A systematic genome-wide analysis of zebrafish protein-coding gene function

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1, 2, 3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3, 4, this number falls considerably short of the more than 22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7, 8, 9, antisense morpholino oligonucleotides10, targeted re-sequencing11, 12, 13, and zinc finger and TAL endonucleases14, 15, 16, 17 have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence18, 19, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis

RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development

Detection and characterization of small insertion and deletion genetic variants in modern layer chicken genomes

Background: Small insertions and deletions (InDels) constitute the second most abundant class of genetic variants and have been found to be associated with many traits and diseases. The present study reports on the detection and characterisation of about 883 K high quality InDels from the whole-genome analysis of several modern layer chicken lines from diverse breeds. Results: To reduce the error rates seen in InDel detection, this study used the consensus set from two InDel-calling packages: SAMtools and Dindel, as well as stringent post-filtering criteria. By analysing sequence data from 163 chickens from 11 commercial and 5 experimental layer lines, this study detected about 883 K high quality consensus InDels with 93 % validation rate and an average density of 0.78 InDels/kb over the genome. Certain chromosomes, viz, GGAZ, 16, 22 and 25 showed very low densities of InDels whereas the highest rate was observed on GGA6. In spite of the higher recombination rates on microchromosomes, the InDel density on these chromosomes was generally lower relative to macrochromosomes possibly due to their higher gene density. About 43-87 % of the InDels were found to be fixed within each line. The majority of detected InDels (86 %) were 1-5 bases and about 63 % were non-repetitive in nature while the rest were tandem repeats of various motif types. Functional annotation identified 613 frameshift, 465 non-frameshift and 10 stop-gain/loss InDels. Apart from the frameshift and stopgain/loss InDels that are expected to affect the translation of protein sequences and their biological activity, 33 % of the non-frameshift were predicted as evolutionary intolerant with potential impact on protein functions. Moreover, about 2.5 % of the InDels coincided with the most-conserved elements previously mapped on the chicken genome and are likely to define functional elements. InDels potentially affecting protein function were found to be enriched for certain gene-classes e.g. those associated with cell proliferation, chromosome and Golgi organization, spermatogenesis, and muscle contraction. Conclusions: The large catalogue of InDels presented in this study along with their associated information such as functional annotation, estimated allele frequency, etc. are expected to serve as a rich resource for application in future research and breeding in the chicken