Embryonic hematopoiesis modulates the inflammatory response and larval hematopoiesis in Drosophila

Abstract

Recent lineage tracing analyses have significantly improved our understanding of immune system development and highlighted the importance of the different hematopoietic waves. The current challenge is to understand whether these waves interact and whether this affects the function of the immune system. Here we report a molecular pathway regulating the immune response and involving the communication between embryonic and larval hematopoietic waves in Drosophila. Down-regulating the transcription factor Gcm specific to embryonic hematopoiesis enhances the larval phenotypes induced by over-expressing the pro-inflammatory Jak/Stat pathway or by wasp infestation. Gcm works by modulating the transduction of the Upd cytokines to the site of larval hematopoiesis and hence the response to chronic (Jak/Stat over-expression) and acute (wasp infestation) immune challenges. Thus, homeostatic interactions control the function of the immune system in physiology and pathology. Our data also indicate that a transiently expressed developmental pathway has a long-lasting effect on the immune response.

Temporal specificity and heterogeneity of the fly immune cells’ transcriptional landscape

“…Surprisingly, most LM markers such as Myospheroid (Mys or L4) (Irving et al, 2005), Misshapen (Msn) (Braun et al, 1997), Cher (or L5) (Rus et al, 2006) and Atilla (or L1) (Honti et al, 2009) were also detected at significant levels in the hemocytes from both stages, suggesting that they are expressed at basal levels in normal hemocytes and are strongly induced in LM. Finally, Gcm is involved in hemocyte development in the early embryo (stage 8-10) (Bernardoni et al, 1997) and is no longer expressed by E16 (Bazzi et al, 2018). Accordingly, Gcm transcripts are barely detected in E16 and WL transcriptomes (levels < 40 normalized read count).…”

“…We hence optimized the infestation protocol to obtain a rather uniform response and developed a protocol of mild infestation that maximize the survival rate of the Drosophila OregonR hosts (see methods, Figure S6A ). Our protocol maximizes the number of larvae containing a single wasp egg (Bazzi et al, 2018). In these conditions, we observe first the production of LM and a decrease of PL, which suggests that the PL trans-differentiate into LM from the resident and circulating pool of hemocytes (24 h after infestation).…”

“…Following this, the lymph glands histolyse (>90% of lymph gland histolysed 48 h after infestation) and the hemocytes proliferate ( Figure S6B-D ). By 72 h after infestation (which corresponds to the time of hemocyte collection for the sc RNA seq), all lymph glands are histolysed and the hemolymph contains hemocytes (PL, CC and LM) from the two hematopoietic waves ( Figure S7A-F ) (Bazzi et al, 2018). It is important to note that the WI dataset covers only the cells that have not encapsulated the wasp egg.…”

“…For the phagocytosis assay on NimC1/P1 negative hemocytes, 20 third instar larvae were bled in Schneider medium supplemented with PTU and were treated with latex beads 0.50 μm diluted 1/500 for 2, 5 or 10 min. Cells were then fixed and labelled with Rabbit anti-Srp(Bazzi et al, 2018), mouse anti-P1(Vilmos et al, 2004). We then used the secondary antibodies Cy5 Goat Anti-Mouse IgG (Jackson ImmunoResearch Labs Cat# 115-177-003, RRID:AB_2338719) and Cy3 Goat Anti-Rabbit IgG (Jackson ImmunoResearch Labs Cat# 111-165-144, RRID:AB_2338006) and DAPI.…”

“…The response to wasp infestation involves the embryonic hemocytes that differentiate from the procephalic mesoderm (1 st wave of hematopoiesis) (Tepass et al, 1994), as well as the hemocytes that originate from the lymph gland, the site of the 2 nd hematopoietic wave. While in not infested (NI) conditions, the lymph gland histolyses and releases hemocytes in circulation during the pupal life, upon wasp infestation (WI), it undergoes precocious histolysis so that both lymph gland and embryo derived hemocytes populate the larva (Banerjee et al, 2019; Bazzi et al, 2018; Letourneau et al, 2016). Our single cell RNA sequencing assay identifies the same number of PL clusters as that observed in normal conditions, showing that the PL from the first and from the second hematopoietic wave share the same features.…”