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The hes6 retroviral insertion results in a strongly hypomorphic allele A Schematic of the genomic organization of the hes6 mutant locus. The proviral insertion is located towards the 3′ end of the first exon of the gene. Positions of primers used for genotyping and cDNA analysis are indicated by arrows. B Sequence of the retrovirally inserted hes6 locus. Black: 5′UTR, green: exon one coding sequence, red: retroviral sequence, blue: predicted translation. The retroviral insertion creates several stop codons (bold) in frame with the wildtype hes6 translation. This gives rise to a predicted protein that terminates before the bHLH domain, which mediates DNA-binding and dimerization. The genomic organization of the locus was determined by sequencing, but we cannot rule out that alternative splicing or read-through might yield some translation of longer polypeptides than the 19 amino acid fragment shown. C Embryos from an incross of heterozygous carriers were in-situ stained with a hes6 riboprobe. Strong (C) and weak staining patterns (C′) are observed in a mendelian ratio (60/82 and 22/82, respectively), indicating that the retroviral insertion results in a strong reduction of hes6 expression. D PCR on poly-dT primed cDNA indicates absence of mature hes6 mRNA in the mutant (lanes 1 and 2), but points towards the presence of a poly-A-tailed transcript containing the provirus (lanes 3 and 4), explaining the weak staining seen in C′. E Previous studies performing hes6 knockdown reported segmentation defects upon loss of hes6 function [1]. To test whether we could reproduce these results, wildtype and hes6 mutant embryos were injected with MO3-hes6 [2] or left uninjected and grown to an equivalent of 34 hpf at 33°C or 20°C. In-situ staining with the myotome boundary marker cb1045 reveals posterior segmentation defects (red arrowheads) in mutant and injected embryos. Note that segmentation phenotypes were of different penetrance and severity (see also Table S1 below). Injections with MO1-hes6 [1] gave similar results (not shown, see also Table S1 below). Scale bar 300 μm.
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