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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-
GFP. 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-GFP
embryos. MHC-GFP 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.
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