Fernando Casares

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881–885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770–775; Frayling TM, et al. (2007) Science 316:889–894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing β-cells and glucagon-producing α-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

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.

Organ Specification-Growth Control Connection: New In-sights From the Drosophila Eye-Antennal Disc

The eye–antennal disc of Drosophila is serving a guiding role in the studies of how eye identity is specified, as well as how the retina is patterned. However, this system also holds a great potential for studying the coordination between organ growth and specification when various distinct organs form from a common primordium. The eye–antennal disc gives origin not only to the compound eye but also to the head capsule, ocelli, maxillary palp, and antenna, and these organs develop bearing constant size proportions with each other. Here, we review recent results that have shed light on the mechanisms that control the specification and growth of organs of the eye–antennal disc and discuss how these controls are intertwined during the development of neighboring organs to ensure their constant shape and relative sizes. Developmental Dynamics 232:673–684, 2005.

The colorectal cancer risk at 18q21 is caused by a novel variant altering SMAD7 expression

Recent genome-wide scans for colorectal cancer (CRC) have revealed the SMAD7 (mothers against decapentaplegic homolog 7) gene as a locus associated with a modest, but highly significant increase in CRC risk. To identify the causal basis of the association between 18q21 variation and CRC, we resequenced the 17-kb region of linkage disequilibrium and evaluated all variants in 2532 CRC cases and 2607 controls. A novel C to G single nucleotide polymorphism (SNP) at 44,703,563 bp was maximally associated with CRC risk (P = 5.98 x 10(-7); > or =1.5-fold more likely to be causal than other variants). Using transgenic assays in Xenopus laevis as a functional model, we demonstrate that the G risk allele leads to reduced reporter gene expression in the colorectum (P = 5.4 x 10(-3)). Electrophoretic mobility shift assays provided evidence for the role of Novel 1 in transcription factor binding. We propose that the novel SNP we have identified is the functional change leading to CRC predisposition through differential SMAD7 expression and, hence, aberrant TGF-beta signaling.

meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye

During eye development, retinal progenitors are drawn from a multipotent, proliferative cell population. In Drosophila the maintenance of this cell population requires the function of the TALE-homeodomain transcription factor Hth, although its mechanisms of action are still unknown. Here we investigate whether members of the Meis gene family, the vertebrate homologs of hth, are also involved in early stages of eye development in the zebrafish. We show that meis1 is initially expressed throughout the eye primordium. Later, meis1 becomes repressed as neurogenesis is initiated, and its expression is confined to the ciliary margin, where the retinal stem population resides. Knocking down meis1 function through morpholino injection causes a delay in the G1-to-S phase transition of the eye cells, and results in severely reduced eyes. This role in cell cycle control is mediated by meis1 regulating cyclin D1 and c-myc transcription. The forced maintenance of meis1 expression in cell clones is incompatible with the normal differentiation of the meis1-expressing cells, which in turn tend to reside in undifferentiated regions of the retinal neuroepithelium, such as the ciliary margin. Together, these results implicate meis1 as a positive cell cycle regulator in early retinal cells, and provide evidence of an evolutionary conserved function for Hth/Meis genes in the maintenance of the proliferative, multipotent cell state during early eye development.

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes

Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor–encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.

Transphyletic conservation of developmental regulatory state in animal evolution

Specific regulatory states, i.e., sets of expressed transcription factors, define the gene expression capabilities of cells in animal development. Here we explore the functional significance of an unprecedented example of regulatory state conservation from the cnidarian Nematostella to Drosophila, sea urchin, fish, and mammals. Our probe is a deeply conserved cis-regulatory DNA module of the SRY-box B2 (soxB2), recognizable at the sequence level across many phyla. Transphyletic cis-regulatory DNA transfer experiments reveal that the plesiomorphic control function of this module may have been to respond to a regulatory state associated with neuronal differentiation. By introducing expression constructs driven by this module from any phyletic source into the genomes of diverse developing animals, we discover that the regulatory state to which it responds is used at different levels of the neurogenic developmental process, including patterning and development of the vertebrate forebrain and neurogenesis in the Drosophila optic lobe and brain. The regulatory state recognized by the conserved DNA sequence may have been redeployed to different levels of the developmental regulatory program during evolution of complex central nervous systems.

Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon

Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

BackgroundThe zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians.ResultsIn this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern.ConclusionsThe absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations.

The genital disc of Drosophila melanogaster.

Abstract The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development.