Biology subjects

Manzanares, M.

Publications and source records attributed to Manzanares, M..

11 recordsLinked to original sources

bioRxiv2026

DNA replication is dispensable for developmental progression, but required for heterochromatin organization at mouse zygotic genome activation

Early mammalian development progresses through a small number of cleavage divisions with extensive transcriptional and epigenetic reprogramming that result in the very first lineage decisions, leading to blastocyst formation. Although classical embryological studies suggested that developmental progression can occur independently of normal cell division, the extent to which DNA replication contributes to these processes remains unclear. In particular, whether replication is required simply for proliferation or also for establishing the cellular states that regulate stage-specific gene expression is unknown. Here we show that mouse preimplantation development can proceed without DNA replication, but it is required to maintain proper heterochromatin organization at the 2-cell stage, the time of zygotic genome activation. The inhibition of DNA replication with aphidicolin blocked cleavage divisions without preventing zygotic genome activation, embryo morphogenesis, activation of lineage-specification factors, or the establishment of stage-specific transcriptional programs. However, replication arrest selectively altered the expression of developmental genes associated with late-replicating, Polycomb-enriched chromatin domains, and also reduced global levels of H3K27me3 and H3K9me3. These findings indicate that developmental timing in the early embryo is largely uncoupled from DNA replication and cell-cycle progression. Instead, DNA replication contributes to transcriptional fidelity by establishing or maintaining repressive chromatin states during the extensive genome reprogramming that accompanies zygotic genome activation.

developmental biology↗

bioRxiv2025

The Paraphyletic Origins of Genetic Resistance to Cabbage Stem Flea Beetle in Brassica oleracea

The cabbage stem flea beetle (CSFB) poses a growing threat to winter Brassica crops in Europe, yet the genetic basis of resistance remains poorly understood. To clarify the genetic architecture and evolutionary origins of resistance to CSFB adult feeding, we conducted a genome-wide association study (GWAS) by combining high-throughput pool-sequencing and a large-scale non-choice feeding assay on 113 Brassica oleracea accessions from wild (or feral) populations and major domesticated morphotypes. We demonstrate that resistance displays moderate heritability with a predominantly polygenic basis, revealing strong phenotypic divergence among morphotypes: B. oleracea var. capitata was generally susceptible, whereas var. botrytis and wild populations showed markedly higher resistance. Despite this polygenic background, we identified a major-effect candidate QTL on chromosome C01 with strong enrichment of resistance alleles in wild populations and susceptible alleles in var. capitata. Genome-wide F-statistics and heterozygosity scans revealed a recent selective sweep at this locus in wild lineages. Considering current evidence for the feral origin of contemporary "wild" populations, our results suggest that resistance evolved after domestication and subsequent feralization, independently of resistance in var. botrytis. This paraphyletic distribution underlines the critical importance of integrating demographic history into quantitative genetic analyses of domesticated plant systems.

genetics↗

bioRxiv2025

NR6A1 is essential for neural crest cell specification, formation and survival

Neural crest cells (NCC) are a migratory progenitor cell population considered unique to vertebrates. Derived from the neuroepithelium during early embryogenesis, NCC contribute to nearly every tissue and organ system throughout the body, and disruptions in NCC development can result in congenital disorders, termed neurocristopathies. Despite decades of research, we have a poor understanding of the cellular mechanisms and signals that govern mammalian NCC formation. We discovered nuclear receptor superfamily 6 group member 1 (NR6A1/GCNF/RTR), is a novel, critical regulator of mammalian NCC specification, formation and survival. Nr6a1 is expressed throughout the neuroepithelium in mouse embryos from E8.0 to E9.5 and briefly in newly delaminated NCC. Nr6a1 loss-of-function perturbs anterior cranial NCC formation and survival, and results in the complete agenesis of migratory NCC caudal to the first pharyngeal arch. Using mouse ESC and human iPSC differentiation into NCC, chromatin immunoprecipitation, and multiomic approaches, we demonstrate that these phenotypes are associated with perturbation of NCC specification (Foxd3, Sox9, Sox10) and epithelial-mesenchymal transition (EMT; Snail1, Zeb2), in concert with persistent expression of pluripotency-associated factors (Oct4 and Nanog) in the neuroepithelium. Conditional deletion revealed that Nr6a1 is required during mid-late gastrulation, demonstrating that murine NCC specification occurs earlier than previously thought. Consistent with these observations, in vivo overexpression of Oct4 in gastrulating mouse embryos disrupts NCC specification and formation. Therefore, Nr6a1 is essential for mammalian NCC formation and moreover, may function as a bimodal switch repressing pluripotency-associated factors in the neuroepithelium, while concomitantly activating NCC specifiers and regulators of EMT.

developmental biology↗

bioRxiv2021

Systematic in vivo interrogation identifies novel enhancers and silencers associated to Atrial Fibrillation

Cis-regulatory elements control gene expression in time and space and their disruption can lead to pathologies. Reporter assays allow the functional validation of enhancers and other regulatory elements, and such assays by means of the generation of transgenic mice provide a powerful tool to study gene regulation in development and disease. However, these experiments are time-consuming and, thus, their performance is very limited. Here, we increase the throughput of in vivo mouse reporter assays by using a piggyBac transposon-based system, and use it to decode the regulatory landscape of atrial fibrillation, a prevalent cardiac arrhythmia. We systematically interrogated ten human loci associated to atrial fibrillation in the search for regulatory elements. We found five new cardiac-specific enhancers and implicated novel genes in arrhythmia through genome editing and three-dimensional chromatin analysis by 4C-seq. Of note, functional dissection of the 7q31 locus identified a bivalent regulatory element in the second intron of the CAV1 gene differentially acting upon four genes. Our system also detected negative regulatory elements thanks to which we identified a ubiquitous silencer in the 16q22 locus that regulates ZFHX3 and can outcompete heart enhancers. Our study characterizes the function of new genetic elements that might be of relevance for the better understanding of gene regulation in cardiac arrhythmias. Thus, we have .established a new framework for the efficient dissection of the genetic contribution to common human diseases.

genomics↗

bioRxiv2021

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF

The eukaryotic genome is tightly packed inside the nucleus, where it is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors that bind DNA, such as the multi-zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but the impact of such structure on genome function during the first stages of mammalian development is still unclear. Here, we show that deletion of the Ctcf gene in mouse embryos impairs the correct establishment of chromatin structure, but initial lineage decisions take place and embryos are viable until the late blastocyst stage. Furthermore, we observe that maternal CTCF is not necessary for development. Transcriptomic analyses of mutant embryos show that the changes in metabolic and protein homeostasis programs that occur during the progression from the morula to the blastocyst depend on CTCF. Yet, these changes in gene expression do not correlate with disruption of chromatin structure, but mainly with proximal binding of CTCF to the promoter region of genes downregulated in mutants. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but mostly as independent processes.

developmental biology↗

bioRxiv2020

Enhancer priming by H3K4 methylation safeguards germline competence

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naive pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

genomics↗

bioRxiv2019

STAG2 cohesin is essential for heart morphogenesis

The distinct functions of cohesin complexes carrying STAG1 or STAG2 need to be unraveled. STAG2 is commonly mutated in cancer and germline mutations have been identified in cohesinopathy patients. To better understand the underlying pathogenic mechanisms, we here report the consequence of Stag2 ablation in mice. STAG2 is largely dispensable in adults and its tissue-wide inactivation does not lead to tumors but reduces fitness and affects both hematopoiesis and intestinal homeostasis. STAG2 is also dispensable for murine embryonic fibroblasts in vitro. In contrast, null embryos die by mid gestation showing global developmental delay and heart defects. Histopathological analysis and RNA-sequencing unveiled that STAG2 is required both for proliferation and regulation of cardiac transcriptional programs and in its absence, secondary heart field progenitors fail to enter the heart tube. These results provide compelling evidence on cell- and tissue-specific roles of the two cohesin complexes and how their dysfunction contributes to disease.

developmental biology↗

bioRxiv2019

A Human Accelerated Region participates in early human forebrain patterning and expansion

The expansion of the mammalian brain is associated with specific developmental processes; however, not much is known about how evolutionary changes participated in the acquisition of human brain traits during early developmental stages. Here we investigated whether enhancers active during the phylotypic stage show human-specific genomic divergence which could contribute to the evolutionary expansion of the forebrain. Notably, we identified an active enhancer containing a human accelerated region (HAR) located in the Chromosome 14q12, a region enriched with neurodevelopmental genes, such as Foxg1, Nkx2.1 and Nova1. Reporter analysis revealed that the human variant is active in the forebrain in transgenic mice and that it has stronger enhancer activity than the mouse or chimpanzee versions. Humanization of the mouse enhancer variant in transgenic mice and in mouse organoids resulted in an expansion of Foxg1 expressing domains in the forebrain early neural progenitors with a bias towards dorsal identities. Overall, our results suggest that human-specific mutations in critical regulatory elements controlling early brain development impact the expansion and patterning of the forebrain.

developmental biology↗

bioRxiv2019

Nanog regulates Pou3f1 expression and represses anterior fate at the exit from pluripotency.

Pluripotency is regulated by a network of transcription factors that maintains early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development its expression is restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses the anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting the neural fate by repressing Pou3f1 at the onset of gastrulation.

developmental biology↗

bioRxiv2019

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3 end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs.

developmental biology↗

bioRxiv2018

Transitions in cell potency during early mouse development are driven by Notch

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naive pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

developmental biology↗