Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at an enormous rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in vertebrate model organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1-3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3,4, this number falls significantly short of all >22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7-9, antisense morpholino oligonucleotides10, targeted re-sequencing11-13 and zinc finger and TAL endonucleases14-17 have made significant contributions to our understanding of the biological activity of vertebrate genes, but the number of genes studied again falls well short of the >26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies is particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Enabled by a well-annotated zebrafish reference genome sequence18,19, high-throughput sequencing and efficient chemical mutagenesis, we describe an active project that aims to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Thus far we have identified potentially disruptive mutations in more than 38% of all known 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 1000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

MicroRNAs (miRNAs) are produced by the Dicer1 enzyme; the role of Dicer1 in vertebrate development is unknown. Here we report target-selected inactivation of the dicer1 gene in zebrafish. We observed an initial build-up of miRNA levels, produced by maternal Dicer1, in homozygous dicer1 mutants, but miRNA accumulation stopped after a few days. This resulted in developmental arrest around day 10. These results indicate that miRNA-producing Dicer1 is essential for vertebrate development.

One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.

Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat (LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50 hu255H mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50 hu255H mutants. The SDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli.