Figure 5.

RhoGAP68F acts downstream of dAP-2 to promote apical constriction and epithelial invagination at presumptive joints. (A–B’) Wild type and (C–D’) Dll>RhoGAP68F RNAi leg imaginal disc stained for E-cad (white in A-A’ and C-C’, Red in B-B’ and D’-D) to mark cell outlines, and for dAP-2 (green in B-B’ and D’-D) to mark segment boundaries at ~4h after puparium formation (APF). (B and D) Grazing sections at the plane of the ZA (B’ and D’) and mid-saggital sections to reveal the apicobasal axis of the epithelium. (A-B’) The concentrically folded leg imaginal disc telescopes-out along the proximodistal (PD) axis and the epithelium of presumptive joints invaginates by apical constriction to initiate joint morphogenesis. dAP-2 accumulates at high levels in the distal part of the joint and at lower level in the proximal part. Note that proximal joints are more articulated compared to distal joints at this stage. (C–D’) Legs depleted for RhoGAP68F elongate along the PD axis but form either shallow or no invaginations at presumptive joints (asterisks in D’ indicate shallow invaginations). dAP-2 expression remains largely intact in these legs. In a small number of segments we detect small gaps or thinning (arrowheads in D) of the stripe of dAP-2 expression reflecting mild patterning defects. However, epithelial invaginations were either shallow or altogether missing despite the proper expression of dAP-2 in most segments indicating that RhoGAP68F acts downstream of dAP-2 to promote apical constriction and epithelial invagination.

(E–G) RhoGAP68F acts in parallel to JNK-reaper mediated apoptosis to promote tarsal joint morphogenesis. Arrowheads point to the Ptc domain. Expression of a puc-lacZ reporter in (E) wild type, (F) ptc>RhoGAP68FRNAi and (G) ptc>Necd (dominant negative N receptor). (E) The puc-lacZ reporter is expressed at high levels in tarsal joints 2–5 (F) Expression of RhoGAP68FRNAi with ptc-GAL4 led to a modest inhibition of epithelial invagination in the Ptc domain. However, puc-lacZ expression was not affected. . (G) In contrast, expression of Necd with ptc-GAL4 to inhibit N signaling in the Ptc domain led to the downregulation of puc-lacZ expression in the Ptc domain.