Fig. 4

Runx1 levels detected by ISH are not affected in lmo4a mutants. (A) TALENs were designed to a region (blue) ?20?bp downstream of the lmo4a translation start site (green). Isolated mutant alleles carry 5?bp deletions (?25-29) (red gaps) upstream of the conserved LIM domains (orange), resulting in a frameshift after S8. The resulting mutant protein is predicted to lack the LIM domains, including the crucial S39 (dark red). (B) Representative images of ISH for runx1 in 28?hpf wild-type (blue), heterozygous (green) and lmo4a^{?25-29/?25-29} (orange) embryos, showing the expression in the dorsal aorta. (C) 2% agarose gel showing representative genotypes of wild-type (WT), heterozygous (HET) and mutant (MUT) lmo4a embryos, distinguished by RFLP. Yellow: wild-type 258?bp+87?bp bands, pink: 340?bp mutant band. First lane from the left: 100?bp DNA ladder. (D) Quantification of the runx1 mRNA signal, detected by ISH, from 28?hpf wild-type (n=15), heterozygous lmo4a^+/? (het) (n=34) and lmo4a^{?25-29/?25-29} mutant (n=18) embryos from one clutch shows no significant difference in runx1 pixel intensity among the different genotypes (ANOVA, P>0.6). The coefficients of variation are 41%, 38% and 37% for wild-type, heterozygote and mutant groups, respectively. Blue, green and orange data point correspond to the example images from panel B. The bars represent mean±s.d. (E) Boxplots displaying normalised runx1 mRNA levels (2^??Ct) in single wild-type (blue; n=12) and lmo4a^{?25-29/?25-29} (mut, orange; n=12) embryos, measured by qRT-PCR, showing decreased levels of runx1 in the mutants compared to wild type. *P<0.05 (t-test).