Figure 1. Cytosolic Ca²⁺ levels regulate blood progenitor maintenance.

Shown are primary lymph gland lobes from wandering 3^rd instar larvae except (C, early 2^nd) and (D, mid 2^nd instar). Error bars in the graphs represent standard deviation. Scale bar: 50μm. See also Figure S1.

(A) Three distinct zones of the lymph gland: PSC functions as hematopoietic niche (blue, Antp staining) and maintains undifferentiated progenitors of the Medullary Zone (MZ, green; dome-Gal4, UAS-2xEYFP). The outermost Cortical Zone (CZ) contains differentiating hemocytes (red; Hml^Δ– RFP). (B) Schematic representation of wild-type lymph gland. Blue: PSC; green: MZ; peach: intermediate progenitors expressing mixed markers (Krzemien et al., 2010); red: CZ. Blue arrow: Hh signal; Red arrow: ADGF-A signal.

(C–E) Blood progenitors maintain elevated levels of intracellular Ca²⁺.

In (C–E), the GCaMP Ca²⁺ sensor (Ubi-Gal4; UAS-GCaMP) is ubiquitously expressed using the Ubi-Gal4 driver (see Figure S1A–C).

(C) During early 2^nd instar, high GCaMP (green) activity is seen in all cells of the lymph gland.

(D) Later, differentiation (P1, red) initiates in a small number of cells (arrow) which attenuate GCaMP (green) activity (inset: high magnification).

(E) By late 3^rd instar GCaMP sensor activity is extremely low in mature hemocytes (P1, red) while the MZ continues to display GCaMP sensor activity suggesting that they maintain elevated Ca²⁺ level in their cytosol.

(F–O) High Ca²⁺ level is essential for progenitor maintenance. dome^MZ-Gal4, UAS-2XEYFP expression (green; see Table S1) marks progenitors and P1 (red) marks differentiated cells. Driver used in (F–O): dome^MZ-Gal4. n = # of lymph glands analyzed for statistical analysis (O), percentage of progenitor cells (P1 negative) are counted. p values refer to quantitation shown in (O).

(F) Control (n=5)

(G–K) Lowering Ca²⁺ signaling in the MZ cells leads to reduced maintenance of progenitors.

(G) IP3R^RNAi (p = 2.5E-06; n = 13)

(H) RyR^RNAi (p = 4.2E-05; n = 10)

(I) Orai^RNAi (p = 1.8E-05; n = 10)

(J) CaM^B34Q (Dominant Negative; p = 4.6E-03; n=13)

(K) CaMKII^RNAi (p = 1.3E-05; n=11)

(L–N) Raising Ca²⁺ signaling in the MZ cells leads to enhanced maintenance of progenitors.

(L) IP3R overexpression (p = 3E-02; n = 4)

(M) SERCA^DN (p = 9E-02; n=10)

(N) CaMKII^WT overexpression (p = 1.4E-03; n=13)

(O) Quantitation of the genotypes shown in Figures F–N.