Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells.код для вставкиСкачать
THE ANATOMICAL RECORD 22414-21 (1989) Distribution of Extracellular Matrix in the Migratory Pathway of Avian Primordial Germ Cells LANCE E. URVEN, URSULA K. ABBOTT, AND CAROL A. ERICKSON Departments of Avian Science (L.E.U., U.K.A.) and Zoology (C.A.E.), University of California, Davis, California 95616 ABSTRACT The appearance and distribution of extracellular matrix (ECM) was documented along the migratory route of chicken primordial germ cells (PGCs). The antimouse embryonal carcinoma cell antibody, EMA-1, was used to label PGCs (Urven et al.: Development 103:299-304, 1988). Antibodies against laminin, fibronectin, chondroitin sulfate proteoglycan and collagen type IV were used to label extracellular matrix components. When the PGCs emerged from the epiblast, all four ECM molecules were restricted principally to the basement membrane of the epiblast. Chondroitin sulfate was also located between hypoblast cells during this period. In late germinal crescent stages, when the PGCs entered the lumina of blood vessels, the same ECM molecules were more widespread in the mesoderm and in extracellular spaces. In addition, laminin and collagen type IV were identified on lateral surfaces of ectodermal cells at this stage. When the germ cells moved through the mesenchyme into the germinal ridge, the ECM molecules were found around mesenchymal cells, and, in the cases of laminin, fibronectin and collagen type IV, in the basement membranes of the germinal ridge epithelia. Because the appearance of these ECM components is temporally and spatially correlated with the movement of PGCs, we suggest that early PGC migration may depend on their timely appearance. The extracellular matrix (ECM) provides support; a substratum against which cells can exert traction; and, in some cases, directional cues to migrating cells (Heasman and Wylie, 1981; Turner et al., 1983; Erickson, 1987; Hammarback, 1988). Embryonic systems are particularly useful for studies of the role of the ECM in cell migration. We have been especially interested in the control of primordial germ cell (PGC) migration. PGCs are undifferentiated cells that ultimately give rise to spermatogonia and oogonia. In vertebrates, they originate outside the embryo proper and travel by various routes, depending on the species, to the developing gonads. Avian PGCs serve as a good model for control of embryonic cell migration for several reasons. In birds, PGCs are easily identified on the basis of morphology, in conjunction with either histochemical (Meyer, 1964) or immunological (Pardenaud et al., 1987; Urven et al., 1988) markers. They migrate through a variety of tissue types and display several morphogenetic behaviors. PGCs initially appear in the epiblast and invaginate into the blastocoel (EyalGiladi et al., 1981; Sutasurya et al., 1983; Ginsburg and Eyal-Giladi, 1986; Pardenaud et al., 1987; Urven et al., 1988). Later, as mesoderm invades the anterior and lateral regions of the area pellucida, PGCs are located in the blastocoel, mesoderm and hypoblast of the “germinal crescent.” They then enter the developing extra-embryonic splanchnic blood vessels and are passively carried to the level of the germinal ridges (gonadal anlagen), where they leave the vasculature and migrate actively through the intervening mesenchyme 0 1989 ALAN R. IJSS, INC into the germinal ridge epithelium (Swift, 1914; Meyer, 1964; Clawson and Domm, 1969; Fujimoto et al., 1976). The germinal ridge is the sole migratory target of PGCs, unlike neural crest cells or neurites, which have multiple paths and numerous destinations. ECM molecules found in the migratory pathway of PGCs may act to control their migration. Fibronectin, in particular, has been implicated in the control of PGC migration in amphibians (Cathalot and Brustis, 1986; Wylie and Heasman, 1982), birds (Critchley et al., 1979; England, 19831, and mammals (Alvarez-Buylla and Merchant-Larios, 1986; Donovan et al., 1987). Laminin has also been shown to aid in the adhesion of mouse PGCs in vitro (Donovan et al., 1987), suggesting that it may affect PGC migration, at least in mammals. These and other ECM molecules have been suggested to have roles in the migration of a variety of other cell types as well. Fibronectin has been shown to affect adhesion and migration in vitro of selected cell lines (Goodman and Newgreen, 19851, fibroblasts (Couchman et al., 1982,19831,neural crest cells (Newgreen et al., 1982; Erickson and Turley, 1983; Rovasio et al., 1983; Tucker and Erickson, 1984), involuting mesodermal cells (Boucat et al., 1984a,b), and myogenic cells (Turner et al., 1983). Fibroblasts (Couchman et al., 19831,neurite growth cones (Hammarback et al., 19881, R e c e i v z e p t e m b e r 12, 1988; accepted October 31, 1988. Address reprint requests to Lance E. Urven, Dept. of Population Dynamics, The Johns Hopkins University School of Public Health, 615 N. Wolfe Street, Baltimore, MD, 21205. 15 AVIAN GERM CELL MIGRATION AND ECM DISTRIBUTION TABLE 1. Antibodies used to identify extracellular matrix components in the migratory pathways of avian primordial germ cells Antibody EMA-1 Polyclonal Polyclonal Polyclonal CS-56 Specificity PGC surface glycoproteins Fibronectin Laminin Collagen type IV Chondroitin sulfate proteoglycan Type Mouse IgM Dilution Undiluted Source Dr. E.M. Eddy Goat IgG Rabbit IgG Rabbit IgG Mouse IgM 1:lOO 1:lOO 1:50 1:200 Organon Teknika Dr. H. Kleinman Dr. H. Kleinman ICN neural crest cells (Rovasio et al., 1983; Bilozur and Hay, man of the National Institutes of Health, Bethesda, 19881, and a variety of epithelial and mesenchymal cell Maryland. The antifibronectin is commercially availlines (Goodman and Newgreen, 1985) have also been able (Organon Teknika, One Technology Court, Malshown to adhere to andor migrate on laminin in vitro. vern, PA, 19355) and was made in goat against human Collagen type IV, adsorbed to culture plates, allows plasma fibronectin. The antichondroitin sulfate is a attachment of fibroblasts (Murray et al., 19791, endo- commercial monoclonal antibody specific for the glythelial cells (Herbst et al., 19881,hepatocytes (Ruben et cosaminoglycan portion of native chondroitin sulfate al., 1981), and some permanent cell lines (Aumailley proteoglycan (ICN Immunobiologicals, P.O. Box 1200, and Timpl, 1986). Chondroitin sulfate, on the other Lisle, IL, 60532). Each slide was rinsed twice in PBS, hand, has been shown to decrease cell adhesion in cul- washed for 10 minutes in 0.2 M glycine to quench tures of neural crest cells (Newgreen et al., 1982; Erick- autofluorescence, rinsed again in PBS, and dipped in son and Turley, 1983; Tucker and Erickson, 1984). PBS with 2% bovine serum albumen. Primary antibodWe have examined chicken embryos for the presence ies were applied dropwise to the sections and allowed to of laminin, fibronectin, collagen type IV, and chon- incubate for 1.5 to 2 hours in a humidified chamber, droitin sulfate in the PGC migratory pathway. We followed by two rinses in PBS. The presence of primary have first considered the location of specific ECM mol- antibody binding was indicated by secondary labeling ecules at the onset of migration t o address the possi- using rhodamine isothiocyanate-conjugated antimouse bility that PGC movement may await the presentation antibody (Organon Teknika) to indicate EMA-1, or of suitable substrates. Secondly, we have determined fluorescein-conjugated secondary antibody directed whether any particular components show differential against the appropriate immunoglobulin class (Ordistribution in the PGC pathway. Of the components ganon Teknika) to indicate the anti-ECM antibodies. studied, only fibronectin has been previously examined Secondary antibodies were diluted 1 5 0 in PBS, applied specifically with reference to the avian PGC migratory in the same manner as described above for primary pathway (Critchley et al., 1979; Sanders, 1982; En- antibodies, and incubated for 30 minutes. Control sections were incubated in PBS in place of primary antigland, 1983; Fujimoto and Yoshinaga, 1986). body to test for nonspecific binding of secondary antiMATERIALS AND METHODS bodies and tissue autofluorescence. After rinsing twice White leghorn chicken embryos were dissected from in PBS, slides were mounted in 2% n-propyl gallate in the yolk after 1,2,3,4,and 5 days of incubation at 37°C glycerol (Giloh and Sedat, 1982), then studied and phoand 55% relative humidity. Each embryo was staged tographed with a Leitz Dialux 20 photomicroscope according to the criteria of Hamburger and Hamilton equipped for epif luorescence. (1951; referred to hereafter as H & H stages) and rinsed RESULTS thoroughly in phosphate-buffered saline (PBS) prior to In chicken embryos incubated for 1 day (stages 4-6 fixation for 1hour with 4% paraformaldehyde in PBS. Embryos were then washed three times in PBS before H & H), PGCs are the only EMA-1-positive cells vena 2-hour infiltration, first in PBS containing 15% su- tral to the EMA-1-positive epiblast (Fig. 1A). They are crose, then in PBS containing 30% sucrose. Embryos distributed in the anterior half of the area pellucida in were maintained on a revolving table during fixation, rinses, and infiltrations to enhance penetration of solutions. Embryos were embedded in O.C.T. compound (Miles Scientific Division, Miles Laboratories, Inc., C IV collagen type IV Abbreviations 30W475 North Aurora Road, Naperville, IL 60566) un- ce coelomic epithelium Cs chondroitin sulfate der liquid nitrogen, and stored at -30°C. Serial sections were cut a t 12 pm with a Bright Cry- ec extra-embryonic ectoderm embryo ostat model OTF/AS/MR. Sections were either double- em en endoderm labeled or alternately labeled for PGCs using EMA-1 ep epiblast antibody (Hahnel and Eddy, 1986; Urven et al., 19881, Fn fibronectin generously donated by Dr. E.M. Eddy, and for extra- h hypoblast Ln laminin cellular matrix components using the antibodies listed m mesoderm in Table 1.Antilaminin and anticollagen type IV were me mesenchyme made in rabbit and kindly provided by Dr. H. K. Klein- Schematic diagrams indicate the level of section for each figure. 16 L.E. URVEN ET AL. Fig. 1 . Paraformaldehyde-fixed, frozen sections of gastrula stage (1-day, stage 4-6 H & H) chick embryos immunofluorescently labeled to indicate A) EMA-1-positive PGCs (arrowheads) emerging from the EMA-1-positive epiblast; B) laminin distribution in the same section; C)fibronectin in a comparable section; D) collagen type IV in a comparable section; E) chondroitin sulfate in a n adjacent section to that shown in A and B. Scale bar = 50 fim. 17 AVIAN GERM CELL MIGRATION AND ECM DISTRIBUTION TABLE 2. Immunofluorescent detection of ECM components in the early germinal crescent of chicken embryos at 1 day of incubation (stages 4-6, Hamburger and Hamilton, 1951)' EDiblast ++ ++ ++ ++ Fibronectin Laminin Collagen type IV Chondroitin sulfate Mesoderm + +- HvDoblast - + + + + ,strong fluorescence; + , moderate or scattered fluorescence; -, no fluorescence above background. TABLE 3. Immunofluorescent detection of ECM components in the late germinal crescent of chicken embryos at two days of incubation (stages 9 to 10, Hamburger and Hamilton, 1951)' Ectoderm Mesoderm + + ++ ++ Fibronectin Laminin Collagen type IV Chondroitin sulfate Endoderm - + ++ ++ ++ ++ + + + + , strong fluorescence; + ,moderate or scattered fluorescence; -, no fluorescence above background. TABLE 4. Immunofluorescent detection of ECM components in the germinal ridge area of chicken embryos at three to five days of incubation (stages 15-26, Hamburger and Hamilton, 1951)' Fibronectin Laminin Collagen type IV Chondroitin sulfate Mesenchvme Germinal ridge basement membrane + + ++ ++ ++ ++ + ++ Germinal ridge eDithelium - + + , strong fluorescence; + ,moderate or scattered fluorescence; -, no fluorescence above background. the region called the germinal crescent (Swift, 1914). At these stages, all four ECM components were found principally in the basement membrane of the epiblast (Fig. 1B-E; Table 2). With the exception of collagen type IV, all these antibodies faintly labeled the surfaces of some scattered mesodermal cells. Chondroitin sulfate was seen intermittently within the hypoblast. When PGCs were entering or becoming trapped in germinal crescent blood vessels in 2-day (stages 8-11 H & H) embryos (Fig. 2; Table 3),the ECM components we examined became more widespread in the mesodermal mesenchyme. Laminin and collagen type IV were present in the ectodermal basement membrane as well as on the lateral surfaces of these cells. Some barely detectable labeling occurred in the ectodermal basement membranes stained with antifibronectin and antichondroitin sulfate antibodies. Laminin, collagen type IV, and chondroitin sulfate were scattered on the surfaces of some endoderm cells. By the third day of incubation (stages 15-18 H & H), PGCs were beginning to emerge from blood vessels of the splanchnopleure and from the dorsal aorta at the level of the germinal ridges. They then migrated through the intervening mesenchyme and penetrated the mesothelium near the coelomic angle (Fig. 3A). By the fifth day of incubation (stages 25-26 H & H), many of the PGCs have reached the germinal ridges. Throughout this period, fibronectin and chondroitin sulfate were seen in all mesenchyme a t the level of the germinal ridge, including that near the germinal ridge and in the mesentery (Fig. 3C,E; Table 4). Collagen type IV and laminin were also present among mesenchyme cells, though a t a reduced level as compared to that in basement membranes. Fibronectin, collagen type IV, and laminin were found in the basement membranes of the germinal ridge epithelia. None of the antibodies labeled the apical or lateral surfaces of the germinal ridge epithelium cells. DISCUSSION We have examined the distribution of laminin, fibronectin, collagen type IV, and chondroitin sulfate in the pathway of migrating avian PGCs. All these ECM molecules have been implicated in adhesion and motility of various other cell types. The fibronectin distribution described here is similar to that seen in other studies of the chick gastrula (Critchley et al., 1979;Sanders, 1982; England, 1983). Fibronectin has previously been reported to disappear from the dorsal mesentery at stage 23 H & H, after PGC migration is complete (Fujimoto and Yoshinaga, 1986). In our study, fibronectin could still be found in this tissue as late as stage 26 H & H (5 days). This is the first report to describe the appearance of laminin, collagen type IV, and chondroitin sulfate in the PGC migratory route. All four ECM components occurred in both the germinal crescent and near the germinal ridge. Because the four are essentially codistributed spatially and temporally, any of them may L.E. URVEN ET AL. 18 2 Fig. 2. Paraformaldehyde-fixed, frozen sections of late germinal crescent stage (2day, stage 8-11 H & H) chick embryos immunofluorescently labeled to indicate A) EMA-I-positive PGC (arrowhead) in the endoderm; B) laminin distribution at the left side of the section shown in A; C ) fibronectin in a comparable section; D) collagen type IV in a comparable section; E) chondroitin sulfate in a n alternate section to that shown in A and B. Scale bar=50 pm. AVIAN GERM CELL MIGRATION AND ECM DISTRIBUTION Fig. 3. Paraformaldehyde-fixed, frozen sections of germinal ridge stage (4-day, stage 20-22) chick embryos immunofluorescently labeled to indicate: A) PGCs (arrowheads) in the germinal ridge epithelium; B) laminin distribution in the same section; C ) fibronectin in a comparable section; D) collagen type IV in a comparable section; E) chondroitin sulfate in a n adjacent section to that shown in A and B. Scale bar = 50 pm. 19 20 L.E. URVEN ET AL. have roles as specific substrata required for PGC migration in vivo. Even laminin and collagen type IV, which are considered to be principally associated with basement membranes (Kleinman et al., 19841, are found to some extent among the mesenchyme in the migratory pathway. If the presence of a n ECM component alone conferred directionality to PGCs, we would expect to find one or more of them distributed strictly in the migratory pathway. However, the four ECM components all occurred in mesenchyme of the sclerotome and other areas outside of the migratory pathway a t the level of the germinal ridges. We have not addressed the possibility that quantitative differences in the concentration of ECM components may occur along the PGC migratory pathway. It is possible that substrate concentration gradients may give PGCs directional cues, in addition to the chemotactic guidance proposed by other investigators (Dubois, 1965; Dubois, 1968; Kuwana et al., 1986). In vitro experimental analysis of PGC behavior will be needed to provide data addressing this question. Herbst et al. (1988) demonstrated the feasibility of this approach in their studies of endothelial cell movement on adsorbed gradients of collagen type IV. Our results indicate that PGC migration is coincident with the appearance of at least four ECM molecules. When PGCs first leave the epiblast, migration is limited to some fairly restricted movement on the basement membrane (Critchley et al., 1979; England, 1983) or the hypoblast (Lee et al., 1978). Mesoderm and mesodermal ECM are rare in the germinal crescent at this time. When the PGCs are moving into the splanchnic blood vessels, ECM becomes available throughout the mesenchyme as mesoderm and its associated ECM advance into the region from the primitive streak. More ECM becomes available between the epiblast and splanchnopleure when PGCs must move through the area to enter the blood vasculature. ECM formation is temporally and spatially correlated with the migration of PGCs, and its appearance may allow early PGC movement. Similarly, at the end of the migratory pathway, ECM is plentiful in basement membranes and among the mesenchyme cells through which PGCs travel. ECM is not seen between germinal ridge epithelium cells where the PGCs ultimately settle. Again, active migration is correlated with the presence of ECM, whereas presumably less motile PGCs in the germinal ridge epithelium do not have access to ECM. The migration of PGCs from the germinal crescent to the germinal ridges is clearly correlated with the presence of laminin, fibronectin, collagen type IV, and chondroitin sulfate. These molecules are present outside the PGC migratory pathway, as well, and therefore cannot guide PGCs to the germinal ridge simply on the basis of their presence or absence. Which, if any, of these ECM components may be specifically required for PGC adhesion and locomotion remains to be investigated. Based on present observations, however, it is likely that the departure of the PGCs from the germinal ridge requires the prior appearance of the ECM. ACKNOWLEDGMENTS The authors wish to thank Dr. E.M. Eddy and Dr. H.K. Kleinman for their penerous " gifts of the EMA-1 u ~~ ~~ and antilaminin and anticollagen type IV antibodies, respectively. We greatly appreciate the advice and suggestions of Dr. J.R. McCarrey. This work was supported, in part, by a USDA Competitive Research grant 87-CRCR-1-2301 and by a n NIH Genetics Training grant 5-T32-GM07467. LITERATURE CITED Alvarez-Buylla, A., and H. Merchant-Larios 1986 Mouse primordial germ cells use fibronectin as a substrate for migration. Exp. Cell Res., 165:362-368. Aumailley, M., and R. Timpl 1986 Attachment of cells to basement membrane collagen type IV. J . Cell Biol., 103:1569-1575. Bilozur, M.E. and E.D. 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