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First published online 23 November 2004
doi: 10.1242/jcs.01552

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Function of Rho GTPases in embryonic blood cell migration in Drosophila

Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada

View larger version (109K): [in a new window]   Fig. 1. Characterization of gcm-Gal4- and Coll-Gal4-induced expression pattern. (A-C) gcm-lacZ embryos stained with anti-ß-Gal antibody. ß-Gal is located in the nuclei. (A) The first signs of ß-Gal expression are detected in the hemocyte primordium at stage 8. (B) At early stage 12, ß-Gal is found in migrating macrophages (arrows) and in the glia cells (arrowheads). (C) At stage 17, ß-Gal is still present in macrophages and glia cells. (D-F) In situ hybridization showing the expression pattern of lacZ transcript in gcm-Gal4 UAS-lacZ embryos. (D) lacZ transcript is first observed in the hemocyte progenitors at late stage 8. (E) A stage 12 embryo with lacZ transcript in migrating macrophages (arrows) and glial cells (arrowheads). (F) A close-up of the lateral region of a stage 15 embryo with lacZ expression in macrophages (arrows) and epidermal stripes (vertical rows of labeled cells). (G-I) gcm-Gal4 UAS-lacZ embryos stained with anti-ß-Gal antibody. ß-Gal is located in the cytoplasm. (G) In gcm-Gal4 UAS-lacZ embryos, ß-Gal is not detected until stage 9 in the hemocyte primordium. (H) Subsequently, the expression pattern of ß-Gal mimics that of gcm-lacZ (stage 12 embryo; arrows indicate macrophages and arrowheads indicate glia cells). (I) ß-Gal expression persists in macrophages until late embryogenesis. Shown is a close-up of macrophages in the ventral region of a stage 17 embryo. (J-L) Coll-lacZ embryos stained with anti-ß-Gal antibody. (J) In Coll-lacZ embryos, ß-Gal (nuclear) becomes detectable in macrophages at stage 13, but only in an apparently random fraction of macrophages. (K) ß-Gal is detected in all macrophages by stage 15, which also display a more intense staining. This expression persists until stage 17 (L). (M-O) In situ hybridization of Coll-Gal4 UAS-lacZ embryos with a lacZ-specific probe. (M) lacZ transcript is initially detected in macrophages at stage 13, but only in a random subset of cells. (N) At stage 15, all macrophages express the lacZ transcript. The salivary glands also express lacZ (arrow). (O) lacZ expression in macrophages continues in stage 17 embryos. (P-R) Coll-Gal4 UAS-lacZ embryos stained for ß-Gal. (P) ß-Gal is first observed in stage 13 embryos in a subset of macrophages. (Q) At stage 15, all macrophages are ß-Gal positive, and ß-Gal persists until stage 17 (R).

View larger version (165K): [in a new window]   Fig. 2. Migrating macrophages extend lamellipodia and filopodia. Time-lapse sequences of live embryos in which macrophages are labeled with gcm-Gal4 UAS-mGFP. (A-J) Focus is on macrophages migrating between the ventral epidermis and the ventral cord in stage 14 embryos. (K-P) Shown is a macrophage migrating over the yolk sac underneath the amnioserosa in a stage 13 embryo. Migrating macrophages extend wide lamellipodia (arrows in D,G,K). These protrusions are very dynamic and quickly extend, retract and change shape (A-F and I-J). The extension of lamellipodia occurs in the direction of cell movement (e.g. K-M). A macrophage extends a single lamellipodium at a given time. In addition to lamellipodia, macrophages extend thin needle-like protrusions (arrowheads in C,G,J). These filopodia are also very dynamic (B,C). Occasionally, part of the trailing end of the cell is seen to pinch off (K-P). (Q) TEM of a stage 14 wild-type embryo showing a macrophage extending a lamellipodium (arrow). Inside the cell, vesicles containing apoptotic bodies (dark inclusions) can be seen. (R) Whole mount stage 14 gcm-Gal4 UAS-lacZ embryo stained with anti-ß-Gal antibody (red) and anti-peroxidasin (green). All bars, 10 µm.

View larger version (109K): [in a new window]   Fig. 3. Normal Rac activity is essential for macrophage migration. Embryos were stained with anti-Peroxidasin antibody to label macrophages. (A,C,E,G) Stage 13 embryos; (B,D,F,G) stage 15 embryos. (A) In wild-type stage 13 embryos, macrophages have migrated from the anterior and posterior regions toward the middle along the ventral cord. Part of the ventral abdominal region of the embryo is still devoid of macrophages (arrows). (B) At late embryogenesis, macrophages are evenly distributed throughout a wild-type embryo. (C,D) Expression of Rac1N17 under the control of the gcm-Gal4 causes an arrest of macrophage migration. Only a few macrophages move anteriorly and posteriorly for short distances. (E,F) Expression of Rac1V12 arrests macrophage migration and most macrophages remain in the anterior region forming a cluster around the foregut. (G,H) Expression of Rac1L89 causes a delay in macrophage migration. These embryos show a larger macrophagefree area ventrally in the stage 13 embryo (G; area between arrows) than wild-type embryos. A ventral region devoid of macrophages persists also at later stages in Rac1L89-expressing embryos (H; arrows). (I-K) Whole-mount embryos were stained with anti-Cqr antibody (red). Confocal images of Cqr expression were superimposed with differential interference contrast images to reveal cell profiles. (I) Cqr is a macrophage-specific scavenger receptor that labels the plasma membrane and early phagosomes (Franc et al., 1996; Franc et al., 1999). Wild-type macrophages contain approximately four phagosomes per cell (Franc et al., 1999). Macrophages expressing Rac1N17 (J) or Rac1V12 (K) show normal expression of Cqr, as seen within the dense cluster of macrophages surrounding the foregut. These cells contain few or no phagosomes.

View larger version (141K): [in a new window]   Fig. 4. Rac activity is essential to maintain normal morphology and distribution of macrophages in late embryos. The activity of Rac was disrupted by expressing mutant Rac1 isoforms under the control of Coll-Gal4. UAS-lacZ was co-expressed to label macrophages and embryos were stained with anti-ß-Gal antibody. (A,C,E,G) Stage 17 embryos; (B,D,F,H), close-up of stage 17 embryos, focusing on the ventral area between the epidermis and the ventral cord. (A,B) Macrophages are evenly distributed in a stage 17 wild-type embryos (A) and have a spindle-like morphology (B). (C,D) Rac1N17 causes macrophages to clump in various areas of the embryo (C), and blocks the formation of cellular protrusions causing macrophages to appear rounder (D). (E,F) Rac1V12 causes strong clustering of macrophages and as a result, large areas of the embryo are devoid of macrophages (E). Rac1V12-expressing macrophages extend longer, more prominent cellular protrusions (F) than wild-type macrophages (B). (G,H) Rac1L89 induces macrophages to cluster (G), but the phenotype is milder than observed with Rac1N17 or Rac1V12. Also Rac1L89-expressing macrophages extend more prominent protrusions (H) as seen upon expression of Rac1V12 (F).

View larger version (72K): [in a new window]   Fig. 5. F-actin accumulation and lamellipodia formation in macrophages with altered Rac activity. Stage 14 embryos stained with anti-peroxidasin (red) to label the macrophages and rhodamineconjugated phalloidin (green) to label F-actin. (A) In wild-type macrophages, F-actin is localized mostly in the cell cortex. (B) Macrophages expressing Rac1N17 show F-actin levels and distribution indistinguishable from wild type. (C) Macrophages expressing Rac1V12 contain strongly elevated levels of F-actin. (D-F) TEM of macrophages at stage 14. (D) Macrophages expressing Rac1N17 are round and have few or no cytoplasmic extensions. The macrophages shown are located close to the foregut and contain only few phagosomes (arrows). (E) Macrophages expressing Rac1V12, which cluster around the foregut, show an increased number of cytoplasmic protrusions, and occasionally contain phagosomes (arrows). (F) Some of the macrophages expressing Rac1V12 contain two nuclei (arrowheads). Bars, 10 µm (D-F).

View larger version (73K): [in a new window]   Fig. 6. Defects in macrophage migration in Rac1, Rac2 and Mtl mutant embryos. All panels show stage 14 embryos double-labeled for anti-peroxidasin (green) to identify macrophages and anti-ß-Gal (red) to identify embryos that carry a TM6 balancer chromosome, carrying wild-type alleles of Rac1, Rac2 and Mtl. (A) Embryo expressing ß-Gal that was derived from a Rac1J10 Rac2 mutant germline clone but is heterozygous for Rac1J10 and Rac2 as it received a paternal copy of TM6. Macrophages show a normal distribution. (B) Homozygous Rac1J10 Rac2 mutant embryo derived from a Rac1J10 Rac2 germline clone. Macrophages have failed to populate the posterior trunk region (between arrows). (C) Homozygous Rac1J10 Rac2 Mtl mutant embryo derived from a Rac1J10 Rac2 Mtl germline clone. Again, macrophages have failed to populate the posterior trunk region (between arrows). Green labeling between arrows represents basement membrane staining by anti-peroxidasin antibody that is less prominent in A and B owing to variations in staining intensities. Bar, 100 µm.

View larger version (70K): [in a new window]   Fig. 7. Effects of altered Cdc42 activity on macrophage. (A,D,G) Stage 15 embryos stained with anti-peroxidasin antibody to label macrophages. Embryos expressing Cdc42N17 (A) or Cdc42V12 (D) under the control of gcm-Gal4 show a wild type distribution of macrophages. A Cdc42V12-expressing embryo has a reduced number of macrophages but they are larger. (G) Embryos with overall reduced Cdc42 activity (Cdc42MZ mutants) show an absence of macrophage migration along the ventral cord. (B,E,H) Transmission electron micrographs of stage 14 embryos. Macrophages expressing Cdc42N17 (B), or Cdc42V12 (E), or macrophages in Cdc42 mutant embryos (H) display wild-type morphology with normal membrane extensions and presence of phagosomes (arrows). Some Cdc42V12-expressing macrophages contain two nuclei (arrowheads in E). (C,F,I) Embryos expressing Cdc42N17 or Cdc42V12 under the control of Coll-Gal4 line. Embryos also carried UAS-lacZ and were stained with anti-ß-Gal antibody. Each panel shows a close-up of a whole-mount stage 17 embryo, focusing on the ventral area between the epidermis and the ventral cord. (C) Cdc42N17-expressing macrophages are evenly distributed and have a wild-type morphology. (F) Cdc42V12-expressing macrophages have a normal distribution, but the cells do not show cytoplasmic protrusions and have a rounded morphology. (I) Wild-type embryo. Bars, 10 µm (B,E,H).

View larger version (55K): [in a new window]   Fig. 8. Macrophage migration mutants identified in the deficiency screen. Stage 13-15 whole-mount embryos stained with anti-peroxidasin (A-F,H,I) or anti-ß-Gal antibodies (G,J,K) to highlight macrophages. Genotypes are indicated on panels.