Biology subjects

Lopez-Rios, J.

Publications and source records attributed to Lopez-Rios, J..

9 recordsLinked to original sources

bioRxiv2025

Functional architecture of cardiac TF regulatory landscapes in control of mammalian heart development

Congenital heart disease (CHD), the most common human birth defect, often results from disruptions in gene regulatory networks (GRNs) that control cardiac lineage specification and cell type identity during heart development. A conserved core set of cardiac transcription factors (TFs) orchestrates these processes through combinatorial interactions that are cell type-specific and tightly regulated across space and time. However, the genomic enhancer architecture that integrates upstream effectors to establish precise cardiac TF dosage and downstream transcriptional output remains largely unresolved. Here, we assessed the functional necessity of five developmental heart enhancer modules previously linked to the regulation of Gata4 and Hand2, core cardiac TFs exhibiting overlapping roles in myocardial and endocardial development. While individual enhancer deletions in mouse embryonic hearts revealed a surprising degree of transcriptional resilience, a subset of Gata4 enhancers proved indispensable for embryonic progression in a genetically compromised background. To achieve higher precision in cardiac cell type-specific enhancer prediction, we applied single-nucleus multiome profiling, enabling the delineation of cardiac cistromes underlying heart morphogenesis. By integrating this resource with deep learning applications, site-directed transgenesis, and chromatin conformation modeling, we mapped the cardiac enhancer repertoire and regulatory signatures that orchestrate Hand2 dynamics across distinct cardiac compartments and lineages. Genome editing further revealed an essential role for the Hand2-upstream regulatory interval (H2-URI) in transcriptional control of endocardial lineage effectors and, consequently, trabecular network formation and cardiac cushion patterning. Together our findings highlight substantial resilience in the cis-regulatory architectures governing cardiac TF dynamics and demonstrate that combinatorial integration of upstream lineage identities across modular enhancer landscapes establishes the cardiac cell type-specific programs driving heart morphogenesis. These results advance the reconstruction of cardiac GRNs and enhance the functional interpretation of CHD-associated variants.

developmental biology↗

bioRxiv2025

Neotenic transcriptomic features in the adult turquoise killifish brain

Aging is one of the main challenges facing modern society. Understanding the cellular processes that occur in the later stages of life is essential to address age-related diseases. Although aging is a global process that affects the entire body, the brain is one of the most sensitive organs and is affected by numerous degenerative diseases. In this study, we investigate cell types and genes that are particularly sensitive to the aging process. To this end, we performed a time-course single-cell RNAseq experiment series in the brain of the killifish, the vertebrate model with the shortest lifespan, which makes it an excellent model for aging studies. Our analysis reveals that non-glial progenitor cells are among those populations that change the most between young and old animals. Furthermore, we identify specialized stromal clusters that seem to support a primitive hematopoietic program in the brain, which is active only during the embryonic stages in other vertebrate species. Our results show that the expression of embryonic genes in the adult brain appears to be a general feature in killifish, and that several cell populations in the adult killifish brain show a higher level of similarity to the zebrafish embryonic populations than to adult ones. Our study suggests that adult killifish maintain a neotenic gene expression status in the brain that may help in sustaining their characteristic high proliferation and metabolic rates, as well as combat the detrimental effects of this high metabolism on cells, especially at advanced ages.

genomics↗

bioRxiv2024

Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

genomics↗

bioRxiv2023

The cellular landscape of the endochondral bone highlights its multipotent and immunomodulatory features during the transition to extrauterine life

The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison to the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis, and hematopoiesis. Our data also suggests dynamic inter- and intra-compartment interactions as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

cell biology↗

bioRxiv2022

Widespread Increase in Enhancer-Promoter Interactions during Developmental Enhancer Activation in Mammals

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here, we investigate the 3D conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. 61% of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general significance of enhancer- promoter physical proximity for developmental gene activation in mammals.

genomics↗

bioRxiv2022

Uncovering Hidden Enhancers Through Unbiased In Vivo Testing

Transcriptional enhancers are a predominant class of noncoding regulatory elements that activate cell type-specific gene expression. Tissue-specific enhancer-associated chromatin signatures have proven useful to identify candidate enhancer elements at a genome-wide scale, but their sensitivity for the comprehensive detection of all enhancers active in a given tissue in vivo remains unclear. Here we show that a substantial proportion of in vivo enhancers are hidden from discovery by conventional chromatin profiling methods. In an initial comparison of over 1,200 in vivo validated tissue-specific enhancers with tissue-matched mouse developmental epigenome data, 14% (n=286) of active enhancers did not show canonical enhancer-associated chromatin signatures in the tissue in which they are active. To assess the prevalence of enhancers not detectable by conventional chromatin profiling approaches in more detail, we used a high throughput transgenic enhancer reporter assay to systematically screen over 1.3 Mb of mouse genomic sequence at two critical developmental loci, assessing a total of 281 consecutive 5kb regions for in vivo enhancer activity in mouse embryos. We observed reproducible enhancer-reporter activity in 88 tissue-specific elements, 26% of which did not show canonical enhancer-associated chromatin signatures in the corresponding tissues. Overall, we find these hidden enhancers are indistinguishable from marked enhancers based on levels of evolutionary conservation, enrichment of transcription factor families, and genomic positioning relative to putative target genes. In combination, our retrospective and prospective studies assessed only 0.1% of the mouse genome and identified 309 tissue-specific enhancers that are hidden from current chromatin-based enhancer identification approaches. Our findings suggest the existence of tens of thousands of active enhancers throughout the genome that remain undetected by current chromatin profiling approaches and are an unappreciated source of additional genome function of import in interpreting growing whole human genome sequencing data.

genomics↗

bioRxiv2022

The little skate genome and the evolutionary emergence of wing-like fin appendages

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

evolutionary biology↗

bioRxiv2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Gene deserts are defined as genomic regions devoid of protein coding genes and spanning more than 500 kilobases, collectively encompassing about 25% of the human genome. Approximately 30% of all gene deserts are enriched for conserved elements with cis-regulatory signatures. These are located predominantly near developmental transcription factors (TFs) but despite predicted critical functions, the transcriptional contributions and biological necessity of most gene deserts remain elusive. Here, we explore the cis-regulatory impact of a gene desert flanking the Shox2 gene, a TF indispensable for proximal limb, craniofacial and cardiac pacemaker development. Using a functional genomics approach in mouse embryos we identify the gene desert as a hub for numerous Shox2-overlapping enhancers arranged in a globular chromatin domain with tissue-specific features. In accordance, using endogenous CRISPR deletion, we demonstrate that the gene desert interval is essential for Shox2 transcriptional control in developing limbs, craniofacial compartments, and the heart. Phenotypically, gene desert ablation leads to pacemaker-related embryonic lethality due to Shox2 depletion in the cardiac sinus venosus. We show that this role is partially mediated through a distal gene desert enhancer, providing evidence for intra-gene desert regulatory robustness. Finally, we uncover a multi-layered functional role of the gene desert by revealing an additional requirement for stylopod morphogenesis, mediated through an array of proximal limb enhancers (PLEs). In summary, our study establishes the Shox2 gene desert as a fundamental genomic unit that controls pleiotropic gene expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

developmental biology↗

bioRxiv2020

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among median and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3 null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and median fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 downregulation in shh mutant fins rescues fin loss in a manner similar to how Gli3-deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of median fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

evolutionary biology↗