Background: Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. Results: to characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. Conclusion: Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.

Background: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. Results: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. Conclusion: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression.

To elucidate important cellular and molecular interactions that regulate patterning and skeletal development, vertebrate limbs served as a model organ. A growing body of evidence from detailed studies on a subset of limb regulators like the HOXD cluster or SHH, reveals the importance of enhancers in limb related developmental and disease processes. Exploiting the recent genome-wide availability of functionally confirmed enhancer dataset, this study establishes regulatory interactions for dozens of human limb developmental genes. From these data, it appears that the long-range regulatory interactions are fairly common during limb development. This observation highlights the significance of chromosomal breaks/translocations in human limb deformities. Transcriptional factor (TF) analysis predicts that the differentiation of early nascent limb-bud into future territories entail distinct TF interaction networks. Conclusively, an important motivation for annotating the human limb specific regulatory networks is to pave way for the systematic exploration of their role in disease and evolution.

Background: GLI2, a zinc finger transcription factor, mediates Sonic hedgehog signaling, a critical pathway in vertebrate embryogenesis. GLI2 has been implicated in diverse set of embryonic developmental processes, including patterning of central nervous system and limbs. In humans, mutations in GLI2 are associated with several developmental defects, including holoprosencephaly and polydactyly. Results: Here, we demonstrate in transient transgenic zebrafish assays, the potential of a subset of tetrapod-teleost conserved non-coding elements (CNEs) residing within human GLI2 intronic intervals to induce reporter gene expression at known regions of endogenous GLI2 transcription. The regulatory activities of these elements are observed in several embryonic domains, including neural tube and pectoral fin. Moreover, our data reveal an overlapping expression profile of duplicated copies of an enhancer during zebrafish evolution. Conclusions: Our data suggest that during vertebrate history GLI2 acquired a high level of complexity in the genetic mechanisms regulating its expression during spatiotemporal patterning of the central nervous system (CNS) and limbs.

The zinc-finger transcription factor GLI3 acts as a primary transducer of Sonic hedgehog (Shh) signaling in a context-dependent combinatorial fashion. GLI3 participates in the patterning and growth of many organs, including the central nervous system (CNS) and limbs. Previously, we reported a subset of human intronic cis-regulators controlling many known aspects of endogenous Gli3 expression in mouse and zebrafish. Here we demonstrate in a transgenic zebrafish assay the potential of two novel tetrapod-teleost conserved non-coding elements (CNEs) docking within GLI3 intronic intervals (intron 3 and 4) to induce reporter gene expression at known sites of endogenous Gli3 transcription in embryonic domains such as the central nervous system (CNS) and limbs. Interestingly, the cell culture based assays reveal harmony with the context dependent dual nature of intra-GLI3 conserved elements. Furthermore, a transgenic zebrafish assay of previously reported limb-specific GLI3 transcriptional enhancers (previously tested in mice and chicken limb buds) induced reporter gene expression in zebrafish blood precursor cells and notochord instead of fin. These results demonstrate that the appendage-specific activity of a subset of GLI3-associated enhancers might be a tetrapod innovation. Taken together with our recent data, these results suggest that during the course of vertebrate evolution Gli3 expression control acquired a complex cis-regulatory landscape for spatiotemporal patterning of CNS and limbs. Comparative data from fish and mice suggest that the functional aspects of a subset of these cis-regulators have diverged significantly between these two lineages.

The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs. © 2013 Japanese Society of Developmental Biologists.

Background: Human genome is enriched with thousands of conserved non-coding elements (CNEs). Recently, a medium throughput strategy was employed to analyze the ability of human CNEs to drive tissue specific expression during mouse embryogenesis. These data led to the establishment of publicly available genome wide catalog of functionally defined human enhancers. Scattering of enhancers over larger regions in vertebrate genomes seriously impede attempts to pinpoint their precise target genes. Such associations are prerequisite to explore the significance of this in vivo characterized catalog of human enhancers in development, disease and evolution. Results: This study is an attempt to systematically identify the target gene-bodies for functionally defined human CNE-enhancers. For the purpose we adopted the orthology/paralogy mapping approach and compared the CNE induced reporter expression with reported endogenous expression pattern of neighboring genes. This procedure pinpointed specific target gene-bodies for the total of 192 human CNE-enhancers. This enables us to gauge the maximum genomic search space for enhancer hunting: 4 Mb of genomic sequence around the gene of interest (2 Mb on either side). Furthermore, we used human-rodent comparison for a set of 159 orthologous enhancer pairs to infer that the central nervous system (CNS) specific gene expression is closely associated with the cooperative interaction among at least eight distinct transcription factors: SOX5, HFH, SOX17, HNF3β, c-FOS, Tal1beta-E47S, MEF and FREAC. Conclusions: in conclusion, the systematic wiring of cis-acting sites and their target gene bodies is an important step to unravel the role of in vivo characterized catalog of human enhancers in development, physiology and medicine. © 2013 Parveen et al; licensee BioMed Central Ltd.