Fig. 7.
Ecdysone signaling is required for late CCAP-EN differentiation. CCAP-GAL4 was used to overexpress dominant-negative EcR-B1/A in CCAP neurons. This overexpression prevented further differentiation of late CCAP-ENs and resulted in a failure of leg extension. (A–D) Leg extension (arrowhead) in pharate adults for each dominant-negative EcR isoform. Control (A) and EcR-B2DN flies (D) had wild-type leg extension, but EcR-ADN (B) or EcR–B1DN (C) expression resulted in failed leg extension. (A′–D′) Pharate adult abdominal VNC showing distribution of all CCAP-GAL4,UAS-nlsEGFP–labeled neurons (green). In control (A′) and in EcR-B2DN (D′) flies, all CCAP neurons expressed CCAP-GAL4, including CCAP-ENs (white arrows), CCAP-INs (arrowheads), and CCAP-PLs (orange arrows). In EcR-ADN (B′) or EcR–B1DN (C′) flies, late CCAP-ENs lose CCAP-GAL4 expression. Early CCAP-ENs (white arrows) and CCAP-PLs (orange arrows) are unaffected. Segmental midline CCAP projections (Fig.1 and Fig. S1) were used as a reference to confirm that all remaining CCAP-ENs were within T3–A4 segments. (E and F) Early L2 VNCs double-labeled (with fluorophore splits of boxed area) for Bursα (red) and Dac (green), showing A1–A7 hemisegments. (E) (Upper) In hemisegments A5–A7 there was only a single CCAP neuron expressing Bursα. (Lower) Magnified view of boxed region in E shows that those neurons lack Dac expression (arrowhead), confirming them to be CCAP-IN. (F) (Upper) hs-ftz-f1 animals were heat shocked at late L1. CCAP neuron doublets expressing Bursα were observed in every hemisegment from T3–A7. (Lower) Magnified view of boxed region shows close-up of A6 hemisegment. The additional Bursα-expressing CCAP neuron (arrow) expressed Dac, indicative of a late CCAP-EN that had differentiated precociously. Genotypes: (A and A′) CCAP-GAL4, UAS-nlsEGFP/+; (B and B′) CCAP-GAL4, UAS-nlsEGFP/UAS-EcR-ADN ; (C and C′) CCAP-GAL4, UAS-nlsEGFP/UAS-EcR-B1DN; (D and D′) CCAP-GAL4, UAS-nlsEGFP/UAS-EcR-B2DN ; (E) CCAP-GAL4,UAS-nlsEGFP/+; (F) hs-ftz-f1/hs-ftz-f1.