Drosophila Embryonic Hemocytes Produce Laminins to Strengthen Migratory Response

Abstract

The most prominent developmental function attributed to the extracellular matrix (ECM) is cell migration. While cells in culture can produce ECM to migrate, the role of ECM in regulating developmental cell migration is classically viewed as an exogenous matrix presented to the moving cells. In contrast to this view, we show here that Drosophila embryonic hemocytes deposit their own laminins in streak-like structures to migrate efficiently throughout the embryo. With the help of transplantation experiments, live microscopy, and image quantification, we demonstrate that autocrine-produced laminin regulates hemocyte migration by controlling lamellipodia dynamics, stability, and persistence. Proper laminin deposition is regulated by the RabGTPase Rab8, which is highly expressed and required in hemocytes for lamellipodia dynamics and migration. Our results thus support a model in which, during embryogenesis, the Rab8-regulated autocrine deposition of laminin reinforces directional and effective migration by stabilizing cellular protrusions and strengthening otherwise transient adhesion states.

Keywords: Drosophila; cell migration; extracellular matrix; hemocytes; lamellipodia dynamics; laminins.

Graphical abstract

Figure 1

Laminins Are Required for Proper Hemocyte Migration around the Hindgut and Over the VNC and for Lamellipodia Dynamics and Persistence (A–F) Stills taken from live imaging of GFPMoe-expressing hemocytes migrating around the hindgut in control (A–C) and LanB1 mutant embryos (D–F). (G) Tracking individual hemocytes reveals a significant decrease in the velocity of hemocytes from LanB1 mutant embryos (p < 0.001). (H and I) Graph showing a significant decrease in the velocity of hemocytes from LanB1 embryos compared to control on both the (H) ventral (p < 0.0001) and (I) dorsal sides of the VNC (p < 0.01). (I and J) Hemocytes from control (I) and LanB1 (J) embryos. (K and L) Graphs showing the lamellipodial area of (K) control (n = 6) and (L) LanB1 (n = 7) hemocytes measured at 30-s intervals over a 30-min time period. (M and N) Average lamellipodial area (M) and lamellipodial area change (N) per hemocyte from control and LanB1 embryos (n = 6 and 7 hemocytes from 3 different embryos per genotype, respectively). (O and P) Radial diagrams illustrating lamellipodia distribution in control (O) and LanB1 embryos (P). Scale bars represent 50 μm (A–F) and 20 μm (I and J).

Figure 2

Hemocytes Provide Autocrine Laminins as a Substrate for Their Movement (A–C) Still images taken from live imaging of transplanted hemocytes from (A) control embryos (red) into control embryos (green), (B) LanB1 embryos (red) into control embryos (green), and (C) control embryos (red) into LanB1 embryos (green). (A′–C′) Tracking analysis of (A)–(C), respectively. (D) Quantification of hemocyte migration speed in the transplanted embryos of the indicated genotypes. (E–G) Hemocytes from control embryos in control (E) and LanB1 mutant embryos (G) and hemocytes from LanB1 in control embryos (F). (H) Graph showing lamellipodia area of transplanted hemocytes relative to controls (hemocytes from control embryos transplanted in control embryos). Scale bars represent 30 μm (A–C) and 20 μm (E–G).

Figure 3

LanB1-GFP Distribution around Hemocytes and the VNC (A) Dorsal view of a fixed stage 10 control embryo expressing a LanB1-GFP fosmid stained with anti-GFP (green) and anti-Srp (red) antibodies. LanB1-GFP can be found around migrating hemocytes (arrowhead). (B–D) Still images taken from live LanB1-GFP; srpH > mChMoe embryos. Hemocytes entering the tail in stage 10 embryos (B). LanB1-GFP can be found in track-like arrays around hemocytes (arrowhead). Dorsal view of a stage 14 embryo showing LanB1-GFP decorating the BM surrounding the VNC (C). Representative images of hemocytes over the VNC of a stage 14 embryo during a FRAP experiment (D). Scale bars represent 20 μm (A), 15 μm (B and C), and 10 μm (D).

Figure 4

The RabGTPase Rab8 Is Required for Proper Hemocyte Migration (A and B) Ventral view of fixed stage 13 embryos expressing mChMoe in hemocytes (srpH > mChMoe) and Rab8YFP (A) or Rab10YFP (B) stained with anti-RFP (red) and anti-GFP (green) antibodies. (C–F) Lateral view of fixed stage 13 embryos expressing UAS-mChMoe and UAS-Rab8^DN (C), UAS-Rab10^DN (D), or a UAS-RNAi (F) against stratum under the control of the srpH-Gal4 driver and staining with an anti-RFP antibody. Stage 13 crag mutant embryo expressing mChMoe in hemocytes (E). (G) Quantification of hemocyte migration phenotype over the VNC in embryos of the indicated genotype. (H) Quantification of migration speed in hemocytes expressing Rab8^DN compared to hemocytes from control and LanB1 embryos during random migration. The statistical significance of differences was assessed with a t test; ^∗∗∗p < 0.0001 and ^∗∗p < 0.005. Scale bars represent 50 μm.

Figure 5

Rab8 Regulates LanB1-GFP Secretion and Lamellipodia Dynamics (A) Lateral view of fixed stage 15 embryos expressing mChMoe in hemocytes (srpH > mChMoe) and LanB1-GFP stained with anti-RFP (red) and anti-GFP (green) antibodies. (B) Role of Rab8 on regulating laminin secretion was assessed by co-expressing UAS-Rab8^DN. (C) Quantification of LanB1-GFP intensity in the VNC of embryos expressing UAS-Rab8^DN in hemocytes. (D and E) Control (D) and Rab8^DN-expressing (E) hemocytes. (F–I) Average lamellipodial area (F), lamellipodial area change (G), and radial diagrams illustrating the distribution of lamellipodia in control (H) and Rab8^DN-expressing hemocytes (I). Scale bars represent 50 μm (A and B) and 20 μm (D and E).