PMC

Over-expression of WntD blocks Dorsal protein activation independently of Cactus. a,c, Wild type embryos stained with antibodies to Dorsal (a), or Twist (c). b,d, Embryos from females carrying P[nos-Gal4:VP16] and P[UASp-wntD] transgenes, stained with antibodies against Dorsal (b), or Twist (d). inset b, Total Dorsal protein levels (assayed by western blot) are equivalent in wild type embryos (lane 1) and those from P[nos- Gal4:VP16]/P[UASp-wntD] females (lane 2). e-h Cuticles of embryos with the maternal genotypes: wild type (e); P[nos-Gal4:VP16]/P[UASp-wntD] (f); cact¹/cact⁴ (g); and cact¹/cact⁴ ; P[nos-Gal4:VP16]/P[UASp-wntD] (h).

wntD knockout flies exhibit ectopic Dorsal activation. a, “Ends-out” knockout targeting scheme, illustrating how a white mini-gene was used to interrupt the wntD open reading frame. b, Southern blot of Sma-I digested genomic DNA, confirming proper integration of targeting construct. c, Anti-WntD Western blot of lysate from wild type and wntD^KO1 embryos (arrow indicates size of WntD protein). d,e, yw (d) and yw; wntD^KO1 (e) embryos stained with antibodies against Dorsal. Arrows show point of ventral-most nuclear Dorsal seen in control embryos. f,g adult female yw (f) and yw; wntD^KO1 (g) flies. Arrowheads mark sites of ectopic melanization.

wntD mutants show an aberrant response to microbial infection. a, One week old adult yw (squares, n=60), yw; wntD^KO1 (circles, n=57), yw; dl¹/dl⁴; wntD^KO1 (gray triangles, n=56), and yw; dl⁴/+; wntD^KO1 (gray crosses, n=57) were injected with a dilute culture of Listeria monocytogenes, and survival was monitored. Log rank tests indicate that wntD mutant curve is significantly different from the other three, with p<0.0001. b,c, Real-time PCR was used to monitor diptericin (b) and drosomycin (c) mRNA levels in yw (white bars) and yw; wntD^KO1 (gray bars) adults following injection with Micrococcus luteus. Results are means and s.e.m. Asterisks (*) denote significance level of p<0.05.

wntD is expressed with D/V polarity, and is under the control of Toll signaling. a-e, In situ hybridization to wntD mRNA in wild type embryos (a,b,e) and those derived from Toll^10b mothers (c,d). Wild type expression is seen in the ventral poles at stage 5 (a), presumptive mesoderm at stage 6 (b), and neurogenic ectoderm at stage 9 (e). Stronger, expanded wntD expression is seen in Toll^10b-derived embryos at stages 5 and 6 (c,d). f,g Close-up ventral views (anterior left) of stage 9 wild type embryos stained for wntD mRNA (f) or with anti-WntD antibody (g). Arrows indicate examples of WntD antigen detected multiple cell diameters away from wntD-expressing cells. Scale bars indicate 50 μm. All embryos here and henceforth oriented anterior left, ventral down, unless otherwise indicated