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 approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases 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 genes. 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 sequence, 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.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution.

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-ß 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BACKGROUND: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations. RESULTS: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward. CONCLUSIONS: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.

Identification of the zebrafish maternal and paternal transcriptomes.

Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.

Multi-allelic phenotyping--a systematic approach for the simultaneous analysis of multiple induced mutations.

The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease.

Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling.

Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR-Gpr1 was chosen as a prototype to evolve novel glucose sensors. We demonstrate superior variant representation and several unique hits in the synthetic library compared to the PCR generated library. Application of this mutational approach further builds the possibilities of synthetic biology in tuning of a response to known ligands and in generating biosensors for novel ligands.

Identification of a plant isoflavonoid that causes biliary atresia.

Biliary atresia (BA) is a rapidly progressive and destructive fibrotic disorder of unknown etiology affecting the extrahepatic biliary tree of neonates. Epidemiological studies suggest that an environmental factor, such as a virus or toxin, is the cause of the disease, although none have been definitively established. Several naturally occurring outbreaks of BA in Australian livestock have been associated with the ingestion of unusual plants by pregnant animals during drought conditions. We used a biliary secretion assay in zebrafish to isolate a previously undescribed isoflavonoid, biliatresone, from Dysphania species implicated in a recent BA outbreak. This compound caused selective destruction of the extrahepatic, but not intrahepatic, biliary system of larval zebrafish. A mutation that enhanced biliatresone toxicity mapped to a region of the zebrafish genome that has conserved synteny with an established human BA susceptibility locus. The toxin also caused loss of cilia in neonatal mouse extrahepatic cholangiocytes in culture and disrupted cell polarity and monolayer integrity in cholangiocyte spheroids. Together, these findings provide direct evidence that BA could be initiated by perinatal exposure to an environmental toxin.

High-throughput target-selected gene inactivation in zebrafish.

There is an increasing requirement for efficient reverse genetics in the zebrafish, Here we describe a method that takes advantage of conventional mutagenized libraries (identical to ones used in forward screens) and re-sequencing to identify ENU-induced mutations in genes of interest. The efficiency of TILLING (Targeting Induced Local Legions IN Genomes) depends on the rate of mutagenesis in the library being screened, the amount of base pairs screened, and the ability to effectively identify and retrieve mutations on interest. Here we show that by improving the mutagenesis protocol, using in silico methods to predict codon changes for target selection, efficient PCR and re-sequencing, and accurate mutation detection we can vastly improve current TILLING protocols. Importantly it is also possible to use this method for screening for splice and mis-sense mutations, and with even a relatively small library, there is a high chance of identifying mutations across any given gene.

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation.

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.

Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.

Mouse ENU mutagenesis programmes have yielded a series of independent mutations on proximal chromosome 4 leading to dominant head-bobbing and circling behaviour due to truncations of the lateral semicircular canal of the inner ear. Here, we report the identification of mutations in the Chd7 gene in nine of these mutant alleles including six nonsense and three splice site mutations. The human CHD7 gene is known to be involved in CHARGE syndrome, which also shows inner ear malformations and a variety of other features with varying penetrance and appears to be due to frequent de novo mutation. We found widespread expression of Chd7 in early development of the mouse in organs affected in CHARGE syndrome including eye, olfactory epithelium, inner ear and vascular system. Closer inspection of heterozygous mutant mice revealed a range of defects with reduced penetrance, such as cleft palate, choanal atresia, septal defects of the heart, haemorrhages, prenatal death, vulva and clitoral defects and keratoconjunctivitis sicca. Many of these defects mimic the features of CHARGE syndrome. There were no obvious features of the gene that might make it more mutable than other genes. We conclude that the large number of mouse mutants and human de novo mutations may be due to the combination of the Chd7 gene being a large target and the fact that many heterozygous carriers of the mutations are viable individuals with a readily detectable phenotype.

Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection.

This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node-tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence was derived from 1978 clones of the Endoderm library. These yielded 1440 distinct cDNAs, of which 123 proved to be novel in the mouse. In situ hybridization analysis was carried out on 160 of the cDNAs, and of these, 29 (18%) proved to have restricted expression patterns.