Fig. 5. In vivo validation of Fit1 and Fit2 using dsRNA injection.

(A-E) Micrographs of whole-mount in situ hybridizations of Drosophila embryos with Dig-labeled antisense probes specifically targeting Fit1 (A-C) and Fit2 (D,E), oriented anterior to the left. (A,D) Lateral view of stage 14 embryos. (B) Dorsal view of a stage 16 embryo focusing on visceral muscle and somatic body wall muscles. (C) High-magnification image showing Fit1 somatic body wall muscle expression. Arrowhead, somatic body wall muscles; arrow, visceral gut muscles. (E) Lateral view of a stage 16 embryo. (F-J) Fluorescence micrographs of stage 17 embryos carrying MHC-τGFP. dsRNAs targeting (F) lacZ (2 μg/μl), (G) mys (2 μg/μl), (H) Fit1 (2 μg/μl), (I) Fit2 (2 μg/μl) and (J) Fit1 (1 μg/μl) + Fit2 (1 μg/μl) were injected into MHC-τGFP embryos. MHC-τGFP allows visualization of all somatic muscles, as shown in F, where the embryos were injected with a negative control dsRNA targeting lacZ (n=67, none showed muscle phenotypes). Note that severely rounded muscles are present in the embryos injected with dsRNAs targeting mys (G) (100% penetrance, n=87, where n is the number of embryos injected) and Fit1 + Fit2 (J) (96% penetrance, n=150), whereas dsRNAs targeting either Fit1 (H) or Fit2 (I) alone only caused some muscles to round up (short arrows in H,I). Long arrows in H-J point to the ventral acute muscles that are still present as fibers in H and I, but round up in J. Scale bars: 50 μm in A for A-E, in F for F-J.