Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier

Abstract

Drosophila immune response involves three types of hemocytes ('blood cells'). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells.

Figure 1. Col Expression during Lymph Gland Ontogeny

(A) Col expression in lymph gland precursors is first observed in two separate clusters of cells (black arrows) in the dorsal-most mesoderm of thoracic segments T2 and T3 at stage 11 (stages according to Campos-Ortega and Hartenstein [1997]). Col expression in the head region is ectodermal (parasegment 0) and related to its function in head segmentation (Crozatier et al. 1999). (B and C) The clusters of Col-expressing cells get closer between stage 12 and early stage 13 (B) before coalescing (C). (D and E) Col expression becomes progressively restricted to the posterior-most cells of the forming lymph glands (arrowhead) during stage 14, as shown by the partial overlap between Odd-skipped (Odd) and Col expression. (F and G) Enlarged view of lymph glands after completion of embryogenesis, stage 16. Col expression marks the prospective PSC (Lebestky et al. 2003) in a dorsal-posterior position (arrowheads). (H) Schematic representation of Col expression in the lymph glands and pericardial cells in stage 16 embryos. (I) A srp^6G mutant embryo arrested at stage 13. Col is expressed in the presumptive lymph gland primordium (black arrow), although it is not possible to distinguish between high and low levels of expression. All embryos are oriented anterior to the left. (A–C), (G), and (I) are lateral views; (D–F) are dorsal views. (B), (C), and (E–G) are higher magnifications of the dorsal thoracic region. White arrows in (A) and (I) indicate Col expression in a developing dorsal muscle (Crozatier and Vincent 1999).

Figure 2. col Requirement for Lamellocyte Differentiation

(A–C) 4′,6-diamidino-2-phenylindole (DAPI) staining of hemocytes from wt (A and B) and from col¹ (C) third instar larvae. (A) Uninfected larva; (B) and (C) infected larvae. Plasmatocytes (inset in [A]) are always present, whereas lamellocytes (inset in [B]) are detected in the hemolymph of wt (B) but not col¹ (C) larvae 48 h after infestation by L. boulardi. In col¹ mutants, the wasp eggs are not encapsulated (white arrows) and develop into larvae (bottom right organism in [C]). (D–F) Lamellocytes expressing the P-lacZ marker l(3)06949 (Braun et al. 1997) surround the wasp eggs in wt larvae (D), are completely absent in infected col¹ mutant larvae (E), and differentiate in the absence of wasp infection following enforced Col expression in hematopoietic cells (srpD-Gal4/UAS-col larvae) (F). (G) srpD-Gal4/UAS-col pupa showing the presence of melanotic tumors. Bars: 50 μm.

Figure 3. Col Expression in Lymph Glands of Third Instar Larvae

(A and B) Col is expressed in the primary lobes, in a posterior cluster of cells (arrow), and in a variable number of secondary lobes. Low expression is also detected in some pericardial cells (asterisks), the significance of which remains unknown. PI, propidium iodide. (C) Schematic representation of the lymph glands and Col expression in late third instar larvae. (D and E) Col expression 24 h (D) and 48 h (E) after wasp infection; despite strong cell proliferation, including in secondary lobes, Col expression remains unchanged (black arrow). (F–H) Overlap between Ser-lacZ (Bachmann and Knust 1998) and Col expression in PSC cells; note a few scattered Ser-expressing cells that do not stain for Col. Bars: 50 μm (A, B, D, and E) ; 10 μm (F–H).

Figure 4. PSC-Specific Gene Expression Is Dependent upon Col Activity

PSC-specific expression of col, Ser-lacZ, and Ser (arrowhead in [A], [C], and [E]) is lost in col¹ mutant larvae (B, D, and F); only Ser expression in scattered cells is maintained (arrow in [E] and [F]). Bar: 50 μm.

Figure 5. Col-Expressing Cells Play an Instructive Role in Lamellocyte Production

Expression of the crystal cell marker doxA3 (Waltzer et al. 2003) (A, B, and G); of the lamellocyte markers α-ps4 (M. Meister, unpublished data) (C–F and H) and L1 (Asha et al. 2003) (J); and of Col (I and J); in wt (A, C, and E), col loss-of-function mutant (B, D, and F), and srp-Gal4/UAS-col (G–J) larvae. In (E) and (F), larvae were taken 48 h after infestation. An increased number of doxA3-positive cells (B) parallels the absence of lamellocyte differentiation (F) in col¹ mutant lymph glands. Conversely, lamellocyte differentiation and a reduced number of doxA3-positive cells are observed upon enforced Col expression (G and H). Double staining for Col and L1 shows that Col-expressing cells and differentiating lamellocytes do not overlap in the lymph gland. (I) shows ectopic Col expression compared to expression in the PSC (arrowhead; not visible in [J]). Antibody and in situ probes are indicated on each panel. In all panels, larvae are oriented with the head to the left: a single primary lobe is shown, with sometimes a few secondary lobes. Bar: 50 μm.

Figure 6. A Model for Lamellocyte Specification

(A) A model for the induction of lamellocyte differentiation in the Drosophila lymph glands in response to wasp parasitization. Col enables PSC cells to respond to a primary signal (S1) that is likely emitted by plasmatocytes upon their encounter with a parasite (Russo et al. 1996; Meister 2004). As a result, the PSC cells send a secondary signal (S2) that causes prohemocytes to develop into lamellocytes. Notch (N) signalling instructs a fraction of prohemocytes to become crystal cells (Duvic et al. 2002; Lebestky et al. 2003). The circular arrow indicates that increased proliferation leading to increased numbers of crystal cells and lamellocytes follows parasitization (Sorrentino et al. 2002). (B) Schematic view of hematopoiesis in Drosophila and mouse. Left: Lymph gland cells contain two types of hematopoietic cells, PSC cells and uncommitted precursors. These precursors can give rise to either plasmatocytes or crystal cells. Crystal cell precursors can also give rise to lamellocytes upon receiving a signal from the PSC cells expressing Col (dotted arrows); this signalling is itself dependent upon a communication between circulating plasmatocytes and the PSC (A). Right: In mice, hematopoietic stem cells (HSC) give rise to common myeloid precursors (CMP) and common lymphoid precursors (CLP) (adapted from Orkin [2000] and Schebesta et al. [2002]). Signalling between CMP- and CLP-derived cells is an essential component of adaptive immunity. Col and EBF functions, in Drosophila and vertebrate hematopoiesis, respectively, suggest an ancestral role in their conferring on a subset of hematopoietic cells the ability to respond to signals from circulating immune supervisors (generically designated here as macrophages) and to provide a secondary line of defence against specific intruders.