Figure 1.

dMusashi loss of function provokes Sima-dependent growth defects and enhances tracheal sprouting. (A) Layout of the Drosophila melanogaster sima transcript (FBtr0344374). A predicted Musashi binding element (MBE) occurs in the 3′ UTR of all the species analyzed of the Drosophilidae family. Minimal binding sequences for Msi (UAG and GUAG) are highlighted in black boxes, while the linker region between them is shown in grey. The nucleotides non-conserved between species are shown in red. (B) musashi homozygous mutants (msi¹) display prolonged larval development, resulting in delayed pupariation in comparison to their wild type siblings (w¹¹¹⁸), while in msi¹, sima⁰⁷⁶⁰⁷ double homozygotes, normal pupariation timing is restored (error bars represent SEM; n = 3 with a minimum of 60 individuals analyzed per genotype at each point of the curve; *P < 0.05 and **P ≤ 0.01; Student's t test compared with msi¹). (C) Whereas musashi (msi¹) homozygous mutants and wild type third instar larvae attain similar size before entering pupariation, fga⁹ homozygotes are smaller. fga⁹ msi¹ double homozygous mutants reach the third larval instar but are remarkably smaller than fga⁹ homozygotes and never pupariate. In fga⁹ msi¹ sima⁰⁷⁶⁰⁷ triple homozygous third instar larvae, normal growth is rescued. (D) Quantification of final body volume of third instar larvae in the experiment depicted in panel C. Error bars represent SEM (n = 3 with a minimum of 25 larvae analyzed per genotype in each experiment; different letters indicate statistical differences with a P < 0.05 in a one-way ANOVA with a Bonferroni post-hoc test). (E) Photographs of the morphology of a terminal cell of the third dorsal branch of a third instar larva in different genotypes or oxygen concentrations are shown. Numbers indicate individual subcellular extensions of more than 1 μm diameter, the ‘‘thick terminal branches’’ (TTBs). Thinner terminal branches may ramify from TTBs but these were not counted. Ramification of terminal cells is enhanced in hypoxic (5% O₂ 16 h) wild type control larvae (dSRF> white RNAi), as well as in individuals with dMsi loss of function in these cells (dSRF > msiRNAi; white RNAi) maintained in normoxia. Sima knockdown, along with dMsi silencing, leads to rescue of the normal ramification pattern. (F) Quantification of the results shown in panel E. Error bars represent SEM (n = 3 with a minimum of 25 larvae analyzed per genotype in each experiment; **P ≤ 0.01; ***P ≤ 0.001; Student's t test).