Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response

Abstract

Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, our studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow.

Graphical abstract

Figure 1

Adult Hematopoietic Hubs Drosophila (A and B) Hemocyte clusters (A) in the abdominal segments on either side of dorsal midline (Dm) are closely associated with dorsal vessel (DV) (B). (C–C''') 3D reconstruction of a fillet showing the position of four abdominal hemocyte clusters (green) above DV (red). Horizontal rotation angles are as mentioned. (C') Lateral view of (C), whereas (C'') and (C''') are rotation of (C') toward dorsal side. (D) Position of hemocyte cluster 1 with relation to DV. (E) Transverse section (dotted line through D) showing the cluster is nested in a groove created by body wall muscle (BWM) and transverse heart muscle (THM). LHM, longitudinal heart muscle. (F) Location of hemocyte cluster with relation to dorsal cuticle (cyan). (G) The hemocytes are dorsal to pericardial cells (PC). Genotype used: w; hand-Gal4,hml-Gal4,UAS-FLP.JD1,UAS-2xEGFP/+;P{Gal4-Act5C(FRT.CD2).P}S/+. (H) Scheme depicting anatomical position of the cluster based on our observation. (I) The hemocytes are fenestrated in a network of Pericardin. (J and L) Compared with control (J), there is a drastic reduction in expression of Pericardin in loh^MB05750 (L). (K and M) The normal clusters of hemocytes (K) get disrupted in loh^MB05750 (M). (N and O) The nested hemocytes are intercalated in Laminin A (N), downregulation of which by mef2-Gal4 affects cluster formation (O). (P and Q) Plasmatocytes in hematopoietic cluster express pxn and crq. (R) In a cluster of w;UAS-GTRACE/gcmGla4;+/+fly, some plasmatocytes (P1) are gcm lineage traced (EGFP). (S) Some hemocytes in the cluster are positive for ZCL2897. (T) Expression of dorothy (green) in the cluster (T). (T'–T'''') Zoomed in regions of T showing that dorothy-positive cells (arrow, green) express low levels of Srp (red). (U) Presence of collier lineage traced cells (EGFP) in the w;UAS-GTRACE/+;kn-Gal4/+cluster. (V) Schematic representation of the cluster and its components. (W) Presence of crystal cells in the cluster. (X–X'') Co-localization of lz-GFP with proPO(red). (X') and (X'') are zoomed in region of (X). (Y) Melanization of crystal cells (arrow). (Y') Melanized crystal cells (arrow) are detected in a position that corresponds to the first cluster of hemocytes in A1. Scale bar represent 100 μm (A and B); 10 μm (T'–T''''); the rest represent 20 μm. See also Figure S1 and Movies S1, S2, and S3.

Figure 2

Hemocyte Progenitors Are Present within the Hematopoietic Hub in Adult (A) Plasmatocyte and crystal cell originate from precursors that express only Srp. (B) Presence of plasmatocytes and few hemocytes that express only Srp (arrow) at 2 dpe in the hub. (B' and B'') Zoomed in regions of (B). Arrows denote cells that express only Srp. (C) At 5 dpe, crystal cells, plasmatocytes, and several Srp positive (arrow) cells are seen in the cluster. (C' and C'') Zoomed in of (C) showing crystal cells (arrowhead) and Srp-positive hemocyte (arrow). (D–G) Temporal kinetics of expressions of Su(H)LacZ (red) and lz-GFP (green) in the cluster. At 2 dpe, only Su(H)LacZ-positive cells are seen (arrow, D). Expression of lz-GFP is turned on in some Su(H)LacZ-positive cells by 3 dpe (arrowhead, E). At 5 (F) and 7 (G) dpe, while the number of lz-GFP (double arrowhead) expressing cells increase, a concomitant decrease in Su(H)LacZ-positive cells (arrow) is observed. (H–I') Expressions of Srp (gray) and Su(H)LacZ (red) in the cluster. (H and H') A cell (arrow) that has high levels of Srp expression also expresses Su(H)LacZ. (I and I') With stabilization of Su(H)LacZ (red) expression, the Srp expression declines (I and I'). (J–L) Crystal cell number compared with control (J) drastically reduces on knocking down Notch expression (K), while its overexpression causes huge increment. (M) Quantitative analysis of (J)–(L). (N) hml expression in differentiated cells of primary (1°) lobe of the lymph gland. (O) Loss of Pvr in hml cells leads to complete differentiation of 1° lobe and while tertiary (3°) and quaternary (4°) lobe cells (arrows) remain undifferentiated. (P) hml-Gal4, UAS-GFP hematopoietic hub houses Srp (red) hml (green) and Hnt (cyan) expressing cells. (Q) Only few hml (green) cells and Hnt (cyan) cells are seen in UAS-PvrRNAi; hml-Gal4, UAS-GFP hub, which has plenty of Srp (red, arrow) only cells. (R) collier expresses (green) in PSC, 2° lobe and the 3° and 4° lobes of late third instar larval lymph gland. (S–S'') col-Gal4 lineage traced cells (green) and Srp (gray) expression in the cluster. (S') and (S'') are zoomed in region of S showing collier lineage traced cells that are either high (arrowhead) or low (arrow) in Srp expression. (T and T') collier lineage traced cells (EGFP) in w; UAS-GTRACE/+;kn-Gal4/+ hub are either P1 (gray, membrane) or Hnt (gray, nucleus) or lack both Hnt and P1 expression (arrows in T–T'). (U–U'') A crystal cell (Hnt) or plasmatocyte (P1) can arise from collier lineage traced cells (EGFP). (U') and (U'') are zoomed in image of U. (V) Potential of Srp positive col lineage traced cells in the hematopoietic hub. Oenocyte (Oe) Fb, fat body, PC, pericardial cell. Scale bar represents 20 μm. Error bar denotes SE. See also Figure S2.

Figure 3

Evidence of Post-larval Hematopoiesis in Drosophila (A) Hematopoietic hub of adult fly. Box indicates the area of imaging. (B and B') hml lineage traced (EGFP) and live (RFP) expression in the hub at 5 dpe of w; hml-Gal4/UAS-GTRACE; +/+ flies. (C–D'') Zoomed-in regions of B showing the plasmatocytes that are either lineage traced or lineage traced as well as actively expressing hml or only have live hml expression (only RFP, arrows). (E–F') Activation of G-TRACE construct was prevented by following the timeline (E') in w; UAS-GTRACE/+; notch-Gal4/ P{tubP-GAL80^ts}2 fly. (F) and (F') are higher magnification of (E). (G–H''') N lineage trace and live expression in crystal cells (Hnt) of adult hub (G) where activation of G-TRACE by notch-Gal4 following timeline (G') was done. (H–H''') Zoomed in images of (G). (I–K') A subset of plasmatocytes (P1) in hub was N lineage traced (I) where G-TRACE was activated by notch-Gal4 following timeline (I'). (J) and (J') are zoomed in of (I). (K and K') Another example. (L) Scheme depicting the surge of hematopoiesis based on our observation. Scale bar represents 20 μm. See also Figure S3.

Figure 4

The Hematopoietic Hub Is Functionally Active (A–E) Plasmatocytes number in the hub at 2–35 dpe. (F) Quantitative analysis of the data in (A)–(E). (G) Comparative account of plasmatocytes number present in adult circulation at 2 and 5 dpe. (H–H'') Plasmatocytes in the hub can phagocytose E. coli (H). (H') and (H'') are further zoomed in images of (H). (I–I'') While fat body (Fb) cells incorporate BrdU (red, I') in adult fly, hemocytes of hub do not. (J) Only on infection BrdU incorporation is seen. (J' and J'') Zoomed in region of (J). (K) Quantification of BrdU incorporation in the hub of control (blue) and infected flies (red). (L) Scheme depicting potential of the hematopoietic hub. Scale bar represents 20 μm; 5 μm (H''). Error bar denotes SE. See also Figure S4.