Fig. S4

Fig. S4

The her1^hu2124 and the her7^hu2625 alleles lead to full loss of her1 and her7 function, respectively. (A) Schematic representation of the genomic organization of the her1 locus. Boxes represent exons, and lines represent introns (distances not to scale). An asterisk indicates the approximate position of a nonsense mutation in the hu2124 allele that was generated by ENU mutagenesis [28] at the Hubrecht laboratory (Netherlands). Carriers of the her1^hu2124 allele are referred to as her1 mutant in this work and were homozygous viable and fertile. The mutant stop codon disrupts the bHLH domain, which is encoded within the first three exons. (B) Sequencing trace from heterozygous carriers of the hu2124 allele. The C-to-T exchange is evident, changing the codon from Ser to stop. (C) To study whether her1^hu2124 lead to full loss of her1 function, wildtype (wt) and her1 mutants were injected with a combination of her1 targeted morpholino antisense oligonucleotides (MOs) or left uninjected, grown to 34 hpf, and stained with the myotome boundary marker cb1045. her1MO injection into wt and the her1 mutant results in partially penetrant anterior segmentation defects similar to the uninjected her1 mutant. Scale bars, 300 μm (big panels) and 50 μm (insets). (D) The percentage of defective posterior boundaries for each segment along the anterior trunk was determined in groups of embryos treated as in (C). Combining the mutant allele and MO-mediated knockdown does not increase the penetrance or severity of segmentation defects, suggesting that her1 function is fully lost in all three conditions. Data are pooled from two (wt) or three (her1 mutant) independent experiments. (E) Schematic representation of the genomic organization of the her7 locus. Boxes represent exons, and lines represent introns (distances not to scale). An asterisk indicates the approximate position of the nonsense mutation in the hu2625 allele that was generated by ENU mutagenesis [28] at the Hubrecht laboratory (Netherlands). Carriers of the her7^hu2625 allele are referred to as her7 mutant in this work, and homozygous carriers were viable and fertile. The premature stop codon in her7 mutants is located within the HLH domain that mediates dimerization between bHLH proteins. (F) Sequencing trace from heterozygous carriers of the hu2625 allele. The A-to-T exchange is evident, changing the 38^th codon of the Her7 protein from Lys to stop. (G) wt and her7 mutant embryos were injected with her7 targeted MOs or left uninjected, grown to 34 hpf, and stained with the myotome boundary marker cb1045. Segmentation defects posterior to a similar axial level (arrowhead) are evident in her7MO injected wt embryos as well as uninjected and injected her7 mutant embryos. Scale bar 300 μm. (H) The Anterior Limit of Defects (ALD) [75] was scored in groups of her7MO injected wt embryos and injected or uninjected her7 mutant embryos to exactly quantify the severity of the segmentation defects. Combination of MO-mediated knockdown and the her7 mutant allele shifts the ALD anteriorly by less than one segment. Although it cannot be excluded that this slight shift is due to the knockdown of some residual her7 activity in the mutant, these data suggest that her7 function is almost completely absent in homozygous carriers of the her7^hu2526 allele. Data were pooled from two independent experiments; error bars indicate standard deviation.