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

西班牙鼹鼠（Talpa occidentalis）肢体形态的进化基因组学

• 批准号: 460195844
• 负责人: Professor Dr. Stefan Mundlos
• 金额: --
• 依托单位: Institut für Medizinische Genetik und Humangenetik (CVK)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/460195844?language=en
• 关键词: Evolutionary; genomics; limb; morphology; Spanish

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.

突变是遗传疾病和表型多样性的原因，但也是进化新奇的来源。虽然对致病基因组变异的全基因组搜索现已进入常规测试，但在进化环境中识别和表征基因组变化仍然是一项挑战。在这里，我建议调查的基因组基础上的肢体形态的鼹鼠（塔尔帕oceanentalis）极端进化适应的一个例子。基于我们先前在破译鼹鼠间性的分子基础方面的工作，我们将专注于调节基因组的变化，并寻找改变基因调节的足迹，因为这些可能是进化新奇的来源。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Hi-C以鉴定TADs，鉴定共线性的断裂，并鉴定加速区域（真实的等人，Science 2020）。我们还收集了来自鼹鼠的胚胎肢，用于进一步分析E11.5、13.5、17.5期小鼠等同物。使用这些数据和组织，我们将：1）通过批量RNA-seq进行表达分析，并使用ChIP-seq针对各种组蛋白标记和ATAC-seq针对开放染色质在发育中的鼹鼠前肢和后肢的3个阶段（对应于肢体发育和图案形成的重要时间点）中鉴定活性调节区域（E11.5、13.5和17.5）; 2）从E13.5鼹鼠前肢和后肢产生单细胞（sc）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。将数据与已经产生的同等阶段的小鼠肢体进行比较; 3）进行综合数据分析以鉴定候选基因/基因座。我们将使用在我们以前的项目中生成的数据，并通过目标1和2中生成的批量和单细胞数据来改进过滤; 4）重新设计小鼠的基因组变化。将在体内验证目标3中区域的识别，以识别生物学相关的变化。我们将使用我们高效的CRISVar系统在小鼠ES细胞中产生基因组重排和选定重排和增强子元件的敲入。小鼠将通过ES细胞聚集产生，并评估基因表达变化和表型。总的来说，我们将应用无偏倚的全基因组方法来识别涉及鼹鼠肢体特定形态的基因组变体，并测试它们在小鼠中的作用。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跨物种的基因表达。这些见解也将是有价值的了解人类骨骼疾病的病理机制。