Activation of Drosophila hemocyte motility by the ecdysone hormone

Summary Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.

. Schematic representation of developmental ecdysone pulses and known hemocyte activity. Schematic representation of the ecdysone pulses that occur during the development of Drosophila melanogaster from embryonic to pupal life stages adapted from Thummel, 2001. The blue line shows the amounts of ecdysteroid (left Y axis) going through a series of peaks (pulses), following a temporal pattern. Red line, right Y axis, shows the mean cell migration velocity of hemocytes (mm/min) (Wood et al., 2006;Moreira et al., 2011;Sampson and Williams, 2012b) as a measure of hemocyte activity and motility. High activity and motility coincide with higher titres of ecdysteroid at embryogenesis and at the onset of metamorphosis.  . Possible model of the molecular mechanism employed by ecdysone to induce cell activity. This figure represents a schematic model of various intra-cellular signalling events that leads to the activation of hemocytes, and increased motile behaviour, as derived from our results and published work regarding ecdysone. From the point that ecdysone is present in the media, the following steps occur: (1) Extra-cellular ecdysone (Ecd) diffuses and binds to the EcR-B2/USP receptor complex in the cell nucleus.
(2) The receptor-ligand complex now binds to respective EcRE promoters to decrease the expression of rhoGAP16F.
(3) By a decrease in rhoGAP16F expression, less Rac1 GAP is produced leading to a reduction in the overall concentration of this Rac1 GAP therefore shifting the Rho-GTPase switch toward more Rac1-GTP active molecules.
(4) Ecdysone affects PI3K, whether directly or indirectly, symbolised by the dashed line, to increase levels of ATP and PiP 3 by autophagy. (5) PiP 3 now relocates to the plasma-membrane edge to allow for (6), the localisation of GTP-bound GTPases -Rac1 and Cdc42. The presence of GTP-Rac1 and GTP-Cdc42 induces signalling cascades that ultimately lead to (7), the polymerisation and extension of actin and tubulin, respectively, utilising the excessive pool of ATP, for actin polymerisation, produced from step 4. Movie 1. Time lapse of Late LIII hemocytes in vivo. A late LIII w; pxn_GAL4-UAS_GFP; crq_GAL4-UAS_GFP larva was mounted on a glass slide using double-sided tape. The time lapse, of GFP-labelled hemocytes, was made by taking a Z-stack, with a slice interval of 4-6 mm, every 3 minutes for 1.5 hours. The video shows that at this stage, hemocytes are predominantly attached to the integument and there are very few hemocytes in circulation.
Movie 2. Early stage LIII Or-R hemocytes in ex vivo culture on collagen IV matrix. This live time-lapse is of a group of early stage LIII Or-R hemocytes that have not experienced a pulse of ecdysone in vivo or ex vivo conditions. The time lapse, of hemocytes in bright field, was constructed from live cell conditions with a frame rate of 1 frame/15 seconds taken over a minimum of a 20 minute time period.
Movie 3. WPP Or-R hemocytes ex vivo on collagen IV matrix. This live time-lapse is of a group of WPP Or-R hemocytes that have experienced an ecdysone pulse in vivo at the onset of metamorphosis. Live data collection was conducted ex vivo after hemocyte isolation. This time-lapse, of hemocytes in bright field, was constructed in the same way as in supplementary material Movie 2.
Movie 4. Time lapse of WPP haemocytes in vivo. An early w; pxn_GAL4-UAS_GFP; crq_GAL4-UAS_GFP WPP (1 h APF) was mounted on a glass slide using double-sided tape. The time lapse, of GFP-labelled hemocytes, was made by taking a Z-stack, with a slice interval of 4-6 mm, every 3 minutes for a minimum of 30 minutes. The video shows that at this stage, hemocytes are leaving the dorsal patches at the integument and migrating towards target tissues.
Movie 5. Early stage LIII Or-R hemocytes isolated and incubated ex vivo with ecdysone hormone. Early stage LIII hemocytes were incubated for 3.5 hours with 10% (v/v) ecdysone. This live time-lapse, of hemocytes in brightfield, was conducted, after the incubation period, for a minimum of 2 hours at a frame rate of 1 frame/15 seconds.
Movie 6. Control UAS-EcRB2 (F645A) only LIII hemocytes incubated ex vivo with ecdysone hormone. Best representative early stage LIII hemocytes from UAS-EcR (F645A) only controls were isolated and incubated with 10% (v/v) ecdysone ex vivo. Hemocytes were incubated for 3.5 hours and then live timelapse, of these hemocytes in brightfield, was conducted after incubation period for a minimum of 20 minutes at a frame rate of 1 frame/15 seconds.
Movie 7. He_GAL4 driven UAS-EcRB2 (F645A) LIII hemocytes isolated and incubated with ecdysone ex vivo. Best representative early stage LIII hemocytes from He_GAL4 expressed UAS-EcRB2 (F645A) time-lapsed after 3.5 hours ecdysone incubation ex vivo. This live time-lapse, of hemocytes in brightfield, was conducted after incubation period for a minimum of 20 minutes at a frame rate of 1 frame/15 seconds.