JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila

Abstract

The lymph gland is the major site of hematopoiesis in Drosophila. During late larval stages three types of hemocytes are produced, plasmatocytes, crystal cells, and lamellocytes, and their differentiation is tightly controlled by conserved factors and signaling pathways. JAK/STAT is one of these pathways which have essential roles in vertebrate and fly hematopoiesis. We show that Stat has opposing cell-autonomous and non-autonomous functions in hemocyte differentiation. Using a clonal approach we established that loss of Stat in a set of prohemocytes in the cortical zone induces plasmatocyte maturation in adjacent hemocytes. Hemocytes lacking Stat fail to differentiate into plasmatocytes, indicating that Stat positively and cell-autonomously controls plasmatocyte differentiation. We also identified the GATA factor pannier (pnr) as a downstream target of Stat. By analyzing the phenotypes resulting from clonal loss and over-expression of pnr in lymph glands, we find that Pnr is positively regulated by Stat and specifically required for the differentiation of plasmatocytes. Stat and Pnr represent two essential factors controlling blood cell maturation in the developing lymph gland and exert their functions both in a cell-autonomous and non-cell-autonomous manner.

Figure 1. Differentiation of hemocytes within the cortical zone (CZ)

During the third instar larval stage the cortical prohemocytes (pxn∷GFP, green) proliferate and differentiate into crystal cells (expressing PPO, cytoplasmic marker, pink) and plasmatocytes (P1, membrane marker, red). Confocal projections, z-sections (on the top of each projection, A-C’), and the schematic representation of a mature lymph gland (D) show that the gradual increase in plasmatocyte numbers on the surface of the CZ coincides with its expansion. During the same time the number of crystal cells also increases, but they are distributed throughout all layers of the CZ. Cells in the PSC are stained with anti-Antp (nuclear, red). (E, F) Projections of lymph glands expressing pxn∷Stat92e RNAi show drastic increase of plasmatocytes and crystal cells in the CZ during the mid-third instar (10-18 hours before pupariation, E, E’) and late third instar larval stages (2-6 hours before pupariation, F, F’) compared to wild type (A-C’). Plasmatocyte maturation is induced in the inner layers of the CZ (F, z-section). (G, G’) Projection of a lymph gland expressing pxn∷pnr RNAi; few fully differentiated plasmatocyte are detected, while crystal cells are increase in 30% of cases. DNA is shown in blue. Scale bars are 10 μm.

Figure 2. Cell-autonomous and non-autonomous functions of Stat92e

(A-C) Confocal crossections trough lymph glands with Stat92e³⁹⁷ MARCM clones and (D-F) Stat92eRNAi flip-out clones. (A, B, and E). Loss of Stat in the clones (green) located in the inner part of the CZ enhances the maturation of adjacent hemocytes into plasmatocytes (membrane marker, red) and sometimes crystal cells (cytoplasmic, pink). Loss of Stat in the clones located at the surface of the CZ (C and F) causes delay in plasmatocyte maturation within the clone, but does not affect crystal cell differentiation (PS4, read in G). (H-L) Over-expression of UAS-hop in flip out clones induces lamellocytes differentiation in a non-cell-autonomous manner. Confocal cross-sections of lymph gland lobe show that differentiated lamellocytes (red) appear near the clones (H) and on the surface of the lobe (I). 4-5 days after induction of clones, the lymph glands burst, the number of circulating hemocytes increases, and lamellocytes form aggregates (K and L). Lamellocytes are elongated cells and have larger nuclei than plasmatocytes (visualized with F-actin, red). Both wild type blood cells (non-green) and hemocytes that express hop (labeled with GFP, green) are increased in numbers and differentiate into lamellocytes. (J) Control blood smear shows low number of blood cells; rare wild type flip-out clones are labeled with GFP (green). DNA is shown in blue. Scale bars are 10 μm.

Figure 3. Cell-autonomous function of pnr in lymph gland development and terminal differentiation of plasmatocytes

(A-D’) Confocal cross-sections through the surface layers of lymph glands with pnr^VX6 MARCM clones. (A, B) pnr^VX6 cells (green) fail to differentiate into plasmatocytes (P1, red). (C) Clonal loss of Pnr does not affect early stages of hemocyte differentiation, illustrated by Pxn (red) expression within the clone (green), nor does it affect crystal cell maturation (D, D’, PPO, purple). Confocal cross-sections of lymph glands from mid-third instar (10-18 hours before pupariation) wild type (E) and late third instar (2-4 hours before pupariation) pnr^MD237 (F, G) lymph glands. In pnr^MD237 the expression of the early differentiation marker Pxn (compare E and F, red) in the CZ is not altered, but further expansion of the CZ and plasmatocyte maturation are inhibited (no P1 staining in G). (H, I) pnr does not affect lamellocyte differentiation upon wasp infection. Flat, elongated lamellocytes are visualized by staining with L1 antibody (patchy red) and Phalloidin (green). DNA is shown in blue. DNA is shown in blue. Scale bars are 10 μm.

Figure 4. Pnr-α overexpression in the lymph glands

(A-C) Ectopic expression of UAS-pnr-α in random flip-out clones (A, B), and in the CZ (C; pxn-GAL4, UAS-GFP, UAS-pnr-α, green) causes a small organ size, and lack of plasmatocytes in lymph glands of larvae 2-4 hours before pupariation. (D-F) Plasmatocyte maturation (P1, membrane, red) in CZ is restored by pnr-α expression in Stat92e knock down clones. Expression of pnr-α in Stat92e knock down clonesdoes not modify the non-cell–autonomous phenotype of Stat (D,E; elevated differentiation in adjacent cells), and suppress Stat92e cell autonomous phenotype. Note high level of plasmatocyte marker (membrane, red) in the clone cells (green) on the CZ surface (F, compare to Fig. 2F). DNA is shown in blue. Scale bars are 10 μm.

Figure 5. Stat positively regulates pnr expression in the lymph glands

(A-B’) Confocal crossections trough second instar and early third instar stage lymph glands expressing pnr-LacZ (red). Loss of Stat in Stat92eRNAi flip-out clones (green) causes the reduction of pnr-driven LacZ expression. The reduction is seen in second instar lymph glands (A,A’) and in secondary lobes of early third instar larvae (B, B’).

Figure 6. Lymph gland cortical zone signaling