purpose

Insertional Insertional mutagenesis in mutagenesis in

zebrafish rapidly zebrafish rapidly identifies genes identifies genes

essential for early essential for early vertebrate vertebrate

developmentdevelopmentGregory Golling, Adam Amsterdam, Gregory Golling, Adam Amsterdam, Zhaoxia Sun, Marcelo Antonelli, Zhaoxia Sun, Marcelo Antonelli,

Ernesto Mldonado, Wenbiao chen, Ernesto Mldonado, Wenbiao chen, Shwan Burgess, Maryann Haldi, Karen Shwan Burgess, Maryann Haldi, Karen Artzt, Sarah Farrington, Shuh-yow Lin, Artzt, Sarah Farrington, Shuh-yow Lin, Robert M. Nissen and Nancy HopkinsRobert M. Nissen and Nancy Hopkins

PurposePurpose

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

BackgroundBackgroundForward Genetic ScreenForward Genetic Screen

- to identify mutations that produce a - to identify mutations that produce a certain phenotype, then identify the certain phenotype, then identify the mutated genemutated gene

- to identify the genes necessary for - to identify the genes necessary for embryonic development in vertebratesembryonic development in vertebrates

- formation of functional organs in - formation of functional organs in zebrafish can be analyzedzebrafish can be analyzed

Background contBackground cont

From large-scale chemical From large-scale chemical mutagenesis…mutagenesis…

- 2,400 genes are essential for the - 2,400 genes are essential for the normal development of a zebrafish normal development of a zebrafish embryo embryo

- 800 genes for specific or localized - 800 genes for specific or localized defectsdefects

- 1,600 genes for less specific - 1,600 genes for less specific phenotypes and recurring syndromes.phenotypes and recurring syndromes.


To clone chemically mutated genesTo clone chemically mutated genes

- positional cloning- positional cloning

- candidate gene approach- candidate gene approach

- only about 50 mutants have been - only about 50 mutants have been identified.identified.

- could represent only a fraction of - could represent only a fraction of the types of genes for specific the types of genes for specific developmentdevelopment


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

MethodsMethods

1.1. MutagenesisMutagenesis

2.2. Genotyping embryosGenotyping embryos

3.3. cDNA cloningcDNA cloning

4.4. Alcian blue stainingAlcian blue staining

5.5. Access numbersAccess numbers

Methods contMethods cont

1.1. MutagenesisMutagenesis

- Moloney murine leukemia-based - Moloney murine leukemia-based retroviral vector as a mutagenretroviral vector as a mutagen

- retrovirus used as a mutagen to - retrovirus used as a mutagen to simplify the cloning processsimplify the cloning process


2. Genotyping embryos2. Genotyping embryos

- sorted embryos into phenotypically - sorted embryos into phenotypically wildtype and mutant groupswildtype and mutant groups

- genotyped by PCR- genotyped by PCR

- by Southern blot- by Southern blot


3. cDNA cloning3. cDNA cloning

- clone DNA flanking proviral insert - clone DNA flanking proviral insert by inverse PCRby inverse PCR

- to obtain the rest of cDNA, used - to obtain the rest of cDNA, used either RT-PCR and RACEeither RT-PCR and RACE



- RT-PCR methodology- RT-PCR methodology

http://www.bio.davidson.edu/courseshttp://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html/Immunology/Flash/RT_PCR.html

- RACE (rapid amplification for - RACE (rapid amplification for cDNA ends)cDNA ends)

: used SMART RACE: used SMART RACE



- to confirm that the correct junction - to confirm that the correct junction fragment have been clonedfragment have been cloned

: linkage analysis: linkage analysis

: RT-PCR or in situ hybridization : RT-PCR or in situ hybridization


4. Alcian blue staining4. Alcian blue staining

- cationic dyes- cationic dyes

- detect a cartilage- detect a cartilage

5. Accession numbers5. Accession numbers

ResultsResults

Classification of mutant phenotypesClassification of mutant phenotypes

- mutants are grouped according to - mutants are grouped according to phenotypic defectsphenotypic defects

- used alcian blue and acridine orange- used alcian blue and acridine orange

- classifications are preliminary- classifications are preliminary

: indistinguishable from the : indistinguishable from the phenotypes phenotypes spectrum by ENU spectrum by ENU mutagenesismutagenesis

Results contResults cont


-- mutants with unique and specific mutants with unique and specific defects from chemical mutagenesisdefects from chemical mutagenesis

- mutants with general defects from mutants with general defects from insertional mutagenesisinsertional mutagenesis



- mutations in various genes produce - mutations in various genes produce phenotypes involving cartilagephenotypes involving cartilage

: hi954: hi954


- mutation with pigmentation - mutation with pigmentation abnormalitiesabnormalities

: disrupted genes encoding : disrupted genes encoding proteins proteins associated with associated with cytoplasmic organellescytoplasmic organelles

: hi577a, hi923, hi 1207, hi112: hi577a, hi923, hi 1207, hi112

:hi1463:hi1463

-hi2499a-hi2499a


- hi2092- hi2092


- Hi904Hi904


Genes required for early vertebrate Genes required for early vertebrate developmentdevelopment

- cause of mutants with nonspecific - cause of mutants with nonspecific developmental defectsdevelopmental defects

- cause of mutants with specific - cause of mutants with specific developmental phenotypesdevelopmental phenotypes


Genes required for early vertebrate Genes required for early vertebrate developmentdevelopment

- 20% of the identified mutants had - 20% of the identified mutants had unpredicted biochemical functionunpredicted biochemical function

- proteins differ in degree of novelty- proteins differ in degree of novelty

: hi459: hi459


Gene required for early vertebrate Gene required for early vertebrate developmentdevelopment

- for all the genes, there is a clearly - for all the genes, there is a clearly identifiable human ortholog or a human identifiable human ortholog or a human gene with some similarity that can be gene with some similarity that can be identifiedidentified

- some do not have orthologs in D. - some do not have orthologs in D. melanogaster or in yeastmelanogaster or in yeast

- some are highly conserved from yeast to - some are highly conserved from yeast to mammalsmammals

DiscussionDiscussion

- the first large-scale, unbiased view of the the first large-scale, unbiased view of the required genes for early development of required genes for early development of vertebrate embryovertebrate embryo

- Cloned most mutated genes within two weeksCloned most mutated genes within two weeks- Some genes are important in growth control Some genes are important in growth control

as well as human diseasesas well as human diseases- Impossible to estimate the total number of Impossible to estimate the total number of

mutable genes by retroviral insertion. mutable genes by retroviral insertion. - Will have isolated insertional mutations in Will have isolated insertional mutations in

around 450-500 genes out of 2,400 genes. around 450-500 genes out of 2,400 genes.