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

• Applicant: Professor Dr. Stefan Mundlos
• Subject Area: General Genetics and Functional Genome Biology ; Evolutionary Cell and Developmental Biology (Zoology) ; Human Genetics
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 460195844

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.

• DFG Programme: Research Grants