the active migration of drosophilaprimordial germ cells

3495Development 121, 3495-3503 (1995)Printed in Great Britain © The Company of Biologists Limited 1995DEV3273

The active migration of Drosophila primordial germ cells

Mariusz K. Jaglarz1 and Kenneth R. Howard2,3

Roche Institute of Molecular Biology, Roche Research Center, 340 Kingsland Street, Nutley, NJ 07110, USA1Present address: Department of Zoology, Jagiellonian University, R. Ingardena 6, 30-060 Krakow, Poland2E-mail: [email protected] from July 1996: University College London, Gower Street, London WC1E 6BT

We describe our analysis of primordial germ cell migrationin Drosophila wild-type and mutant embryos using highresolution microscopy and primary culture in vitro. Duringmigratory events the germ cells form transient interactionswith each other and surrounding somatic cells. Both in vivoand in vitro they extend pseudopodia and the accompany-ing changes in the cytoskeleton suggest that actin poly-merization drives these movements.

These cellular events occur from the end of the blasto-derm stage and are regulated by environmental cues. Weshow that the vital transepithelial migration allowing exitfrom the gut primordium and passage into the interior of

the embryo is facilitated by changes in the structure of thisepithelium.

Migrating germ cells extend processes in different direc-tions. This phenomenon also occurs in primary culturewhere the cells move in an unoriented fashion at substra-tum concentration-dependent rates. In vivo this migrationis oriented leading germ cells to the gonadal mesoderm. Wesuggest that this guidance involves stabilization of states ofan intrinsic cellular oscillator resulting in cell polarizationand oriented movement. Key words: primordial germ cells, cell migration, gonadogenesis,Drosophila

SUMMARY

INTRODUCTION

In many invertebrate and vertebrate animals, the formation ofprimordial germ cells (PGCs) is spatially and temporarilyseparated from the somatic gonad (Nieuwkoop and Sutasurya,1979, 1981). Often PGCs form early during development anddisplay elaborate behavior including an active search for thesomatic gonad. In Drosophila melanogaster PGCs form at theposterior pole of the embryo in a process driven by interactionof the centrosome, actin and myosin (reviewed in Schejter andWieschaus, 1993). Initially, PGCs lie outside the blastoderm andduring gastrulation are included in the amnio-proctodeal invagi-nation. This invagination elongates during germ band extensionto form the primordia of both the hindgut and posterior midgut(PMG), which develops at the distal, blind end of the invagina-tion (reviewed in Skaer, 1993). By early embryonic stage 10,PGCs lie packed at the end of the PMG. Subsequently, theyleave its lumen and associate with the somatic gonad.

These events were first described by Rabinowitz (1941) andSonnenblick (1941). More recently further studies of PGCmigration have used specific markers for the PGCs and vitaldyes in living embryos (Hay et al., 1988; Jaglarz and Howard,1994; Warrior, 1994). These studies show that the somatictissue regulates the time of exit of the PGCs (reviewed in Weiand Mahowald, 1994).

Earlier morphological studies of this process have suggestedeither that exit occurs by formation of a discontinuity in thegut tissue or by PGCs insinuating themselves through the gutepithelium (Campos-Ortega and Hartenstein, 1985 and refer-ences therein). Here we resolve this question showing that

there is a novel and specific interaction between the PGCs andthe cells of the PMG and that the PGC transendodermalmigration is permitted by changes in the structure of the PMG.

MATERIALS AND METHODS

For light and electron microscopy (EM), embryos were collected at25°C on apple juice plates and staged under oil. After chorion removalin 50% commercial bleach (Clorox), the embryos were extensivelywashed with distilled water and fixed at the interface of 50% glu-taraldehyde and heptane for 10 minutes, transferred to sticky tape andthe vitelline membranes removed manually with a tungsten needleunder 2.5% glutaraldehyde in either 0.1 M phosphate or cacodylatebuffer, pH 7.2. The embryos were fixed for 2 hours in glutaraldehydeand after several washes postfixed in 1% OsO4 in the same buffer.After dehydration in a graded series of ethanol and acetone theembryos were embedded in Polybed 812 (Polysciences). Sectionswere stained with 1% methylene blue in 1% borax for lightmicroscopy or contrasted with uranyl acetate and lead citrate, andexamined in a JEM 1200 EX, electron microscope at 80 kV.

A total of 75 wild-type and mutant embryos at stages 9-11 werefixed and 5 per stage were picked for sectioning. They were examinedfirst as semithin then ultrathin sections. In areas of interest, serialultrathin sections were examined and interpreted in conjunction withdata from light and confocal microscopes. PGCs are easy to recognizein the EM by lipid-deficient cytoplasm and the presence of polargranules (Allis et al., 1979).

For phalloidin (specific recognition of F-actin, Wulf et al., 1979)and immunostaining, the embryos were collected and staged asdescribed above and fixed with 8% paraformaldehyde. After hand-peeling, the embryos were stained with anti-vasa antibody and

3496 M. K. Jaglarz and K. R. Howard

fluorescein-conjugated secondary reagents (Jackson Immuno Res.).Following several washes in PBS/0.1% Triton X-100, the embryoswere stained with rhodamine-labeled phalloidin (Molecular Probes)for 20 minutes, dehydrated in ethanol, mounted in methyl salicylateand examined with a Bio-Rad MRC 600 confocal microscope,equipped with a Krypton-Argon laser. At least 10 embryos wereoptically sectioned for each embryonic stage (stages 5-16).

In vitro experimentsExtracellular matrix proteins (EMPs) were obtained from Collabora-tive Biomedical Products and prepared according to the manufac-turer’s recommendations. PGCs were removed from embryos atstages 4-7 with glass transplantation needles and placed on a coverslip(coated with the EMPs at different concentrations, Table 1) in a dropof Schneider’s medium (Sigma) supplemented with 10% bovineserum. The coverslip was then carefully transferred to a culture dishand more medium added. In the case of Matrigel, both the thin coatingand the thick gel method were used. In the latter, PGCs were culturedinside the Matrigel. The cells were observed at 18°C and the imagesrecorded using video equipment.

StockshkbA/TM3β-gal; hkb homozygous embryos were identified by virtueof their lack of expression of β-gal and for EM examination by lackof the stomodeum and anterior midgut invaginations (Reuter and

Fig. 1. Optical sections through embryos at various stages of developmevisualize F-actin (red). Both channels are superimposed but in some Figdistribution. (A) The posterior pole of an embryo during blastoderm forF-actin. Arrowhead, F-actin accumulation in the apical region of the blawith variably shaped pseudopodia. (C) Higher magnification of the regiaround the pseudopodia. Arrows, F-actin aggregates. (D) The PMG at sregion of the endodermal epithelium. (E) Prior to leaving the lumen of tThey penetrate the epithelium in several different places. One PGC is se(star) wedged between endodermal cells. Arrowheads, regions of reducetransendodermal migration. Note the irregular shape and polarization ofPGC lined with F-actin; arrowheads, F-actin accumulation in the leadininteraction between cytoplasmic processes of migrating PGCs. Arrows,meet. Bars: A,B,G 10 µm; C, 5 µm; D, 20 µm; E,F 15 µm.

Leptin, 1994); lam A6-26 A101/TM6β-gal; stg7M/TM3; osk301/TM3.OreR was used as the wild-type stock.

RESULTS

Initially PGCs are almost perfectly spherical with a well-developed layer of cortical F-actin (Fig. 1A). Extensivechanges in shape are first seen at the onset of gastrulation whenPGCs become elongated and extend broad pseudopodia (Fig.1B,C). These changes are accompanied by rearrangements ofthe actin cytoskeleton. The cortical layer of F-actin is pro-nounced in the body of the PGCs but thins around thepseudopodia (Fig. 1C). In the cytoplasm of both the pseudopo-dia and the body of the cells, there are punctuate focal accu-mulations of F-actin. As they enter the lumen of the gut pri-mordium during germ band elongation, PGCs are in closecontact with each other and their pseudopodia are extended indifferent directions (Fig. 1D). As germ band elongationproceeds, they pack into the blind end of the PMG.

At this time both the hindgut and posterior midgut primordiaform a single-layered epithelium, with cells closely apposed toeach other (Figs 1D, 2A). In stages 9 and early 10 in the apicalregion of this epithelium, two types of specialized contact are

nt stained with anti-vasa antibody (green) and Rh-phalloidin tos the red channel is shown separately to better illustrate the F-actinmation. Pole cells are spherical with a well developed cortical layer ofstoderm. (B) The posterior pole of a gastrulating embryo. Note PGCson shown in the rectangle in B. The cortical layer of F-actin thinstage 9. Note the intense staining (arrowheads) associated with the apicalhe PMG (stage 10), PGCs maintain contact with each other (arrows).en inside the epithelium (asterisk), another has a large pseudopodiumd phalloidin staining (compare with D). (F) PGCs in the process of the cell passing through the epithelium. Arrow, elongated tail of theg edge. (G) Projection of three optical sections (1µm apart) reveals the fibers of F-actin; star, the area where PGC cytoplasmic extensions

3497Migration of Drosophila primordial germ cells

Semithin sections through the gut primordium at progressively olderarly stage 9. PGCs (asterisks) fill the lumen of the gut primordiumlosely adhere to each other. Arrow, an endodermal cell during a

ion; y, yolk. (B) Early stage 10. Cells of the PMG form a single-elium. Note the cytoplasmic extensions in the luminal surface of theell below a PGC marked with an asterisk. (C) Late stage 10. Two

sks) are visible in different stages of the transendodermal migration.n the intercellular space between the endodermal cells. The other, inween the PMG and the yolk sac. Note large spaces (arrows) which areeen cells only in the distal region of the gut primordium. (D) Apicalen cells of the PMG at stage 9. Arrow, adherens junction; arrowheads, (E) In the apical region of the PMG at stage 10 interdigitatedcells do not adhere closely to each other and intercellular spaces (star)etween them. Bars: A-C 10 µm; D,E 500 nm.

observed: adherens junctions (in the form of zonula adherens)and gap junctions (Fig. 2D). There are also distinct surface spe-cializations of this epithelium. The luminal surface of the cellsof the hindgut primordium are smooth or have only very shortprojections. In contrast, the luminal surface of the cells of thePMG is characterized by the presence of up to 2 µm long cyto-plasmic protrusions which contact PGCs (Figs 2B, 3A,B).These cytoplasmic ‘arms’ contain axial microtubules (Fig. 3B).Fine intercellular spaces between endodermal cellsare observed. (Figs 2B, 3A).

At the extended germ band stage, PGCs arefound inside a cup-shaped formation of endodermalcells in the distal part of the PMG and are separatedfrom each other by cytoplasmic extensions of thesomatic cells (Fig. 3C). At this time the PGCs showa characteristic ‘tear drop’ form, with the side ofthe PGCs in close contact with the surroundingsomatic cells being broad and rounded.

Following this stage, there is a drop in the F-actinlevel in the apical region of the endodermal cellsand the distribution of cellular junctions changes(Fig. 1E). Intercellular spaces between neighboringcells become more pronounced (Fig. 2C) andadherens junctions no longer form continuous beltsaround the cells apices (Fig. 2E).

The transendodermal migration occurs in severalplaces in the blind region of the PMG (Fig. 1E).During this transit, cytoplasmic processes of thePGCs are found in contact with each other (Fig.1F,G). F-actin is variably distributed in the PGCsin the form of rather inconspicuous fibers, whichare best seen when several optical sections arestacked together (Fig. 1G).

PGCs maintain numerous contacts with the sur-rounding endodermal cells as they pass betweenthem (Fig. 4A). In the areas of close membraneapposition, no intercellular space is visible (Fig.4B). In 4 out of 5 cases, the endodermal cells andtheir nuclei in the immediate vicinity of a PGCpseudopodium assume a concave shape (Fig. 4A).

After their exit, PGCs migrate on the endodermand enter the mesoderm. During migration both onthe endoderm and mesoderm, about 20% of PGCsare found with two or three pseudopodia extendedin different directions (Fig. 5A-C). In everyexamined embryo (stage 10-12), we find 3 or 4PGCs with F-actin arranged in a ring of dots at thebase of the pseudopodia and a similar number ofcells with aggregates of F-actin inside the processes(Fig. 5D,E).

Finally, as the PGCs meet the gonadal mesodermand condense into a gonad, they once again show aspherical form and pseudopodia are no longerobserved (Fig. 5F). Interestingly, PGCs notincluded in the forming gonads retain pseudopodia(Fig. 5F).

Mutants studiesThe beginning of the migration of PGCs throughthe PMG coincides with changes in the F-actin dis-tribution and the ultrastructural organization of the

Fig. 2. (A-C)stages. (A) Ewhose cells cmitotic divislayered epithendodermal cPGCs (asteriOne is seen ithe space betpresent betwregion betwegap junction.neighboring are present b

endodermal epithelium. To ask if the migrating PGCs inducethese changes, we examined two mutants: oskar301 (osk) andhuckebein (hkb). In the first mutation, PGCs are absent(Lehmann and Nüsslein-Volhard, 1986); in the second,changes in the gut primordium prevent exit (Jaglarz andHoward, 1994; Warrior, 1994).

Initially, the PMG of osk embryos shows a similar patternof phalloidin staining to wild type: the apical region of the


epithelium is intensely stained (not shown). However, as germband elongation proceeds this staining is reduced in the mostdistal region of the PMG, although it remains strong in thehindgut primordium and proximal part of the PMG (Fig. 6A).In addition, cells in the blind region of the PMG extend cyto-plasmic processes similar to those in wild-type embryos (Fig.7A). Adherens junctions no longer form continuous beltsaround the apical regions of all the endodermal cells and inter-cellular spaces are observed between cells (Fig. 7A).

In hkb embryos, PGCs do not leave the gut primordium atstage 10 and remain inside after germ band retraction (Fig.6B,C). In contrast to wild-type, the apical regions of the cellsof the gut primordium are intensely stained with phalloidinthroughout germ band extension (Fig. 6B) and retraction (Fig.6C). Zonula adherens are well developed between these cells(Fig. 7B). Only fine cytoplasmic processes, which never

exceed 400 nm, are observed on the apical surfaces. Interest-ingly, PGCs in the lumen of the mutant gut primordium are incontact with each other but they never show elaborate interac-tions with the somatic cells (Fig. 7B). In neither osk nor hkbmutants are the cup-shaped cells present.

Prior to PGC exit cells of the PMG undergo the 14th cycleof mitotic divisions (Foe, 1989). If this is necessary for theproper differentiation of the gut primordium the PGCs willnot exit until after this division. To investigate this possibil-ity, we examined string (stg) mutants, which never undergodivision 14 (Edgar and O’Farrell, 1989). In stg embryosPGCs leave the PMG at the same time as in the wild-type andno significant differences in their migration to the gonadalmesoderm were observed. We conclude that the cell cycleclock does not drive the changes necessary for germ cell exitfrom the PMG.

Fig. 3. (A) At the onset ofpenetration of the PMG epithelium,both the body and a cytoplasmicprojection (small star) of a PGC arefound in contact (arrows) with thecytoplasmic ‘arms’ of somatic cells.Arrowhead, adherens junction; m,mitochondria; n, PGC nucleus; largestar, intercellular space betweenendodermal cells. Bar, 1 µm.(B) Higher magnification of a PGCin contact (arrows) with thecytoplasmic extension of anendodermal cell. Arrowheads,microtubules ; m, mitochondria. Bar,400 nm. (C) Different steps of thePGC interaction with endodermalepithelium. Note a concave shape ofthe somatic cells and cytoplasmicprocesses (arrows) separating PGCs.Asterisks, PGCs; curved arrows,areas of contact between PGCs andthe endodermal cells. Bar, 2 µm.


Fig. 4. (A) A PGC with apseudopodium (star) inthe intercellular spacebetween the somatic cells.Note the concave shapeof the endodermal cell (e)in contact with thepseudopodium. Arrows,contact areas between thePGCs and endodermalcells; n, PGC nucleus.Bar, 2 µm. (B) A highermagnification of the areamarked with anarrowhead in A (PGC tothe right). Arrows,closely apposedmembranes of theinteracting cells;arrowhead, microtubule;l, lipid droplet; m,mitochondria; Bar,200 nm.

Behavior of PGCs in primary cultureIn order to determine if PGCs could be motile outside thedeveloping embryo, they were removed prior to gastrulation,placed in a simple primary culture and their behavior examinedusing video. These studies follow earlier work of Allis et al.(1979) who showed that PGCs can be successfully cultured invitro for a short period of time and after transplantation intoembryos are able to give rise to functional germ cells. Here wedemonstrate that these cells not only adhere to all substratathey were presented with (Table 1), but also extend cytoplas-mic processes and translocate significant distances. In contrast,blastoderm cells taken from the mid-dorsal region of a stage 7embryo and cultured under the same conditions do not translo-cate. They do however attach to the surface and extendprocesses (not shown).

Table 1. Substrates used Substrate Substrate concentration [µg/cm2] Mean v

collagen IV 4.07.0

fibronectin 1.02.07.0

laminin 2.03.07.0

Matrigel 2.0*

4.010.050.0150.0

poly-D-lysine 2.04.0

glass coverslip −

The images were recorded every 5 seconds and the mean velocity of migrating

The behavior of the PGCs in culture depends primarily onthe nature of the substratum and its concentration. There is athreshold of concentration above which PGCs adhere toostrongly to the substratum and remain immobile (Table 1).Although PGCs are motile on all substrata, only laminin andMatrigel produced morphology similar to that in fixed embryos(Fig. 8). The migration on all substrata was unoriented. Ifmigrating cells contact each other, they crawl over each otherand continue their migration as cell clusters (Fig. 8D-F).Apparently these cells have higher affinity to each other thanto the substratum.

The migration of PGCs in vitro consists of four steps. First,there is an initial quiescent period that is extended in cellsremoved from early stage 4 embryos, which undergo divisionsbefore displaying motility. Subsequently, PGCs randomly extend

for in vitro experimentselocity [µm/min] Remarks

1.20.02.51.70.0

2.4 / 2.1 second value, velocity for a group of PGCs1.50.0

2.0 / 2.2 * concentration for Matrigel [µg/ml]; second value, velocity for a group of PGCs

1.2 thin coating method0.0 thin coating method0.0 culture inside the gel0.0 culture inside the gel0.20.0

3.0

PGCs was measured for three PGCs from three repetitions of each experiment.


Fig. 5. Optical sections through embryos stained with anti-vasaantibody (green) and Rh-phalloidin (red). (A-C) PGCs migratingon the endoderm. Differences in F-actin distribution coincidewith variable morphological forms of the PGCs. Arrows, localaccumulations of F-actin. (D) Stack of four optical sections (0.5µm apart) showing aggregates (arrowhead) of F-actin arrangedin a ring around the base of the pseudopodium. Two of thesequential sections (red channel) shown at right. (E) PGCsmigrating in the mesoderm. Arrows, different localizations of F-actin. (F) Gonad condensation. Only PGCs incorporated into theforming gonad (asterisk) become round, the remaining cellscontinue to show pseudopodia (arrows). Bars, 10 µm; bar in C,5 µm.

Fig. 6. Optical sections through mutant embryos stained with anti-vasa antibody (green) and Rh-phalloidin (red). (A) The gutprimordium of an oskar301 embryo at stage 10. Note reduced F-actin staining in the distal part of the PMG, although the stainingin the hindgut primordium (hp) and proximal part of the PMG(arrowheads) remains strong.Bar, 20 µm. (B,C) The gutprimordium of a huckebeinembryo at stage 10(A) and 13(B). Intense F-actin staining inthe apical region of the cells ofthe gut primordium (arrowheads)continues throughout differentstages of development. Arrows,aggregates of F-actin. Bars,10 µm.

Fig. 7. (A) The PMG of an oskar301 embryo at early stage 10. Note the lackof the PGCs in the lumen of the PMG. The apical regions of the most distalendodermal cells are extended into cytoplasmic projections (stars, someprojections in cross section) similar to those observed in wild-typeembryos. Arrowheads, adherens junctions; asterisk, intercellular bridge;open stars, intercellular space; l, lipid droplets; m, mitochondria. (B) Thegut primordium of a huckebein embryo at late stage 10. In contrast to wild-type embryos at this stage, well developed zonula adherens (arrowheads)continue to be present between the closely apposed somatic cells. PGCs arein close contact with each other and do not interact with the somatic cells.m, mitochondria; n, PGC nucleus. Bars, 1 µm.


Fig. 8. (A-F) Frames from a time-lapse analysis of PGC behavior on laminin (3.0 µg/cm2), in vitro. Numbers in the upper right hand corner,time (hours : minutes) from the beginning of the experiment; numbers on the cells, the same cell over the course of the experiment. For aboutan hour the PGCs remained spherical and a cell divided and produced cells 6 and 7 (A). Over the next 6 hours PGCs extended pseudopodia (e.g. cells 1,2,4,7 in B and 1,3 in C) and translocated variable distances. Since PGCs migrated back and forth the cell position does not reflect theactual distance of migration. PGCs differed significantly in their migratory behavior. For instance cells 5 and 6 did not translocate.Interestingly, these cells were in contact with cellular debris (star). In contrast, cell 1 migrated a long distance (compare its position in A, C andF) and came in contact with cell 2 (D). After that encounter these two cells crawled over each other and continued to migrate together (E,F).Bar, 20 µm.

Fig. 9. A dorsal view of a stage 14 laminin A mutant embryo stainedwith anti-vasa antibody. Only some PGCs are included in the gonad(arrow), the remaining cells are dispersed in the posterior part of theembryo. Bar, 50 µm.

Fig. 10. Schematic representation of PGC (red) migration throughthe endodermal epithelium of the PMG. Changes occur from left toright. Arrow, adherens junction.

small projections some of which attach to the substratum. This isfollowed either by rolling or cytoplasmic flow into one broadpseudopodium, resulting in polarization of the cell (Fig. 8).

PGCs frequently change the direction of migration and movein a random fashion. They are capable of translocating in thatway for relatively long distances (100 µm or more) withvariable speed (for instance 0.2 µm/minute on poly-D-lysineor 2.4 µm/minute on laminin, see Table 1). When culturedinside Matrigel, PGCs that are embedded in groups crawl overeach other and will exchange places. However they do notpenetrate the gel or disperse.

The possibility that laminin may be necessary for migrationin vivo was investigated by examining the zygotic phenotypeof a null mutant for chain A of Drosophila laminin (for reviewof Drosophila extracellular matrix proteins see Hortsch andGoodman, 1991). In these mutants, an increased number ofPGCs were not incorporated into the gonads and remaineddispersed throughout the embryo (Fig. 9). The defect in PGCmigration in the laminin mutant occurs late during germ bandretraction and this late effect may be a consequence of the per-durance of maternal laminin.

DISCUSSION

Our data allow us to conclude three things about the migrationof Drosophila PGCs. First, that these cells are capable of


moving on their own, that they are likely to do this in vivo andthat the underlying cytoplasmic events are associated withactin polymerization. Secondly, that the orientation of the PGCmigration is likely to be achieved by stabilization of particularquasi randomly generated cytoplasmic extensions and notsimply by instructive cues driving de novo formation of theseextensions. Finally, we show that the transendodermalmigration from the extraembryonic gut lumen to the interior ofthe embryo is mediated by remodeling of the endodermalepithelium and is not driven simply by an activation of thePGCs motile machinery, which has by this time been active forseveral hours.

Drosophila germ cells move actively soon after theirformationOur culture experiments show that the PGCs can migrate bythe extension of pseudopodia and respond to different substratawith different degrees of activity. Furthermore, this migrationis specific in the sense that blastoderm cells do not move underthe same conditions. Two pieces of evidence strongly arguethat these migratory properties are also manifested in vivo.First, blastoderm cells do not migrate after transplantation tothe PMG whilst germ cells do (Jaglarz and Howard, 1994).Secondly, similar migratory morphology is seen in fixedembryos. Interestingly, this migratory activity begins at theblastoderm stage, well before their exit from the PMG (see alsoCounce, 1963; Jura, 1964; Rabinowitz, 1941; Sonnenblick,1941) and it is not the onset of migratory activity that initiatesthe transendodermal movement.

Although PGCs show migratory morphology from late blas-toderm stage on and this activity could help these cells to moveinto the developing gut, we do not propose that this activity issolely responsible for this movement. In fact, blastoderm cellstransplanted before gastrulation at the posterior pole of theembryo are included in the PMG as can inanimate objects likefragments of the transplantation needle (Jaglarz and Howard,1994; Rabinowitz, 1941). However, in all of these cases PGCswere also present and it is possible that their activity pushedthe transplants inside.

Changes in the actin cytoskeletonActin polymerization is involved in the formation ofpseudopodia and movement of many cell types (reviewedin Condeelis, 1993; Stossel, 1993). Association of F-actin withadherens junctions is also well established (Fristrom, 1988).We have described extensive rearrangements of the actincytoskeleton during Drosophila PGC migration. These modi-fications are quite variable in fixed material at any one stageof development, a feature that we attribute to the dynamicnature of these processes. There are also stage-specific changesin these cells, which we suggest reflect their interactions withdifferent environments as they navigate to the gonad. Theaggregates of phalloidin-stained material are of particularinterest and punctate distributions of F-actin, very similar tothose that we see in Drosophila embryos, have been reportedin yeast, hyphae of filamentous fungi, murine macrophages andembryonic cells (Adams and Pringle, 1984; Amato et al., 1983;Lehtonen and Badley, 1980). Interestingly, aggregates of actinare usually associated with those parts of the cell that undergoelongation. In yeast dots of F-actin are concentrated in areasof incipient bud formation and elongation. Moreover, a ring of

actin spots was found at the base of the forming buds (Adamsand Pringle, 1984).

The randomly distributed dots and patches of F-actin maymark actin nucleation sites involved in driving the formationof pseudopodia. However, some of the actin patches that wedescribe may correspond to sites of close plasma membraneapposition between migrating PGCs and their environment.

Interactions between endoderm and the germ cellsWe propose that PGCs in contact with each other in the devel-oping gut actively search their environment by extendingpseudopodia that eventually come into contact with cytoplas-mic extensions of the somatic cells. These contain micro-tubules and may provide a rigid substratum that would guidePGCs centripetally. This initial movement however, wouldnot result in transepithelial exit due to the presence of intactadherens junctions. These junctions are rearranged at the timeof exit and intercellular spaces appear between endodermalcells. We suggest that this results in the formation oflow-resistance paths that permit exit. Similar changes in theadherens junction distribution in the wild-type PMG havebeen reported by Tepass and Hartenstein (1994) and are mostlikely to represent the initial steps in the transition of the PMGepithelium into mesenchyme. These observations areschematically represented in Fig. 10.

There is a possibility that PGCs initiate the changes in thePMG. However, examination of osk mutants shows that theyoccur even when PGCs are absent. Note that, despite theloosening of the epithelium, PGCs do not penetrate the PMGfreely and leave as single cells that are interconnected by cyto-plasmic extensions. This interaction of migrating DrosophilaPGCs with each other has not been previously reported and isreminiscent of the behavior of mouse PGCs where migratingcells link to each other by means of long cytoplasmic processes(Gomperts et al., 1994).

This transendodermal migration bears some similarity to themigration of leukocytes during inflammatory responses. Theinitial weak binding of the leukocytes to the surface of theendothelium is followed by sustained attachment and activa-tion of the cells and only then the migration through the inter-cellular spaces of the endothelium can take place (reviewed inSpringer, 1994).

PGCs always leave the gut primordium in a restricted areaof the PMG. The behavior of PGCs in hkb embryos indicatesthat this area is defined by hkb, which encodes a transcriptionfactor specifying the endodermal cells (Brönner et al., 1994).Lack of hkb product causes transformation of these cells intohindgut-like cells, which retain their epithelial structure. Wepropose that both the preferential adhesion of germ cells to thehkb-dependent epithelium and the changes seen as it developsare caused by hkb target genes, which directly modify cellstructure and surface properties and have yet to be identified,at least in this context.

The regulation of PGC behavior by the endodermal tissue isnot peculiar to Drosophila. In a frog (Rana pipiens), it has beenshown that, although PGCs have inherent migratory properties,they do not leave endoderm until changes occur in theirsomatic environment (Subtelny and Penkala, 1984).

The orientation of migrationWe have suggested that Drosophila PGCs extend processes in


a relatively random fashion. Consequently, the direction ofmigration is determined by which processes mature, somethingthat is regulated by environmental factors. During migration,membranes of PGCs are found in close apposition withmembranes of other cells. These contact zones lack any par-ticular specializations suggesting that they are transient attach-ment points allowing the cells to explore their environment,making new contacts and releasing old ones as they propelthemselves. During transendodermal migration orientation isclearly provided by the structure of the PMG. However, in theother movements of these cells, our ultra structural studies donot identify guidance cues, which are likely to be evident onlyat the molecular level.

Drosophila PGCs in culture do not show contact paralysisof movement, a feature typical for many motile cells in vitro(Abercrombie, 1980). In this respect they behave similarly tomouse PGCs in culture (Stott and Wylie, 1986). Furthermore,our culture experiments demonstrate that the PGCs responddifferently to different substrata and suggest that substratumguidance is in principle possible in this system. However,although laminin promotes migration of Drosophila PGCs invitro, zygotic loss of the laminin A chain gene results in onlysubtle defects in migration. This suggests that laminin mightbe only one of the components necessary for proper migrationof PGCs in vivo. We hope that our in vitro assay will allow usto further elucidate the role of extracellular proteins in PGCmigration.

We would like to thank Drs Corey Goodman, Ruth Lehmann andDetlef Wiegel and the Bloomington stock center for Drosophilastocks. Dr Robin Wharton for the anti-vasa antibody. We are gratefulto Dr Paul Wassarman for kindly sharing with us his EM facilities,Drs Helen Skaer and Kristin White for fixation protocols and MrSteven Mortillo for technical support. We would also like to thank thetwo anonymous referees who provided stimulating and constructivecomments on this manuscript during review. This work was supportedby Hoffmann-La Roche Inc. M. J. wishes to thank all members of theDepartment of Zoology of the Jagiellonian University for making hisextended stay at the Roche Institute possible.

Note added in proof

During review of this manuscript Callaini et al. (1995) reportedsimilar data on aspects of Drosophila germ cell migration:Callaini, G., Riparbelli, M. G. and Dallai, R. (1995). Polecell migration through the gut wall of the Drosophila embryo:analysis of cell interactions. Dev. Biol. 170, 365-375.

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(Accepted 17 August 1995)

the active migration of drosophilaprimordial germ cells

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