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four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

Gerri R. Buckles¹, Cordelia Rauskolb², John Lee Villano^1, and Flora N. Katz^1,*

¹ Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA
² Waksman Institute, Rutgers, The State University, Piscataway, New Jersey 08854, USA
Present address: University of Chicago Medical Center, Section of Medicine, Department of Hematology/Oncology, 5841 South Maryland Ave, MC 2115, Chicago, IL 60627-1470, USA

View larger version (42K): [in a new window]   Fig. 1. Subcellular fractionation of Fj in cultured cells. (A) Schematic diagram of the Fj protein, showing the relevant domains referred to in the text. Tm, transmembrane domain; Ag, approximate extent of the antigen used to generate anti-FjC. (B) Western blot analysis of Fj expression. Lane 1: expression of the C-terminal domain of Fj in bacterial cells. The slowest migrating band, of Mr 63.5x103, represents the intact C-terminal domain. Considerable protein degradation occurs. The arrow indicates a fragment of approximate Mr 20x103, used as the antigen (Ag) to generate anti-FjC. Lanes 2-7: subcellular fractionation of heat-shocked S2:fj cells containing a HS-fj cDNA insertion. S100, 100,000 g supernatant; P4, 4000 g pellet; P100, 100,000 g pellet; pellet (p) and supernatant (s) after treatment of the P100 pellet with either PBS or 0.1 M Na2CO3 (ALK). Lanes 8-11: expression of Fj in intact, washed cells (c) and extracellular medium (s) of S2 cells with (+) or without (-) the HS-fj cDNA (pHS-fj) insertion. The lines at the far left designate Fj-specific bands. The blot was developed with anti-FjC.

View larger version (26K): [in a new window]   Fig. 2. Western blot analysis of Fj expression in larval tissue. ConA-concentrated extracts from third instar larvae of wild-type flies compared with (A) fj mutant flies and (B) transgenic line P60 containing the pHS-fj+ transposon, with or without a 30 minute heat shock. The blots were stained with anti-FjC. All lanes in A and B contain extracts derived from equal numbers of larvae, but blot B was developed for a shorter period of time. The three forms of the Fj protein are indicated by lines to the left of the blots.

View larger version (87K): [in a new window]   Fig. 3. Leg patterning defects resulting from ectopic expression of fj. A,D,F, and G are adult female complete tarsi. E is an adult male leg showing the tarsus, tibia and femur. (A) Wild type. The lines indicate the joints separating the five tarsal segments. (B) fjN7. An incomplete joint is present at T2/3 (arrow). (C) HS-fj. An ectopic joint-like structure is present in the center of the T3 segment (arrow). (D-G) UAS-fj driven by: (D) 69B-Gal4, with truncations and loss of the T2/3 joint; (E) ptc-Gal4, with significant truncation and segment fusions of the tarsus. The arrowhead points to the juncture between the tarsus and the tibia. The sex comb is obvious on the tarsal remnant. (F,G) dpp-Gal4, with representative leg truncation and segment fusions, cuticular abnormalities, and outgrowths (arrows). The outgrowth in F appears to be segmented and both outgrowths contain bristles. The dpp-Gal4 stock alone has no detectable phenotypes (data not shown). Bar in A represents 50 µm in A,D,F, and G; 10 µm in B and C; and 100 µm in E.

View larger version (73K): [in a new window]   Fig. 4. Fj affects segmentation and growth non-autonomously. Portions of tarsi of adult legs containing flip-out clones expressing fj. The fj-expressing clones are marked with yellow and are outlined. (A) Wild type. Arrows indicate that joints are visible around the circumference of the leg. (B) fj-expressing clone in T2-T4. Arrows denote the extent of joint structures, which do not form around the circumference of the leg. Note that joint formation is inhibited far from the cells expressing fj. (C) fj-expressing clone in T2-T3. A leg outgrowth is induced nonautonomously (arrow); the outgrowth does not include any yellow bristles.

View larger version (63K): [in a new window]   Fig. 5. Fj regulates N ligand expression. (A,B) Comparison of fj-lacZ (green) and Ser (red) expression in everting pupal leg. Expression is complementary in tarsal segments T2-4 (inset of T2 and T3) while expression overlaps in T1, where fj-ß-gal expression is lower in cells also expressing Ser (arrow). (C,D) Ser expression (red) in wild-type (C) and fjd1 (D) everting pupal legs. To help identify the tarsal segments, we used a reporter gene construct, I-2.2 (green)(Bachmann and Knust, 1998), which is expressed in T1-4 in a pattern complementary to endogenous Ser. Ser expression is dramatically reduced in T2 of fjd1 while I-2.2 expression remains. (E-H) ptc-Gal4 UAS-GFP UAS-fj mid-third instar leg discs. ptc-Gal4 drives expression of GFP (green) and fj in a stripe along the anterior side of the AP compartment boundary. (E,F) Ser (red in E and white in F) expression is induced in cells adjacent to those expressing fj (arrows). (G,H) Dl (red in G and white in H) expression is also induced in cells adjacent to fj-expressing cells (arrows). (I,J) dpp-Gal4 UAS-GFP UAS-fj mid-third instar leg discs. dpp-Gal4 drives expression of GFP (green) and fj in the anterior compartment. Ser (red in I and white in J) is induced non-autonomously in posterior cells abutting those expressing high levels of fj (arrows). Ser and Dl were visualized by antibody staining.

View larger version (45K): [in a new window]   Fig. 6. Dominant enhancement of the fj phenotype. (A) Table showing genetic interactions with fj hypomorphic alleles. N, number of legs analyzed. Abl-, Df(3L)stj7, a deletion that removes the abl gene. All interactions are statistically significant at P<0.001 by the 2 test (Devore and Peck, 1997). (B) Illustration of ‘complete’ joints (black arrows), ‘partial’ joints (white arrowhead), and ‘fusion’ phenotypes.