function of rho gtpases in embryonic blood cell … › ... › joces › 117 › 26 ›...

IntroductionCell migration is one of the most prominent aspects of animalmorphogenesis. There are many different types of cellmigration events in which cells move on extracellularsubstrates or on the surface of other cells (Montell, 1999;Rorth, 2002; Starz-Gaiano and Montell, 2004; Friedl, 2004).During Drosophila embryogenesis, macrophages migrateextensively to populate the interstitial spaces between organprimordia (Tepass et al., 1994). Embryonic macrophagesappear to be engaged in two main activities: the phagocytosisof apoptotic cells and the secretion of extracellular matrix(ECM) molecules (Evans et al., 2003). While macrophagemigration and cell death in the embryo are largely independentprocesses, it was recently shown that the effective removal ofdead cells from the central nervous system by macrophages iscritical for CNS morphogenesis (Sears et al., 2003). Embryonicmacrophages persist into postembryonic stages and give rise toa substantial portion of the adult blood cell population that isfurther supplemented by hemocytes that derive from the larvallymph glands (Holz et al., 2003; Evans et al., 2003).

Embryonic blood cells (hemocytes) derive entirely from thehead mesoderm (Tepass et al., 1994). Approximately 700of those cells, called plasmatocytes, are migratory and

differentiate into macrophages whereas a small stationarypopulation of 36 hemocytes remain associated with the foregutand give rise to the crystal cells (Lebestky et al., 2000; Evanset al., 2003). The migration of macrophages duringembryogenesis can be subdivided into three phases (Tepass etal., 1994; Cho et al., 2002). In phase I (stages 10 and 11)plasmatocytes initiate motility and scatter locally throughoutthe head region. In phase II (stages 12 to 14) plasmatocytes(which have now started to phagocytose and are thusconsidered macrophages) undergo a large-scale migration,following a few major migration routes, as they emerge fromthe head region to populate the rest of the embryo. At stage 11the germband of the embryo is extended so that the tail islocated next to the head. Macrophages enter the tail and thenare carried with the retracting germband during stage 12 topopulate the posterior of the embryo. As the germband retracts,macrophages also migrate beneath the amnioserosa that coversthe embryo dorsally. Further, macrophages will emanate fromthe head and the tail and migrate along the dorsal and ventralaspects of the ventral nerve cord to populate the ventral trunkregion. A late aspect of the migration of macrophages duringphase II depends on the Pvr/Pvf guidance system (Brückner etal., 2004). In phase III (stages 15-17) macrophages that are

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Hemocyte development in the Drosophila embryo is agenetic model to study blood cell differentiation, cellmigration and phagocytosis. Macrophages, which make upthe majority of embryonic hemocytes, migrate extensivelyas individual cells on basement membrane-coveredsurfaces. The molecular mechanisms that contribute to thismigration process are currently not well understood. Wereport the generation, by P element replacement, oftwo Gal4 lines that drive expression of UAS-controlledtarget genes during early (gcm-Gal4) or late (Coll-Gal4)stages of macrophage migration. gcm-Gal4 is used for liveimaging analysis showing that macrophages extend large,dynamic lamellipodia as their main protrusions as well asfilopodia. We use both Gal4 lines to express dominant-negative and constitutively active isoforms of the RhoGTPases Rac1, Cdc42, Rho1 and RhoL in macrophages,and complement these experiments by analyzing embryosmutant for Rho GTPases. Our findings suggest that Rac1and Rac2 act redundantly in controlling migration andlamellipodia formation in Drosophila macrophages, and

that the third Drosophila Rac gene, Mtl, makes nosignificant contribution to macrophage migration. Cdc42appears not to be required within macrophages but in othertissues of the embryo to guide macrophages to the ventraltrunk region. No evidence was found for a requirement ofRho1 or RhoL in macrophage migration. Finally, toestimate the number of genes whose zygotic expression isrequired for macrophage migration we analyzed 208chromosomal deletions that cover most of the Drosophilagenome. We find eight deletions that cause defects inmacrophage migration suggesting the existence ofapproximately ten zygotic genes essential for macrophagemigration.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/117/26/6313/DC1

Key words: Cell migration, UAS-Gal4 system, Rho GTPases,Hemocyte, Cytokinesis, Macrophage

Summary

Function of Rho GTPases in embryonic blood cellmigration in DrosophilaMagda Paladi and Ulrich Tepass*Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada*Author for correspondence (e-mail: [email protected])

Accepted 21 September 2004Journal of Cell Science 117, 6313-6326 Published by The Company of Biologists 2004doi:10.1242/jcs.01552

Research Article

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scattered through the entire internal space of the embryoretaining vigorous local motility. Macrophages tend tocongregate only at sites of excessive cell death, to phagocytoseapoptotic cells (Abrams et al., 1993; Tepass et al., 1994).

Mechanistically, cell migration is a complex processinvolving the coordinated activity of several cellularcompartments, including the protrusions of cytoplasmicprocesses at the leading edge, the subsequent forwardmovement of the main cell body, and the retraction of thetrailing end of the cell. Traction-generating adhesiveinteractions with the substratum are dynamically regulated asthey are newly established at the leading edge, maintained inthe main cell body and dissolved at the trailing edge of the cell.Adhesion receptors, such as integrins and cadherins, are linkedto the actin cytoskeleton, which generates the forces needed topropel the cell forward (Lauffenburger and Horvitz, 1996; Hall,1998; Pollard and Borisy, 2003; Ridley et al., 2003). SmallGTPases of the Rho family have been identified as keyregulations of adhesion and cytoskeletal dynamics duringmigration (Hall, 1998; Ridley, 2001). Six Drosophila Rhoproteins have been described to date: Cdc42, Rac1, Rac2, Rho1and the two divergent Rho proteins, Rho-like (RhoL) and Mig-2-like (Mtl) (Luo et al., 1994; Harden et al., 1995; Hariharan etal., 1995; Murphy and Montell, 1996; Fehon et al., 1997;Sasamura et al., 1997; Newsome et al., 2000; Ng et al., 2002;Hakeda-Suzuki et al., 2002). Mtl is the Drosophila orthologueof the C. elegans Mig-2 Rho GTPase (Zipkin et al., 1997) and

is structurally similar to both Rac and Cdc42. Functionally, Mtlappears to behave like Rac1 and Rac2 because these threeGTPases act redundantly in regulating dorsal closure and axongrowth and guidance. Mtl, Rac1 and Rac2 are therefore referredto as the Drosophila Rac genes (Ng et al., 2002; Hakeda-Suzukiet al., 2002). A functional similarity between RhoL [which hasalso been referred to as Rac3 (Sasamura et al., 1997)] and otherRho family members has not been established.

The role of Drosophila Rho GTPases in variousdevelopmental processes was initially assayed using two typesof mutations: a mutation that encodes the dominant negative(DN) isoform, which has a reduced affinity for nucleotides andis thought to sequester guanine nucleotide exchange factors(GEFs), preventing them from functioning anywhere in thecell. The second mutation is a constitutively active (CA)isoform, which is permanently bound to GTP (Ridley, 2001).The expression of DN and CA isoforms of Rho GTPases in atissue-specific manner has been a useful tool in elucidating therole of these proteins in developing tissues, particularlybecause the expression of DN forms eliminates the activity ofmaternally and zygotically derived gene products. However,comparisons of phenotypes induced by the expression of DNisoforms and phenotypes of corresponding loss-of-functionmutations have revealed some inconsistencies (Luo et al.,1994; Kaufmann et al., 1998; Genova et al., 2000; Hakeda-Suzuki et al., 2002). In this study we have analyzed thefunction of Rho GTPases in Drosophila embryonic

Journal of Cell Science 117 (26)

Table 1. Genetic strains used in this study except deficiency lines Strain Genotype Description and references

Cdc423 y w, Cdc423/FM6 Fehon et al., 1997Cdc424 y w, Cdc424/FM6 Fehon et al., 1997Cdc426 y w, Cdc426 Fehon et al., 1997Coll-lacZ CyO, P{ry+t7.2=lArB}Cg25CA109.1F2/b1 Adh cn l(2);ry506 BDSCColl-Gal4 CyO, P[Gal4, w+]Cg25C/Bl This studyda-Gal4 w; P{w+mW.hs=GAL4-da.G32}UH1 Wodarz et al., 1995∆2-3 Ki pP P[ry+, ∆2-3]99B BDSCgcm-lacZ P{ry+t7.2=PZ}gcmrA87/CyO; ry506 Giagrande et al., 1993gcm-Gal4 w;P[Gal4, w+]gcm/CyO, ftz-lacZ This studygcm-Gal4 UAS-lacZ w;gcm-Gal4, UAS-lacZ/CyO-ftz-lacZ This studymbcC1 red1 e1 mbcC1/TM3, P{ry+t7.2=ftz-lacZ.ry+}TM3, Sb1 ry BDSCmbcD11.2 mbcD11.2/TM3, P{w+mC=HZR+6.8Xb}JG1, Sb1 BDSCUAS-mGFP; gcm-Gal4, UAS- y1 w;P{w+mC=UAS-mCD8::GFP.L}LL4; gcm-Gal4, This study; mCD8::GFP lines are from Lee and Lou,mGFP/CyO; UAS-mGFP P{w+mC=UAS-mCD8::GFP.L}LL5/CyO; P{w+ 1999

mC=UAS-mCD8::GFP.L}LL6P[Gal4, w+]E132 w,P[Gal4, w+]E132 Halder et al., 1995Rac1J11 Rac2∆ y w; Rac1J11, Rac2∆, FRT80/ TM6 UZ Hakeda-Suzuki et al., 2002; Ng et al., 2002Rac1J10 Rac2∆ y w; Rac1J10, Rac2∆, FRT80/ TM6 UZ Hakeda-Suzuki et al., 2002; Ng et al., 2002Rac1J10 Rac2∆ Mtl∆ y w; Rac1J10, Rac2∆, Mtl∆, FRT80/ TM6 UZ Hakeda-Suzuki et al., 2002; Ng et al., 2002Rac2∆ Mtl∆ y w; Rac2∆, Mtl∆/ TM6 UZ Hakeda-Suzuki et al., 2002; Ng et al., 2002Rho1 Rho172O/TM3, P{ry+t7.2=ftz/lacB} Stutt et al., 1997UAS-Cdc42N17 w; P{w+mC=UAS-Cdc42.N17}3 Luo et al., 1994UAS-Cdc42V12 w; P{w+mC=UAS-Cdc42.V12}LL1 Luo et al., 1994UAS-lacZ w;P{w+mC=UAS-lacZ.B}Bg4-1-2 BDSCUAS-lacZ w;P{w+mC=UAS-lacZ.B}Bg4-2-4b BDSCUAS-Rac1L89 w; P{w+mC=UAS-Rac1.L89}6 Luo et al., 1994 UAS-Rac1N17 y w; P{w+mC=UAS-Rac1.N17}1 Luo et al., 1994UAS-Rac1V12 y w; P{w+mC=UAS-Rac1.V12}1 Luo et al., 1994UAS-Rho1N19 w, UAS-Rho1N19 Stutt et al., 1997UAS-hRhoAV14 w; UAS-hRhoAV14 Harden et al., 1999UAS-RhoLN25 w; P{w+mC=hs-RhoL.N25}AM1 Murphy and Montell, 1996UAS-RhoLV20 w; P{w+mC=UAS-RhoL.V20}AM1 Murphy and Montell, 1996sim th1 st1 cp1 in1 kniri–1 pP sim8/TM3, Sb1 BDSCwild type Oregon R Wild-type strain (BDSC)

All deficiency lines were obtained from the Bloomington Drosophila Stock Center (BDSC) and are described in FlyBase.

6315Macrophage migration in Drosophila

macrophage migration by studying embryos that misexpressDN and CA isoforms of GTPases as well as by analyzingembryos mutant for specific GTPases.

Materials and MethodsGeneticsFlies were raised on standard medium. Crosses were performed at25°C, or at 29°C if Gal4 drivers were involved. Embryos werecollected on yeasted apple juice agar plates and staged according toCampos-Ortega and Hartenstein (Campos-Ortega and Hartenstein,1997). The fly strains used in this study are listed in Table 1. Embryosthat have reduced maternal and no zygotic Cdc42 expression(Cdc42MZ) were derived from Cdc423/Cdc426 or Cdc424/Cdc426

females crossed to wild-type flies. Cdc423 and Cdc424 are null alleleswhile Cdc426 is a hypomorphic allele of Cdc42 (Fehon et al., 1997;Genova et al., 2000). As Cdc42 is located on the X chromosome, 50%of males derived from these females carry a Cdc42 null allele. Doublemutant germline clones for Rac1J10 and Rac2∆ in a wild-type or ahomozygous Mtl∆ mutant background were induced as describedpreviously (Hakeda-Suzuki et al., 2002). Rac2∆ and Mtl∆ are nullalleles while Rac1J10 is a hypomorphic allele (Hakeda-Suzuki et al.,2002). For all experiments at least 30 embryos were evaluated, exceptfor the Rac1J10 and Rac2∆ embryos generated in an Mtl∆ mutantbackground. Of these, we recovered 16 embryos that could beevaluated, from which seven carried a balancer chromosome asindicated by a lacZ marker and thus had normal zygotic Racexpression. Nine embryos were maternally and zygotically mutant forall three Rac genes. Six of those embryos were in stages 13-15 anddisplayed macrophage migration defects while the remaining threeembryos were in stages 16 or 17 and showed normal dispersal ofmacrophages.

Generation of macrophage-specific Gal4 driver linesTwo macrophage-specific Gal4 lines were generated by targeted Pelement replacement, a technique suggested to us by Frank Laski (seealso Gonzy-Treboul et al., 1995; Sepp and Auld, 1999). The Xchromosomal insertion P[Gal4, w+]E132 was used as a donor lineand gcm-lacZ and Coll-lacZ, both carrying the rosy (ry) gene as agenetic marker, as recipient lines. After selecting transposase-inducedautosomal insertions of P[Gal4, w+], potential successful targetedtransposition events were identified by examining the Gal4 expressionpattern in a UAS-nGFP or UAS-lacZ background. Eight out of 21(38%) tested lines were successful conversions of gcm-lacZ to gcm-Gal4 and three out of five (60%) tested lines were conversions of Coll-lacZ to Coll-Gal4. A recombinant chromosome containing both gcm-Gal4 and UAS-lacZ was generated to label macrophages. As Coll-Gal4 is located on the CyO balancer chromosome, genetic crossesinvolving Coll-Gal4 were carried out in the presence of a UAS-lacZinsertion on the third chromosome.

Observations of live embryosFor observations of live embryos they were dechorionated in 50%bleach for 1 minute, rinsed with water and quickly lined up on a 22×40mm coverslip coated with Scotch tape glue. To extract the glue, theScotch tape was soaked in heptane. The heptane evaporates quicklywhen spread on a coverslip, leaving the glue behind. A rim of Vaselinewas placed around the embryos and the depression created was filledwith halocarbon® 56 oil (Halocarbon Product Cooperation). Thecoverslip was attached to a metal microscope slide with a 15×32 mmrectangular gap in its center. Embryos were imaged using an invertedmicroscope attached to a LSM510 Zeiss laser scanning confocalmicroscope. Movies were generated by taking an image every 10-20seconds over 20- to 30-minute time periods.

Immunohistochemistry and histological techniquesAntibody stainings of embryos followed standard protocols. Embryosused for phalloidin stainings were devitellinized by hand instead of amethanol heptane mixture. Primary antibodies used were rabbit anti-β-Galactosidase (β-Gal) (1:1500; Cappel), rabbit anti-Croquemort(1:1000) (Franc et al., 1996), mouse anti-peroxidasin (1:2000)(Nelson et al., 1994). Secondary antibodies were Alexa488, Cy3 andHRP conjugated (1:500; Jackson Laboratories; Molecular Probes).Phalloidin-Oregon Green488 (1:80; Molecular Probes) was used tolabel F-actin. For observations with differential interference contrastmicroscopy, embryos were dehydrated in an ethanol series and thenmounted in a mixture of two-thirds Canada balsam, one-third methyl-salicylate. For observations with laser scanning confocal microscopy,embryos were mounted directly in antifade (70% glycerol in PBScontaining 1 mg/ml p-phenylene diamine). Whole-mount in situhybridization with digoxigenin-labeled DNA probes followedstandard procedures. Transmission electron microscopy was carriedout as described previously (Tepass and Hartenstein, 1994), andultrathin sections were analyzed and photographed on a Hitachi H-7000 transmission electron microscope.

Deficiency screenThe Deficiency Kit collection (Bloomington Drosophila StockCenter) was used to identify loci involved in macrophage migration.Embryos were collected from individual deficiency strains and stainedwith macrophage markers. For all lines bearing deletions on thesecond chromosome and a few lines with deletions on the first or thirdchromosome, the embryos were stained with anti-peroxidasinantibody. In the remaining lines, macrophages were labeled in thebackground of the gcm-Gal4, UAS-lacZ chromosome with an anti-β-Gal antibody. For most deletions on the first and third chromosome,blue balancers were introduced to enable the identification of embryoshomozygous for the deletion. For the remaining lines, homozygousmutant embryos were recognized by a consistent phenotype displayedby approximately 25% of the embryos.

ResultsGeneration of macrophage specific Gal4 driver linesTo manipulate gene expression specifically in migratinghemocytes two Gal4 driver lines that induce expression of UAScontrolled target genes in macrophages were generated. Weused targeted P element replacement, which allows for theexchange of one P element with the sequence of another(Gonzy-Treboul et al., 1995; Sepp and Auld, 1999), to convertenhancer-trap lines that showed expression of lacZ inembryonic hemocytes into Gal4 drivers. gcm-lacZ[(Bernardoni et al., 1997) Fig. 1A-C] and Coll-lacZ [(Bellen etal., 1989) Fig. 1J-L], which carry a P element enhancer-trapinsertion in glia cells missing (gcm) and the collagen IVencoding gene Cg25C, respectively, were chosen as they hadthe most useful expression pattern for this study. In gcm-lacZembryos, β-Gal expression was first detected in the hemocyteprimordium at the end of stage 7 (Fig. 1A) and persisted inmacrophages until late embryogenesis (Fig. 1C). In addition toexpression in macrophages, lacZ is also activated in some glialcells and in a segmentally repeated single-cell-wide stripes ofepidermal cells located in the segmental groves (Fig. 1Band not shown). In Coll-lacZ embryos, β-Gal expression inmacrophages was first detected at stage 13, although onlyweakly in an apparently random subpopulation ofmacrophages (Fig. 1J). The levels of β-Gal expressionincreased by stage 15 and were maintained in macrophages

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Fig. 1. Characterization of gcm-Gal4- and Coll-Gal4-induced expression pattern. (A-C) gcm-lacZ embryos stained with anti-β-Gal antibody. β-Gal is located in the nuclei. (A) The first signs of β-Gal expression are detected in the hemocyte primordium at stage 8. (B) At early stage 12,β-Gal is found in migrating macrophages (arrows) and in the glia cells (arrowheads). (C) At stage 17, β-Gal is still present in macrophages andglia cells. (D-F) In situ hybridization showing the expression pattern of lacZ transcript in gcm-Gal4 UAS-lacZ embryos. (D) lacZ transcript isfirst observed in the hemocyte progenitors at late stage 8. (E) A stage 12 embryo with lacZ transcript in migrating macrophages (arrows) andglial cells (arrowheads). (F) A close-up of the lateral region of a stage 15 embryo with lacZ expression in macrophages (arrows) and epidermalstripes (vertical rows of labeled cells). (G-I) gcm-Gal4 UAS-lacZ embryos stained with anti-β-Gal antibody. β-Gal is located in the cytoplasm.(G) In gcm-Gal4 UAS-lacZ embryos, β-Gal is not detected until stage 9 in the hemocyte primordium. (H) Subsequently, the expression patternof β-Gal mimics that of gcm-lacZ (stage 12 embryo; arrows indicate macrophages and arrowheads indicate glia cells). (I) β-Gal expressionpersists 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-β-Gal antibody. (J) In Coll-lacZ embryos, β-Gal (nuclear) becomes detectable in macrophages at stage 13, butonly in an apparently random fraction of macrophages. (K) β-Gal is detected in all macrophages by stage 15, which also display a more intensestaining. 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 macrophagesexpress 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 β-Gal. (P) β-Gal is first observed in stage 13 embryos in a subset of macrophages. (Q) At stage15, all macrophages are β-Gal positive, and β-Gal persists until stage 17 (R).


until the end of embryogenesis (Fig. 1L). In addition, β-Galexpression was also seen in the fat body. A similar expressionpattern was reported for Cg25C (Lunstrum et al., 1988).

P element replacement was highly efficient for both linesgenerating gcm-Gal4 and Coll-Gal4 (see Materials andMethods). The expression of UAS-lacZ under the control ofgcm-Gal4 and Coll-Gal4 was characterized and compared tolacZ expression of gcm-lacZ and Coll-lacZ (Fig. 1). In gcm-Gal4 UAS-lacZ embryos, lacZ transcript was first detected in

the hemocyte primordium at stage 8 and persisted inmacrophages until stage 15 (Fig. 1D-F). After stage 15, lacZtranscript declines in macrophages. The expression pattern ofβ-Gal protein was similar to that of the lacZ transcript. β-Galwas first detected in the hemocyte progenitors at stage 9,approximately 30 minutes later than in the gcm-lacZ line.Although the lacZ transcript began to disappear frommacrophages at stage 15, β-Gal protein was maintained untilstage 17 (Fig. 1G-I). gcm-Gal4 appears to drive the expression

Fig. 2. Migrating macrophages extend lamellipodia and filopodia. Time-lapse sequences of live embryos in which macrophages are labeledwith 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 extendwide lamellipodia (arrows in D,G,K). These protrusions are very dynamic and quickly extend, retract and change shape (A-F and I-J). Theextension of lamellipodia occurs in the direction of cell movement (e.g. K-M). A macrophage extends a single lamellipodium at a given time. Inaddition 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 macrophageextending a lamellipodium (arrow). Inside the cell, vesicles containing apoptotic bodies (dark inclusions) can be seen. (R) Whole mount stage14 gcm-Gal4 UAS-lacZ embryo stained with anti-β-Gal antibody (red) and anti-peroxidasin (green). All bars, 10 µm.

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of UAS controlled reporter genes uniformly in all migratingmacrophages. In gcm-Gal4 UAS-lacZ embryos both the lacZtranscript and β-Gal were also detected in glial cells andepidermal stripes. Thus, gcm-Gal4 is active in macrophageprogenitor cells and in plasmatocytes/macrophages duringphase I and II of their migration (stages 8-15).

In Coll-Gal4 UAS-lacZ embryos, lacZ transcript and β-Galprotein were first detected at stage 13 in a subset ofmacrophages (Fig. 1M,P). The number of macrophagesexpressing lacZ increased in older embryos, and by stage 15,all macrophages showed lacZ expression (Fig. 1N,Q). Inaddition, lacZ expression in Coll-Gal4 UAS-lacZ embryos wasalso detected in the fat body and in the salivary glands. Thelatter does not correlate with the expression pattern of lacZ inthe original Coll-lacZ line. The activation of reporter genes by

Coll-Gal4 in the salivary glands is likely a secondary effect ofthe Gal4/UAS system, caused by the presence of an hsp70(salivary gland-specific) enhancer sequence in the Gal4construct (Brand and Perrimon, 1993; Gerlitz et al., 2002). Inconclusion, Coll-Gal4 is expressed in macrophages during latephase II and phase III of their migration (stages 13-17). Neitherthe gcm or Coll enhancer-trap lines nor the corresponding Gal4drivers showed expression in crystal cells.

Macrophage morphologyCell migration requires the formation of membrane protrusionsat the leading edge, driven in most cells by actinpolymerization. Motile cells in culture most commonly displaytwo types of protrusions – filopodia and lamellipodia


Fig. 3. Normal Rac activity isessential for macrophage migration.Embryos were stained with anti-Peroxidasin antibody to labelmacrophages. (A,C,E,G) Stage 13embryos; (B,D,F,G) stage 15embryos. (A) In wild-type stage 13embryos, macrophages havemigrated from the anterior andposterior regions toward the middlealong the ventral cord. Part of theventral abdominal region of theembryo is still devoid ofmacrophages (arrows). (B) At lateembryogenesis, macrophages areevenly distributed throughout a wild-type embryo. (C,D) Expression ofRac1N17 under the control of thegcm-Gal4 causes an arrest ofmacrophage migration. Only a fewmacrophages move anteriorly andposteriorly for short distances.(E,F) Expression of Rac1V12 arrestsmacrophage migration and mostmacrophages remain in the anteriorregion forming a cluster around theforegut. (G,H) Expression ofRac1L89 causes a delay inmacrophage migration. Theseembryos show a larger macrophage-free area ventrally in the stage 13embryo (G; area between arrows)than wild-type embryos. A ventralregion devoid of macrophagespersists also at later stages inRac1L89-expressing embryos(H; arrows). (I-K) Whole-mountembryos were stained with anti-Cqrantibody (red). Confocal images ofCqr expression were superimposedwith differential interference contrastimages to reveal cell profiles. (I) Cqris a macrophage-specific scavengerreceptor that labels the plasmamembrane and early phagosomes(Franc et al., 1996; Franc et al.,1999). Wild-type macrophagescontain approximately four phagosomes per cell (Franc et al., 1999). Macrophages expressing Rac1N17 (J) or Rac1V12 (K) show normalexpression of Cqr, as seen within the dense cluster of macrophages surrounding the foregut. These cells contain few or no phagosomes.


(Lauffenburger and Horwitz, 1996). To characterize the shapeand extensions of migrating macrophages, we examinedmacrophages in live embryos using time-lapse confocalmicroscopy. For these observations, macrophages were labeledusing the gcm-Gal4 driver in combination with four to sixcopies of UAS-mGFP, expressing membrane-tethered GFP(Lee and Luo, 1999).

As expected, time-lapse observations revealed thatmacrophages are highly motile cells. Migrating macrophagesextend predominantly one wide, flat cytoplasmic protrusion(Fig. 2A-P; see Movies 1 and 2 in supplementary material) thatresemble the lamellipodia described for motile cells in culture.These lamellipodia are often 15-20 µm in length, about twicethe diameter of the main cell body. The macrophagelamellipodia are highly dynamic, constantly changing shape byrapid extension and retraction. Extension and retraction ofa large lamellipodium is achieved within 5-6 minutes.Lamellipodia extend in the direction of movement, which isillustrated in Fig. 2G-J that shows a macrophage indicated byan asterisk retracting its lamellipodium as the cell body rotatesclockwise, followed by the emergence of a lamellipodium at adifferent site that now becomes the new leading edge andpointing in the direction of movement. A similar scenario isdemonstrated in Fig. 2K-P, in which a lamellipodium can beseen progressively turning toward the left, followed by the cellbody. In addition to lamellipodia, macrophages also extendmultiple thin, needle-like cytoplasmic protrusions similar tofilopodia (Fig. 2G-J; see Movies 1 and 2 in supplementarymaterial). Filopodia protrude from the periphery oflamellipodia and display an exploratory behavior with quickretraction and extension. They were often seen to precede

lamellipodium formation. Alternatively, filopodia-likestructures are often the apparent remnants of collapsinglamellipodia. Lamellipodia are well-preserved inglutaraldehyde-fixed material (Fig. 2Q). In contrast, usingDIC/Nomarski optics or laser confocal microscopy we werenot able to visualize lamellipodia in macrophages fixed withformaldehyde and labeled for cytoplasmic β-Gal, or withantibodies against peroxidasin, which accumulates in theextensive endoplasmic reticulum and other compartments ofthe biosynthetic pathway (e.g. Fig. 2R). In such preparations,most migratory macrophages appear spindle-shaped, whilemacrophages that contain many phagosomes are large, roundedcells. Taken together, these observations show that migratingmacrophages extend dynamic membrane extensions thatare comparable to lamellipodia and filopodia. A largelamellipodium polarized in the direction of movement is thepredominant extension or embryonic macrophages.

Function of Rho GTPases in macrophage migrationTo examine the function of Rho GTPases in embryonicmacrophage migration, the effects of expressing DN and CARho GTPase isoforms under the control of gcm-Gal4 and Coll-Gal4 drivers were examined. The phenotypes observed werecompared to those of embryos carrying mutations in RhoGTPase genes.

RacThe expression of both the DN (Rac1N17) and CA (Rac1V12)isoforms of Rac1 under the control of gcm-Gal4 blocked

Fig. 4. Rac activity is essential to maintain normalmorphology and distribution of macrophages inlate embryos. The activity of Rac was disrupted byexpressing mutant Rac1 isoforms under the controlof Coll-Gal4. UAS-lacZ was co-expressed to labelmacrophages and embryos were stained with anti-β-Gal antibody. (A,C,E,G) Stage 17 embryos;(B,D,F,H), close-up of stage 17 embryos, focusingon the ventral area between the epidermis and theventral cord. (A,B) Macrophages are evenlydistributed in a stage 17 wild-type embryos(A) and have a spindle-like morphology (B).(C,D) Rac1N17 causes macrophages to clump invarious areas of the embryo (C), and blocks theformation of cellular protrusions causingmacrophages to appear rounder (D). (E,F) Rac1V12

causes strong clustering of macrophages and as aresult, large areas of the embryo are devoid ofmacrophages (E). Rac1V12-expressingmacrophages extend longer, more prominentcellular protrusions (F) than wild-typemacrophages (B). (G,H) Rac1L89 inducesmacrophages to cluster (G), but the phenotype ismilder than observed with Rac1N17 or Rac1V12.Also Rac1L89-expressing macrophages extendmore prominent protrusions (H) as seen uponexpression of Rac1V12 (F).

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macrophage migration. Most macrophages expressing Rac1N17

failed to migrate and remained around the foregut forminga tight cluster. This defect persisted until the end ofembryogenesis (Fig. 3C,D). A few macrophages were seen inthe head and tail of the embryos, suggesting that some cellshad the ability to migrate, although they moved only a shortdistance.

Macrophages expressing Rac1V12 showed similar, but moredrastic defects (Fig. 3E,F). Most cells were unable to migrateand remained clustered anteriorly. The number of migratorymacrophages was even smaller than seen upon expression ofRac1N17. Macrophages expressing either DN or CA Rac1 werepositive for macrophage-specific markers such as peroxidasin(Nelson et al., 1994), a basement membrane protein producedby macrophages (Fig. 3A-F) and Croquemort (Cqr) (Franc etal., 1996), a receptor required within macrophages for theefficient uptake of apoptotic cells (Fig. 3I-K), suggesting thatthe phenotype caused by altered Rac1 activity cannot beattributed to defects in cell fate determination. These findingssuggest that the correct regulation of Rac activity is essentialfor macrophage migration.

To address the question of whether Rac function is alsorequired during late embryonic stages when macrophages showlocal motility, the expression of the various Rac1 isoforms wasexamined using the Coll-Gal4 driver that is expressed inmacrophages after they are dispersed throughout the embryo.The expression of Rac1N17 under the control of Coll-Gal4caused the macrophages to cluster at various sites, such as thehead region, around the pharynx, midgut and hindgut, andlaterally along the ventral cord. The deviation from wild typewas evident at stage 16, but became more pronounced duringstage 17 (Fig. 4C,D). Coll-Gal4 UAS-Rac1V12 embryos showedan even more severe clustering of macrophages than observedwith Rac1N17. The clusters appeared larger and tighter andlarge areas of the embryo were devoid of macrophages (Fig.4E,F). In addition to abnormal clustering, macrophagesdisplayed abnormal cell shapes in response to expression ofmutant Rac1 isoforms. Rac1N17 blocked the extension ofprotrusions resulting in rounded cell shape (Fig. 4D), whereasRac1V12 caused macrophages to adopt a more elongated andspindle-like shape compared to wild type, and to display morepronounced extensions (Fig. 4F). These findings suggest thatnormal Rac activity is required throughout embryogenesis tocontrol macrophage motility.

The expression of a third mutant isoform of Rac1, Rac1L89,

in macrophages caused these cells to migrate slower than inwild type although defects were mild in comparison to thosecaused by expression of Rac1N17 and Rac1V12. In wild-typestage 13 embryos when the germband is retracted,macrophages from the head and tail regions migrate toward themiddle of the embryo along the ventral cord. At this stage theanterior and posterior groups of macrophages have not met inthe center of the embryo, and the ventral part of the ventralcord of abdominal segments A3-A5 is devoid of macrophages


Fig. 5. F-actin accumulation and lamellipodia formation inmacrophages with altered Rac activity. Stage 14 embryos stainedwith anti-peroxidasin (red) to label the macrophages and rhodamine-conjugated phalloidin (green) to label F-actin. (A) In wild-typemacrophages, F-actin is localized mostly in the cell cortex.(B) Macrophages expressing Rac1N17 show F-actin levels anddistribution indistinguishable from wild type. (C) Macrophagesexpressing Rac1V12 contain strongly elevated levels of F-actin.(D-F) TEM of macrophages at stage 14. (D) Macrophages expressingRac1N17 are round and have few or no cytoplasmic extensions. Themacrophages shown are located close to the foregut and contain onlyfew phagosomes (arrows). (E) Macrophages expressing Rac1V12,which cluster around the foregut, show an increased number ofcytoplasmic protrusions, and occasionally contain phagosomes(arrows). (F) Some of the macrophages expressing Rac1V12 containtwo nuclei (arrowheads). Bars, 10 µm (D-F).


(Fig. 3A). In subsequent stages, the entire ventral cord iscovered by macrophages (Fig. 3B). Analyzing whenmacrophages meet on the ventrally on the ventral cord providesa good measure to determine the speed of macrophagemigration. Macrophages in embryos that express Rac1L89

under the control of gcm-Gal4 did not spread over the ventralsurface of the ventral nerve cord at stage 13 (Fig. 3G). At stages15, the abdominal segments A3-A7 of these embryos stillremained devoid of macrophages (Fig. 3H). Expression ofRac1L89 under the control of Coll-Gal4 induced someclustering of macrophages, and accentuated the spindle-likeshape of macrophages similar to Rac1V12 (Fig. 4G,H). Previouswork has reported both loss-of-function and gain-of-functionactivities for Rac1L89 (e.g. Luo et al., 1994; Kaufmann et al.,1998). Our observations suggest that Rac1L89 functions as aweak CA isoform in macrophages.

To determine how Rac functions in regulating macrophage

morphology, the filamentous actin (F-actin) content ofmacrophages was analyzed. In wild-type macrophages, lowlevels of F-actin were dispersed around the cell periphery (Fig.5A). The membrane extensions characteristic of macrophagescould not be discerned with this staining procedure.Macrophages expressing Rac1N17 showed wild-type levels ofcortical F-actin (Fig. 5B). In contrast, Rac1V12-expressingmacrophages displayed strongly elevated levels of F-actin (Fig.5C). Marcophages that cluster around the foregut in Rac1N17-and Rac1V12-expressing embryos were examined withtransmission electron microscopy (TEM) to analyze cellmorphology. The expression of Rac1V12 elicits the formationof an excessive number of lamellipodia (Fig. 5E),corresponding to the dramatic accumulation of F-actin,whereas Rac1N17 was found to block the formation ofmembrane extensions (Fig. 5D). TEM preparations alsorevealed that some macrophages expressing Rac1V12, but notmacrophages expressing Rac1N17, contain two nuclei (Fig. 5F).15-20% of the macrophages that expressed Rac1V12 and whosenuclei were visible in the section, were binucleate. The actualfraction of macrophages that contain two nuclei is presumablyhigher as the plane of sectioning may not reveal both nuclei.This observation suggests that activation of Rac blockscytokinesis in macrophage progenitors. In addition, TEMpreparations of embryos expressing either the DN or CA Rac1isoform show that at least some of these macrophages containapoptotic cells (Fig. 5D,E), although the number ofmacrophages that display phagocytotic activity is much lowerthan in wild-type. Taken together, our findings suggest that Racactivity plays a key role in macrophage migration bycontrolling actin organization and lamellipodia formation.

We also analyzed loss-of-function mutations of the threeDrosophila Rac genes. Animals that lack either Rac1, Rac2 orMtl activity are viable and do not show defects in macrophagemigration. Embryos double mutant for any combination of nullmutations of two Rac genes, or triple mutant embryos carryingthe null mutations Rac2∆ and Mtl ∆ as well as the hypomorphicallele Rac1J10 did not display defects in macrophage migration(data not shown). Rac genes have maternal components ofexpression (Hakeda-Suzuki et al., 2002) that could supportnormal macrophage migration even in the absence of zygoticRac expression. Thus, we next analyzed embryos derived fromRac1J10 Rac2∆ germline clones that were homozygous mutantfor Rac1J10 and Rac2∆, and embryos derived from Rac1J10

Rac2∆ germline clones in a Mtl∆ mutant background that werehomozygous mutant for all three Rac genes. The hypomorphicallele Rac1J10 rather than a Rac1 null allele was used in theseexperiments as complete removal of Rac1 and Rac2 activityfrom the germline blocks germline development or earlyembryogenesis (Hakeda-Suzuki et al., 2002). Rac1J10 Rac2∆

maternal and zygotic mutant embryos displayed mild defects inmacrophage migration as macrophages did not disperse into theventral posterior trunk region by stage 14 or 15 (Fig. 6B). Bylate embryogenesis macrophages had dispersed throughout theentire ventral trunk region in these embryos. In contrast, siblingembryos that derived from Rac1J10 Rac2∆ germline clones buthave single paternal wild-type copy of Rac1 and Rac2 show anormal distribution of macrophages (Fig. 6A). Surprisingly,embryos maternal and zygotic mutant for Rac1J10 Rac2∆ andMtl∆ displayed the same macrophage migration defects (Fig.6C) as embryos that lack Rac2 and have reduced Rac1 activity

Fig. 6. Defects in macrophage migration in Rac1, Rac2 and Mtlmutant embryos. All panels show stage 14 embryos double-labeledfor anti-peroxidasin (green) to identify macrophages and anti-β-Gal(red) to identify embryos that carry a TM6 balancer chromosome,carrying wild-type alleles of Rac1, Rac2 and Mtl. (A) Embryoexpressing β-Gal that was derived from a Rac1J10 Rac2∆ mutantgermline clone but is heterozygous for Rac1J10 and Rac2∆ as itreceived a paternal copy of TM6. Macrophages show a normaldistribution. (B) Homozygous Rac1J10 Rac2∆ mutant embryo derivedfrom a Rac1J10 Rac2∆ germline clone. Macrophages have failed topopulate the posterior trunk region (between arrows).(C) Homozygous Rac1J10 Rac2∆ Mtl∆ mutant embryo derived from aRac1J10 Rac2∆ Mtl∆ germline clone. Again, macrophages have failedto populate the posterior trunk region (between arrows). Greenlabeling between arrows represents basement membrane staining byanti-peroxidasin antibody that is less prominent in A and B owing tovariations in staining intensities. Bar, 100 µm.

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(Fig. 6B) – macrophages did not populate the posterior trunkregion by stages 13-15, but showed normal dispersal in lateembryos. These results suggest that Rac1 and Rac2 actredundantly to promote macrophage migration and that Mtldoes not contribute significantly to macrophage motility.

Cdc42The expression of DN Cdc42N17 or CA Cdc42V12 under thecontrol of either gcm-Gal4 or Coll-Gal4 did not interfere withthe migration or distribution of macrophages (Fig. 7).Surprisingly, in embryos that have reduced maternal and nozygotic Cdc42 expression (hereafter referred to as Cdc42MZ

mutant embryos) the ventral cord is not covered bymacrophages. Also the ubiquitous expression of Cdc42N17,using a da-Gal4 driver, causes embryonic defects similar tothose observed in Cdc42MZ mutants including defects inmacrophage migration (data not shown). Ubiquitousexpression of Cdc42V12 elicits defects in the entire embryo sosevere that macrophage dispersal could not be evaluated. Thesefindings suggest that Cdc42 is required in cells other than themacrophages to promote macrophage migration. In Cdc42mutant embryos, germband retraction is defective (Genova etal., 2000). As most of the posterior half of the ventral cord getspopulated with macrophages carried posteriorly during

germband retraction, it was important to establish whetherdefects in germband shortening affect the spatial and temporaldistribution of macrophages. To test this, hindsight (hnt)mutant embryos, which fail to undergo germband retraction(Yip et al., 1997), were analyzed for alterations in macrophagedistribution. These embryos show a normal macrophagedispersal (data not shown), suggesting that germbandretraction, which carries macrophages to the posterior pole ofthe egg in wild type, is not essential for macrophage dispersalthroughout the embryo.

Macrophages expressing either Cdc42N17 or Cdc42V12 underthe control of gcm-Gal4 and Cdc42MZ embryos displayed wild-type cytoplasmic protrusions (Fig. 7B,E,H) and had normal F-actin content (data not shown). Unexpectedly, the expressionof Cdc42V12 under the control of Coll-Gal4 resulted in themacrophages becoming round with only short protrusionsbeing extended (Fig. 7F). In contrast, macrophages expressingCdc42N17 under the control of Coll-Gal4 (Fig. 7C) did notshow an altered morphology, nor did macrophages in Cdc42MZ

mutant embryos (not shown). In addition, the expression ofCdc42V12 under the control of gcm-Gal4 caused an increase inthe size of macrophages and a reduction in their number toapproximately 400 (n=4; Fig. 7D) compared to wild-typeembryos, which have about 700 macrophages (Tepass et al.,1994). Furthermore, TEM analysis of these embryos revealed


Fig. 7. Effects of altered Cdc42 activity on macrophage. (A,D,G) Stage 15 embryos stained with anti-peroxidasin antibody to labelmacrophages. 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 14embryos. Macrophages expressing Cdc42N17 (B), or Cdc42V12 (E), or macrophages in Cdc42 mutant embryos (H) display wild-typemorphology with normal membrane extensions and presence of phagosomes (arrows). Some Cdc42V12-expressing macrophages contain twonuclei (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-β-Gal antibody. Each panel shows a close-up of a whole-mount stage 17 embryo, focusing on the ventral areabetween 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 roundedmorphology. (I) Wild-type embryo. Bars, 10 µm (B,E,H).


a fraction of binucleate cells among macrophages thatexpressed Cdc42V12 (Fig. 7E). This suggests that Cdc42V12,similar to Rac1V12, blocks cytokinesis in hemocyte progenitors,which would account for the enlargement of macrophage cellsand the decrease in macrophage number in those embryos.

Rho1The expression of DN Rho1N19 under the control of either gcm-Gal4, Coll-Gal4 or da-Gal4 did not alter the migration ofmacrophages, nor their distribution or morphology (data notshown). Consistent with these observations, macrophagedefects were not observed in Rho1 mutant embryos. A CAisoform of Rho1 was not available, but a CA isoform of thehuman RhoA had been generated (Harden et al., 1999). Theexpression of this construct under the control of gcm-Gal4 hadno effect on macrophage migration (data not shown). Thus,Rho1 has no apparent role in macrophage migration.

RhoLRhoL/Rac3 was identified as a novel Drosophila Rho protein(Murphy and Montell, 1996; Sasamura et al., 1997). Theexpression of DN RhoLN25 or CA RhoLV20 under the controlof gcm-Gal4 or da-Gal4 did not interfere with the migrationof macrophages. This result was unexpected as RhoL transcriptwas shown to be enriched in macrophages (Sasamura et al.,1997). Interestingly, in embryos with a deficiency that coversthe RhoL locus (Df(3R)by416), macrophages fail to populatethe ventral side of the ventral cord, causing a phenotype similarto that of sim mutants (Fig. 8B,C). This raises the possibilitythat RhoL may function in macrophage migration and that theRhoLN25 and RhoLV20 constructs are ineffective.

A genetic screen for macrophage migration defectsTo identify genes whose zygotic expression is required for

embryonic macrophage migration, a systematic screen ofchromosomal deletions was carried out to reveal mutants inwhich the migration process is disrupted. We analyzed 208deficiencies received from the Bloomington Drosophila StockCenter that cover over 80% of the Drosophila genome.Eighteen of the 208 lines produced mutant embryos with severemorphological defects or an early developmental arrest thatprecluded an evaluation of defects in macrophage migration.We found eight lines that produced embryos with clear defectsin macrophage migration. In one group of mutants thatincludes Df(2L)s1402, Df(3R)e-N19 and Df(2L)ast2,macrophages fail to migrate along the ventral cord, and muchof the ventral abdomen remains free of macrophages (Fig. 8D-F). This phenotype resembles that described for single minded(sim) mutants (Fig. 8B) (Zhou et al., 1995). In a second groupof mutants that include Df(2L)TE29Aa-11, Df(3L)66C-G28,Df(2L)N22-14, Df(3R)mbc-R1 and Df(3R)mbc-30, little if anymigration occurs and most macrophages remain in the anteriorregion, clustered around the foregut (Fig. 8G-K). Thisphenotype is similar to the phenotype induced by theexpression of DN or CA Rac1 under the expression of gcm-Gal4, or the slightly weaker phenotype described for embryosthat lack the Pvr receptor (Cho et al., 2002; Sears et al., 2003).In fact Df(2L)TE29Aa-11 uncovers the Pvr locus. Df(3R)mbc-R1 and Df(3R)mbc-30 are overlapping deficiencies thatuncover myoblast city (mbc) the Drosophila ortholog ofmammalian DOCK180, a known regulator of Rac activityduring cell migration (Erickson et al., 1997; Nolan et al., 1998;Raftopoulou and Hall, 2003). To confirm mbc requirement formacrophage migration we examined embryos homozygousmutant for either of two mbc alleles (mbcC1, mbcD11.2). To oursurprise we found that mbc mutant embryos did not show anyapparent defects in macrophage migration although theydisplayed the previously described defects in muscledevelopment (data not shown). This finding suggests thatDf(3R)mbc-R1 and Df(3R)mbc-30 uncover a gene other thanmbc that is required for macrophage migration.

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-β-Gal antibodies (G,J,K) to highlight macrophages. Genotypes are indicated on panels.

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DiscussionIn the Drosophila embryo, macrophages migrate over theinternal surfaces of organs that are covered with basementmembranes. The space between organs is not filled with matrixaside from the basement membrane (Tepass and Hartenstein,1994; Tepass et al., 1994) suggesting that Drosophilamacrophage migration is more related to a two- rather than athree-dimensional cell culture system. Macrophages show anelongated, polarized morphology typical for many migratingcell types. The leading edge of macrophages is characterizedby a long and broad lamellipodium that often extends twice thelength of the main cell body. From the trailing end, chunks ofcytoplasm may be lost as the cell moves forward. Thelamellipodia of Drosophila embryonic macrophages havepreviously been described using TEM analysis but mis-identified as filopodia (Tepass et al., 1994). Lamellipodiaformation is highly dynamic with a lamellipodium beingextended and retracted within several minutes. Whilemacrophages also extend filopodia, most filopodia-likestructures we have observed appear to be the remnants ofcollapsing lamellipodia. Recent cell culture work onDrosophila S2R+ cells, which are believed to have originatedfrom embryonic hemocytes, also reveals lamellipodia as thepredominant extension. Disruption of SCAR, a downstreamtarget of Rac, in S2R+ cells leads to the collapse oflamellipodia, leaving in their wake tread-like protrusions thatresemble filopodia, but contain a branched filament networkrather than a parallel bundle of filaments (Biyasheva et al.,2004).

We have generated two Gal4 driver lines that are expressedin migrating macrophages of the Drosophila embryo.Combined, both Gal4 lines promote expression of UAS-controlled target genes throughout the entire period ofembryonic macrophage migration. gcm-Gal4 is expressed inhemocyte progenitors and in all macrophages up to at leaststage 15, encompassing phase I and II of macrophagemigration. In contrast, Coll-Gal4 drives expression initially atstage 13 in some macrophages, and high levels of expressionare seen from stage 15 to the end of embryogenesis in allmacrophages, representing phase III of macrophage migration.gcm-Gal4 is also expressed in glial cells and in epidermalstripes, and Coll-Gal4 in the fatbody. Thus, these driver linesare useful tools to analyze the development of macrophagesand some other cell types.

gcm-Gal4 and Coll-Gal4 were used to express DN and CAmutant isoforms of the GTPases Rac1, Cdc42, Rho1 and RhoLin macrophages. Only the DN and CA forms of Rac1 interferewith macrophage migration cell-autonomously. Rac1N17

prevents the formation of lamellipodia while Rac1V12 causesan extensive formation of lamellipodia accompanied byincreased accumulation of F-actin. Also Rac1L89 promoteslamellipodia formation suggesting that this isoform acts as aweak activated form of Rac1. The effects of modulating Rac1activity on actin polymerization, lamellipodia formation andcell migration in Drosophila macrophages are similar to thoseobserved in many other cell types in invertebrates andvertebrates (Hall, 1998; Ridley, 2001), confirming a generalrole for Rac as a key regulator of cell migration.

The analysis of mutant embryos also demonstrates arequirement for Rac activity in macrophage migration.

Embryos that lack Rac2 activity completely and have astrongly reduced Rac1 activity show a clear delay in migration.This defect is not enhanced by the complete loss of the activityof the third Drosophila Rac gene Mtl, suggesting that Mtl doesnot function in macrophage migration. As embryos thatcompletely lack the function of either Rac1 or Rac2 shownormal migration, Rac1 and Rac2 must act redundantly duringthis migration process. The migration defects caused by Racgene mutations are substantially weaker than those elicited bythe expression of Rac1N17. One possible explanation for thisdiscrepancy is that the hypomorphic Rac1J10 allele, which weneeded to use instead of a null allele to generate germlineclones with reduced Rac activity, provides sufficient functionto support largely normal migration. Alternatively, it is possiblethat expression of Rac1N17 not only compromises the activityof Rac1 and Rac2 but also other cellular processes essential formigration, as is apparently the case in planar cell polarity(Hakeda-Suzuki et al., 2002). For example, Rac1, Rac2 andCdc42 may act redundantly in macrophage migration andRac1N17 disrupts the activity of all three GTPases. This, andother potential scenarios need to be addressed in future studies.

How is Rac activity controlled in macrophages? Mbc, theDrosophila DOCK180 ortholog and a known upstreamregulator of Rac in other cell migration processes (Duchek etal., 2001; Raftopoulou and Hall, 2004), has no essential rolein macrophage migration as mbc mutant embryos do notdisplay macrophage migration defects. Pvr, the Drosophilahomologue of the mammalian PDGF/VEGF receptor, is areceptor tyrosine kinase that may act upstream of Rac. Pvr actsupstream of Rac in the migration of border cells in theDrosophila ovarian follicle (Duchek et al., 2001) and itsactivity is required for normal embryonic macrophagemigration (Cho et al., 2002; Sears et al., 2003; Brückner et al.,2004). Pvr acts primarily as a trophic factor in macrophages asmost macrophages undergo apoptosis in Pvr mutant embryos(Brückner et al., 2004). This function of Pvr is mediated largelythrough the Ras/MAPK pathway (Brückner et al., 2004).Consistently, we did not find evidence for macrophage celldeath in Rac mutants or embryos expressing mutant Rac1isoforms. Surviving macrophages in embryos that lack Pvrengage in ‘cannibalistic phagocytosis’, which apparently slowstheir dispersal through the embryo. Blocking programmed celldeath in Pvr mutants prevents macrophage cell death. In theseembryos mild macrophage migration defects are observed(Brückner et al., 2004) that are similar to those we have seenin embryos that lack maternal and zygotic Rac2 and havestrongly reduced activity of Rac1. Thus, the control of Racactivity by Pvr to promote macrophage migration is anattractive possibility.

Expression of DN Rho1 and CA human Rho1 did notinterfere with macrophage migration, nor did null mutations inRho1 compromise migration. To our surprise, we also did notfind an effect of the DN or CA isoforms of Cdc42 – whenspecifically expressed in macrophages – on the migration speedor distribution of these cells, which suggests that Cdc42 is notrequired in macrophages for their migration. Expression ofCdc42V12, similar to Rac1V12, blocks cytokinesis in hemocyteprogenitors indicating that its expression is effective. In othermigrating cells, Cdc42 functions in several processes criticalfor migration including cell polarization, microtubuleorganization and filopodia formation (Etienne-Manneville and



Hall, 2002; Fukata et al., 2003; Raftopoulou and Hall, 2003;Ridley et al., 2003). Blocking Cdc42 function in mammalianmacrophages prevents filopodia formation. These macrophagesare still able to migrate but fail to follow a cytokine gradient(Allen et al., 1997; Allen et al., 1998). If expression ofCdc42N17 in Drosophila macrophages would have similareffects, one might expect migration defects, in particularduring phase II of macrophage motility when large scale, mostlikely guided, migration takes place, which we did not observe.However, macrophages show migration defects in embryos thatexpress Cdc42N17 uniformly in all tissues as well as in Cdc42mutant embryos. In both types of embryos, macrophages failto populate the ventral trunk region. Migration may either besubstantially slower in those embryos or macrophages fail tobe specifically attracted to this region of the embryo. A similarmigration defect has been described for embryos mutant forsim, which lack the midline cells of the central nervous system(Zhou et al., 1995), and for Pvr mutant embryos, ligands ofwhich are expressed in the ventral nerve cord (Cho et al., 2002;Brückner et al., 2004). This raises the possibility that Cdc42plays a role in midline development or the secretion of Pvrligands.

Finally, we have investigated the effect on macrophagemigration of 190 chromosomal deletions that coverapproximately 75% of the Drosophila genome. Additionaldeletions studied, which together cover about 12% of thegenome, were not informative because phenotypes weredifficult to interpret because of severe morphologicalaberrations of the embryo, arrest of embryonic developmentprior to macrophage development or the loss of mesoderm asa consequence of the loss of genes required for mesodermspecification, such as twist and snail. We found eight lines thatshowed defects in macrophage distribution. For statisticalreasons it is likely that most of these deletions uncover only asingle gene that is essential for migration. Mutants withmacrophage migration defects include deletions that uncovergenes previously shown to be required for macrophagedevelopment such as Pvr (Df(2L)TE29Aa-11) (Cho et al.,2002; Sears et al., 2003), gcm and gcm2 [Df(2L)N22-14 andDf(2L)s1402 – both deficiencies uncover gcm and gcm2(Bernadoni et al., 1997; Alfonso and Jones, 2002)]. We note,however, that the migration defects in Df(2L)N22-14 mutantembryos (by contrast to Df(2L)s1402 mutants) appear strongerthan those reported for gcm and gcm2 double mutantssuggesting that this deletion may uncover another gene thatcontributes to macrophage migration. A deletion that uncoverssim was missing from our collection. Interestingly, a deletionthat covers RhoL causes migration defects, raising thepossibility that RhoL is required for macrophage migration,although the expression of a DN form of RhoL did not resultin migration defects. Together, these findings suggest that theDrosophila genome may encode 10-12 genes whose zygoticcomponents of expression are essential for macrophagemigration. This is a relatively small number of genesconsidering the known complexity of the cellular machineryinvolved in cell migration, suggesting that other migrationfactors are either maternally provided or act redundantly.

We thank Frank Laski for suggesting the P element replacementstrategy to us. We are grateful to Lisa and John Fessler, BarryDickson, Liqun Luo, Nathalie Franc, Marek Mlodzek, Denise

Montell, Lynn Cooley, Nick Harden and the Bloomington DrosophilaStock Center for fly stocks and reagents. We thank Jennifer Liaw andRobert Pascal for help in early stages of this project, and we thankDorothea Godt for critical comments on the manuscript. This workwas supported by a grant from the Natural Sciences and EngineeringResearch Council of Canada.

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