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

项目摘要 我们的目标是发现密切相关的物种是如何采用不相关的轴决定因素来指定 前胚动物发育的许多方面在像人类这样不同的物种之间是保守的 和苍蝇一样，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意想不到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 密切相关的物种是理解基因网络可塑性原因的有力途径。这 用脊椎动物模式生物实施该方法是极其困难的，但是可以容易地实现 在昆虫中。蝇类（双翅目）是特别合适的，因为它们包括黑腹果蝇（Drosophila melanogaster）， 发展生物学中的主要模式生物，因为其他双翅目物种可以在 在实验室环境下进行功能研究。我们对多种双翅目模式生物的研究 在上一个资助期间，确定了这些密切相关的物种使用广泛的前 决定因素（AD）建立胚胎极性和前特异性基因表达，最有可能通过 形成具有形态发生素样活性的长距离转录因子梯度。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高度不稳定。 虽然果蝇的AD（Bicoid）是一种经典的形态发生模型，但尚不清楚其形成的机制是否是 行动可以概括，也没有如何进化过渡到Bicoid依赖模式的形成是 办妥了一批这两个问题中的第一个将通过识别和表征染色质和 在蛾Clogmia albipunctata，Cal-Opamat（Aim 1）中AD的基因靶标和在 普通摇蚊Chironomus riparius，Panish（Aim 2）.这将通过检测AD依赖性 使用ATAC-seq，在具有或不具有降低的AD活性的阶段匹配胚胎中的染色质可及性， RNA干扰这些实验将根据需要通过ChIP-seq进行补充，并通过表征表达 和预测的靶基因在体内的功能。第二个问题，关于向Bicoid过渡的问题- 依赖模式的形成，将通过确定前规格的机制来解决， 士兵飞Hermetia illucens（目标3）。选择这种生物是因为它是最容易驯服的 外群到具有二面体的物种的分支。一项试点研究揭示了几个AD候选人，包括希尔-斯陶， Staufen的直系同源物这种RNA结合蛋白结合早期果蝇胚胎中的bicoid mRNA。以识别 Hermetia的AD、前定位和Hil-Stau结合mRNA将通过表达研究确定， 免疫沉淀、测序，并通过RNAi检查它们的功能。确定前- Clogmia，Chironomus和Hermetia的规范机制将导致一个新的框架来解释 模式形成和基因调控的经典模型中的关键调节因子的进化可塑性。

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.