To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.

It is estimated that ∼2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating ∼1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ∼36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ∼80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F 5 . We screened an estimated 760 insertions among F 3 progeny from 92 F 2 families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Agouti-related protein (AGRP) and proopiomelanocortin (POMC) genes encode secreted hypothalamic factors regulated by metabolic state in mammals and are involved in energy homeostasis. The zebrafish, Danio rerio, is a model system for forward genetics in vertebrates: POMC and AGRP in this organism have not been well characterized. Toward this end, AGRP and POMC were cloned from zebrafish. Zebrafish AGRP cDNA encodes a 127-amino-acid protein 36% and 40% identical to human and mouse AGRP, respectively. Zebrafish POMC cDNA encodes a 222-amino-acid preprohormone. Sequence identity to the mammalian ortholog is about 50%. Zebrafish AGRP and POMC transcripts were detected at 24 h post-fertilization (hpf) by RTPCR, and in situ hybridization demonstrated zebrafish AGRP mRNA exclusively in hypothalamus and POMC mRNA in hypothalamus and pituitary. Fasting did not alter POMC transcript levels, while AGRP transcripts were significantly upregulated. The ratio of AGRP/POMC transcripts in adult brain was increased up to threefold by fasting. These results demonstrate that the melanocortin system is regulated by metabolic state in zebrafish, and this finding thus provides a vertebrate system for the genetic analysis of the role of the melanocortin system in energy homeostasis.

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