Fly Embryo Stages Professional Phagocytes Also

The Drosophila life cycle consists of a number of stages - embryogenesis, three larval stages, a pupal stage, and the adult stage.

Scale bars represent 20 m ( aa, b, c, ee ), 10 m ( e, ff ), or 5 m ( b, f ).

Here, we present our identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development.

These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults.

Our enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene ( Calnexin14D ) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by the Calnexin14D -associated enhancer.

Finally, we show that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers.

Taken together this demonstrates macrophage heterogeneity in Drosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use of Drosophila to study macrophage heterogeneity in vivo.

These processes include the dissemination and maintenance of tissue resident populations ( Gordon and Plddemann, 2017 ) and the ability to adopt a spectrum of different activation states (termed macrophage polarisation), which can range from pro-inflammatory (historically termed as M1-like) to anti-inflammatory, pro-healing (M2-like) macrophage activation states ( Martinez and Gordon, 2014; Murray, 2017 ).

Therefore, the absence of an adaptive immune system may restrict the diversity of macrophage populations in more simple organisms that possess only an innate immune system.

However, the fact that macrophage markers can be highly divergent, even when comparing mammals as closely related as mice and humans ( Murray and Wynn, 2011 ), has hampered investigation of whether this is indeed the case, indicating a need for alternative markers and approaches.

Given these intricacies, it is clear that we need to better understand the fundamental components and pathways responsible for the specification of different macrophage subtypes, particularly in vivo.

Recently, the macrophage-first hypothesis has been proposed, re-emphasising the idea that acute signals can polarise macrophages ahead of the involvement of T cells ( Wiegertjes et al., 2016 ).

Consequently, organisms without a fully developed adaptive immune system represent intriguing models in which to examine this idea and better understand macrophage heterogeneity in vivo.

Fruit fly blood is specified in two waves an embryonic wave in the head mesoderm and in the larval lymph gland, with those cells released at the end of larval development ( Gold and Brckner, 2015 ).

Blood cell proliferation has also been shown to occur in haematopoietic pockets attached to the larval body wall ( Leito and Sucena, 2015; Makhijani et al., 2011 ).

These waves of haematopoiesis generate three types of blood cell (also referred to as hemocytes): plasmatocytes, crystal cells, and lamellocytes.

However, despite these evolutionarily-conserved commonalities, the plasmatocyte lineage has, until very recently, been considered a homogeneous cell population.

Drawing on a collection of reporter lines (; Kvon et al., 2014 ), we have identified regulatory elements that define novel plasmatocyte subpopulations in vivo.

We show that these molecularly distinct subpopulations exhibit functional differences compared to the overall plasmatocyte population and that the proportion of cells within these subpopulations can be modulated by external stimuli such as increased levels of apoptosis.

Furthermore, we show that misexpression of a gene associated with a subpopulation-specific enhancer element is able to modulate plasmatocyte behaviour in vivo, thereby identifying novel effector genes of plasmatocyte subpopulation function.

Together our findings reveal that macrophage heterogeneity is a fundamental and evolutionarily conserved characteristic of innate immunity that pre-dates the development of the adaptive immune system.

This significantly extends the utility of an already powerful genetic model system and provides further avenues to understand regulation of innate immunity and macrophage heterogeneity.

For instance, imaging the inflammatory responses of plasmatocytes to epithelial wounds, we find that some cells close to injury sites rapidly respond by migrating to the wound, while other neighbouring cells fail to respond ( Figure 1a; Video 1 ).

We also find that plasmatocytes exhibit variation in their expression of well-characterised plasmatocyte markers such as crq-GAL4 ( Figure 1bb; Franc et al., 1996; Stramer et al., 2005 ) and display a broad diversity in their migration speeds within the embryo (random migration at stage 15; Figure 1cd ).

Fly Embryo Stages Professional Phagocytes Also

These professional phagocytes also display differences in their capacities to phagocytose apoptotic cells with some cells engulfing many apoptotic particles, whereas others engulf very few, if any ( Figure 1e ).

Furthermore, phagocytosis of microorganisms by larval hemocytes also varies significantly from cell-to-cell in vitro ( Figure 1f ).

These differences within the plasmatocyte lineage led us to hypothesise that this cell population is more heterogeneous than previously appreciated.