Fig. 2.
Proper CMI expression levels are required for normal development. (A) The cmi gene region, location of the P[EPgy2]EY06424 insertion and genomic deletions in the cmi P-excision mutants. RT-PCR primer sites are indicated by arrows. (B) Transcript analyses of cmi and drev in wild-type (OregonR), P[EY06424] and cmi mutant (cmi1 and cmi2) larvae. The ribosomal gene rp49 served as an RNA control. Quantitative RT-PCR (right panel) confirmed the complete loss of cmi mRNA in the cmi1 mutant. Asterisk indicates no mRNA detected. (C) Retained and malformed mouth hooks in cmi mutant larvae. Mutants display malformed hooks in L2 and occasionally L2 hooks are retained in L3. (D) Necrotic patches in cmi mutant larvae (arrows). Hemocyte counts were analyzed in wild-type (WT; OregonR, OR), cmi-IR, trr-IR and HA-cmi staged larvae (before/after ecdysone pulse). Shown are average hemocyte counts per field. Significant differences (P≤0.01) were only observed comparing OR and HA-cmi hemocyte counts at both stages. (E) Depletion and overexpression of cmi in Drosophila embryos following P[GawB]69B-GAL4-dependent RNAi (cmi-IR) and overexpression (HA-cmi). CMI was detected using αCMI or αHA antibodies. Expression of the HA-cmi transgene was determined at varying growth temperatures (°C). Tubulin levels served as loading controls (αTub). (F) Expression of cmi in cultured S2 cells following cmi and trr RNAi. Knockdown of cmi was verified by western blot and qRT-PCR. (G) Proper CMI levels are essential for normal development. Left panels: Positions of veins in wild-type fly wings (OregonR) and upon cmi depletion (cmi-IR) and overexpression (HA-cmi) using the wing-specific C765-GAL4 driver. L2-L5, longitudinal veins; ACV, anterior crossvein; PCV, posterior crossvein. Note the shortening of veins with reduced cmi (arrowheads) and ectopic veins with elevated cmi levels. Right panels: Modest overexpression of HA-cmi at 22°C leads to additional pattern defects including ommatidial disruptions (arrows) and ectopic sex combs (arrow) on the male distal prothoracic tarsi (middle panels, enlarged in inset). RNAi depletion of cmi in developing eye discs using ey-GAL4 at 25°C results in reduced eye size and missing antennae (arrow, lower right). Error bars indicate s.e.m.