By Golling et. al

1. Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development Presented by: Pam Lincoln By Golling et. al

2. Background • Previously, they used ethylnitrosourea (ENU) to make single base pair mutations • When they found a mutant of interest they tried to isolate the gene • Isolation of the mutation was challenging and often required previous knowledge of the molecular pathways involved

3. Introduction • Forward genetic screens are used to isolate genes necessary for embryonic development • Previous screens have shown about 800 genes can be mutated to give relatively specific or localized defects during development • Another 1600 genes can be mutated to give less specific phenotypes • In five and half years, from hundreds of mutants, the genes underlying only about 50 mutants have been reported • Using a new technique, mutated genes can be identified in as little as two weeks

4. Methods Mutagenesis: • They used a Moloney murine leukemia-based retroviral vector as a mutagen for stable transfer of exogenous genes and VSV-G envelope protein to infect a wide range of organisms • They injected blastula stage embryos with the retrovirus • Founders were bred together and F1 fish with multiple insertions were used to generate an F2 generation • Transgenic F2 were inbred and F3 were examined for mutations

5. Methods Finding the Gene: • A Southern was performed with DNA from embryos and adults which were mated. They identified the band common to all phenotypic embryos • They performed inverse PCR with appropriate primers and isolate the PCR product of the appropriate size

6. Methods (cont) Genotyping Embryos: • Embryos from heterozygous parents were sorted into phenotypically wildtype and mutant groups • They genotyped 24 embryos in each group using PCR • Wildtype and Mutants yielded different PCR products Alcian Blue staining

7. Results Identification of retrovirus-induced mutations Obtaining the Gene: • When an insert is potentially linked to a mutant phenotype, the DNA flanking the insert is cloned using inverse PCR. About 50% of the time a candidate gene is found by homology search in the NCBI database • In additional cases small chromosomal walks are used to obtain and sequence more DNA • This yields a candidate gene 2/3 of the time

8. ResultsIdentification of retrovirus-induced mutations Confirmation: • To confirm that the correct gene was cloned they performed linkage analysis • Mutant embryos are homozygous, while wildtype embryos are heterozygous or non-transgenic • 24 mutants and 24 wildtype embryos were genotyped in most cases • If there are no recombinants and no other insertions, the insert is considered to be the cause of the mutation • RT-PCR or in situ hybridization was used as further evidence that the gene was disrupted by the appropriate insertion

9. ResultsClassification of mutant phenotypes

10. ResultsClassification of mutant phenotypes • The first 75 mutants identified were listed • Mutants were grouped by phenotypic defects based on examination under a low powered microscope • Classifications are considered preliminary • Mutants were grouped in two classes, specific and general • Specific mutations include those in which phenotypes are observed in the brain, eyes, jaw, arches or cartilages, midline, ear, fins, liver, gut, kidney, muscle, pigment, body shape, etc. • General mutations include those with extensive cell death in the central nervous system, a small head and eyes, show retardation and those that show several defects • Common and general defects were discarded in chemical mutagenesis screens

11. ResultsClassification of mutant phenotypes • Hi954 - a mutant that results from the disruption of a gene encoding UDP-glucuronic acid decarboxylase • Cartilage cells do not stain, but can be seen in cross-section • Similar to an ENU-induced mutant jekyll

12. ResultsClassification of mutant phenotypes • Hi2092 in the gene caudal causes shortened trunk and tail with no yolk extension • Hi923 shows reduced pigmentation in the body and eyes due to a mutation in a subunit of vacuolar ATP synthase

13. ResultsGenes required for early vertebrate development

14. ResultsGenes required for early vertebrate development • Two classes of mutants, broad and specific • They suggest that genes required for protein synthesis, RNA processing, DNA replication and chromatin assembly give rise to non-specific mutations • Genes required for transcription factors, receptors and ligands give rise to mutants with specific developmental phenotypes • 20% of mutants give rise to genes for which a biochemical function cannot be predicted

15. Discussion • ENU provided the first broad view of the types of embryonic and early larval mutants that can be obtained and allowed estimates of the number of genes required for development • However, this method had a bias toward, receptors, ligands and transcription factors • The method described in this paper presents a less biased view of the genes required for development • Insertional mutagenesis makes the task of cloning the gene much faster and easier but it initially requires substantially more work to isolate the same number of mutations as ENU

16. Further Reading Mullins, M. (2002). Building-blocks of embryogenesis. Nat Genet 31, 125-126. Amsterdam, A. et al. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, 2713-2724 (1999). http://www.genesdev.org/cgi/content/full/13/20/2713