Figure 7. Requirement of Decapentapelagic (Dpp) from the PSC for hematopoietic stem cell maintenance in Drosophila.

(A) The maintenance signal for progenitors: Hedgehog (green; n = 7) starts to express in lg at about 18 hr AEH. (B–C') Lamin C (red) is expression in the niche/PSC (green) in 8 hr and 18 hr lg. (D–D") pMad (red) labeling at 8 hr AEH is enriched in HSCs (N-GFP; n = 6). Also see Figure 7—figure supplement 1C–C". (E) shows that MadGFP expression is predominantly absent from the PSC (F–G’) In first instar lymph gland, HSCs can be visualized by E(spl)mβ -CD2 (red, [F]). Downregulation of Dpp function in the niche causes precocious loss of this HSC marker (E(spl), red, [G]). F'–G' represent scheme of results from (F–G). Also see Figure 7—figure supplement 1D. (H–I) shows that in a late third instar, above genotype causes a 3-fold reduction in the size of the lymph gland (P1, red, n = 15, p=3.98092E-10, two tailed unpaired Student’s t-test) in comparison to the control in H. However, CZ cells (P1 positive: plasmatocytes, red, [H–I]) are present in above genotype. (J–K') Attenuation of Mad expression by Mad^RNAiin the HSCs (K) also causes premature loss of HSCs (E(spl)mβ-CD2, red, N = 15; 18 hr). J'–K' represent scheme of results from (J–K). Also see Figure 7—figure supplement 1D. (L–M) depicts that this genotype in a third instar stage also results in 3.6 fold reduction in the lymph gland size in comparison to the control in (L), although P1 positive cells are still detectable (red, n = 15, p=7.27E-10, two tailed unpaired Student’s t-test; [M]). Also see Figure 7—figure supplement 1E and G and Figure 7—figure supplement 1K–L. (N–Q) The hetero-allelic mutant combination dpp^d12/dpp^d14 (n = 12; p=4.75E-11, two tailed unpaired Student’s t-test; [N]) or temperature sensitive mutant combination (dpp^ts/dpp^d12; n = 12; p=3.76329E-10, two tailed unpaired Student’s t-test; [O]) causes a 3.6 and a 4.7 fold decrease in the size of the lymph gland respectively. Dpp receptor Thickveins (Tkv) mutant animals (tkv¹/tkv⁷; n = 12; p=7.78E-11, two tailed unpaired Student’s t-test; [P]) as well as Mad deficiency (mad¹²/mad^1-2; n = 12; p=3.81811E-11, two tailed unpaired Student’s t-test; [Q]) exhibit a similar decrease in the size. Like RNAi genotypes, in all classical loss of function, an analogous phenotype is seen. (P1, red; [N–Q]; compare with [L]). Also see Figure 7—figure supplement 1H–J (R) Quantification of the results from H–I and L–Q. Average numbers of cells per lobe are indicated. (S) Schematic representation of Dpp function in HSC maintenance. Dpp from the Antp expressing PSC is transported to pMad expressing HSCs (also expressing STAT, N, Hth), near the dorsal vessel to activate its receptor Tkv, leading to the nuclear translocation of Mad that maintains HSCs. Thus, loss of either Dpp from the PSC (G) or Mad (K) or loss of Tkv (P) from the N expressing cells results in precocious loss of HSCs. Scale bar = 5 μm for A-E and 20 μm for rest. Error bars=S.D. Figure 7 has one figure supplement.

Figure 7—figure supplement 1. Involvement of Dpp signaling in HSC maintenance.

(A–B) show Upd3 expression ([A], green, n = 12) in a subset of PSC cells not earlier than 18 hr AEH, while (B) shows Wingless (Wg, red, n = 16) expression throughout first instar lymph gland. (C–C") shows another representative example of pMad enrichment in HSCs (N-GFP) (red, n = 6) (D) Quantitative analysis of effect of Dpp or Mad loss on HSCs, as seen by change in nuclear size. Nuclear size of first row of big cells near the DV decreases from 19.8 mm² to 15.19 mm² (n = 40 cells, p=5.46748E-19, two tailed unpaired Student’s t-test) in Mad loss and 14.78 mm² (n = 40 cells, p=9.66379E-19, two tailed unpaired Student’s t-test) in Dpp loss from the HSC and PSC respectively. (E–G) Expressing dpp ^RNAi([F], n = 12) in the niche or Mad^RNAi([G], n = 15) in the HSC, during the critical window of late 16 of embryogenesis to 18 hr AEH, causes a drastic reduction in the size of the lymph gland but does not affect medullary-cortical zonation as seen by Cubitus Interruptus (Ci, Red) labeling, in comparison to the control in [E]. (H–I) show presence of medullary zone (Ci, Red, compare with [E])in hetero-allelic mutant combination of dpp (dpp^d12/dpp^d14 (n = 10; [H]) as well as Mad deficiency (mad¹²/mad^1-2; n = 9; I]), although the size of the lymph gland is drastically reduced. (J) Analogous results are obtained in tkv⁷/tkv¹(Ci, Red, compare with [E]; n = 12). (K) shows wild type E(spl)mβ-CD2 expression as visualized by CD2 (red) staining in a third instar lg. (K'–K") shows that E(spl)mβ-CD2 expression overlaps with differentiating blood cell marker- Hemolectin (Hml; green).(L) shows that down-regulation of Mad from HSC during late embryo to 18 hr AEH has no effect on the E(spl)mβ-CD2 expression in the differentiated hemocytes of a late third instar lg (n = 6, compare with [K]). (M) Quantitative analysis of niche number in dpp loss (a short window of stage 16 of embryogenesis to 18 hr AEH) from the niche in 1st instar is comparable to control (p=0.856283596, n = 10, two tailed unpaired Student’s t-test) In contrast, classical loss of function genotype of dpp (dpp^d12/d14) revealed a significant increment in niche cell count when compared to wild-type (p=7.49838E-07, n = 10, two tailed unpaired Student’s t-test). Scale bar 5 μm (A–C") and 20 μm (E–L). Error Bars= S.D. Genotypes are shown on top of corresponding panels. DAPI marks the nucleus.