Zebrafish were maintained in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act 1986, under project licence 80/2192, which was reviewed by The Wellcome Trust Sanger Institute Ethical Review Committee.
Heterozygous F2 fish were randomly incrossed and upon egg collection F2 adults were fin clipped and kept as isolated breeding pairs. For each family we aimed to phenotype 12 pairs, over 3 weeks of breeding. Each clutch of eggs, which was labelled with the breeding pair ID, was sorted into three 10cm petri dishes of ~50 embryos each. Embryos were incubated at 28.5°C. Previous mutagenesis screens were used as a reference for the phenotyping 27 (link),28 (link). Those phenotypes studied were: day 1 – early patterning defects, early arrest, notochord, eye development, somites, patterning and cell death in the brain; day 2 – cardiac defects, circulation of the blood, pigment (melanocytes), eye and brain development; day 3 – cardiac defects, circulation of the blood, pigment (melanocytes), movement and hatching; day 4 – cardiac defects, movement, pigment (melanocytes) and muscle defects; day 5 – behaviour (hearing, balance, response to touch), swim bladder, pigment (melanocytes, xanthophores and iridophores), distribution of pigment, jaw, skull, axis length, body shape, notochord degeneration, digestive organs (intestinal folds, liver and pancreas), left-right patterning. In the first round of the phenotyping, all phenotypic embryos were discarded. At 5 dpf, >48 phenotypically wild-type embryos were harvested. Embryos were fixed in 100% methanol and stored at −20°C until genotyping was initiated. In the second round, F2s that were heterozygous for a suspected causal mutation were re-crossed. All phenotypes observed in those clutches of embryos were counted, documented and photographed. Phenotypic embryos were fixed in 100% methanol and at 5 dpf 48 phenotypically wild-type embryos were also collected. The first round genotyping results were assessed using a Chi-squared test with a p-value cut off of <0.05. If the number of homozygous embryos was above the cut-off (i.e. in the expected 25% ratio), the allele was deemed to not cause a phenotype within the first 5 dpf. If the number of homozygous embryos was below the cut-off, the allele was carried forward into the second round of phenotyping. In the second round, we aimed to genotype 48 embryos for each phenotype, ideally from multiple clutches. An allele was documented as causing a phenotype if the phenotypic embryos were homozygous for the allele. We allowed up to 10% of embryos for a given phenotype to not be homozygous, to account for errors in egg collection. Such alleles were outcrossed for further genotyping with F4 embryos at a later date. Where possible, alleles were also submitted to complementation tests.
Heterozygous F2 fish were randomly incrossed and upon egg collection F2 adults were fin clipped and kept as isolated breeding pairs. For each family we aimed to phenotype 12 pairs, over 3 weeks of breeding. Each clutch of eggs, which was labelled with the breeding pair ID, was sorted into three 10cm petri dishes of ~50 embryos each. Embryos were incubated at 28.5°C. Previous mutagenesis screens were used as a reference for the phenotyping 27 (link),28 (link). Those phenotypes studied were: day 1 – early patterning defects, early arrest, notochord, eye development, somites, patterning and cell death in the brain; day 2 – cardiac defects, circulation of the blood, pigment (melanocytes), eye and brain development; day 3 – cardiac defects, circulation of the blood, pigment (melanocytes), movement and hatching; day 4 – cardiac defects, movement, pigment (melanocytes) and muscle defects; day 5 – behaviour (hearing, balance, response to touch), swim bladder, pigment (melanocytes, xanthophores and iridophores), distribution of pigment, jaw, skull, axis length, body shape, notochord degeneration, digestive organs (intestinal folds, liver and pancreas), left-right patterning. In the first round of the phenotyping, all phenotypic embryos were discarded. At 5 dpf, >48 phenotypically wild-type embryos were harvested. Embryos were fixed in 100% methanol and stored at −20°C until genotyping was initiated. In the second round, F2s that were heterozygous for a suspected causal mutation were re-crossed. All phenotypes observed in those clutches of embryos were counted, documented and photographed. Phenotypic embryos were fixed in 100% methanol and at 5 dpf 48 phenotypically wild-type embryos were also collected. The first round genotyping results were assessed using a Chi-squared test with a p-value cut off of <0.05. If the number of homozygous embryos was above the cut-off (i.e. in the expected 25% ratio), the allele was deemed to not cause a phenotype within the first 5 dpf. If the number of homozygous embryos was below the cut-off, the allele was carried forward into the second round of phenotyping. In the second round, we aimed to genotype 48 embryos for each phenotype, ideally from multiple clutches. An allele was documented as causing a phenotype if the phenotypic embryos were homozygous for the allele. We allowed up to 10% of embryos for a given phenotype to not be homozygous, to account for errors in egg collection. Such alleles were outcrossed for further genotyping with F4 embryos at a later date. Where possible, alleles were also submitted to complementation tests.
