Periodic Reporting for period 1 - 3D-REVOLUTION (The impact of 3D regulatory landscapes on the evolution of developmental programs)
Reporting period: 2023-07-01 to 2025-12-31
Understanding how genomes evolve to generate phenotypic diversity remains a central challenge in biology. In vertebrates, tissues and organ formation is controlled by developmental programs, which are genetically encoded plans that determine when and where genes are turned on or off. Developmental programs do not only comprise the action of genes, but also of three-dimensional (3D) regulatory landscapes, which enables communication between promoters and non-coding regulatory elements. Previous studies, including our own, have shown that alterations in 3D regulatory landscapes can lead to abnormal phenotypes or evolutionary novelties. Yet, how such landscapes evolve and influence developmental programs remains understudied.
By integrating single-cell omics, 3D chromatin interaction profiling and genome editing, the 3D-REVOLUTION project will investigate how changes in 3D regulatory landscapes refine vertebrate developmental programs. We focus on gonadal sex determination, a critical process for species perpetuation that also exhibits a remarkable evolutionary diversity. This aspect makes sex determination an ideal model for exploring the balance between conservation and innovation in the context of genome evolution.
This project pursues four specific objectives:
1. To define the core and variable transcriptional networks of sex determination across tetrapod clades.
2. To investigate the evolutionary dynamics of cis-regulatory elements controlling sex determination networks
3. To determine the impact of 3D chromatin organization on the evolution of sex determination
4. To develop innovative inter-species transgenic methods to test the causal role of 3D regulatory landscape variation in vivo.
This project will boost our understanding of the mechanisms by which regulatory mutations contribute to phenotypic variation. Our focus on sex determination is relevant in an evolutionary context, as climate change can profoundly impact this process and affect species viability. Moreover, the generated knowledge may be applied to investigate sex-related conditions with unknown molecular origins in humans, such as Differences of Sex Development (DSD).
By integrating single-cell omics, 3D chromatin interaction profiling and genome editing, the 3D-REVOLUTION project will investigate how changes in 3D regulatory landscapes refine vertebrate developmental programs. We focus on gonadal sex determination, a critical process for species perpetuation that also exhibits a remarkable evolutionary diversity. This aspect makes sex determination an ideal model for exploring the balance between conservation and innovation in the context of genome evolution.
This project pursues four specific objectives:
1. To define the core and variable transcriptional networks of sex determination across tetrapod clades.
2. To investigate the evolutionary dynamics of cis-regulatory elements controlling sex determination networks
3. To determine the impact of 3D chromatin organization on the evolution of sex determination
4. To develop innovative inter-species transgenic methods to test the causal role of 3D regulatory landscape variation in vivo.
This project will boost our understanding of the mechanisms by which regulatory mutations contribute to phenotypic variation. Our focus on sex determination is relevant in an evolutionary context, as climate change can profoundly impact this process and affect species viability. Moreover, the generated knowledge may be applied to investigate sex-related conditions with unknown molecular origins in humans, such as Differences of Sex Development (DSD).
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.
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.
Our comparative approach has been effective at identifying novel regulators of sex determination, such as Meis genes (Mota-Gomez et al, in 2nd revision) or the mir17-92 microRNA cluster (Hurtado et al, Nat Comms, 2024). This is relevant as the number of sex determining factors identified in the latest years has been very low, an aspect that hinders the molecular diagnosis for human DSD. Our results show how genomics approaches can reveal novel insights on biological processes .
This project also shift the focus from genes towards the role of the non-coding genome and of 3D chromatin organization on developmental programs. It is becoming evident that mutations affecting those regulatory layers is quite relevant for species evolution, as their associated effects tend to be tissue-specific and less detrimental than those of coding mutations. The identification of a novel non-coding regulatory region at the Fgf9 locus is a good example for this (Mota-Gomez et al, in 2nd revision). In transgenic mouse experiments, we demonstrate that the deletion of this region causes male-to-female sex reversal, overcoming the embryonic lung lethality associated with the gene inactivation.
A prominent limitation of evolutionary studies is the difficulties in performing functional experiments for non-classical model species, which are essential to establish causality links. In this project, we develop advance transgenic methods to compare the function of orthologous enhancer sequences in vivo, and to replace 3D regulatory landscapes between species. Such approaches are well suited to adress the impact of regulatory mutations in evolutionary studies, or to generate more accurate animal models of human diseases. As such, the technological developments associated with this project provide a toolkit with broad applicability in genomics, developmental biology, and biotechnology.
This project also shift the focus from genes towards the role of the non-coding genome and of 3D chromatin organization on developmental programs. It is becoming evident that mutations affecting those regulatory layers is quite relevant for species evolution, as their associated effects tend to be tissue-specific and less detrimental than those of coding mutations. The identification of a novel non-coding regulatory region at the Fgf9 locus is a good example for this (Mota-Gomez et al, in 2nd revision). In transgenic mouse experiments, we demonstrate that the deletion of this region causes male-to-female sex reversal, overcoming the embryonic lung lethality associated with the gene inactivation.
A prominent limitation of evolutionary studies is the difficulties in performing functional experiments for non-classical model species, which are essential to establish causality links. In this project, we develop advance transgenic methods to compare the function of orthologous enhancer sequences in vivo, and to replace 3D regulatory landscapes between species. Such approaches are well suited to adress the impact of regulatory mutations in evolutionary studies, or to generate more accurate animal models of human diseases. As such, the technological developments associated with this project provide a toolkit with broad applicability in genomics, developmental biology, and biotechnology.