PUBLICATION

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes

Authors
Novodvorsky, P., Watson, O., Gray, C., Wilkinson, R.N., Reeve, S., Smythe, C., Beniston, R., Plant, K., Maguire, R., M K Rothman, A., Elworthy, S., van Eeden, F.J., Chico, T.J.
ID
ZDB-PUB-151028-6
Date
2015
Source
PLoS One   10: e0141611 (Journal)
Registered Authors
Chico, Tim J., Elworthy, Stone, Gray, Caroline, van Eeden, Freek, Wilkinson, Robert
Keywords
Embryos, Zebrafish, Blood flow, Morpholino, Phenotypes, TALENs, Mutagenesis, Transcription factors
MeSH Terms
  • Animals
  • Morpholinos/genetics
  • Morphogenesis/genetics*
  • Kruppel-Like Transcription Factors/antagonists & inhibitors
  • Kruppel-Like Transcription Factors/biosynthesis
  • Kruppel-Like Transcription Factors/genetics*
  • Zebrafish/genetics
  • Zebrafish/growth & development
  • Signal Transduction
  • Gene Expression Regulation, Developmental
  • Humans
  • Mutation
  • Zebrafish Proteins/antagonists & inhibitors
  • Zebrafish Proteins/biosynthesis
  • Zebrafish Proteins/genetics*
  • Hematopoietic System/growth & development*
  • Genotype
  • Cardiovascular System/growth & development*
(all 18)
PubMed
26506092 Full text @ PLoS One
Abstract
The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development.
Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.
The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.
Genes / Markers
Marker Marker Type Name
dll4GENEdelta-like 4 (Drosophila)
gapdhGENEglyceraldehyde-3-phosphate dehydrogenase
hey2GENEhes-related family bHLH transcription factor with YRPW motif 2
klf17GENEKruppel like factor 17
klf2aGENEKruppel like factor 2a
klf2bGENEKruppel like factor 2b
klf4GENEKruppel like factor 4
mir126aMIRNAGmicroRNA 126a
mitfaGENEmelanocyte inducing transcription factor a
mpv17GENEmitochondrial inner membrane protein MPV17
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Figures
Figure Gallery (12 images) / 2
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
a9
    Complex
    hu2117
      Point Mutation
      m145
        Point Mutation
        s916TgTransgenic Insertion
          sh317
            Small Deletion
            ubs1TgTransgenic Insertion
              w2
                Point Mutation
                y1TgTransgenic Insertion
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                  Human Disease / Model
                  No data available
                  Sequence Targeting Reagents
                  Target Reagent Reagent Type
                  klf2aTALEN2-klf2aTALEN
                  klf2bMO3-klf2bMRPHLNO
                  tnnt2aMO1-tnnt2aMRPHLNO
                  1 - 3 of 3
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                  Fish
                  Antibodies
                  Name Type Antigen Genes Isotypes Host Organism
                  Ab1-klf2polyclonal
                    Rabbit
                    Ab3-actbpolyclonal
                      Rabbit
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                      Orthology
                      No data available
                      Engineered Foreign Genes
                      Marker Marker Type Name
                      EGFPEFGEGFP
                      mCherryEFGmCherry
                      1 - 2 of 2
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                      Mapping
                      No data available