Figure 3.
Abi regulates hematopoietic homeostasis by activating Notch signaling. (A and B) Abi is required for activation of Notch signaling in hemocytes of the Hml lineage. (A) Confocal images of dorsal hemocyte clusters (abdominal segment A7) in WT, abi5/Df, and HmlΔ-GAL4,abi5/UAS-HA-abi,Df (abi5/Df, HmlΔ > abi) late-third instar larvae carrying HmlΔ-DsRed (pseudocolored magenta) and the Notch signaling reporter NRE-GFP (green). Larvae were stained with an anti-Lz antibody (blue). (B) Numbers of NRE-GFP+ hemocytes counted and classified as Hml+Lz−, Hml+Lz+, or Hml−Lz+. (C and D) Transheterozygous interaction between abi and Ser or Notch (N). (C) Bright-field images of WT, N55e11/+; abi5/+, and abi5,+/+,SerRX82 late-third instar larvae (heated at 70°C for 10 min). Dorsal views of the two most posterior segments (A7 and A8). (D) Numbers of heat-blackened crystal cells in the A7 and A8 segments of the indicated genotypes. (E and F) Mutations in abi suppress the excess crystal cell phenotype caused by the overexpression of full-length N, but not NICD. (E) Bright-field images of HmlΔ-GAL4/+, HmlΔ-GAL4/UAS-N (HmlΔ > N), UAS-NICD/+; HmlΔ-GAL4/+ (HmlΔ > NICD), abi5/Df, HmlΔ-GAL4,abi5/UAS-N,Df (abi5/Df, HmlΔ > N), and UAS-NICD/+; HmlΔ-GAL4,abi5/+,Df (abi5/Df, HmlΔ > NICD) late-third instar larvae (heated at 70°C for 10 min). Dorsal views of the two most posterior segments (A7 and A8). (F) Numbers of heat-blackened crystal cells in segments A7 and A8. The genotypes analyzed additionally include HmlΔ-GAL4/UAS-Ser (HmlΔ > Ser) and HmlΔ-GAL4,abi5/UAS-Ser,Df (abi5/Df, HmlΔ > Ser). (G) Confocal images of peripheral hemocytes from WT, N55e11/+; abi5/+, and abi5,+/+,SerRX82 late-third instar larvae stained with an anti-L1 antibody (green) and DAPI (blue). Arrowheads indicate L1+ lamellocytes. (H) Percentage of L1+ lamellocytes among all peripheral hemocytes (total DAPI count). (I and J) Overexpression of NICD, but not full-length Notch, completely suppresses the excess lamellocyte phenotype caused by abi mutations. (I) Confocal images of peripheral hemocytes from late-third instar larvae of the indicated genotypes stained with an anti-L1 antibody (green) and DAPI (blue). Arrowheads indicate L1+ lamellocytes. (J) Percentage of L1+ lamellocytes among all peripheral hemocytes (total DAPI count). Data represent the mean ± SEM. n = 12 larvae. Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test. Comparisons are with WT or HmlΔ-GAL4/+ unless otherwise indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Scale bars: 20 μm (A, G, and I); 200 μm (C and E).

Abi regulates hematopoietic homeostasis by activating Notch signaling. (A and B) Abi is required for activation of Notch signaling in hemocytes of the Hml lineage. (A) Confocal images of dorsal hemocyte clusters (abdominal segment A7) in WT, abi5/Df, and HmlΔ-GAL4,abi5/UAS-HA-abi,Df (abi5/Df, HmlΔ > abi) late-third instar larvae carrying HmlΔ-DsRed (pseudocolored magenta) and the Notch signaling reporter NRE-GFP (green). Larvae were stained with an anti-Lz antibody (blue). (B) Numbers of NRE-GFP+ hemocytes counted and classified as Hml+Lz−, Hml+Lz+, or Hml−Lz+. (C and D) Transheterozygous interaction between abi and Ser or Notch (N). (C) Bright-field images of WT, N55e11/+; abi5/+, and abi5,+/+,SerRX82 late-third instar larvae (heated at 70°C for 10 min). Dorsal views of the two most posterior segments (A7 and A8). (D) Numbers of heat-blackened crystal cells in the A7 and A8 segments of the indicated genotypes. (E and F) Mutations in abi suppress the excess crystal cell phenotype caused by the overexpression of full-length N, but not NICD. (E) Bright-field images of HmlΔ-GAL4/+, HmlΔ-GAL4/UAS-N (HmlΔ > N), UAS-NICD/+; HmlΔ-GAL4/+ (HmlΔ > NICD), abi5/Df, HmlΔ-GAL4,abi5/UAS-N,Df (abi5/Df, HmlΔ > N), and UAS-NICD/+; HmlΔ-GAL4,abi5/+,Df (abi5/Df, HmlΔ > NICD) late-third instar larvae (heated at 70°C for 10 min). Dorsal views of the two most posterior segments (A7 and A8). (F) Numbers of heat-blackened crystal cells in segments A7 and A8. The genotypes analyzed additionally include HmlΔ-GAL4/UAS-Ser (HmlΔ > Ser) and HmlΔ-GAL4,abi5/UAS-Ser,Df (abi5/Df, HmlΔ > Ser). (G) Confocal images of peripheral hemocytes from WT, N55e11/+; abi5/+, and abi5,+/+,SerRX82 late-third instar larvae stained with an anti-L1 antibody (green) and DAPI (blue). Arrowheads indicate L1+ lamellocytes. (H) Percentage of L1+ lamellocytes among all peripheral hemocytes (total DAPI count). (I and J) Overexpression of NICD, but not full-length Notch, completely suppresses the excess lamellocyte phenotype caused by abi mutations. (I) Confocal images of peripheral hemocytes from late-third instar larvae of the indicated genotypes stained with an anti-L1 antibody (green) and DAPI (blue). Arrowheads indicate L1+ lamellocytes. (J) Percentage of L1+ lamellocytes among all peripheral hemocytes (total DAPI count). Data represent the mean ± SEM. n = 12 larvae. Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test. Comparisons are with WT or HmlΔ-GAL4/+ unless otherwise indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Scale bars: 20 μm (A, G, and I); 200 μm (C and E).

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