Lineage Tracing of Lamellocytes Demonstrates Drosophila Macrophage Plasticity Martin Stofanko, So Yeon Kwon, Paul Badenhorst* Institute of Biomedical Research, University of Birmingham, Edgbaston, England Abstract Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of qimmune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as ‘‘non-self’’. Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. We show here that over-expression of the CoREST-interacting transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. We detect double-positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, we have marked plasmatocytes and show by lineage tracing that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, our data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge. Citation: Stofanko M, Kwon SY, Badenhorst P (2010) Lineage Tracing of Lamellocytes Demonstrates Drosophila Macrophage Plasticity. PLoS ONE 5(11): e14051. doi:10.1371/journal.pone.0014051 Editor: Francois Schweisguth, Institut Pasteur, France Received July 14, 2010; Accepted October 26, 2010; Published November 19, 2010 Copyright: 2010 Stofanko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Medical Research Council and Cancer Research UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: p.w.badenhorst@bham.ac.uk Introduction Leukocytes play a critical role in the innate immune responses of humans and invertebrates. Like their human counterparts, Drosophila leukocyte-like cells (termed hemocytes) neutralize invading pathogens through phagocytosis, encapsulation and melanization. Three hemocyte cell types have been shown to occur (reviewed in [1–3]). Of these, the most abundant is the plasmatocyte, which accounts for 95% of circulating hemocytes. Plasmatocytes are professional macrophages that are capable of removing foreign material and apoptotic cells by phagocytosis [4]. Less abundant are crystal cells, which comprise approximately 5% of hemocytes. Crystal cells are responsible for melanin synthesis during pathogen encapsulation and are required for wound healing and coagulation [5]. The final hemocyte type is a large flattened cell, the lamellocyte. These are responsible for encapsu- lating material recognized as ‘‘non-self’’. Lamellocytes are seldom detected in larval hemolymph in the absence of immune challenge. However, large numbers differentiate either upon infestation by parasitic wasps [6,7], or in a number of melanotic mutant strains [8,9]. Two waves of hematopoiesis occur. First, during embryonic development hemocytes arise from the head mesoderm [10,11] and subsequently populate the entire embryo. Following hatching of the larva, these hemocytes of embryonic origin persist and replicate in the larval hemolymph, while a second wave of hematopoiesis occurs in the larval lymph gland [11–13]. Hemocytes generated in the lymph gland are usually liberated at metamorphosis and contribute to the pupal and adult hemocyte populations [11]. As Drosophila exhibit an open circulatory system, hemocytes circulate in hemolymph (blood) that freely bathes all organs. In larva, movement of the hemolymph is mediated by contractions of the dorsal vessel and by peristaltic movements of the body. In a mature third instar larva approximately 5000 hemocytes occur, of which two thirds freely circulate in the hemocoel, while the remainder attach to the inner surface of the integument [12]. Both plasmatocytes and crystal cells are detected in these distinct sessile compartments [12,14]. The function of these sessile compartments is unclear, although it has been proposed that they provide a progenitor pool for lamellocytes [15]. Current models for the origin of the three hemocyte types propose that plasmatocytes, crystal cells and lamellocytes represent distinct lineages that arise separately from a common stem cell or prohemocyte [1–3,12]. The transcriptional hierarchy controlling plasmatocyte and crystal cell development is well characterized. In embryos, initial hemocyte determination requires the GATA factor Serpent (Srp) [16]. Subsequent differentiation into plasma- tocytes is regulated by the transcription factors Glial cells missing (Gcm) and Gcm2 [17]. In contrast, differentiation into crystal cells is triggered by expression of the AML-1/Runx1 homologue Lozenge [18]. Lamellocyte differentiation can be triggered by activation of a number of signalling pathways including the JAK/ PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e14051