Identification of RNA binding motif proteins essential for cardiovascular development

Background We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. Results To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. Conclusion Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation

Identification of RNA binding motif proteins essential for cardiovascular development

Background: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. Results: To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. Conclusion: Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation

A Rare Myelin Protein Zero (MPZ) Variant Alters Enhancer Activity In Vitro and In Vivo

expression. variants. that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.CepidFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNP

Manipulating Mitotic Recombination in the Zebrafish Embryo Through RecQ Helicases

RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activity, and demonstrate that zebrafish RecQ genes have conserved function in suppressing mitotic recombination

Rbm24a and Rbm24b Are Required for Normal Somitogenesis

<div><p>We recently demonstrated that the gene encoding the RNA binding motif protein 24 (RBM24) is expressed during mouse cardiogenesis, and determined the developmental requirement for its zebrafish homologs Rbm24a and Rbm24b during cardiac development. We demonstrate here that both Rbm24a and Rbm24b are also required for normal somite and craniofacial development. Diminution of <i>rbm24a</i> or <i>rbm24b</i> gene products by morpholino knockdown resulted in significant disruption of somite formation. Detailed <i>in situ</i> hybridization-based analyses of a spectrum of somitogenesis-associated transcripts revealed reduced expression of the cyclic muscle pattering genes <i>dlc</i> and <i>dld</i> encoding Notch ligands, as well as their respective target genes <i>her7</i>, <i>her1</i>. By contrast expression of the Notch receptors <i>notch1a</i> and <i>notch3</i> appears unchanged. Some RBM-family members have been implicated in pre-mRNA processing. Analysis of affected Notch-pathway mRNAs in <i>rbm24a</i> and <i>rbm24b</i> morpholino-injected embryos revealed aberrant transcript fragments of <i>dlc</i> and <i>dld</i>, but not <i>her1</i> or <i>her7</i>, suggesting the reduction in transcription levels of Notch pathway components may result from aberrant processing of its ligands. These data imply a previously unknown requirement for Rbm24a and Rbm24b in somite and craniofacial development. Although we anticipate the influence of disrupting RBM24 homologs likely extends beyond the Notch pathway, our results suggest their perturbation may directly, or indirectly, compromise post-transcriptional processing, exemplified by imprecise processing of <i>dlc</i> and <i>dld</i>.</p></div

Notch-signaling pathway transcripts are depleted in somites of <i>rbm24a</i>MO and <i>rbm24b</i>MO embryos.

<p><i>ISH</i> images of the PSM of dorsally oriented 13–15 somite and laterally oriented 24 hpf ctrlMO, <i>rbm24a</i>MO and <i>rbm24b</i>MO embryos. 13–15 somite embryos are shown in 2 different temporal stages of development. Spatial expression of <i>dlc</i> (A–F), <i>dld</i> (G–L), <i>her1</i> (M–R) and <i>her7</i> (S–X) and <i>tbx16</i> (Y–DD). Asterisks highlight cycling expression. n = 8–12 embryos stained per time-point with no more than 1 deviating from the displayed expression. arrow, regions of interest for PSM expression.</p

Genome-wide identification of conserved regulatory function in diverged sequences

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term “covert.

Aberrant <i>dlc</i> and <i>dld</i> splice forms are detectable in cDNA from <i>rbm24a</i>MO and <i>rbm24b</i>MO embryos.

<p>RT-PCR experiments to amplify <i>dlc</i> and <i>dld</i> mRNA transcripts using total cDNA generated from 13 somite uninjected, ctrlMO, <i>rbm24a</i>MO and <i>rbm24b</i>MO embryos (n = 50 embryos per condition). RT-PCR for <i>dlc</i> transcript yielded a fragment of correct length for all conditions and an additional short fragment for <i>rbm24a</i>MO and <i>rbm24b</i>MO cDNA (A). <i>dlc</i> pre-mRNA, <i>dlc</i> mRNA and <i>dlc</i> short 1 fragment sequences are depicted graphically to scale. RT-PCR for <i>dld</i> transcript yielded a fragment of correct length for all conditions and two additional short fragments for <i>rbm24a</i>MO and <i>rbm24b</i>MO cDNA (B). <i>dld</i> pre-mRNA, <i>dld</i> mRNA and <i>dld</i> short 1 and <i>dld</i> short 2 fragments are depicted graphically to scale. 13 somite and 24 hpf uninjected, ctrlMO, <i>rbm24a</i>MO and <i>rbm24b</i>MO embryos were used to generate cDNA used for qRT-PCR. All transcripts were assayed in triplicate per cDNA sample. Transcript levels were normalized to <i>elfalpha</i> transcript levels and fold change for each transcript is shown as compared to uninjected embryos. An asterisk denotes statistically significant fold changes compared to uninjected. * = p<0.05; ** = p<0.01; *** = p<0.001. black arrows, short <i>dlc</i> and <i>dld</i> fragments; boxes, exons; thick black lines, introns; thin black lines, wild type splicing, dashed lines, aberrant splicing.</p