Evolutionary genomics of limb morphology in the Spanish mole (Talpa occidentalis)

突变是遗传疾病和表型多样性的原因，也是进化新颖性的来源。虽然对致病基因组变异的全基因组搜索现在已进入常规测试，但在进化环境中识别和表征基因组变化仍然是一个挑战。在这里，我建议研究鼹鼠（Talpa occidentalis）肢体形态的基因组基础，作为极端进化适应的一个例子。基于我们之前破译鼹鼠间性的分子基础的工作，我们将把重点放在调控基因组的变化和寻找改变基因调控的足迹上，因为这些可能是进化新颖性的来源。为此，我们在之前的研究中生成了西班牙鼹鼠的完整染色体基因组，进行了Hi-C鉴定TADs，鉴定了共链断裂，并鉴定了加速区（Real等）。科学2020)。我们还收集了鼹鼠的胚胎肢体，用于进一步分析E11.5, 13.5, 17.5期小鼠当量。利用这些数据和组织，我们将：1)通过bulk RNA-seq进行表达分析，并使用ChIP-seq识别鼹鼠前肢和后肢发育3个阶段的各种组蛋白标记和开放染色质的活性调控区域，这些区域对应于肢体发育和模式化的重要时间点（E11.5, 13.5和17.5）；2)从E13.5摩尔前肢和后肢生成单细胞RNA-seq和sc- atac，以鉴定肢体特异性细胞群、细胞命运轨迹和调控事件。这些数据将与已经生成的同等阶段的小鼠肢体进行比较；3)对候选基因/基因座进行综合数据分析。我们将使用上一个项目中生成的数据，并根据Aim 1和Aim 2中生成的批量和单细胞数据对过滤进行细化；4)重新设计小鼠基因组变化。Aim 3中区域的鉴定将在体内进行验证，以确定与生物学相关的变化。我们将利用我们高效的CRISVar系统在小鼠胚胎干细胞中产生基因组重排和选择重排和增强元件的敲入。通过es细胞聚集产生小鼠，并评估基因表达变化和表型。总的来说，我们将采用一种无偏倚的全基因组方法来鉴定与鼹鼠肢特定形态有关的基因组变异，并测试它们在小鼠中的影响。这将有助于理解进化如何利用基因组变异，调控变化如何转化为表型，以及调控景观如何控制物种间的基因表达。这些见解对于理解人类骨骼疾病的病理机制也很有价值。

Mutations are the cause of genetic disease and phenotypic diversity but also the source of evolutionary novelty. While the genome-wide search for disease-causing genomic variants has now entered routine testing, the identification and characterization of genomic changes in an evolutionary setting remain a challenge. Here I propose to investigate the genomic basis of limb morphology in moles (Talpa occidentalis) as an example of extreme evolutionary adaption. Based on our previous work in deciphering the molecular basis of mole intersexuality, we will focus on changes in the regulatory genome and search for footprints of altered gene regulation, as these are the likely source of evolutionary novelty. For this purpose, we generated in our previous study a full chromosome genome of the Spanish mole, performed Hi-C for identification of TADs, identified breaks of synteny, and identified accelerated regions (Real et al. Science 2020). We also collected embryonic limbs from moles for further analysis of stages E11.5, 13.5, 17.5 mouse equivalent. Using these data and tissue we will: 1) perform expression analysis by bulk RNA-seq and identify active regulatory regions using ChIP-seq for various histone marks and ATAC-seq for open chromatin in the 3 stages of developing mole fore and hind limbs that correspond to important time points of limb development and patterning (E11.5, 13.5 and 17.5); 2) generate single-cell (sc) RNA-seq and sc-ATAC from E13.5 mole fore and hind limbs to identify limb-specific cell populations, cell fate trajectories and regulatory events. The data will be compared to already generated mouse limbs of the equivalent stage; 3) perform integrative data analysis for the identification of candidate genes/loci. We will use the data generated in our previous project and refine the filtering by the bulk and the single-cell data generated in Aim 1 and 2; 4) re-engineer genomic changes in mice. The identification of regions in Aim 3 will be validated in vivo to identify changes that are biologically relevant. We will use our efficient CRISVar system to generate genomic rearrangements and a knock-ins of selected rearrangements and enhancer elements in mouse ES cells. Mice will be generated by ES-cell aggregation and evaluated for gene expression changes and phenotype.Collectively, we will apply an unbiased genome-wide approach to identify genomic variants involved in the specific morphology of mole limbs and test their effect in mice. This will help to understand how evolution uses genomic variations, how regulatory changes translate into phenotypes and how regulatory landscapes control gene expression across species. These insights will also be valuable for understanding the pathomechanisms of human skeletal disease.