Fatma O. Kok

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

Targeted chromosomal deletions and inversions in zebrafish

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

Zebrafish rest regulates developmental gene expression but not neurogenesis

The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 ‘silencer elements’, which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors.

Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations. Summary: Genome-editing approaches in zebrafish reveal that distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.

Construction and Application of Site-Specific Artificial Nucleases for Targeted Gene Editing

Artificial nucleases have developed into powerful tools for introducing precise genome modifications in a wide variety of species. In this chapter the authors provide detailed protocols for rapidly constructing zinc finger nucleases (ZFNs) and TALE nucleases (TALENs) and evaluating their activity for the targeted generation of InDels within the zebrafish genome.

CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function

MicroRNAs (miRNAs) are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Here we explore the use of CRISPR/Cas9 as a powerful tool to delineate the function and regulation of miRNA families and clusters. We focused on four miRNA clusters composed of miRNA members of the same family, homo-clusters or different families, hetero-clusters. Our results highlight different regulatory mechanisms in miRNA cluster expression. In the case of the miR-497~195 cluster, editing of miR-195 led to a significant decrease in the expression of the other miRNA in the cluster, miR-497a. Although no gene editing was detected in the miR-497a genomic locus, computational simulation revealed alteration in the three dimensional structure of the pri-miR-497~195 that may affect its processing. In cluster miR-143~145 our results imply a feed-forward regulation, although structural changes cannot be ruled out. Furthermore, in the miR-17~92 and miR-106~25 clusters no interdependency in miRNA expression was observed. Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool that can uncover novel mechanisms of clustered miRNA regulation and function.

Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis

Cell‐type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild‐type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity. Developmental Dynamics 239:548–558, 2010.

Correction: Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors

There was an error published in Development 143 , [3785-3795][1]. In the Materials and Methods section we provided an incorrect serine residue number for the map2k2b mutation. The corrected section of text is as follows: An activated myc-tagged form of MEK was generated from zebrafish map2k2b by

A Platform for Reverse Genetics in Endothelial Cells

The recent development of programmable nucleases has the potential to revolutionize biological sciences. In particular, the Cas9 nuclease, which functions as a component of the clustered regularly interspaced short palindromic repeats (CRISPR) system in bacteria,1 has proven to be a highly efficient tool for genome editing in a wide range of model organisms, including mouse, zebrafish, Drosophila , and Caenorhabditis elegans .2–5 Application of Cas9 also allows straightforward genetic manipulations in cultured cells and is efficient enough to perform genome-wide screens in cell lines.6,7 However, applying genome editing tools in this manner in vascular biology is challenging because of the widespread use of primary cell cultures, which have a limited lifespan and are difficult to use for clonal analysis. Fortunately, studies by Abrahimi et al in this issue describe several solutions that facilitate the application of Cas9 in cultured endothelial cells. Together, these technical advances provide a valuable platform to enable straightforward and robust reverse genetic analysis in endothelial cells. Article, see p 121 The discovery of RNA interference8 and its derived or related applications (eg, short hairpin RNAs and small interfering RNAs) revolutionized the analysis of gene function in both model organisms and cell culture.9 However, these approaches often only partially decrease gene function and can have widespread off-target effects leading to false-positives.10 Accordingly, Abrahimi et al chose to apply a more definitive genetic approach by directly introducing targeted deletions into …

Crispr/Cas9 gene editing reveals novel tertiary constraints in clustered mirna processing

Introduction MicroRNAs (miRNAs) play an important role in the cellular function. They often form families, with members sharing high sequence homology, a property that hampers miRNA research as there is a lack of elegant tools for specific miRNA manipulation. Objective To establish a reliable workflow for miRNA inhibition using genome editing. Methods and Results This study focused on miR-195, a member of the miR-15 family and employed the CRISPR-Cas9 system. To this end we generated mouse vascular smooth muscle cells (VSMCs) stably expressing Cas9 nuclease. Cells were then transfected with an in vitro transcribed single guide RNA targeting the miR-195 stem loop. T7 endonuclease I assay (T7EI) and Sanger sequencing confirmed efficient editing. QPCR demonstrated effective decrease of miR-195 but not of miR-15a and miR-16, two highly expressed members of the miR-15 family in VSMCs. Surprisingly the expression of miR-497 was also decreased in edited cells. Noteworthy, miR-195 and miR-497 form a miRNA cluster and are co-transcribed as a primary miRNA. No gene editing was detected by T7EI and sequencing of the mir-497 genomic locus. Computational simulation predicted that mutations of the miR-195 stem loop led to changes in the three dimensional structure of the primary miR-497~195 transcript that could affect its processing to mature miRNAs. Similar findings were obtained the miR-143~145 cluster that encodes miR-143 and miR-145a, two miRNAs that do not belong to the same family, show no sequence homology and play a pivotal role in vascular biology. Specific targeting of the mir-145a locus effectively inhibited the expression of both miR-143 and miR-145a while no genomic editing was observed for the mir-143 locus. Noteworthy, the expression of Carmn, a long non coding RNA in the vicinity of the miR-143~145 cluster that constitutes an independent transcription unit did not differ in miR-145a edited cells confirming that only the primary miRNA transcript is affected. On the contrary, gene editing in the miR-17~92 and miR-106b~25 clusters, two miRNA clusters with a key function in the cardiovascular system, resulted in targeted miRNA inhibition. MiR-18a and miR-25 were targeted on each cluster, respectively. Specific editing only for the intended miRNA locus was observed and QPCR quantification indicated inhibition of the edited miRNA. No effect on the expression of other miRNAs occurred, both for cluster miR-17~92 and miR-106b~25. Detailed analysis of the gene editing in the four clusters revealed that the unintended inhibition of miRNA expression in the cluster coincides with disruption of sequence motifs of the terminal loop of the targeted hairpin, suggesting that these elements are critical for the maturation not only of individual hairpins but the entire primary transcript in miRNA clusters. Conclusions CRISPR/Cas9 emerged as a powerful tool that can offer novel insights into the role of miRNAs in cardiovascular diseases.