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The establishment of segmentation in the Drosophila leg

Waksman Institute, Rutgers the State University of New Jersey, Piscataway, NJ 08854, USA

View larger version (117K): [in a new window]   Fig. 1. Expression of SER is established progressively. (A) In early third instar leg discs (72 hours AEL), a ring of SER expression is detected in the coxa. (B) At 78 hours AEL, a new ring of SER expression arises in the femur. (C) By 84 hours AEL, SER expression is detected in four domains, two of which are in the tarsal region (ta2 and ta5). (D) By mid third instar (96 hours AEL), SER is expressed in at least six prospective segments. It is unclear whether the new tarsal ring corresponds to ta1 or ta3; therefore, both the ta2 and the new ring are designated as ta. (E,F) Ultimately, by late third instar (120 hours AEL) SER is expressed in one ring per segment and expression continues during pupal stages. co, coxa; fe, femur; ta2,tarsal segment 2; ta5, tarsal segment 5; ti, tibia.

View larger version (77K): [in a new window]   Fig. 2. Expression of SER relative to the leg gap genes. SER expression (red) was compared with that of HTH, DAC, and DLL. (A,B) The coxa ring of SER (arrow) arises in cells proximal to those expressing DAC (A, green), within cells expressing HTH (B, green). SER expression is at the cell membrane and HTH expression is nuclear, so overlap in expression is not seen in all focal planes. Arrow in inset points to two cells obviously expressing both HTH and SER. (C-C’’’) Femur SER expression (bracket) arises within proximal DAC-expressing cells (blue). DLL expression (green) does not overlap with the femur SER expression. (D,E) At early-mid third instar, two sites of SER expression arise in the tarsus. A ring of expression (arrow) is observed distal to cells expressing DAC (D, green), but within cells expressing DLL (E, green). (F-F’’’) Expression of SER in the tibia (arrow) arises in cells expressing both DAC (blue) and DLL (green). DAC and DLL overlap in turquoise in C and F.

View larger version (106K): [in a new window]   Fig. 3. DAC positively regulates SER expression. (A-A’’) ptcGAL4 UASdac UASGFP results in a cell autonomous induction of SER expression (red, arrow) in early third instar leg discs. (B) ptcGAL4 UASdac adult leg with segmental fusions. fe, femur. (C-C’’) dac mutant clone showing a cell autonomous loss of SER expression in the femur (arrow). (E-E’) dac mutant clone in the tibia results in a cell autonomous loss of SER expression, except at the proximal edge of the clone (arrow). (F) dac homozygous mutant leg disc with greatly reduced SER expression in the femur (arrow). (D,G) dac mutant clones (outlined) cause segment fusions in adult legs. (D) Femur-tibia fusion. (G) Fusion extending from tarsal segments1-3. Arrows indicate where normal joints would lie (partially visible by upper arrow); asterisks denote apical bristles, which normally lie just proximal to tarsal segment borders.

View larger version (73K): [in a new window]   Fig. 4. HTH regulates early SER expression both cell autonomously and nonautonomously. All panels are leg discs from ptcGAL4 UAShth UASGFP larvae; SER in red. (A,A’) Ectopic expression of HTH does not affect SER expression in the coxa. (B,B’) Ectopic expression of HTH cell autonomously represses SER expression in the femur (arrow), and cell nonautonomously induces SER (arrowheads). (C) Adult leg displaying bifurcation at the level of the femur (fe). (D-D’’) Ectopic expression of HTH cell autonomously represses DAC expression (white arrow). In the anterior compartment, ectopic HTH results in an induction of DAC expression (black arrow), while in the posterior compartment, ectopic HTH does not affect DAC expression in those cells in which SER was induced (arrowhead).

View larger version (97K): [in a new window]   Fig. 5. DLL represses SER expression. (A-A’’) ptcGAL4 UASDll UASGFP early third instar leg discs. Ectopic DLL expression in the femur (arrow) results in a cell autonomous loss of SER and DAC expression. (B,B’) ptcGAL4 UASDll UASGFP results in repression of SER expression in tarsal segment 5 (arrow). (C) ptcGAL4 UASDll adult leg with tarsal segment fusions (bracket). (D-D’’) Dll Minute clone results in the cell autonomous induction of SER expression within the tarsus (arrow).

View larger version (58K): [in a new window]   Fig. 6. DAC acts through minimal Ser enhancer elements. (A) Schematic of the Ser locus with genomic regions used for the lacZ reporter constructs indicated as SerlacZ2.2 (2.2 kb fragment) and SerlacZ1.9 (1.9 kb fragment) (Bachmann and Knust, 1998). (B-B’’) Expression of SerlacZ1.9 (red) is first detected in the prospective femur. ß-Galactosidase expression overlaps with DAC (blue) and lies distal to HTH (green). (C-C’’) FLP-out clones of dac (marked with GFP, green) result in cell autonomous induction of SerlacZ1.9 expression (red, arrow). (D) Expression of SerlacZ2.2 in tarsal segments 1-4 (green) is complementary to endogenous SER expression (red). (E,E’) FLP-out clones of dac (marked with GFP, green) result in cell autonomous repression of SerlacZ2.2 expression (red, arrow).

View larger version (90K): [in a new window]   Fig. 7. DAC regulates the progressive expression of DL and fringe. (A-C) DL expression (red) occurs progressively with more rings of expression added as development proceeds. (D) DL (red) and SER (green) expression are coincident within the coxa of early third instar leg discs (arrow). (E,F) ptcGAL4 UASdac UASGFP early third instar leg disc. DL expression is cell autonomously induced by DAC (arrow). (G) fringe expression at early third instar. Expression is visible in a single proximal ring (arrow). (H) fringe expression at early-mid third instar. Expression is observed in three rings and a central spot. (I) ptcGAL4 UASdac expression results in the induction of fringe expression in a stripe along the AP axis of the developing leg (arrow).

View larger version (25K): [in a new window]   Fig. 8. Summary model for the establishment of segmentation in the Drosophila leg. Although SER and leg gap gene expression occurs within the same cells, their expression is depicted here as two separate bars. For each panel, the top bar incicates the expression of the leg gap genes and the bottom bar denotes where SER expression (in red) is induced relative to this expression, with proximal on the left and distal on the right. Expression of HTH is in dark blue; DLL in yellow; DAC in light blue; DAC and DLL overlap in green; and HTH, DAC and DLL overlap in purple. Position of eventual leg segment borders shown in gray. Similar regulatory interactions are thought to induce segmental DL and fringe expression. Note that the model, as drawn here, depicts an increase in leg disc size during development, but the amount of growth and expression domains are not necessarily to scale. See text for further details of model. (A) Early third instar (72 hours AEL). HTH induces SER expression within the coxa, while DLL represses SER expression in more distal regions. (B) Early third instar (78 hours AEL). DAC induces SER expression within the femur, while HTH-expressing cells produce a signal (X) that nonautonomously induces SER within the femur. (C) Early-mid third instar (84 hours AEL). DLL represses SER expression, yet two sites of expression are observed within the tarsus. Other genes expressed within the tarsus (tarsal factors, TF), such as SS and/or BAB, may induce expression here. At early third instar SS expression overlaps with DLL, while later DLL expression extends more proximally. BAB has graded expression in tarsal segments 1-4. (D) Mid third instar (96 hours AEL). DAC induces SER expression within the tibia, perhaps by overcoming repressive effects of DLL. New tarsal rings are also induced, depicted here in tarsal segment three; however, the precise segmental order in which tarsal expression arises is not known. (E) Late third instar (120 hours AEL). HTH, DAC and DLL are hypothesized to function together to induce SER within the trochanter. All tarsal segments also now express SER. (F) Adult leg, with leg segment borders shown relative to the schematic shown in E. Leg segment borders (the joints) form just distal to the sites of Notch ligand expression, and do not always correspond to boundaries of leg gap gene expression. co, coxa; fe, femur; ta1-5,tarsal segments 1-5; ti, tibia; tr, trochanter. The claw forms at the distal tip of the leg.