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.

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.

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.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.