22 research outputs found

    Zebrafish paf1 is required for neural crest specification

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    SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo

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    SH2-domain-containing inositol phosphatase 2 (SHIP2) belongs to a small family of phosphoinositide 5-phosphatases that help terminate intracellular signaling initiated by activated receptor tyrosine kinases. Mammalian SHIP2 is viewed primarily as an attenuator of insulin signaling and has become a prominent candidate target for therapeutic agents that are designed to augment insulin signaling. Despite this view, no signaling pathway has yet been demonstrated as being affected directly by SHIP2 function in vivo, and in vitro studies indicate that the protein may function in multiple signaling pathways. Here, we analyze the role of a SHIP2 family member in the early zebrafish embryo where developmental and gene expression defects can be used to assay specific signaling pathways. The zebrafish ship2a transcript is maternally supplied, and inhibiting the expression of its protein product results in the expansion of dorsal tissue fates at the expense of ventral ones. We show that the developmental defects are the result of perturbation of fibroblast growth factor (FGF) signaling in the early embryo. Loss of Ship2a leads to an increased and expanded expression of outputs of FGF-mediated signaling, including FGF-dependent gene expression and activated mitogen-activated protein kinase (MAPK) signaling. Our findings demonstrate that Ship2a attenuates the FGF signaling pathway in vivo and functions in the establishment of normal tissue patterning in the early embryo. We suggest that modulation of FGF signaling may be a principal function of SHIP2 in mammals

    Identification of rare genetic variants for rotator cuff tearing and repair in high-risk pedigrees

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    Background: Common genetic variants with small effect sizes have been associated with rotator cuff tearing although very few rare, highly penetrant variants have been identified. The purpose of this pilot study was to identify dominant coding variants that segregated with affected individuals in pedigrees at high risk for rotator cuff tears (RCTs). We hypothesize that rare variants contribute to symptomatic RCTs and that they can be identified in related cases with a full-thickness tear requiring surgical management. Methods: We used the Utah Population Database to identify pedigrees that exhibited a significant excess of individuals who had undergone surgical repair of a full-thickness RCT. We analyzed whole exome sequence analysis to identify rare coding variants in 9 independent affected cousin pairs (first or second cousins) who had undergone arthroscopic surgery for repair of a full-thickness RCT (mean age at diagnosis 68 years). Validation of association of the candidate variants with risk for rotator cuff tearing was accomplished utilizing data from the UK Biobank and a separate cohort of unrelated cases of full-thickness RCTs. Results: A total of 82 rare (minor allele frequency <0.005) coding variants were identified as shared in at least one cousin pair affected with full-thickness rotator cuff tearing belonging to a high-risk pedigree, which included variants in RUNX1, ADAM12, TGFBR2, APBB1, PDLIM7, LTBP1, MAP3K4, and MAP3K1. Analysis of 39 of these variants with data available in the UK Biobank (3899 cases with rotator cuff injury and 11,697 matched controls; mean case age 59.9 years) identified a significant association with the APBB1 gene (OR = 2.37, P = .007, uncorrected). The PDLIM7 allele was found to be in significant excess in RCT cases in a separate cohort of Utah patients with full-thickness RCTs (10 carriers out of 458 independent, unrelated patients; minor allele frequency of 0.022) compared to a minor allele frequency of 0.0058 for the European (non-Finnish) control population rate (749 carriers out of 128612 tested) (chi-square test: 19.3 [P < .001]). Discussion: The analysis of closely related individuals with confirmed full-thickness RCTs from high-risk pedigrees has identified 82 rare, shared candidate genetic predisposition coding variants. Association of the PDLIM7 allele with risk for tear was confirmed in an independent cohort of RCTs. Further analysis of the variant alleles is required for confirmation of these genes in rotator cuff tearing

    Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish

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    The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca2+ handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca2+ handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca2+ handling in the heart through its interaction with phosphatidylinositols

    Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

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    <div><p>The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.</p> </div

    TALEN activity at homologous target sequences.

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    <p>(A) Comparison of the <i>ryr3</i> gene sequences targeted by the <i>ryr3-ex5</i> TALEN and homologous sequences of the <i>ryr1a</i> and <i>ryr1b</i> genes. <i>ryr3</i> sequences bound by the <i>ryr3-ex5</i> TALEN are denoted in color, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002861#pgen.1002861.s001" target="_blank">Figure S1</a>: Red letters indicate the Left and Right RVD array binding sites of the TALEN monomer components; the entire target site is defined as including both the nucleotides recognized by RVD array and the 5′ T that interacts with the N-terminal portion of the TALEN. Vertical lines indicate bases that are identical between the <i>ryr3-ex5</i> TALEN target and the gene sequences of homologues; *'s indicate non-identical bases. (B) Uninjected WT embryos (n = 23, grey curves) and WT embryos injected with 100 pg total <i>ryr3-ex5</i> TALEN RNAs (n = 24, blue curves) were analyzed by HRMA at 1 dpf for the presence of newly induced mutations. The gDNA of each embryo was examined in parallel for mutations at <i>ryr3</i>, <i>ryr1a</i>, or <i>ryr1b</i>. Whereas every injected embryo had <i>ryr3</i> mutations, no mutations were detected in the <i>ryr1a</i> or <i>ryr1b</i> genes. (C) Comparison of the <i>ryr1a</i> gene sequences targeted by the <i>ryr1a-ex6</i> TALEN and homologous sequences of the <i>ryr3</i> and <i>ryr1b</i> genes. <i>ryr1a</i> sequences bound by the <i>ryr1a-ex6</i> TALEN are denoted in color, as in (A). Identical and non-identical bases are indicated as in (A). (D) Uninjected WT embryos (n = 23, grey curves) or WT embryos injected with 100 pg total <i>ryr1a-ex6</i> TALEN RNAs (n = 24, red curves) were analyzed by HRMA at 1 dpf for the presence of newly induced mutations. Whereas no mutations at the <i>ryr3</i> locus were detected in these embryos, every injected embryo had mutations in the <i>ryr1a</i> and <i>ryr1b</i> genes.</p

    Induction of somatic mutations with TALENs.

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    <p>(A–D, Upper panels) HRMA detection of targeted mutations in WT embryos that had been injected at the 1 cell stage with RNAs encoding TALENs directed against the <i>golden</i> (A), <i>ryr3</i> (B), <i>tbx6</i> (C), or <i>ryr1a</i> (D) gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002861#pgen.1002861.s001" target="_blank">Figure S1</a> for target sites). gDNA was isolated from individual uninjected or TALEN RNA-injected 1–2 dpf embryos and subjected to HRMA. Each curve is the melting profile of re-annealed amplicons generated from a single embryo. LightScanner Call-IT Software (Idaho Technology) was used to identify melt curves that differed significantly from WT. The results shown here reflect a single experiment, but results from all injections are tablulated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002861#pgen-1002861-t002" target="_blank">Table 2</a>. Newly induced DNA polymorphisms at the targeted loci were evident in all but one injected embryo (red curves, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002861#pgen-1002861-t002" target="_blank">Table 2</a>), and even <i>gol-ex2</i> TALEN RNA-injected embryos that did not exhibit patches of pigmentless tissue had induced <i>golden</i> mutations as detected by HRMA (A, purple curves). (A–C, Lower panels) TALEN-induced sequence alterations at targeted loci. Genomic sequences bordering the targeted loci were amplified from embryonic gDNA samples, cloned, and sequenced. Examples of recovered alleles (purple and pink boxed sequences indicate Left and Right RVD binding sites of the TALENs, respectively; red indicates sequence alterations) indicate that insertion/deletion (indel) mutations centered at the TALEN target sites had been induced in somatic tissues of embryos.</p
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