Early stages in male germ cell differentiation in the mouse

APMIS 106: 127-133. l 9YN Printed in Di~niniirk , All riRhts riwrved

Copj ,r ight 0 A P M I S 1998

NPUUS ISSN 0903-4641

Early stages in male germ cell differentiation in the mouse Review article

ROBERT ANDERSON,'.' MARTIN GARCIA-CASTR0,3* JANET HEASMAN,'-2 and CHRISTOPHER WYLIE2

'Department of Cell Biology and Neuroanatomy, Institute of Human Genetics, 'Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, USA, 3Wellcome/CRC Institute for

Developmental Biology and Cancer, Cambridge, England

Anderson, R., Garcia-Castro, M., Heasman, J. & Wylie, C. Early stages in male germ cell differentiation in the mouse. Review article. APMIS 106: 127-133, 1998.

Primordial germ cells arise during gastrulation and migrate from the hindgut into the gonad primor- dium during early organogenesis. In this article, we discuss factors that control migration, proliferation and targeting of the PGCs. In particular we discuss how changes in adhesiveness control germ cell positioning in the gonad, and the molecules involved.

Key words: Primordial germ cell; migration; gonadal development

Christopher Wylie, Department of Pediatrics, University of Minnesota School of Medicine, Box 206 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455, USA.

Carcinoma in situ (CIS) is a pre-invasive malignant condition of the testis, characterised by islands of aneuploid cells in the seminiferous tubules that express molecular markers characteristic of early germ line cells. In most cases the functions of these markers in germ line differentiation are unknown, as are the effects of their expression later during germ cell differentiation. Despite the central importance of germ cells to biology and medicine (lO-l5% of couples of childbearing age prove to be infertile), little is known about the molecular basis of germ cell differentiation, nor is it clear if and how abnormali- ties of early germ cell differentiation are significant causes of testicular pathology in the adult. This paper will briefly review the early differentiation of the germ line in the mouse embryo, with emphasis on germ cell migration. It should, however, be empha- sised that significant advances in this subject are being made by studies of other model organisms, including the fruitfly Drosophilu and the nematode C. elegans .

* Present address: California Institute of Technology, Beckman Institute, Division of Biology 139 74, Pasadena, CA 91 125, USA.

Primordial germ cells (PGCs) are the progenitors of male and female gametes. Sexually indifferent PGCs are first identifiable in the early mouse embryo at gastrulation, embryonic day 7.5 (E7.5). At this time, a small population of PGCs can be dis- tinguished by the expression of alkaline phosphatase (Chiquoine 1954; Ginsburg et al. 1990) and Oct-4 (Yeom et al. 1996). Two days later (E9.9, PGCs are incorporated into the endoderm of the hindgut. At this time or slightly later, PGCs express several distin- guishing antigens. These include the receptor tyrosine kinase c-kit (Godin et al. 1991; Orr-Urtreger et al. 1990), the lacto-series carbohydrates SSEA-1 (Le"), Ley and 4C9 (Yoshinuga et ul. 1991), the globo-series carbohydrates SSEA-3, and Forssman, as well as fu- cosylated lactosaminoglycans (Fenderson, et ul. 1990; Cooke et al. 1993). The structures of several of these antigens are shown in Table 1. The functions of these carbohydrate antigens remain obscure, although SSEA-1 has been implicated as a homophilic cell adhesion molecule (Bird & Kimber 1984). Closely related carbohydrates are known ligands for the se- lectin family of receptors (Hemmerich & Rosen 1994). Given that many PGC markers are also expressed by germ cell tumours, the function and transcriptional

127

ANDERSON c/ ul

TABLE 1. Structure o f PGC curbohvdrutcs. (Fenderson et al.) Lacto-series SSEA-I (Le”) GalpI+4G lcNAc~I+3Gal~l-+4Glc~l+R

3 t

Fucal

Ley FUCal+2GaIp 1 +4GlcNAcP 1 +3Galp 1 +4Glc@I +R 3 t

Fucal

Globo-series Forsman GalNAcal+3GalNAcPI ~3Galal+4Galpl+4Glc~l+R SSEA-3 GalPl+3GalNAcpI +3Galal+4GalPI -+4Glc@l+R

control of their expression is worthy of continued study.

At about E10, PGCs begin to emigrate from the hindgut endoderm into the surrounding mesoderm. Early emigrants, or “pioneer” PGCs, appear to migrate directly from the hindgut to the adjacent urogenital ridges. The journey is not as trivial for later PGCs, as early migration coincides with hindgut de- scent into the coelomic cavity. Therefore, later emerging PGCs must migrate dorsally through the hindgut mesentery to reach their destination. PGCs do not migrate individually, but contact each other by extending long filopodial processes (Gomperts et ul. 1994). Regardless of their route, most PGCs eventually come to rest on the developing basement membrane of the urogenital ridges. Here PGCs adhere to each other, forming large cellular aggre- gations. At this stage, ectopic PGCs undergo apoptosis (Pesce et al. 1993). In both male and female embryos, the migratory phase is one of substantial proliferation, as the population of PGCs in- creases from less than 100 to approximately 25,000 cells (Tam & Snow 1981). Although male-specific gene expression is detectable earlier (Ross; et ul. 1993), sexual differentiation is morphologically first evident at E12.5. At this stage, the male gonad is marked by sex cord assembly and obvious vascu- larisation. At E13, male germ cells enter mitotic ar- rest, whereas female germ cells begin to enter meiosis. There is now substantial evidence that PGC behaviour is influenced by signals released by, or expressed on, the surfaces of neighbouring somatic cells. Two pieces of work suggested this. First, it was shown that PGCs survive in culture when plated on feeder cells (Donovun et ul. 1986); and second, culture medium conditioned by genital ridges was not only a chemo-attractant but also increased PGC numbers (Godin, et al. 1991). In sub- sequent work to identify the factors released by

128

genital ridges, many purified factors were shown to affect PGC behaviour. These include Steel Factor, Interleukin-4, Transforming Growth Factor cx basic Fibroblast Growth Factor, cytokines of the Leuke- mia Inhibitory Factor family, Retinoic Acid, and Pituitary Adenylate Cyclase Activating Peptide (Donovan 1998). A cuveut of these in vitro experiments is the fact that receptors can be activated by non-cognate ligands, especially if the exogenous ligand is related to the cognate one. It is important, therefore, to prove the requirement for a particular ligand (or its receptor) genetically. Through genetics it was shown that expression of the tyrosine kinase receptor c-kit is absolutely required by PGCs (Nocku et u1. 1989). The ligand for c-kit, Steel Fac- tor, is expressed by somatic cells adjacent to migrating PGCs in soluble and membrane-bound forms (Anderson et ul. 1990). Genetic analyses of other molecules are needed to define better their role in PGC behaviour, although the pleiotropic effects of some knockouts will necessitate the use of tissue- specific knockouts. Control of PGC proliferation and survival is an area of active investigation. and several articles in this issue are devoted to this subject.

The migratory ability of PGCs in culture is tem- porary. PGCs migrate actively on feeder cells in vijro, if explanted at a migratory stage, but lose this ability if taken later (Donovun et (11. 1986). In vitro, PGC markers (e.g. SSEA-I) begin to disappear as migration ends. Finally, PGCs explanted during postmigratory stages aggregate together in small clusters. All of these observations correlate with the phenotype of PGCs in vivo, and suggest that these aspects of PGC behaviour may be autonomous.

The mechanisms of murine PGC migration have been difficult to study, as no germ cell-derived cell lines appear to migrate in vitro. Thus, all experiments must be performed with PGCs taken directly from

MALE GERM CELL DIFFERENTIATION IN MOUSE

the embryo. In this situation, it is difficult to isolate PGCs from their somatic neighbours. Further, unlike several other cells types, including neural crest cells, PGCs will not migrate in the absence of a feeder cell monolayer. Despite these limitations, we and others are beginning to understand how PGCs migrate in vitro and in vivo.

The extracellular matrix (ECM) serves as the scaf- folding for the migration of several stem cell popula- tions in development, including neural crest cells and myoblasts (Lallier et al. 1994; Yao et al. 1996). The route of PGC migration is rich in interstitial laminin and fibronectin (Fujimoto et al. 1985; Garcia-Castro (in press). Both of these ECM molecules have been shown to support the migration of several cell types in vitro. Interstitial laminin is scarce in the urogenital ridges, where most laminin appears to be concen- trated on the basement membrane of the coelomic cavity. This corresponds to the area where PGCs dis- continue migration. The localisations of fibronectin and laminin make them attractive candidates for involvement in PGC movement.

The migration speed of a cell can be determined, in part, by the strength of its interactions with the

Fig. 1. Schematic diagram of EHS Laminin (Yurchenco & Julian 1994). The circles denote globular domains, the arrow indicates the domain of laminin to which PGCs adhere most avidly.

ECM. For example, a migrating smooth muscle cell adheres less avidly to fibronectin and collagen IV than a stationary one (Stone et al. 1993). Early quali- tative studies demonstrated that PGCs bind to ECM molecules in vitro (DeFelici & Dolci 1989; ffrench- Constanr et al. 191 I ) . However, these studies were not able to detect small changes in PGC adhesiveness nor were they easily reproducible. To assess quantitatively PGC binding to individual ECM molecules, we used a modified McClay assay (Garcia-Castro et al. (in press). In this approach, partially purified PGCs are plated on ECM-coated wells, incubated, and sub- jected to a centrifugal force for a given time. Subse- quently, adherent and non-adherent PGCs are counted by use of a soluble alkaline phosphate product and spectrophotometry. This assay is both quantitative and highly sensitive.

Using this approach, we showed that PGCs adhere to laminin, fibronectin and collagen IV (Gar- cia-Casrro et al. 1997). PGC binding to collagen IV was found to be stable before, during and after migration. However, PGC adhesion to laminin and fibronectin decreased during the stage of active migration. PGC-fibronectin adhesion was further diminished during early gonad assembly, whereas PGC-laminin adhesion increased at this stage. The fact that PGCs change their interactions with fibronectin and laminin during migration strengthens the hypothesis of ECM involvement in this process. It also suggests modulation of the receptors expressed by PGCs.

In an early attempt to characterise PGC receptors that bind to the ECM, blocking studies and cation- dependence were tested. Peptides containing the peptide motif Arg-Gly-Asp (RGD), a known integrin binding domain, partially inhibited the binding of PGCs to laminin and fibronectin but not to collagen IV. It should be noted, however, that not all integrins bind through this motif. Heparin partially blocked adhesion of PGCs to laminin only. Finally, chelation of divalent cations resulted in the partial reduction of PGC binding to laminin.

Engelbreth-Holm-Swarm (EHS) laminin is a heterotrimeric molecule consisting of u l , 81 and y l chains (see Fig. I ) . This molecule contains multiple unique cell adhesion sites, as well as domains for ECM binding and self-assembly ( Yurchenco & Julian 1994). This complexity can be overcome by the use of proteolytic fragments of laminin, which typically contain one or two known functional domains in iso- lation from the rest of the molecule. We found that PGCs bound most avidly to a proteolytic fragment corresponding to the globular C-terminus of the laminin ul chain (see Fig. l), a known cell and heparin- binding site (Yurchenco & Cheng 1993). This may ex- plain why PGC binding to whole laminin is partially heparin-sensitive. Small peptides corresponding to

129

ANDERSON et ul.

Fig. 2. Distribution of laminin in an E12.5 male gonad. (A) Low power micrograph of gonad and mesonephros, stained for EHS laminin. Magnification of gonad, stained for (B) alkaline phosphatase, and (C) laminin a l . * sex cord: arrow. alkaline phosphatase-positive PGCs (used with permission from Garcia-Casrro ef ul. (in press).

this region of the molecule preferentialy supported the adhesion of PGCs (Skubitz et ul. 1991). This in- teraction was completely heparin-sensitive.

Given the affinity of PGCs for laminin ctl, we performed immunohistochemistry with monoclonal antibodies specific for this subunit. We found that the laminin ctl chain is highly expressed in the early gonad, despite the relative scarcity of the molecule in the rest of the embryo (see Fig. 2 ) (Garcia-Custro et ul. (in press)). It is interesting to note that mutation of the laminin a1 gene in Drosophilu disrupts PGC migration and assembly (Juglurz & Howard 1995). Given this fact, together with our data, it is temping to speculate that laminin a1 has a similar role in the mouse. Further experimentation is clearly needed.

In the mouse embryo, the integrin family of hetero- dimeric glycoprotein receptors has been shown to be required for the migration of several cell types. For example, the 01 integrin is required for haemato- poetic precursor migration into the fetal liver (Hirsch et ul. 1996). We are currently undertaking an exhaus- tive survey of known integrin subunits on the surface of PGCs during and after migration. We are also examining the possibility that germ cells express novel integrins and/or non-integrin ECM receptors. Using genetics (targeted gene deletion) and cell culture, we can then evaluate the function of identified molecules in vivo and in vitro. Our hypothesis is that these molecules will not only be required for migration, but will be involved in PGC survival and proliferation.

PGCs undergo a striking change in phenotype as

migration ends. At this time, PGCs begin preferen- tially to adhere to each other. We have shown that this is not simply a result of cell division (Gonipcrts et u1. 1994). Homotypic cell-cell adhesion is the hall- mark of cadherin expression in several types of cells. Given the striking morphology o f PGCs in the early gonad, we are actively investigating the potential expression and function of cadherins on PGCs.

Recent work in the fruitfly Drosophilu mehogas ter has identified several novel somatic genes required for proper PGC migration, homing and gonad assembly. It is likely that at least some of these genes will have similar functions in mammals. For example, the Wun protein has been shown to control orderly PGC emi- gration from the hindgut (Zhung et ul. 1997). Perhaps a Wunun-like protein is responsible for the midline predilection of extragonadal germ cell tumours.

CONCLUSION

PGCs migrate actively during development. During migration, PGCs require growth factors from adjacent somatic cells to survive and proliferate. How- ever, PGCs are able to migrate and coalesce indepen- dently of this somatic environment. Like other migrating cell types, PGCs are surrounded by, and adhere to, ECM molecules during migration. Finally, we are investigating the possibility that PGCs aggregate with each other in a cadherin-dependent man- ner.

130


We would like to thank the Wellcome Trust, The Harrison Fund, The Institute of Human Genetics, the NIH (HD33440- Ol), and CONACyT Mexico, and the Life and Health In- surance Medical Research Fund for financial support of this work.

REFERENCES

Anderson, D., Lyman, S., Baird, A.. Wignall, J.. Eisenman, J., Rauch, C.. March, C., Boswell, H. , Gimpel, S.. Co- sman, D. & Williams, D.: Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63:

Bird, J. & Kimber, S.: Oligosacharides containing the fu- cose linked a(1-3) and a(1- 4) to N-acetylglucosamine cause decompaction of mouse morulae. Developmental Biology 104: 449469, 1984.

Chiquoine, A,: The identification, origin and migration of the primordial germ cells of the mouse embryo. Anat. Rec. 118: 135-146, 1954.

Cooke, J., Godin, I., ffrench-Constant. C., Heasman, J. & Wylie, C.: Culture and manipulation of germ cells. Methods Enzymol. 22.5: 37-77, 1993.

DeFelici, M. & Dolci, S.: In vitro adhesion of mouse fetal germ cells to extracellular matrix components. Cell Different. Develop. 26: 87-96, 1989.

Donovan, P., de Miguel, M., Cheng, L. & Resnick, J. L.: Primordial germ cells, stem cells and testicular cancer. APMIS 106: 134-141, 1998.

Donovan, P., Stott, D.. Cairns, L., Heasman. J. & Wylie, C.: Migratory and postmigratory mouse primordial germ cells behave differently in culture. Cell 44: 831- 838, 1986.

Fenderson. B., Eddy, E. & Hakomori, S.-I.: Glycoconjug- ate expression during embryogenesis and its biological significance. BioEssays 12: 173-1 79, 1990.

ffrench-Constant, C.. Hollingsworth, A, , Heasman, J. & Wylie, C.: Response to fibronectin of mouse primordial germ cells before, during and after migration. Develop- ment 113: 1365-1373, 1911.

Fujimoto, T. , Yoshinaga, K. & Kono, I.: Distribution of fibronectin along the pathway of primordial germ cells in mice. Anat. Rec. 211: 271-278, 1985.

Garcia-Castro, M., Heasman. J. & Wylie. C.: A rapid and quantitative assay of adhesiveness of mouse primordial germ cells that can be applied to many types of early embryonic cells. Methods in Cell Science 19: 1-9, 1997.

Garcia-Castro. M.. Anderson, R., Heasman, J. & Wylie. C.: Interactions between germ cells and extracellular matrix glycoproteins during migration and gonad assembly in the mouse embryo. J. Cell Biol. In press.

Ginsburg, M. , Snow, M. & McLaren, A,: Primordial germ cells in the mouse during gastrulation. Development 110: 521-528, 1990.

Godin, I., Wylie, C. & Heasman, J.: Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture. Develop- ment 108: 357-363, 1990.

Godin, I., Deed, R.. Cooke, J.. Zsebo, K., Dexter, M. & Wylie, C.: Effects of the steel gene product on mouse primordial germ cells in culture. Nature 3.52: 807-809, 1991.

175-183, 1990.

Gomperts, M. , Garcia-Castro, M., Wylie, C. & Heasman. J.: Interactions between primordial germ cells play a role in their migration in mouse embryos. Develop- ment 120: 135-141, 1994.

Hemmerich, S. & Rosen, S.: 6'-Sulfated Sialyl Lewis' is a major capping group of GlyCAM-I. Biochemistry 33: 483M835, 1994.

Hirsch, E., Iglesias, A,, Potocnik, A , , Hartmann. U. & Fiissler, R.: Impaired migration but not differentiation of haematopoietic stem cells in the absence of pl integrins. Nature 380: 171-175, 1996.

Jaglarz, M. & Howard, K.: The active migration of Droso- phila primordial germ cells. Development 121: 3495- 3503, 1995.

Lallier, T., Deutzmann, R., Perris, R. & Bronner-Fraser. M.: Neural crest interactions with laminin: structural requirements and localization of the binding site for a lp1 integrin. Developmental Biology 162: 451464, 1994.

Nocka, K., Majumder. S., Chabot, B., Ray, P., Cervone, M. , Berstein, A . & Besmer, P.: Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice - evidence for an impaired c-kit kinase in mutant mice. Genes Dev. 3: 816-826, 1989.

Orr-Urtreger, A, , Avivi, A, , Zimmer, Y,, Givol, D., Yarden, Y. & Lonai, P.: Developmental expression of c-kit, a proto-oncogene encoded at the W locus of the mouse. Development 109: 91 1-923, 1990.

Pesce, M. , Farrace, M. , Piacentini, M. , Dolci, S. & De Fel- ici, M.: Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by supressing programmed cell death (apoptosis). Development 118:

Rossi, P., Dolci, S., Albanasi. C., Grimaldi, P. & Geremia, R.: Direct evidence that the mouse sex-determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in adult testes. Molec. Repro. Develop. 34: 369-373, 1993.

Skubitz, A., Letourneau, P.. Wayner, E. & Furcht. L.: Syn- thetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the p l integrin subunit. J. Cell Biol. 11.5: 1137-1148, 1991.

Stone, J., Quinn, J., Albelda, S. & Lauffenberger, D.: Maximal migration of human smooth muscle cells on fibronectin and type-IV collagen occurs at an inter- mediate attachment strength. J. Cell Biol. 122: 729- 737, 1993.

Tam, P. & Snow, M.: Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. J. Embryol. Exp. Morph. 64: 133-147, 1981.

Yao, C., Ziober, A., Sutherland, A., Mendrick, D. & Kram- er, R.: Laminins promote the locomotion of skeletal myoblasts via the a7 integrin receptor. J. Cell Sci. 109: 3 139-3 1.50, 1996.

Yeom, Y., Fuhrmann, G., Ovitt. C., Brehm, A , , Ohbo. K., Gross, M., Hubner. K. & Scholer, H.: Germline regula- tory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122: 881-894, 1996.

Yoshinaga, K., Muramatsu, H. & Muramatsu, T.: Immuno- histochemical localization of the carbohydrate antigen 4C9 in the mouse embryo: a reliable marker of mouse primordial germ cells. Differentiation 48: 75-82, 1991.

131

1089-1094, 1993.

ANDERSON e/ al.

Yurchenco, P. & Cheng, Y.-S.: Self-assembly and calcium- binding sites in laminin. J. Biol. Chem. 268: 17826- 17299, 1993.

Yurchenco, P. & Julian, 0. R.: Basal lamina assembly. Curr. Biol. 6 ; 674681, 1994.

Zhang, N . , Zhung, J . , Purcell, K.. Cheng. Y. & How,cirti, K.: The Drosophila protein Wunen repels migrating germ cells. Nature 385: 6467 . 1997.

DISCUSSION

R. Short (Australia): Do gonocytes migrate by the vascular route or only through tissue planes?

C. C. Wylie ( U S A ) : Germ cells have never been seen within blood vessels.

A. Timmer (The Netherlands): There is a stem cell factor in the haematopoietic system which modules the function of integrins, and there is probably a stem cell factor which has a role in the survival and proliferation of mouse primordial germ cells. Is there also a stem cell factor which regulates the function of a6pl integrin?

C. C. Wylie: Without stem cell factor the germ cells die, and they also require function p l integrin which suggests that the factor is acting through regulation of integrins.

I. Damjunov (USA): Is the programmed expression of various integrins on germ cells fixed temporally or can it be induced under the influence of the ex- ternal factors in the environment, or is there inter- action between the two? Are germ cells different if placed in a different environment?

C. C. Wylie: This is not known. It would be interesting to see if different culture conditions could in- duce altered integrin expression patterns.

R. Chuganti (USA): Do all germ cells complete the migration or are any left behind, and if so, what happens to them?

C. C. Wylie: Many are left behind and I am not sure what proportion d o not reach the genital ridges, but this is a substantial proportion and can be found at later stages in fetal development in the kidney and adrenal gland where some will enter meiosis. Then they just disappear and die.

R. Short: They also migrate to the brain.

M . Peru (Australia): Heparan sulphate proteo- glycans have a role in adhesion. Is there any evi-

dence that they are critical for the presentation of growth factors to these cells?

C. C. Wylie: No.

A . Timmer (The Netherlands): (in collaboration with J. W. Oosterhuis, H. Schrufordt Koops, D. Th. Sleijier, E. G. E. de Vries & W. Timens): By interacting with cell adhesion molecules, extracellular matrix (ECM) proteins may have a pro- found influence on the morphology, behaviour and function of tumour cells. Among the different cell adhesion molecules that have affinity for ECM proteins, integrins are the most widely studied. We examined by immunohistochemistry different components of 34 testicular non-seminomas in order to determine whether the distribution of integrin subunits and composition of the ECM was corre- lated to differentiation into the embryonal (embryonal carcinoma, teratoma) or extraembryonal (yolk sac tumour, choriocarcinoma) direction. Em- bryonal carcinomas demonstrated variable expression of al, a2, a3, a5, a6, pl and p4 integrin subunits. The different tissues in teratomas demonstrated characteristic integrin profiles (epithelia: al, a2, a3, a5, a6, pl, and 4; cartilage: a3, a5, a6, and p l ; smooth muscle: al, a3, a5, a6 and pl). Yolk sac tumours showed expression of a3, a5, a6, and p l integrin subunits. In choriocarcin- omas, integrin subunits were heterogeneously expressed. Subpopulations of cytotrophoblastic cells could be identified by their integrin profile. Cyto- trophoblastic cells were characterised by p4 integrin subunit expression, not present on syncytio- trophoblastic giant cells. The differences in distribution of ECM proteins between embryonal carcinomas, teratomas, yolk sac tumours and cho- riocarcinomas were relative. Our demonstration of the heterogeneity of integrin subunit expression and composition of the ECM in testicular non-seminomas. suggests a relationship with differentiation and biological behaviour. Future studies will be required to elucidate whether integrin subunit expression is a reflection of the process of differentiation itself (inside-out signalling), or whether differentiation is affected by the pattern of integrin expression (outside-in signalling)

132


R. Anderson (USA): (in collaboration with J. Heas- man & C. C. W y k ) : Primordial germ cells (PGSs) are the founder cells for adult egg and sperm. During development in the mouse, PGCs migrate from the base of the allantois (E8.5) to the urogenital ridges (El 1.5), where they become incorporated into the developing gonad. At this time PGCs cease to migrate, both in vitro and in the embryo. We hypothesise that PGCs accomplish this change in phenotype by al- tering expression of cell surface receptors during development. Previously, we and others have shown that PGCs bind to the extracellular matrix proteins laminin, fibronectin and collagen IV. We chose to

look at receptors that have been shown in other cell types to bind these molecules. To study PGC gene expression, we dissected El 2.5 gonads, purified PGCs by fluorescence-activated cell sorting (FACS), and analysed mRNA expression by RT-PCR. We were able to purify PGCs to greater than 99% purity, as evidenced by alkaline phosphatase activity and the profile of mRNA expression. Using this technique, we found that E12.5 PGCs express specific integrins. This expression was verified by immunofluorescense. Further, we have shown that PGC integrin expression changes during migration, and that in culture, integrin function is required for proper gonad formation.

133

Documents

Early stages in male germ cell differentiation in the mouse