Figure 3.

dMusashi downregulates Sima dependent transcription. (A) Expression of the Sima-inducible reporter HRE-LacZ in wild type, fga⁹or msi¹ mutant embryos maintained in normoxia or exposed to two different hypoxic conditions; arrows indicate groups of tracheal cells that express the reporter with maximal sensitivity, and arrowheads indicate groups of dorsal cells where the reporter expression is more variable but occurs consistently when HIF-dependent transcription is especially strong. In wild type embryos, the reporter is only expressed in strong hypoxia (5% O₂ 5h), while in fga⁹ mutants expression can be detected even in normoxia (21% O₂), and in msi¹ mutants it is already detectable in mild hypoxia (8% O₂ 5 h); fga⁹ msi¹ double homozygous mutants display overall enhanced expression of the reporter in normoxia in comparison to fga⁹ single mutants. (B) Expression of three Sima target genes was analyzed by RT-qPCR in wild type, musashi loss-of-function (msi¹ homozygous mutants or larvae with the heteroallelic combination msi¹/Df(3R)6203) or in msi¹ sima⁰⁷⁶⁰⁷double homozygous mutant third instar larvae maintained in normoxia. In both msi loss-of-function backgrounds (msi¹ or msi¹/Df(3R)6203), expression of the three Sima target genes, fgaB, sequoia and spermidine oxidase, is higher than in wild type controls or in msi¹ sima⁰⁷⁶⁰⁷double mutants. Error bars represent SEM (n = 3; different letters indicate statistical differences with a P < 0.05 in a one-way ANOVA with a Bonferroni post-hoc test).