José Bessa

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.

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.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

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.

Restricted teashirt expression confers eye-specific responsiveness to Dpp and Wg signals during eye specification in Drosophila.

In Drosophila, the eye primordium is specified as a subdomain of the larval eye disc. Here, we show that the Zn-finger transcription factor teashirt (tsh) marks the region of the early eye disc where the eye primordium will form. Moreover, tsh misexpression directs eye primordium formation in disc regions normally destined to form head capsule, something the eye selector genes eyeless (ey) and twin of eyeless (toy) are unable to do on their own. We present evidence that tsh induces eye specification, at least in part, by allowing the activation of eye specification genes by the wingless (wg) and decapentaplegic (dpp) signaling pathways. Under these conditions, though, terminal eye differentiation proceeds only if tsh expression is transient.

Zinc-finger paralogues tsh and tio are functionally equivalent during imaginal development in Drosophila and maintain their expression levels through auto- and cross-negative feedback loops

teashirt (tsh) and tiptop (tio) are two Drosophila gene paralogues encoding zinc‐finger transcription factors. While tsh is an important developmental regulator, tio null individuals are viable and fertile. Here, we show that tio and tsh have coincident expression domains in the imaginal discs, the precursors of the adult body, and that both genes show similar functional properties when expressed ectopically. Furthermore, tio is able to rescue the development of tsh mutants, indicating that both genes are functionally equivalent during imaginal development. Of interest, the transcriptional regulation of tio and tsh is linked by a negative feedback loop. This mechanism might be required to maintain a tight control on the total levels of tio/tsh and could help explaining why Drosophila keeps an apparently dispensable gene. Developmental Dynamics 238:19–28, 2009.

Differences in enhancer activity in mouse and zebrafish reporter assays are often associated with changes in gene expression

BackgroundPhenotypic evolution in animals is thought to be driven in large part by differences in gene expression patterns, which can result from sequence changes in cis- regulatory elements (cis- changes) or from changes in the expression pattern or function of transcription factors (trans- changes). While isolated examples of trans- changes have been identified, the scale of their overall contribution to regulatory and phenotypic evolution remains unclear.ResultsHere, we attempt to examine the prevalence of trans- effects and their potential impact on gene expression patterns in vertebrate evolution by comparing the function of identical human tissue-specific enhancer sequences in two highly divergent vertebrate model systems, mouse and zebrafish. Among 47 human conserved non-coding elements (CNEs) tested in transgenic mouse embryos and in stable zebrafish lines, at least one species-specific expression domain was observed in the majority (83%) of cases, and 36% presented dramatically different expression patterns between the two species. Although some of these discrepancies may be due to the use of different transgenesis systems in mouse and zebrafish, in some instances we found an association between differences in enhancer activity and changes in the endogenous gene expression patterns between mouse and zebrafish, suggesting a potential role for trans- changes in the evolution of gene expression.ConclusionsIn total, our results: (i) serve as a cautionary tale for studies investigating the role of human enhancers in different model organisms, and (ii) suggest that changes in the trans environment may play a significant role in the evolution of gene expression in vertebrates.

Several Cis-regulatory Elements Control mRNA Stability, Translation Efficiency, and Expression Pattern of Prrxl1 (Paired Related Homeobox Protein-like 1)

Background: The mechanisms that control the Prrxl1 expression are poorly understood. Results: Several regulatory elements present in Prrxl1 alternative promoters are functionally characterized, including a binding motif for Phox2b required for Prrxl1 expression in visceral sensory neurons. Conclusion: We define diverse regulatory modules, which control the spatiotemporal expression of Prrxl1 in nociceptive neurons. Significance: A new mechanism involved in the ganglion specific action of Prrxl1 is described. The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5′-UTR variants, named 5′-UTR-A, 5′-UTR-B, and 5′-UTR-C. These 5′-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.

sox21a directs lateral line patterning by modulating FGF signaling

The development of organs composed by repeated functional units is, in many cases, accomplished by the transition of cells from a progenitor to a differentiation domain, triggering a reiterated developmental program. Yet, how these discrete fields are formed during development is still a largely unresolved question. The posterior lateral line (pLL), a sensory organ present in fish and amphibians, develops from a primordium that migrates along the flanks of the animal periodically depositing neuromasts, the pLL functional units. In zebrafish (Danio rerio), the developmental program of the pLL is triggered by the transit of progenitor cells from a Wnt to a Fgf signaling domain. It has been proposed that these two fields are defined by the antagonistic activity of these two signaling pathways, but how they are formed and maintained is still an open question in the development of the pLL. In this work, we show that sox21a, an HMG ‐box transcription factor, is expressed within the Fgf domain. We demonstrate that, while the Fgf signaling pathway do not control sox21a, knockdown of sox21a causes impairment of Fgf signaling, expansion of the Wnt signaling domain and disruption of neuromast development. These results suggest that sox21a is a key player in the pLL primordium patterning, fine‐tuning the border of the Fgf and Wnt signaling domains.

A mobile insulator system to detect and disrupt cis-regulatory landscapes in vertebrates

In multicellular organisms, cis-regulation controls gene expression in space and time. Despite the essential implication of cis-regulation in the development and evolution of organisms and in human diseases, our knowledge about regulatory sequences largely derives from analyzing their activity individually and outside their genomic context. Indeed, the contribution of these sequences to the expression of their target genes in their genomic context is still largely unknown. Here we present a novel genetic screen designed to visualize and interrupt gene regulatory landscapes in vertebrates. In this screen, based on the random insertion of an engineered Tol2 transposon carrying a strong insulator separating two fluorescent reporter genes, we isolated hundreds of zebrafish lines containing insertions that disrupt the cis-regulation of tissue-specific expressed genes. We therefore provide a new easy-to-handle tool that will help to disrupt and chart the regulatory activity spread through the vast noncoding regions of the vertebrate genome.