An unexpected link between notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila

Abstract

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila melanogaster larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. Here we address the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. We show that Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference knockdown of N, Serrate, and neuralized (neur), and twin clone analysis of a N null allele, we show that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany N(RNAi)-induced lamellocyte differentiation and lobe dispersal. Our results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo.

Keywords: Drosophila; Notch; complex genetics; complex immunity; infection; innate immunity; parasite; reactive oxygen species; resistance; tolerance.

Figure 1

Notch intracellular domain expression in third instar lymph glands. (A) Variable Notch expression (red) in the cortical cells of a mid-third instar lymph gland. (A′ and A′′) Magnification shows weak Notch signal in Hml > GFP-positive cells, whereas some Hml > GFP-negative cells have high punctate cytoplasmic staining (arrow). (B–B′′) In the medulla, high anti-Notch staining colocalizes with the Dome > mCD8-GFP membrane signal (arrow). (C–C′′) Notch also colocalizes with Antp > mCD8-GFP signal in an early third instar lymph gland niche (arrow). (D–D′′) Notch staining is lower or is not detected in large lamellocytes (Alexa 488-phalloidin positive, green) in wild-type mid-third instar lymph glands from wasp-infected animals. Lamellocytes are rarely found in uninfected wild-type lymph glands (see Figure 6H). Hoechst (blue) stains DNA.

Figure 2

Ubiquitous and localized N activity in the larval lymph glands. (A–D) Anti-β-galactosidase staining detects moderate ubiquitous expression of Su(H)-lacZ reporter transgene in lobes of first, second, or third instar larvae. Higher expression is detected in a few scattered cells, presumed crystal cells, in lobes from the third instar animals. (Also see Figure 6).

Figure 3

Ubiquitous Notch reporter activity depends on functional N product. Lymph glands from N^ts2/Y; Su(H)-lacZ larvae at permissive (A and B) or nonpermissive (C and D) temperature, stained with anti-β-galactosidase antibody (red). Moderate Notch activity at permissive temperature (A and B) is significantly reduced at nonpermissive temperature (C–E). Standard deviation is shown.

Figure 4

A non-cell-autonomous role for N in crystal cell development. (A) Anterior lobes of Antp > mCD8-GFP lymph gland stained with anti-pro-PO antibody (red) marks crystal cells in control lobes. (B) Antp > NICD lobes stained with anti-pro-PO antibody (red) have more crystal cells compared to control lobes. (C) A significant reduction in crystal cell number is observed in Antp > N^RNAi lobes. Samples were taken from mid-third instar animals. LG, lymph gland. Standard error and P-value are shown.

Figure 5

A cell-autonomous role for the Notch intracellular domain in crystal cell development. (A–B′′) hsFLP; actin > NLS-GFP control clones marked with GFP are negative for pro-PO (red). High magnification of the defined areas A′ and A′′ from panel A and defined areas B′ and B′′ in panel B are shown. (C–D′′) Most cells in hsFLP; actin > NLS-GFP, NICD clones express pro-PO (red), but not Nimrod C (P1). High magnification of the defined areas C′ and C′′ in panel C, and D′ and D′′ in panel D are shown. Hoechst channel is omitted in B and D for clarity.

Figure 6

Notch activity is reduced after wasp infection. Anti-β-galactosidase antibody (red)-stained lobes from Su(H)-lacZ animals costained with anti-pro-PO (green) to detect coincidence of high Notch activity in crystal cells (stars). Cells expressing Su(H)-lacZ only make up 25.3% of all labeled cells; cells positive for pro-PO only constitute 37.3% of all labeled cells. The remainder labeled cell population (37.3%) are double positive (n = 406 cells from 20 lobes). Control animals did not experience wasp attack (A–C′), or animals were exposed to wasps (D–F) before dissection. Upon infection, Notch activity decreases (D–G). Standard deviation is shown in G. (H and I) Lamellocyte differentiation in Su(H)-lacZ lobes is normal. Anterior lobes are from uninfected Su(H)-lacZ (H) or infected (I) animal. Lamellocytes are labeled with phalloidin tagged with Alexa Fluor-647 (white), which also labels the dorsal vessel.

Figure 7

Lymph glands from N^ts animals are competent for lamellocyte differentiation. Lymph gland lobes (A–F), or circulating cells in the hemolymph (C–F, bottom insets), stained with rhodamine-phalloidin to detect the presence of lamellocytes in N^ts1 (A–D) or N^ts2 (E and F) mutants reared at permissive (A and C) or nonpermissive (B and D–F) temperatures are shown.

Figure 8

Notch^RNAi correlates with lamellocyte differentiation and lobe dispersal. Supernumerary lamellocytes are detected with msnf9-cherry reporter (B) or with rhodamine phalloidin (D) in Antp > N^RNAi lobes (B and D–D′′) and not in control lobes (A and C–C′′). Lamellocyte differentiation accompanies lobe dispersal (D–D′′). Hemolymph smears from control (E) and Antp > N^RNAi (F) animals stained with rhodamine-phalloidin show circulating lamellocytes in experimental but not control samples.

Figure 9

Lamellocyte differentiation is induced by neur^RNAi and Ser^RNAi. (A) Control Antp > GFP and (B and B′) Antp > neur^RNAi lymph glands stained with anti-L1 antibody (red). L1-positive lamellocytes are found in the cortex adjacent to satellite niche cells (green). (C) Control Antp > GFP and Antp > Ser^RNAi (D and D′) lymph glands stained with rhodamine-phalloidin. F-actin-rich lamellocytes (red) are adjacent to the GFP-positive niche cells. Hoechst stains DNA (blue). (E) Niche size is unaffected by knockdown of N pathway components. Left: Average number of GFP-positive cells per niche (n ≥ 20) are not significantly different in control (Antp > mCD8-GFP and Antp > y w) lobes and experimental (RNAi of N, Ser, neur or Su(H)) lobes. Right: The average area of GFP-positive cells per niche (n ≥ 20) is not significantly different between control and experimental lymph glands. Standard deviations for both parameters are shown.

Figure 10

N^RNAi–flp-out clones encourage lamellocyte differentiation and lobe dispersal. (A and C) Control lobes after induction of somatic recombination with NLS–GFP-positive cells but without N^RNAi, do not induce lamellocyte differentiation (A) or lobe dispersal (C). The edge of the lobe in C is continuous (arrows), suggesting the presence of an intact basement membrane. (B and D) Clones with NLS–GFP and N^RNAi induce lamellocytes (B, arrow) and lobe shows a discontinuous edge, indicating disrupted basement membrane (D).

Figure 11

Notch clones reveal non-cell-autonomous function in lamellocyte differentiation. (A) Schematic of twin clones (genotypes labeled) and lamellocytes in the cortex of an N^55e11/N⁺ anterior lobe. The schematic corresponds to an analysis of 14 z-stacked confocal images, each of 0.8-µm thickness; merged z-stacks are shown in B–C′′. (B) A mutant (N^55e11/N^55e11) clone, marked by the absence of β-galactosidase and a wild-type (N⁺/N⁺) clone detected by high levels of β-galactosidase (red). Lamellocytes positive for integrin β are also β-galactosidase-positive (yellow), and these cells are found outside the clone boundary (B–C′′). The region of interest in B is shown at a higher magnification in C–C′′, where double-positive N⁺/N⁺ lamellocytes with large, thin morphologies are more clearly evident. Hoechst stains DNA (blue).

Figure 12

N^RNAi induces high levels of ROS in blood cells, but not in the niche. ROS staining of freshly dissected lymph glands of control (Antp > mCD8-GFP, A–A′′), experimental (Antp > mCD8-GFP, N^RNAi) (B–B′′), and (Antp > mCD8-GFP, NICD) (C–C′′) animals. High ROS is detected in the medulla and cortex of Antp > mCD8-GFP, N^RNAi lymph glands. ROS levels are low or undetectable in the niche of all three genotypes (A′′, B′′, and C′′).