Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance

Abstract

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.

Figure 1. Regulation of progenitor quiescence by differentiating hemocytes mediated by Pvf1/Pvr and Adgf-A signaling

(A) A representative lymph gland primary lobe from a mid-second instar larva (left image) consisting of a posterior signaling center (PSC, yellow), progenitors (green), and a few differentiating cells (red). A primary lobe from a later-staged wandering third instar larva (right image) shows three distinct zones: the PSC (yellow), which functions as the niche for the maintenance of progenitors (green) within the medullary zone (MZ), and the peripheral cortical zone (CZ) region, comprised of differentiating blood cells (red). (B-G) Cell proliferation profile in lymph glands from mid-second instar larvae analyzed by BrdU (red) incorporation. (B) Control lymph glands (genotype: Hml-gal4) at this stage have only a few proliferating cells (9 +/- 6 cells, N=6). (C) Induction of cell death in the differentiating cells by expression of Hid and Reaper (Hml-gal4 UAS-hid UAS-rpr) causes an increase in the number of BrdU positive cells (red), indicative of the loss of quiescence among the progenitors (compare with B). A similar loss of quiescence occurs upon (D) down-regulation of Pvf1 in the PSC using Pvf1^RNAi (Antp-gal4 UAS-2xEGFP UAS-Pvf1^RNAi), (E) down-regulation of Pvr in the differentiating cells using Pvr^RNAi (Hml-gal4 UAS-Pvr^RNAi; 39 +/- 7 cells, N=5, p<0.004 when compared to B), or (F) down-regulation of Adgf-A in the differentiating cells using Adgf-A^RNAi (Hml-gal4 UAS-Adgf-A^RNAi). (G) Over-expression of Adgf-A can suppress the proliferation phenotype due to loss of Pvr^RNAi (Hml-gal4 UAS-Pvr^RNAi UAS-Adgf-A; 17 +/- 7 cells, N=8, p<0.03 when compared with E). (H) The color scheme of the model (right) corresponds to the zones represented in the schematic of the lymph gland (left). Maintenance of progenitor quiescence within the MZ requires Pvf1 from the PSC, Pvr function in the CZ, and Adgf-A from the CZ functioning downstream of Pvr. (I-N) Expression of Pvf1 and Pvr in the developing lymph gland. TOPRO-3 (in K-M, blue) marks nuclei. (I and M) Mid-second instar lymph glands from control animals (Hml-gal4 UAS-2xEGFP); (J-L′ and N) Third instar lymph glands. (I) Pvf1 expression is seen as small punctae present at high levels in the PSC (arrowhead) and dispersed in the rest of the lymph gland (arrow). (J) Pvf1 (red, J) expression in the PSC co-localizes with Hedgehog (green, J′). The overlap is shown in yellow (J″). (K) Within the lymph gland proper, Pvf1 (red punctae, K) co-localizes with Rab11 (green punctae, K′), a marker for transcytosis vesicles. The overlap is shown in yellow (K″). (L) Pvr mutant clones (non-green) were generated within wild-type tissue (green) and stained for Pvf1 (red punctae). Pvf1 is present in the Pvr⁺ tissue (arrow) but is strongly reduced in the clones lacking Pvr (arrowhead). (M, N) Temporal analysis of Pvr expression. (M) The first differentiating cells (green, Hml-gal4 UAS-2xEGFP) show strong upregulation of Pvr expression (red, inset). (N) At later stages, Pvr expression is relatively high in the CZ as compared to the MZ, and is lacking in the PSC (arrowhead).

Figure 2. Role of STAT downstream of Pvr

For uniformity, differentiating hemocytes are shown in red even if they are marked with EGFP (Hml-gal4 UAS-2xEGFP). Lymph glands shown are from wandering third-instar larvae. TOPRO3 marks nuclei (blue). (A) Control. Normal Hml-gal4 expression pattern (Hml, red). (B) Pvf1^RNAi expression in the PSC (green; Antp-gal4 UAS-2xEGFP UAS-Pvf1^RNAi). All non-PSC cells of the lymph gland express Peroxidasin (Pxn, red). (C) Pvr^RNAi expression in the CZ (Hml-gal4 UAS-2xEGFP UAS-PvrR^NAi) causes MZ progenitors to differentiate (Hml, red), although Hedgehog (green) expression in the PSC (arrowhead) remains normal. (D and D′) Pvr-mutant clones (Pvr^C2195/Pvr^C2195, non-green cells) differentiate normally as judged by their ability to phagocytose FluoSphere beads (red), similar to neighboring wild type tissue (green). (E) Hedgehog expression (red, inset) is unaffected when Pvf1^RNAi is expressed in the PSC (green; Ser-gal4 UAS-2xEGFP UAS-Pvf1^RNAi); yellow represents co-localization of Hedgehog and Ser-gal4 expression. (F) Expression of Ras^DN in differentiating hemocytes (Hml-gal4 UAS-2xEGFP UAS-Ras^DN) does not affect progenitor fate. (G-I) Loss of STAT function in differentiating cells causes progenitor differentiation (Hml, red). This phenotype can be induced by expressing either (G) Stat92E^RNAi (Hml-gal4 UAS-2xEGFP UAS-Dcr-2 UAS-Stat92E^RNAi), (H) Stat92E^DN (Hml-gal4 UAS-2xEGFP UAS-Stat92E^DN), or (I) dPIAS (Hml-gal4 UAS-2xEGFP UAS-dPIAS). (J) Combined single-copy-loss of Pvr and Stat92E (Pvr^C2195/+; Stat92E0⁶³⁴⁶/+). All progenitors express Pxn (red). (K) As a control, expression of Stat92E^act (Ekas et al., 2010) in differentiating cells has no effect on progenitor fate (Hml-gal4 UAS-2xEGFP UAS-Stat92^ΔNΔC) (L) Co-expression of Stat92E^act and Pvr^RNAi (Hml-gal4 UAS-2xEGFP UAS-Stat92Er UAS-PvrR^NAi) suppresses the progenitor differentiation phenotype caused by Pvr^RNAi alone (compare with C). (M) Stat92E mutant clones (Stat92E⁰⁶³⁴⁶/Stat92E⁰⁶³⁴⁶) within the MZ (lacking green, demarcated by white dots) do not cause ectopic differentiation (lack of red within the clones). (N) Blocking Domeless function in MZ cells (FLP-out gal4 UAS-dome^DN clones, green, see methods for details, demarcated by white dots), does not cause them to differentiate (P1, red). (O) JAK mutant animals (hop^M38/hop^MSV1) do not exhibit ectopic differentiation (P1, red) of progenitors. (P) Expression of JAK^RNAi specifically in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-hop^RNAi) does not cause MZ progenitor differentiation (P1, red). (Q1-9) In the third instar, lymph glands exhibit a small fraction of cells that express a reporter of STAT activity (green, 10X Stat92E-GFP). These cells are negative for domeless (dome-MESO-lacZ; Q1), proliferate (PH3; Q2), and express Pvr^act (Q3-4) and Hml (Hml-ga4, UAS-lacZ; Q5), but lack differentiation markers: Pxn (Q6), Lz (Q7), ProPO (Q8) and P1 (Q9). Cells appear yellow due to co-localization of the STAT reporter and Hml (anti-|3-gal), Pvr^act, or PH3. (R) Schematic representation of STAT function in progenitor maintenance. Pvf1 from the PSC is transported to the differentiating cells in the CZ to activate its receptor Pvr, leading to the activation of STAT that, in turn, generates the CZ signal necessary for the maintenance of the progenitors in the MZ. Thus, loss of either Pvf1 from the PSC (in B), or loss of Pvr (in C) or STAT (in G-I) from the CZ results in proliferation and differentiation of MZ progenitors.

Figure 3. Adgf-A functions downstream of STAT in hematopoietic progenitor maintenance

Third instar lymph glands are shown. Differentiating hemocytes are marked in red, with nuclei marked with TOPRO3 (blue). For uniformity, hemocytes in A, C, F and G are shown in red although they are genotypically Hml-gal4 UAS-2xEGFP. (A) Normal expression pattern of Hml-gal4, showing differentiating cells (Hml, red). The MZ cells lack markers of differentiation and are marked by TOPRO3 (blue). (B) Adgf-A^karel/Adgf-A^karel mutant lymph gland. All progenitors differentiate and express Pxn (red, compare pattern with A). (C) Expression of Adgf-A^RNAi in differentiating hemocytes (Hml-gal4 UAS-2xEGFP UAS-Adgf-A^RNAi) causes differentiation of progenitors (Hml, red; compare with A). (D) Combined single-copy-loss of Pvr and Adgf-A (Pvr^C2195/+; Adgf-A^karel/+). All progenitors differentiate and express Pxn. (E) Combined single-copy-loss of STAT and Adgf-A (Stat92E⁰⁶³⁴⁶/+ Adgf-A^karel/+). All progenitors differentiate and express Pxn. (F) Co-expression of Adgf-A and Pvr^RNAi (Hml-gal4 UAS-2xEGFP UAS-Pvr^RNAi UAS-Adgf-A) suppresses the progenitor differentiation phenotype caused by Pvr^RNAi (compare with Fig. 2C). (G) Co-expression of Adgf-A and STAT^DN (Hml-gal4 UAS-2xEGFP UAS-STAT^DN UAS-Adgf-A) suppresses the progenitor differentiation phenotype caused by STAT^DN (compare with Fig. 2H). (H) Schematic representation of Adgf-A function in progenitor maintenance. Expression of Adgf-A from the differentiating cells, mediated by Pvf1/Pvr and STAT signaling, functions as the CZ signal. Therefore, loss of Adgf-A in the CZ (as in C) causes progenitor differentiation, and over-expression of Adgf-A in the CZ (in F and G) suppresses mutant effects of Pvr and STAT. The earlier steps are as described in Figure 2S.

Figure 4. Adenosine signaling regulates hematopoietic progenitor quiescence

In A-B and D-G, the progenitor cell population is marked with dome-gal4, UAS-2xEGFP (green). In A-N, differentiating hemocytes are shown in red. All lymph glands shown are from wandering third instar larvae. (A-G) Role of the adenosine transporter ENT3, the receptor AdoR, downstream signalling component G_α, and the adenylate cyclase Rutabaga. (A) Control lymph gland showing the normal expression pattern of dome-gal4 UAS-2xEGFP (green) in progenitors and P1 (red) in differentiating cells. (B) ENT3^RNAi expression in progenitors (dome-gal4 UAS-2xEGFP UAS-ENT3^RNAi) causes a reduction in MZ size (compare with A) and a corresponding increase in the CZ. (C) ENT3^RNAi expression in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-ENT3^RNAi) does not cause progenitor differentiation. (D) AdoR^RNAi expression in progenitors (dome-gal4 UAS-2xEGFP UAS-AdoR^RNAi) causes their expansion and reduction of differentiating cells (compare with A). (E) AdoR overexpression in progenitors (dome-gal4 UAS-2xEGFP UAS-AdoR) causes their differentiation (compare with A). (F) G_αi overexpression in progenitor cells (dome-gal4 UAS-2xEGFP UAS-G_αi) blocks their differentiation. (G) Rut^RNAi expression in progenitors (dome-gal4 UAS-2xEGFP UAS-Rut^RNAi) causes their expansion and reduces differentiating cells. (H-J) AdoR function downstream of Pvr. (H) Control AdoR mutants (AdoR^KGex/AdoR^KGex) develop a small lymph gland but with normal zonation. (I) Pvr^RNAi expression in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-Pvr^RNAi) causes loss of progenitors. (J) Mutation in AdoR (AdoR^KGex/AdoR^KGex) partially suppresses the loss of progenitors caused by Pvr^RNAi in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-Pvr^RNAi, compare with Fig 2C). (K-N) AdoR and Pvr together control expression of Adgf-A in differentiating cells. (K) AdoR^RNAi expression in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-AdoR^RNAi) causes progenitors to differentiate (red). (L) PKA^RNAi expression in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-PKA^RNAi) causes progenitors to differentiate (red). (M) Co-expression of Adgf-A with AdoR^RNAi in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-AdoR^RNAi UAS-Adgf-A) suppresses the progenitor differentiation phenotype caused by AdoR^RNAi alone (compare with K). (N) Co-expression of Adgf-A with PKA^RNAi in differentiating cells (Hml-gal4 UAS-2xEGFP UAS-PKA^RNAi UAS-Adgf-A) suppresses the progenitor differentiation phenotype caused by PKA^RNAi alone (compare with L). (O-P) Activation of Pvr is dependent upon the presence of AdoR. (O) A wild-type (w¹¹¹⁸) lymph gland showing the expression pattern of activated (phosphorylated) Pvr (Pvr^act, red). The red staining on the left is in the heart (dorsal vessel, DV). (P) In AdoR^KG03964ex/AdoR^KG03964ex lymph glands expression of Pvr (total protein, Pvr^reg, green) is normal. However, Pvr^act expression (red) is entirely missing from the lymph gland (compare with red staining in O). The red staining on the left is in the dorsal vessel (DV). (Q) Adenosine signaling in the lymph gland. Adenosine signaling through AdoR in progenitors promotes their proliferation and differentiation. This process is kept in check by reduction of available extracellular adenosine. Therefore, in the progenitors, reduction of the adenosine transporter (in B) or increase in the adenosine receptor (in E) causes differentiation. Also in the progenitors, loss of the adenosine receptor (in O) or its downstream components (in F and G) causes expansion of this compartment at the cost of the CZ. In differentiating cells, Pvr collaborates with AdoR to maintain Adgf-A expression. Therefore, loss of adenosine receptor or its downstream components in the CZ (in K and L) causes progenitor differentiation and this phenotype is suppressed by overexpression of Adgf-A.

Figure 5. Interaction between PSC and CZ signals

In A-E, progenitors are marked with dome-gal4 UAS-2xEGFP (green). In A-H, the differentiating cells are shown in red. All lymph glands shown are from wandering third instar larvae. (A) Control lymph gland showing the normal expression pattern of dome-gal4 UAS-GFP (green) in progenitors and P1 (red) in differentiating cells. (B-E) Role of PKA in progenitor maintenance. Loss of PKA function in progenitors causes their expansion and a corresponding reduction of differentiating cells, as shown by expressing (B) the regulatory domain of PKA (dome-gal4 UAS-2xEGFP UAS-PK^mR*), which functions as a sink for cAMP, (C) PKA ^EP2132 (dome-gal4 UAS-2xEGFP UAS-PKA^EP2132) which functions as a dominant-negative, and (D) PKA^RNAi (dome-gal4 UAS-2xEGFP UAS-PKA^RNAi). (E) Gain of PKA function in progenitors through the expression of the constitutively active PKA^mC* (dome-gal4 UAS-2xEGFP UAS-PKA^mC*) causes their differentiation. Compare with A. (F-H) PKA opposes the function of Adgf-A in progenitor maintenance. (F) Normal control PKA^EP2132/+. (G) Control Adgf-A^karel/Adgf-A^karel. Hematopoietic progenitors differentiate. (H) PKA^EP2132/+; Adgf-A^karel/Adgf-A^karel. The progenitor differentiation phenotype due to loss of Adgf-A is suppressed by single copy loss of PKA (compare with G). (I-J) Regulation of Ci^activated by PKA and AdoR. (I-I′) FLP-out cell clones (green in I, see materials and methods) that reduce PKA function (by expressing PKA^EP2132) exhibit high levels of Ci^activated (Ci^act) expression (shown in grayscale, I′). (J) FLP-out cell clones (green) that reduce AdoR function (by expressing AdoR^RNAi) also exhibit elevated levels of Ci^act (shown in grayscale) expression (J′). (K) The niche and CZ signals function together to regulate the levels of Ci^act necessary for progenitor maintenance in the MZ. Note that Ci is activated not only upon loss of PKA (in I) but also upon loss of the adenosine receptor (in J) thus creating a link between Hedgehog and adenosine signaling.