Systematic expression and loss-of-function analysis defines spatially restricted requirements for Drosophila RhoGEFs and RhoGAPs in leg morphogenesis

Abstract

The Drosophila leg imaginal disc consists of a peripheral region that contributes to adult body wall, and a central region that forms the leg proper. While the patterning signals and transcription factors that determine the identity of adult structures have been identified, the mechanisms that determine the shape of these structures remain largely unknown. The family of Rho GTPases, which consists of seven members in flies, modulates cell adhesion, actomyosin contractility, protrusive membrane activity, and cell-matrix adhesion to generate mechanical forces that shape adult structures. The Rho GTPases are ubiquitously expressed and it remains unclear how they orchestrate morphogenetic events. The Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs), which respectively activate and deactivate corresponding Rho GTPases, have been proposed to regulate the activity of Rho signaling cascades in specific spatiotemporal patterns to orchestrate morphogenetic events. Here we identify restricted expression of 12 of the 20 RhoGEFs and 10 of the 22 Rho RhoGAPs encoded in Drosophila during metamorphosis. Expression of a subset of each family of RhoGTPase regulators was restricted to motile cell populations including tendon, muscle, trachea, and peripodial stalk cells. A second subset was restricted either to all presumptive joints or only to presumptive tarsal joints. Depletion of individual RhoGEFs and RhoGAPs in the epithelium of the disc proper identified several joint-specific genes, which act downstream of segmental patterning signals to control epithelial morphogenesis. Our studies provide a framework with which to understand how Rho signaling cascades orchestrate complex morphogenetic events in multi-cellular organisms, and evidence that patterning signals regulate these cascades to control apical constriction and epithelial invagination at presumptive joints.

Figure 1. A subset of RhoGEFs and RhoGAPs is expressed in motile cell populations

(A) Cdep (GEF); expression is restricted to internal tendon precursor cells. (B) drm; expression is restricted to each true leg joint and to the internal tendon precursors (marked by arrowheads). (C) RhoGEF CG8557; expression is detected in a subset of adepithelial cells that form muscle below the surface epithelium. (D) RhoGEF4; expression is detected in a primary tracheal tube at the periphery of the leg. Tracheal antibody 2A12 highlights a tracheal tube in inset. (E–G) RhoGEF CG15611; Expression is detected in the disc stalk of the (E) leg disc, (F) antenna, (F) and in part of the ventral pleura in the wing (marked by arrowheads). These cell populations connect the imaginal disc to the larval epidermis and contribute to disc eversion, migration and fusion during metamorphosis to promote disc closure.

Figure 2. A subset of RhoGEFs and RhoGAPs is restricted to all presumptive leg joints or only to presumptive tarsal joints

The leg imaginal disc gives rise to five true segments moveable by muscle: the coxa, trochanter, femur, tibia and tarsus. The tarsus is further subdivided into five non-musculated tarsal segments and a distal claw. (A–D) Genes restricted to all leg joints. (E-J) Genes restricted to tarsal joints. (A) CG30115 (RhoGEF). (B) vav (RhoGEF). (C) RhoGEF64C. (D) Ephexin (RhoGEF); expression spans the joint and several cell diameters proximal and distal to the joint. (E) RhoGAP5A. (F) RhoGAP68F (G). RhoGAP100F. (H) RhoGEF2; RhoGEF2-lacZ shown in inset. (I) RhoGAP71E; expression is stronger in proximal joints that are more articulated compared to distal joints that are less articulated, RhoGAP71E-lacZ shown in inset. (J) CdGAPr; CdGAPrlacZ shown in inset. A secondary stripe of expression is detected across each tarsal segment (marked by arrowheads) in B and E.

Figure 3. Expression of joint-specific RhoGEFs and RhoGAPs requires the proper patterning of tarsal segments

(A) Wild type adult tarsus. (B) Dll>dAP-2 RNAi; depletion of dAP-2 function with Dll-GAL4 resulted in shortened tarsus with fused joints that recapitulates a strong dAP-2 loss-of-function phenotype. (C) dpp is expressed along the AP compartment boundary where the ptc-GAL4 driver is active. (D–E) ptc>dAP-2 RNAi; depletion of dAP-2 in the Ptc domain resulted in repression of (D) RhoGEF64C and (E) Ephexin in a narrow sector marked by arrows. (F–I’) ptc>N^ecd; inhibition of N receptor signaling in the Ptc domain resulted in the repression of (F) RhoGAP68F, (G) RhoGAP5A, (H-H’) a RhoGAP71E-lacZ reporter and (I-I’) dAP-2 in a narrow sector marked by arrows. (H-H’) The expression of RhoGAP71E-lacZ was repressed in the anterior compartment (marked by Ci expression) along the AP compartment boundary. This narrow sector corresponds to the Ptc domain suggesting that N signaling promotes the expression of RhoGAP71E cell-autonomously. (I-I’) dAP-2 was repressed cell-autonomously in the Ptch domain validating the efficacy of the N^ecd transgene used in this assay (marked by UAS-GFP expression).

Figure 4. A subset of RhoGEFs and RhoGAPs is required for joint morphogenesis

(A–E) Adult tarsi; (A) wild type; the tarsal region of adult legs is subdivided into 5 tarsal segments (t1–t5) and the distal claw (Cl). (B) Dll>CG33275 RNAi (RhoGEF), (C) Dll>RhoGAP5A RNAi and (D) Dll>RhoGAP68F RNAi. (B–D) Depletion of a subset of RhoGEFs and RhoGAPs by RNAi inhibits tarsal joint morphogenesis but does not adversely affect the growth and differentiation of leg segments. Arrowheads in B–D point to partially formed joints shown at higher magnification in insets. All the tarsal joints and tarsal segment (t1-t5) can be accounted for suggesting that the primary defect is in the progression of the process. Note that legs in B and D are slightly shorter and thicker than wild type reflecting a mild defect in axis elongation. (E) Dll>P{EP}RhoGAP68F^P3152, Ectopic expression of RhoGAP68F also led to the formation of partially formed joints. Arrow in E points to such a joint.

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.