Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation

Abstract

Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS(2). The functional significance of this difference in ROS level in the two progenitor types remains unresolved. Here we show that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. We conclude that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. Our results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response.

Figure 1. Reactive Oxygen Species Profile of third instar lymph glands

(a) Schematic diagrams of late third instar (upper panel) and early second instar (lower panel) lymph glands. The second instar lymph gland consists mostly of the progenitor population, which by late third instar, becomes restricted to the central domain of the primary lobe, referred to as the Medullary Zone (MZ). At least three differentiated cell types can be distinguished: plasmatocytes, crystal cells and lamellocytes. Lamellocytes are rarely found in wild-type lymph glands as they are only induced upon infection. All three differentiated cell types are largely restricted to the Cortical Zone (CZ). The third instar lymph gland is comprised of several lobes; primary lobes are found in the most anterior region, and are followed posteriorly by two or more smaller lobes, referred to as secondary and tertiary lobes respectively. (b) The progenitor population in the MZ show elevated ROS levels (red). The dotted outlines of lymph gland lobes in all panels are based on images acquired at high laser power. (c) The expression of the MZ marker, dome-gal4, UAS-2xEYFP (green; genotype abbreviated on the panel as dome>GFP for clarity) overlaps with the ROS dye (red) in cells of the MZ (therefore yellow). (d) As in panel (b), the progenitor population in the MZ show elevated ROS levels (red). (e) hml^Δ-gal4, UAS-2xEGFP is restricted to cells in the CZ (green). Most of the cells that are marked by hml^Δ-gal4, UAS-2xEGFP are low in ROS (therefore green) when compared to cells in the MZ (red). A ring of hml^Δ-gal4, UAS-2xEGFP expressing cells can be seen along the edge of the MZ that are both GFP and ROS positive (therefore yellow). These appear to be cells in a state of transition between the stem-like and the differentiated cell fate. (f) Unlike in previous panels, the red color here marks P1 expression in differentiated plasmatocytes in the CZ. By late third instar, the expression of dome-gal4, UAS-2xEYFP (green) is restricted to the MZ and the cells in the CZ (red) downregulate this marker. (g) Overexpression of the antioxidant protein (GTPx-1) in the progenitor cell compartment (genotype: dome-gal4, UAS-2xEYFP; UAS-Gtpx1) results in a pronounced reduction in the number of cells that express the P1 marker (red). Some cells occupying the CZ region continue to express dome-gal4, UAS-2xEYFP while many others downregulate this marker without yet expressing the differentiation marker P1. (h) In the hypomorphic (weak allele) sod2/sod2 homozygotes, in which the level of expression of a major ROS scavenger is reduced, P1 expression is expanded and can be found throughout the lymph gland (red), rather than being restricted to the CZ. This image is generated from the optical sections acquired from the central part of the gland. Scale bars : 50µm.

Figure 2. Increased ROS production triggers precocious differentiation of the multipotent progenitors

In all panels, the progenitor population expresses the MZ marker, dome-gal4, UAS-2xEYFP (green). In panels (d–g), the green channel has been omitted for clarity. The two genotypes used in these panels are control lymph glands (dome-gal4, UAS-2xEYFP), abbreviated as wild-type (WT), and experimental lymph glands which express a RNAi construct to ND75 (dome-gal4, UAS-2xEYFP; UAS-RNAiND75), abbreviated as ND75^RNAi. Scale bars :50µm. (a) P1 is not expressed (note absence of red) in early second instar WT lymph glands. (b) P1 expression (red) is robustly induced in early second instar ND75^RNAi lymph glands. (c–e) Disruption of ND75 triggers precocious differentiation. (c) In WT third instar lymph glands, plasmatocytes marked with P1 (red) and crystal cells with ProPO (gray) are restricted to the CZ. These differentiated cell types are rarely if ever found in secondary and tertiary lobes. (d) In third instar ND75^RNAi lymph glands, there is a dramatic increase in P1 (red) and ProPO (gray) expressing cells, throughout the primary, as well as in the secondary and tertiary lobes (tertiary lobes are shown in Supplementary Fig. 4). (e) Lamellocytes, marked by L1 (red) are prominently seen in third instar ND75^RNAi lymph glands. Crystal cells are shown in gray. Lamellocytes are rarely found in secondary lobes. (f, g) Scavenging ROS suppresses differentiation associated with ND75 disruption. Overexpression of Gtpx-1 in ND75^RNAi lymph glands (in f and g) potently suppresses differentiation into all three lineages as there is a decrease in P1 (red in f), ProPO (gray in g) and L1 (red in g) expression. Compare (f, g) with (d, e). Controls for titration of GAL4 are shown in Supplementary Figure 6.

Figure 3. Disrupting JNK signaling suppresses the ROS-dependent differentiation Phenotype

The progenitor population also expresses the MZ marker, dome-gal4, UAS-2xEYFP (green), in panels (a–d), but this has been omitted for clarity. Lymph glands that express a RNAi construct to ND75 (dome-gal4, UAS-2xEYFP; UAS-RNAiND75), are abbreviated as ND75^RNAi. Scale bars: 50µm. (a, b) JNK signaling is activated upon ROS increase. puc-lacZ expression (red) in WT lymph glands (a) and ND75^RNAi lymph glands (b). puc-lacZ, which is a transcriptional reporter of JNK signaling is dramatically elevated in ND75^RNAi cells. (c, d) JNK signaling is required for triggering differentiation associated with ND75 disruption. Expressing a dominant negative construct of JNK in the precursor population ameliorates the effect of complex I disruption as the number of plasmatocytes (red in c) crystal cells (gray in c and d) and lamellocytes (red in d) are reduced virtually to WT levels. Compare (3c, d) with (2d, e) (e) Suppression of the number of crystal cells formed in ND75^RNAiUAS-DNbsk lymph glands relative to ND75^RNAi lymph glands. Error bars are s.e.m and n = 10.

Figure 4. FoxO activation and Polycomb downregulation phenocopy aspects of the ROS induced differentiation

In all panels, the progenitor cells express the MZ marker, dome-gal4, UAS-2xEYFP (green), omitted in some panels for clarity. Lymph glands from dome-gal4, UAS-2xEYFP larvae were used as wild-type controls (abbreviated WT). Lymph glands which express a RNAi construct to ND75 in the progenitor cells (dome-gal4, UAS-2xEYFP; UAS-RNAiND75), are abbreviated as ND75^RNAi. Scale bars :40µm. (a, b) Disruption of ND75 leads to induction of the FoxO reporter, thor-lacZ. WT lymph glands (a) do not express thor-lacZ (absence of red). The asterisk in a, points to thor-lacZ expression in the ring gland, adjacent to the lymph gland which serves as an internal control. thor-lacZ expression is significantly induced in ND75^RNAi lymph glands (b). (c, d) Disruption of ND75 leads to expression of the polycomb reporter. The polycomb reporter (red) is not expressed in WT lymph glands (c), but is induced in ND75^RNAi lymph glands (d). (e–g) FoxO overexpression causes an increase in plasmatocytes and crystal cells, but has virtually no effect on lamellocytes. (e) Overexpression of FoxO in the progenitor cells (dome-gal4, UAS-2xEYFP; UAS-foxo) causes their premature differentiation into plasmatocytes as shown for earlier than normal P1 staining (red) in a second instar lymph gland. Compare with Figure 2a. (f) Progenitor cells expressing FoxO in the MZ of the third instar lymph gland also initiate extensive differentiation into plasmatocytes (red). In addition, there is ectopic differentiation in the secondary lobes (arrow, 2°). (g) FoxO expression in the MZ results in an increase in the number of crystal cells (gray). However, only a few isolated L1-positive cells (red) are evident even in late third instar lymph glands. This image is acquired at twice the magnification of the other panels to highlight the few lamellocytes (red). (h) RNAi-mediated downregulation of the expression of two polycomb proteins, Enhancer of polycomb, E(Pc) and polyhomeotic proximal (Ph-p), leads to a robust increase in lamellocytes, that stain for L1 (red). (i, j) When FoxO and the RNAi construct to E(Pc) are expressed together in the MZ progenitors there is an increase in all three mature cell markers. Co-expression of FoxO and an RNAi construct to E(Pc) trigger the full differentiation phenotype associated with complex I disruption as there is an increase in the number of plasmatocytes (red in i), crystal cells (gray in j) and lamellocytes (red in j).