Dynamicity in the expression of Cubitus interruptus in the developing lymph gland. The genotypes are mentioned on the top of the relevant panels. (A–A’’) Morphogen Hh (Antibody, red) expresses in the hematopoietic niche (visualized by Antp-Gal4, UAS-GFP). (A’ and A’’) Higher magnification of the hematopoietic niche shown in A. (B and B’) Detection of extracellular Hh (red) expression in the progenitors (MZ) by detergent-free permeabilization during antibody labeling. (B’’) Higher magnification of a selected area in B (Antp-GFP marks the niche). (C–F) The level of Hh at the source is critical for progenitor maintenance. Progenitor maintenance declines with the decrease in the number of niche cells by string RNAi (Compare C, C’ with D) or Hh is downregulated by hh RNAi (compare C with E–F). P value for F = 2.832 × 10−8. Error bars: SD. The timeline of tub-GAL80ts inactivation adopted in all the above experiments is illustrated in Figure S2C. (G–K) As the lymph gland grows, the number of Hh-expressing cells in the niche increases with time. (G–J’) Ci-155 (activated Ci) profiling in 60, 72, 96, and 106 hr AEH. (K) The quantitative analyses of the increment in the number of Hh-expressing niche cells 48, 60, 72, 96, and 106 hr AEH. (L) The quantitative analysis of the Ci intensity throughout development. An increase in Ci expression occurs with the increment in niche number (K) until 72 hr AEH. Despite the increase in the number of Hh-expressing cells post 72 hr AEH (K), a decline in Ci-155 expression is evident in the progenitors (M). P value for 60 hr compared to 40 hr = 4.128 × 10−5, P value for 72 hr compared to 60 hr = 8.521 × 10−2, P value for 96 hr compared to 72 hr = 4.108 × 10−7, and P value for 106 hr compared to 96 hr = 6.019 × 10−3. (N and O) Compared to control (N) overexpression of activated Ci in the progenitors results in an increase in their number postponing their differentiation (O). (P–P’’) Ci-155 expression (red) is limited to Ance (green)-expressing progenitor cells. A drastic reduction in Ance or Ci-155 occurs in the CZ. (Q and R) Quantitative analyses of Ci-155 level in Ance+ (1, 2, and 3) and Ance– (4) progenitor population illustrating the decline in Ci-155 level in Ance− progenitors. The 21 spots analyzed in total are selected based on their distance from Hh-expressing niche and are equidistant from each other. (S) A scheme depicting our hypothesis that a decline in Ci-155 level initiates differentiation. The yellow dotted line marks the whole of the lymph gland in B’, C’, E’, and P–P’’, while the white dotted line marks the progenitors in C’ and E’. The nuclei are marked with DAPI (blue) in B, B’’, C, D, and E. Figures A–F and N–O are of 96 hr AEH. In G–J, the age of the larvae is mentioned in the respective panels. P–P’’ is 72 hr AEH. P and H are identical lymph glands, where three antibodies were analyzed in three different channels: Ci-155 (red), Ance (blue), and GFP to trace Hh expression. For clarity, the Ance expression is represented in green in P. Error Bars = SD. See also Figure S2. Bar, 20 μm.