Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by the loss of microRNA-92a (miR-92a) expression rather than changes in svb. We used RNAseq of second (T2) pupal legs (20-28 h after puparium formation) to compare the expression of trichome GRN genes between strains with a large and a small naked valley. We also compared the function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. Moreover, we used ATAC-seq of second (T2) pupal legs (20-28 h after puparium formation) to identify the pupal leg enhancers of svb. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution. Overall design: We used RNAseq to compare gene expression in second legs between two Drosophila melanogaster strains and ATAC-Seq to identify enhancers of certain genes. We used three replicates each of ca. 80 second (T2) pupal legs (20-28 h after puparium formation) of two different Drosophila melanogaster strains (ebony[4],white ocelli[1], rough[1] and Oregon R) and extracted RNA or chromatin from them. Library preparation for RNAseq and paired end (75 bp) seqencing with an Illumina HiSeq 4000 were done by Edinburgh Genomics. For ATAC-Seq Chromatin was tagmented using Tn5 transposase and library preparation was performed using a protocol modified after Buenrostro et al. (2013) DOI:10.1038/NMETH.2688. Paired end (50 bp) seqencing with an Illumina HiSeq 2500 was done by the Transcriptome and Genome Analysis Laboratory Goettingen.

趋同表型演化通常由其基础基因调控网络（gene regulatory networks, GRNs）内特定节点的反复突变所导致。这类演化‘热点’位置的基因被认为能够在产生最少多效性后果的前提下，对表型施加最大化影响。这一结论催生了如下假说：若能充分解析某一基因调控网络的细节，则演化路径具备可预测性。果蝇属物种中幼虫毛突的反复演化丢失，是由shavenbaby（svb）基因表达的缺失所引发的。svb同时也是腿部毛突发育所必需的基因，但黑腹果蝇T2股骨‘裸谷’区域毛突的演化新生，并非由svb的变异导致，而是源于microRNA-92a（miR-92a）表达的缺失。本研究对化蛹后20~28小时的第二对（T2）蛹腿进行RNA测序（RNA-seq），以比较‘裸谷’面积大小各异的两个黑腹果蝇品系间毛突相关基因调控网络基因的表达差异。本研究同时对比了幼虫与腿部毛突基因调控网络中各组分的功能，以探究为何在这两类发育背景下，毛突模式演化的遗传基础存在差异。此外，本研究对化蛹后20~28小时的T2蛹腿进行转座酶可及性测序（ATAC-seq），以鉴定svb在蛹腿中的增强子区域。本研究发现，这两类毛突基因调控网络在所使用的基因种类，以及共有基因的调控模式与功能上均存在关键差异。这些基因调控网络的差异解释了为何svb的突变不太可能参与腿部毛突的演化，以及miR-92a如何成为该背景下关键的演化开关。本研究结果表明，不同发育背景下基因调控网络的异质性，以及某一形态特征是丢失还是新生，共同决定了基因调控网络中驱动形态改变的演化节点位置。因此，本研究结果对于理解演化路径及其可预测性具有重要意义。实验设计概述：本研究通过RNA测序比较了两个黑腹果蝇品系的第二对腿部的基因表达情况，并通过转座酶可及性测序（ATAC-seq）鉴定特定基因的增强子区域。我们分别采集了两个黑腹果蝇品系（ebony[4]、white ocelli[1]、rough[1]以及Oregon R）的约80个化蛹后20~28小时的T2蛹腿，各设置3个生物学重复，并从中提取RNA与染色质。RNA测序的文库构建及Illumina HiSeq 4000平台的双端（75 bp）测序由爱丁堡基因组学中心完成。转座酶可及性测序的染色质样品使用Tn5转座酶进行标签化，文库构建参照Buenrostro等人（2013）DOI:10.1038/NMETH.2688的方案修改完成。Illumina HiSeq 2500平台的双端（50 bp）测序由哥廷根转录组与基因组分析实验室完成。