Our work is currently focused on the following aspects:
1.Comparative single-cell transcriptomics across tetrapod species
We are generating and analyzing single-cell RNA-seq datasets from gonadal tissues of representative mammalian (mouse, rabbit), bird (chicken), reptile (turtle), and amphibian (african and western clawed frogs) species. This cross-clade atlas of sex-determining cell types enables the identifications of conserved transcriptional modules that are essential for sex determination. Furthermore, this comparative approach led us to identify factors that are specific of particular species, such as temperature-dependent turtles (Acemel et al, in preparation) or rabbits (Barbera et al, in preparation). Through fruitful collaborations, we have also employed single-cell transcriptomics to report the first microRNA cluster involved in sex determination (Hurtado et al, Nat Comms 2024; collaboration Barrionuevo and Jiménez labs), to identify mechanisms of germ cell specification in turtles (Hatkevich et al, PNAS 2024; collab. Capel lab) or mechanisms associated with the emergence of the bat wing (Schindler et al, Nat Eco Evol, 2025; collab Real and Mundlos labs)
2. Mapping cis-regulatory elements and investigating their evolutionary turnover
Through single-cell ATAC-seq datasets from gonadal tissues, we are mapping the cis-regulatory landscapes associated with sex determination across tetrapod clades. By applying novel deep learning algorithms, we infer and compare species-specific regulatory codes. This approach is particularly useful to understand how variations in the non-coding genome may lead to altered expression patterns and phenotypes. We also made progress with developing a CRISPR-based in vivo dual enhancer reporter system that allows o compare the regulatory activities of orthologous sequences in transgenic mice.
3. Investigating evolutionary variation in 3D chromatin organization
In collaboration with the Marti-Renom lab, we introduced METALoci, a novel approach to reconstruct and compare 3D regulatory landscapes (Mota-Gómez et al, in 2nd revision). By applying this methodology during mouse sex determination, we identified a novel non-coding regulatory region for the pro-testicular factor Fgf9, as well as a novel role for Meis genes during sex determination. By using antibody-based methods to isolate gonadal populations, we are generating Hi-C datasets from non-mammalian species to investigate how variations in 3D chromatin organization impact the evolution of sex determination. Additional efforts of the lab focus on investigating how mechanisms of 3D chromatin organization have emerge during vertebrate evolution (Astica et al., in preparation).
4. Interspecies replacement of 3D regulatory landscapes.
We are applying innovative transgenic approaches to exchange 3D regulatory landscapes between species. Based on the knowledge from the previous aims, we are introducing regulatory mutations that are specific of particular species, in the mouse genome. By generating transgenic mice, we can test it such mutations have a functional impact on sex determination networks. These approaches are currently being employed to validate the mechanisms that activate early estrogenenesis on rabbits, or those driving the emergence of novel sex determining factors in turtles.
