Ana Fernández-Miñán

Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis

The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the noncoding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here, we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that (1) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression, (2) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog, and (3) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation, suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage.

Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary.

The existence of specialised regulatory microenvironments or niches that sustain stable stem cell populations is well documented in many tissues. However, the specific mechanisms by which niche support (or stromal) cells govern stem cell maintenance remain largely unknown. Here we demonstrate that removal of the Jak/Stat pathway in support cells of the Drosophila ovarian niche leads to germline stem cell loss by differentiation. Conversely, ectopic Jak/Stat activation in support cells induces stem cell tumours, implying the presence of a signal relay between the stromal compartment and the stem cell population. We further show that ectopic Jak/Stat signalling in support cells augments dpp mRNA levels and increases the range of Dpp signalling, a Bmp2 orthologue known to act as a niche extrinsic factor required for female germline stem cell survival and division. Our results provide strong evidence for a model in which Jak/Stat signalling in somatic support cells regulates dpp transcription to define niche size and to maintain the adjacent germline stem cells in an undifferentiated state.

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish

The identification and characterization of the regulatory activity of genomic sequences is crucial for understanding how the information contained in genomes is translated into cellular function. The cis‐regulatory sequences control when, where, and how much genes are transcribed and can activate (enhancers) or repress (silencers) gene expression. Here, we describe a novel Tol2 transposon‐based vector for assessing enhancer activity in the zebrafish (Danio rerio). This Zebrafish Enhancer Detector (ZED) vector harbors several key improvements, among them a sensitive and specific minimal promoter chosen for optimal enhancer activity detection, insulator sequences to shield the minimal promoter from position effects, and a positive control for transgenesis. Additionally, we demonstrate that highly conserved noncoding sequences homologous between humans and zebrafish largely with enhancer activity largely retain their tissue‐specific enhancer activity during vertebrate evolution. More strikingly, insulator sequences from mouse and chicken, but not conserved in zebrafish, maintain their insulator capacity when tested in this model. Developmental Dynamics 238:2409–2417, 2009.

Integrin signaling regulates spindle orientation in Drosophila to preserve the follicular-epithelium monolayer.

Epithelia act as important physiological barriers and as structural components of tissues and organs. In the Drosophila ovary, follicle cells envelop the germline cysts to form a monolayer epithelium. During division, the orientation of the mitotic spindle in follicle cells is such that both daughter cells remain within the same plane, and the simple structure of the follicular epithelium is thus preserved. Here we show that integrins, heterodimeric transmembrane receptors that connect the extracellular matrix to the cells cytoskeleton [1, 2], are required for maintaining the ovarian monolayer epithelium in Drosophila. Mosaic egg chambers containing integrin mutant follicle cells develop stratified epithelia at both poles. This stratification is due neither to abnormal cell proliferation nor to defects in the apical-basal polarity of the mutant cells. Instead, integrin function is required for the correct orientation of the mitotic apparatus both in mutant cells and in their immediately adjacent wild-type neighbors. We further demonstrate that integrin-mediated signaling, rather than adhesion, is sufficient for maintaining the integrity of the follicular epithelium. The above data show that integrins are necessary for preserving the simple organization of a specialized epithelium and link integrin-mediated signaling to the correct orientation of the mitotic spindle in this epithelial cell type.

Extensive conservation of ancient microsynteny across metazoans due to cis-regulatory constraints.

The order of genes in eukaryotic genomes has generally been assumed to be neutral, since gene order is largely scrambled over evolutionary time. Only a handful of exceptional examples are known, typically involving deeply conserved clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we report the first systematic survey of microsynteny conservation across metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of unrelated genes that have remained tightly physically linked in diverse lineages across over 600 million years of evolution. Integrating sequence conservation, gene expression data, gene function, epigenetic marks, and other genomic features, we provide extensive evidence that many conserved ancient linkages involve (1) the coordinated transcription of neighboring genes, or (2) genomic regulatory blocks (GRBs) in which transcriptional enhancers controlling developmental genes are contained within nearby bystander genes. In addition, we generated ChIP-seq data for key histone modifications in zebrafish embryos, which provided further evidence of putative GRBs in embryonic development. Finally, using chromosome conformation capture (3C) assays and stable transgenic experiments, we demonstrate that enhancers within bystander genes drive the expression of genes such as Otx and Islet, critical regulators of central nervous system development across bilaterians. These results suggest that ancient genomic functional associations are far more common than previously thought-involving ∼12% of the ancestral bilaterian genome-and that cis-regulatory constraints are crucial in determining metazoan genome architecture.

Numb/Numbl-Opo Antagonism Controls Retinal Epithelium Morphogenesis by Regulating Integrin Endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

The developmental epigenomics toolbox: ChIP-seq and MethylCap-seq profiling of early zebrafish embryos

Genome-wide profiling of DNA methylation and histone modifications answered many questions as to how the genes are regulated on a global scale and what their epigenetic makeup is. Yet, little is known about the function of these marks during early vertebrate embryogenesis. Here we provide detailed protocols for ChIP-seq and MethylCap-seq procedures applied to zebrafish (Danio rerio) embryonic material at four developmental stages. As a proof of principle, we have profiled on a global scale a number of post-translational histone modifications including H3K4me1, H3K4me3 and H3K27ac. We demonstrate that these marks are dynamic during early development and that such developmental transitions can be detected by ChIP-seq. In addition, we applied MethylCap-seq to show that developmentally-regulated DNA methylation remodeling can be detected by such a procedure. Our MethylCap-seq data concur with previous DNA methylation studies of early zebrafish development rendering this method highly suitable for the global assessment of DNA methylation in early vertebrate embryos.

Comparative epigenomics in distantly related teleost species identifies conserved cis-regulatory nodes active during the vertebrate phylotypic period

The complex relationship between ontogeny and phylogeny has been the subject of attention and controversy since von Baers formulations in the 19th century. The classic concept that embryogenesis progresses from clade general features to species-specific characters has often been revisited. It has become accepted that embryos from a clade show maximum morphological similarity at the so-called phylotypic period (i.e., during mid-embryogenesis). According to the hourglass model, body plan conservation would depend on constrained molecular mechanisms operating at this period. More recently, comparative transcriptomic analyses have provided conclusive evidence that such molecular constraints exist. Examining cis-regulatory architecture during the phylotypic period is essential to understand the evolutionary source of body plan stability. Here we compare transcriptomes and key epigenetic marks (H3K4me3 and H3K27ac) from medaka (Oryzias latipes) and zebrafish (Danio rerio), two distantly related teleosts separated by an evolutionary distance of 115-200 Myr. We show that comparison of transcriptome profiles correlates with anatomical similarities and heterochronies observed at the phylotypic stage. Through comparative epigenomics, we uncover a pool of conserved regulatory regions (≈700), which are active during the vertebrate phylotypic period in both species. Moreover, we show that their neighboring genes encode mainly transcription factors with fundamental roles in tissue specification. We postulate that these regulatory regions, active in both teleost genomes, represent key constrained nodes of the gene networks that sustain the vertebrate body plan.

A role for the chaperone Hsp70 in the regulation of border cell migration in the Drosophila ovary

Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. Here, we show that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, we provide biochemical and genetic evidence for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, our results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function.

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP

BackgroundRecent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown.ResultsTo characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity.ConclusionsOur data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.