Figure 1. Dif-dependent Toll/NF-κB signaling is required for correct timing of lymph gland dispersal in response to wasp parasitism.
(A, B, D, E) Differential interference contrast (DIC) microscopy of lymph gland anterior lobes 30 hr post-parasitism. Lymph glands were classified into three groups (see Materials and methods): Disrupted (*) in wt (wild type, (A) or dl1 mutant (E); intact in pll2/pll7 mutant (B); disrupting in Dif1 mutant (D). Representative lymph glands are shown. Ring gland, RG; cardiac tube, CT; anterior lobe, AL; posterior lobe, PL. (C, F, G) Quantification of lymph gland disruption (%). The error bars correspond to SEM, ***p<0.001 and ns (not significant) (Pearson’s Chi-squared test). In this and subsequent similar analyses, numbers of lymph glands analyzed are indicated on histograms. (H, I) DIC microscopy of lymph glands in Dif1 (H) and pll2/pll7 (I) mutants 48 hr post-parasitism. Disrupted (*) and disrupting (arrows) lymph glands are shown. (J–M) Perlecan/Trol (white) immunostaining in control (J, L) and Dif1 anterior lobes (K, M). (N–S) αPS4 (green) immunostaining labels lamellocytes in lymph glands in wt (N, Q), Dif1 (O, R) and pll2/pll7 (P, S) mutants. 20 hr post-parasitism, more than 65% (n = 24) of wt lymph glands displayed lamellocytes, whereas none (n = 24) or less than 15% (n = 18) contained lamellocytes in Dif1 and pll2/pll7 mutants, respectively. 30 hr post-parasitism, Dif1 and pll2/pll7 mutant lymph glands have lamellocytes, whereas most wt lymph glands have disrupted. In this and subsequent figures (unless specified), the scale bar represents 20 µm and nuclei are labeled with TOPRO3 or Draq5.
Figure 1—figure supplement 1. Dif but not Dorsal is required for proper lymph gland disruption in response to wasp parasitism.
