Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development Gregory Golling, Adam Amsterdam, Zhaoxia Sun, Marcelo Antonelli, Ernesto Mldonado, Wenbiao chen, Shwan Burgess, Maryann Haldi, Karen Artzt, Sarah Farrington, Shuh-yow Lin, Robert M. Nissen and Nancy Hopkins

Purpose To identify the genes required for early development in vertebrate over a relatively short period

Background Forward Genetic Screen - to identify mutations that produce a certain phenotype, then identify the mutated gene - to identify the genes necessary for embryonic development in vertebrates - formation of functional organs in zebrafish can be analyzed

Background cont From large-scale chemical mutagenesis… - 2,400 genes are essential for the normal development of a zebrafish embryo genes for specific or localized defects - 1,600 genes for less specific phenotypes and recurring syndromes.

Background cont To clone chemically mutated genes - positional cloning - candidate gene approach - only about 50 mutants have been identified. - could represent only a fraction of the types of genes for specific development

Background cont Insertional mutagenesis - gene tagging - easier to identify genes using PCR - faster to identify the retrovirally mutated genes - unbiased approach to identifying the genes - no need to select mutants - not biased towards known genes - found a lot of genes

Methods 1. Mutagenesis 2. Genotyping embryos 3. cDNA cloning 4. Alcian blue staining 5. Access numbers

Methods cont 1. Mutagenesis - Moloney murine leukemia-based retroviral vector as a mutagen - retrovirus used as a mutagen to simplify the cloning process

Methods cont 2. Genotyping embryos - sorted embryos into phenotypically wildtype and mutant groups - genotyped by PCR - by Southern blot

Methods cont 3. cDNA cloning - clone DNA flanking proviral insert by inverse PCR - to obtain the rest of cDNA, used either RT- PCR and RACE

Methods cont 3. cDNA cloning - RT-PCR methodology logy/Flash/RT_PCR.html logy/Flash/RT_PCR.html - RACE (rapid amplification for cDNA ends) : used SMART RACE

Methods cont 3. cDNA cloning - to confirm that the correct junction fragment have been cloned : linkage analysis : RT-PCR or in situ hybridization

Methods cont 4. Alcian blue staining - cationic dyes - detect a cartilage 5. Accession numbers

Results Classification of mutant phenotypes - mutants are grouped according to phenotypic defects - used alcian blue and acridine orange - classifications are preliminary : indistinguishable from the phenotypes spectrum by ENU mutagenesis

Results cont Classification of mutant phenotypes -mutants with unique and specific defects from chemical mutagenesis - mutants with general defects from insertional mutagenesis

Results cont Classification of mutant phenotypes - mutations in various genes produce phenotypes involving cartilage : hi954

Results cont - mutation with pigmentation abnormalities : disrupted genes encoding proteins associated with cytoplasmic organelles : hi577a, hi923, hi 1207, hi112

:hi1463

-hi2499a

Results cont - hi2092

Results cont - Hi904

Results cont Genes required for early vertebrate development - cause of mutants with nonspecific developmental defects - cause of mutants with specific developmental phenotypes

Results cont Genes required for early vertebrate development - 20% of the identified mutants had unpredicted biochemical function - proteins differ in degree of novelty : hi459

Results cont Gene required for early vertebrate development - for all the genes, there is a clearly identifiable human ortholog or a human gene with some similarity that can be identified - some do not have orthologs in D. melanogaster or in yeast - some are highly conserved from yeast to mammals

Discussion - the first large-scale, unbiased view of the required genes for early development of vertebrate embryo - Cloned most mutated genes within two weeks - Some genes are important in growth control as well as human diseases - Impossible to estimate the total number of mutable genes by retroviral insertion. - Will have isolated insertional mutations in around genes out of 2,400 genes.