SUMMARY 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 twenty 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 eighty percent 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.
There was an error published in Development 143, 3785-3795.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 mutating serines 221 and 225 to aspartic acid (Schramek et al., 1997) by PCR, followed by BP cloning to generate pME-myc-act-map2k2b.In addition, we have since verified that the pME-myc-act-map2k2b plasmid used in the original studies and submitted to Addgene contains the correct sequence. This plasmid contains several nucleotide changes in comparison to the GenBank reference sequence (accession number: NM_001128281). However, all of these changes have been noted in a previous annotation of single nucleotide polymorphisms in the zebrafish and are naturally occurring variants (Butler et al., 2015).We apologize for any confusion that this may have caused and we thank the researchers who discovered the error for bringing it to our attention. ABSTRACTVascular 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.
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.
SUMMARYThe 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 nucleasemediated 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.
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.
Aims Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. Methods and results A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. Conclusion A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.
Endothelial cells emerge from the atrioventricular canal (AVC) to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrβ is first expressed in the epicardium around the AVC and later becomes localized mainly in the mural cells. Pdgfrβ mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single cell RNA sequencing analyses identified Pdgfrβ+ cells as epicardium derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrβ, although smooth muscle gene expression is downregulated. We find that pdgfrβ expression dynamically changes in EPDCs of a regenerating heart. Differential gene expression analyses of pdgfrβ+ EPDCs and mural cells suggest that they express genes important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrβ+ cells during heart regeneration. However, pdgfrβ but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrβ+ cells are essential for coronary development and heart regeneration.
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