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


RESULTS
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.


CONCLUSIONS
The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Peter
P
Novodvorsky
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Oliver
O
Watson
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Caroline
C
Gray
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Robert N
RN
Wilkinson
Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Scott
S
Reeve
Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.
Carl
C
Smythe
Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.
Richard
R
Beniston
Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Karen
K
Plant
Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Richard
R
Maguire
Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Alexander
A
M K Rothman
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.
Stone
S
Elworthy
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.
Fredericus J M
FJ
van Eeden
The Bateson Centre, University of Sheffield, Sheffield, United Kingdom; Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
Timothy J A
TJ
Chico
D000818:Animals; D002319:Cardiovascular System / Q000254:growth & development*; D018507:Gene Expression Regulation, Developmental; D005838:Genotype; D006413:Hematopoietic System / Q000254:growth & development*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D009024:Morphogenesis / Q000235:genetics*; D060172:Morpholinos / Q000235:genetics; D009154:Mutation; D015398:Signal Transduction; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
C436494:Klf2a protein, zebrafish; D051741:Kruppel-Like Transcription Factors; D060172:Morpholinos; D029961:Zebrafish Proteins