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  current news   Press   selected story    
     
  28th October 2011  
  Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates
 
 




Authors
Jin BEN1, Stone ELWORTHY2, Ashley Shu Mei NG1, Freek van EEDEN1 and Philip W. INGHAM1,2,3

1 - Developmental and Biomedical Genetics Group, Institute of Molecular & Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Republic of Singapore.
  
2 - MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
    
3 - Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543
  
Published in Development 138, 4969-4978 (2011) doi:10.1242/dev.070862

Abstract
Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia, and aberrant Hh signalling (manifested by the expanded domains of engrailed and ptc1 expression in the somites); the reduction of nkx2.2 expression in the neural tube; symmetric pectoral fins; cyclopic eyes; and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZtalpid3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer’s vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Gli processing in zebrafish and reveal the conservation of Talpid3 function during vertebrate evolution.


Figure Legends: Fin and head abnormalities in MZta3mutants

(A,B) 2dpf MZta3 embryos displaying hindbrain hemorrhage (arrow) and mild cyclopia(B).

(C,D) Pectoral fins from Mta3 and MZta3 larvae stained with Alcian Blue, showing loss of asymmetry in mutant (D).

(E-G) Cranial blood vessels marked by the fli::GFP transgene; background had been removed in lower monochrome panels for ease of observation. Note the mis-branched and merged vessels of the cerebellar central artery in the MZta3 embryos (F and G), beneath the hemorrhagic area. Posterior Mesencephalic Central Artery is indicated by *. An: anterior; Po: posterior; Di: distal; Pr: proximal; L: left; R: right


For more information of Philip INGHAM’s laboratory, please click here.