The cis-regulatory architecture of a gene desert controlling pleiotropic Shox2 expression and cardiac pacemaker development

Trabajo presentado en Weinstein Meeting, celebrado en Marsella (Francia) del 12 al 14 de mayo de 2022

Genetic dissection of the Ptch1 locus to uncover the regulatory mechanisms conferring evolutionary plasticity to a pleiotropic gene

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en Sevilla (España) del 18 al 21 de septiembre de 2023.Peer reviewe

Dissection of Ptch1 cis-regulatory robustness in the context of artiodactyl limb evolution

How genes are regulated during development and how this regulation has changed during evolution are two of the most appealing questions of modern biology. Indeed, variation of cis-regulatory elements (CRE) is considered to be one of the main mechanisms underlying morphological evolution. The limb is an excellent model to study both of these processes: its development is genetically well understood and it has adopted a wide range of different morphologies during evolution to adapt to different forms of locomotion (Zuniga, 2015; Petit et al., 2017). Most particularly, I am interested in the evolutionary loss and reduction of digits that happened in artiodactyls, as extant members of this order only have 2 or 4 digits and therefore represent a deviation from the ancestral pentadactyl state of tetrapods (Polly, 2007). Ptch1, the receptor of the SHH pathway has been shown to play a central role in these evolutionary processes in bovines (Bos taurus). Indeed, failure to up-regulate this gene contributes to the evolutionary loss of anterior-posterior polarity in the limbs of these animals. In this thesis, I first dissected the mouse Ptch1 cis-regulatory landscape and identified three new limb enhancers of Ptch1, increasing the total number of candidate elements regulating this gene in the limb to six. In particular, I identified a distant CRE located 385 kb upstream of the Ptch1 gene body that drives reporter expression in the posterior-distal limb bud mesenchyme similar to the endogenous Ptch1 gene and the previously identified LRM enhancer. Individual and combined knockout of cis-regulatory elements in the mouse using the CRISPR/Cas9 technology led to little variation in Ptch1 expression in the limb, suggesting that Ptch1 regulation is exceptionally robust. To this robustness also likely contributes the upregulation of Ptch1 itself in response to a dose diminution. Additionally, I analyzed the limb development of the pig (Sus scrofa), another artiodactyl and showed that the evolutionary mechanism underlying pig and bovine limb development were similar. The expression of known genes involved in limb development displayed a similar loss of molecular anterior-posterior polarity in pig and in bovine. This loss of asymmetry is paralleled by changes in the SHH pathway: Ptch1 is more restricted posteriorly in pig than in mouse and Gli1, anteriorized. Furthermore, the evolutionary reduction of digits 2 and 5 in pig was linked with a premature downregulation of AER-FGFs signaling over the primordia of these digits compared with the central ones, providing an explanation to the reduction of lateral digits in pig versus their complete loss in bovines. Next, I also used functional genomics approaches to obtain a comprehensive and unbiased picture of the regulatory variation underlying morphological evolution of the pig limb in comparison to the my reference model, the mouse. In particular, I resorted to ATAC-seq to catalog all regions from the pig and mouse genomes harboring regulatory activity during limb development. This analysis showed that that ∼35% of accessible regions in one of the species are evolutionary conserved at the sequence level, but closed in the other species, suggesting a high level of regulatory re-wiring. Furthermore, many of these alterations are in close proximity to genes that encode components of the major morphological pathways governing limb development such as those of FGF, SHH, WNT or BMP. This study provides a baseline for gene-centric studies that address how regulatory variation impacts on morphological evolution of the limb in artiodactyls. Finally, I used lacZ reporter assays in transgenic mice to address the activity of the main limb regulatory elements identified in the Ptch1 cis-regulatory landscape in artiodactyls, in comparison to the corresponding mouse constructs. This analysis revealed that that some regulatory elements had functionally degenerated in the artiodactyl lineage (e.g. the LRM enhancer), while other CREs had retained a similar activity in mouse and artiodactyls (egg. E385 enhancer) despite >95 Myr of evolutionary distance. Combined with the thorough molecular analysis of pig limb bud development, these results establish a link between the loss of anterior-posterior polarity and the partial degeneration of the Ptch1 cis-regulatory landscape, which likely originated in the common ancestor of the pig and bovine lineages

Functional Genomics and the Evolutionary Diversification of the Pig Limb

Trabajo presentado en British Society for Developmental Biology Annual Spring Meeting, celebrado en Warwick (Inglaterra) del 15 al 18 de abril de 2018

Functional genomics and the evolutionary diversification of the artiodactyl limb

Resumen del póster presentado a la 14th International Limb Development and Regeneration Conference, celebrada en Edimburgo (UK) del 23 al 26 de julio de 2017.-- et al.Recent efforts in mapping the regulatory genome have uncovered that loci with essential functions in development typically contain complex cis‐regulatory landscapes. Such complexity is required to achieve precise spatio‐temporal control of gene expression, as developmental genes are frequently required in several tissues and time points. A general concept in the field is that alterations in the regulation of these pleiotropic and conserved developmental genes are a main mechanism underlying the evolution of morphology. Morphogenesis of the vertebrate limb is a genetically well‐understood process that can be used as a paradigm to gain insight into the mechanisms of transcriptional regulation during both developmental and evolutionary processes. The pig (Sus scrofa), a new arising model in biomedical research, is an artiodactyl (even‐toe ungulate) in which the extremities are specialized for cursorial locomotion, thanks to various modifications of the distal limb skeleton. The pig limb displays loss and reduction of digits, which are largely symmetrical, elongated and with hoofed phalanges. Here we report a detailed morphological and molecular analysis of pig limb development. In particular, we have analyzed the expression of several genes with essential functions in patterning and growth of the limb bud, such as 5'Hoxd genes and components of the SHH, BMP and FGF signaling pathways and their transcriptional targets. These studies have revealed that the pig limb bud displays a loss of anterior‐posterior polarity in the distal mesenchyme that contains the digit progenitor cells. In addition, we have combined transcriptomics in pig and mouse limb buds with the cataloging of regions with regulatory potential using ATAC‐seq, which identifies regions of open chromatin. The ultimate aim of our studies is to determine if evolutionary alterations in the cis‐regulatory networks that control limb development have contributed to the acquisition of specific morphological traits observed in the pig appendicular skeleton.Peer reviewe

Dissection of the Ptch1 regulatory landscape in mice through iterative deletions using CRISPR/Cas9

Trabajo presentado en el UNIA Workshop "CRISPR-Cas: From Microbiology to Biomedicine", celebrado en Baeza (Jaén) el 02 de noviembre de 2021

Gene regulatory and expression differences between mouse and pig limb buds provide insights into the evolutionary emergence of artiodactyl traits

Digit loss/reductions are evolutionary adaptations in cursorial mammals such as pigs. To gain mechanistic insight into these processes, we performed a comparative molecular analysis of limb development in mouse and pig embryos, which revealed a loss of anterior-posterior polarity during distal progression of pig limb bud development. These alterations in pig limb buds are paralleled by changes in the mesenchymal response to Sonic hedgehog (SHH) signaling, which is altered upstream of the reduction and loss of Fgf8 expression in the ectoderm that overlaps the reduced and vestigial digit rudiments of the pig handplate, respectively. Furthermore, genome-wide open chromatin profiling using equivalent developmental stages of mouse and pig limb buds reveals the functional divergence of about one-third of the regulatory genome. This study uncovers widespread alterations in the regulatory landscapes of genes essential for limb development that likely contributed to the morphological diversion of artiodactyl limbs from the pentadactyl archetype of tetrapods.This research has been funded by the Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2017-82974-P to J.L.-R.); the “Unidad de Excelencia María de Maeztu 2017–2021” (MDM-2016-0687); an SNF Doc.Mobility fellowship (to V.T.); SNF grant 310030B_166685 and ERC advanced grant INTEGRAL ERC-2015-AdG, Project ID 695032 (to R.Z.); and the University of Basel.Peer reviewe

Dissecting the cisregulatory robustness of Ptch1 in Iimb development and evolution

Trabajo presentado en el Workshop: From genes to organisms: transcriptional control in development and disease, celebrado en Baeza (España) del 28 al 30 de octubre de 2019

Gene regulatory and expression changes in developing pig limbs hallmark the evolutionary emergence of artiodactyl traits

Trabajo presentado en EMBO Workshop: Limb Development and Regeneration: New Tools for a Classic Model System, celebrado en Barcelona (España), del 2 al 5 de julio de 2019

Dissecting the cis-regulatory robustness of Ptch1 in limb development and evolution

Trabajo presentado en EMBO Workshop: Limb Development and Regeneration: New Tools for a Classic Model System, celebrado en Barcelona (España), del 2 al 5 de julio de 2019