇Clogia albipot tata，Cal-Opamat(Aim 1)中AD基因靶点的研究 常见的河摇蚊，Panish(目标2)。这将通过测试依赖AD来完成 使用ATAC-SEQ和ATAC-SEQ研究有或没有AD活性降低的阶段匹配胚胎中染色质的可及性 RNAi。这些实验将根据CHIP-SEQ的需要进行补充，并通过对表达式进行表征 以及体内预测的靶基因的功能。第二个问题，关于向双曲面的过渡- 相关图案的形成，将通过确定前规范的机制在 士兵驾驶Hermetia IlLucens(瞄准3)。之所以选择这种有机体，是因为它是最容易驯服的 外群到具有双半乳突的物种支系。一项初步研究揭示了几个AD候选人，包括Hil-Stau， 史陶芬的正字同源词。这种RNA结合蛋白能结合果蝇早期胚胎中的双核糖核酸。要确定 Hermetia的AD、前定位和hil-Stau结合的mRNAs将通过表达研究确定，共同- 免疫沉淀、测序和它们的功能将通过RNAi进行检测。前部的测定- Clogia、Chironomus和Hermetia的具体机制将产生一个新的解释框架 模式形成和基因调控的经典模型中关键调控因子的进化可塑性。

PROJECT SUMMARY Our goal is to discover how closely related species adopted unrelated axis determinants for specifying the anterior embryo. Many aspects of animal development are conserved between species as different as humans and flies, but some key regulators change very rapidly over the course of evolution for unknown reasons. Explaining such unexpected plasticity in developmental gene networks will help us understand the basic science of developmental robustness and congenital disease in all animals, including humans. Comparing multiple closely related species is a powerful approach for understanding causes of plasticity in gene networks. This approach is extremely difficult to implement with vertebrate model organisms but can be readily accomplished in insects. Flies (Diptera) are particularly suitable because they include Drosophila melanogaster, one of the leading model organisms in developmental biology, and because other dipteran species that can be cultured in a laboratory setting are amenable to functional studies. Our research in a variety of dipteran model organisms during the previous funding period established that these closely related species use a broad range of anterior determinants (ADs) for establishing embryo polarity and anterior-specific gene expression, most likely through the formation of long-range transcription factor gradients with morphogen-like activity. This discovery enables us to examine why key developmental regulators can be highly unstable in evolution. While Drosophila’s AD (Bicoid) is a classic morphogen model, it is not known whether its mechanism of action can be generalized nor how the evolutionary transition to Bicoid-dependent pattern formation was achieved. The first of these two questions will be addressed by identifying and characterizing chromatin and gene targets of the AD in the moth fly Clogmia albipunctata, Cal-Opamat (Aim 1) and of the AD in the common midge Chironomus riparius, Panish (Aim 2). This will be done by testing for AD-dependent chromatin accessibility in stage-matched embryos with or without reduced AD activity, using ATAC-seq and RNAi. These experiments will be complemented as needed by ChIP-seq, and by characterizing the expression and function of predicted target genes in vivo. The second question, concerning the transition to Bicoid- dependent pattern formation, will be addressed by determining the mechanism of anterior specification in the soldier fly Hermetia illucens (Aim 3). This organism was chosen because it is the closest tractable outgroup to the clade of species with bicoid. A pilot study revealed several AD candidates, including Hil-Stau, the ortholog of Staufen. This RNA-binding protein binds bicoid mRNA in early Drosophila embryos. To identify Hermetia’s AD, anterior-localized and Hil-Stau-binding mRNAs will be determined by expression studies, co- immunoprecipitation, sequencing, and their function will be examined by RNAi. The determination of the anterior- specification mechanisms of Clogmia, Chironomus, and Hermetia will result in a new framework for explaining evolutionary plasticity of a key regulator in a classic model for pattern formation and gene regulation.