THE ANATOMICAL RECORD 246535-544 (1996) Sex-Dependent Expression of Placental (P)-Cadherin During Mouse Gonadogenesis Department of LI-HSIEN LIN AND ROBERT M. DEPHILIP Cell Biology, Neurobwlogy, and Anatomy, Ohio State University College of Medicine, Columbus, Ohio ABSTRACT Background: Placental (PI-cadherin is one of a family of cell adhesion molecules that participate in embryonic sorting and organogenesis. In previous work, P-cadherin was localized to Sertoli cells in the mouse testis as early as postnatal day 1. This early postnatal localization raised questions about when P-cadherin first appeared in the embryonic testis and whether P-cadherin was expressed differentially in the embryonic testis and ovary. Methods: The localization of P-cadherin, epithelial (Ehcadherin,and Miillerian inhibiting substance was determined in frozen sections of mouse gonads between embryonic days 10.5 and 18 using indirect immunohistochemistry. Alkaline phosphatase reactivity was used to identify germ cells. Results: The expression of P-cadherin was traced back to the indifferent stage of gonadogenesis where uniform distribution was observed in the indifferent gonad of both sexes. However, after sexual differentiation, the expression of P-cadherin in the testis was localized to Sertoli cells in the testicular cords, while its expression in the ovary fell below detectable levels. Conclusions: The localization of P-cadherin in the male and female indifferent gonad is similar and cannot be used to distinguish the future testis and ovary. The localization of P-cadherin in the testis after sexual differentiation suggests a role for P-cadherin in testicular cord formation. The common temporal pattern of P-cadherin and Miillerian inhibiting substance expression in Sertoli cells is consistent with a shared regulatory mechanism. o 1996 Waey-Liss, Inc. Key words: Cadherins, Cell adhesion molecules, Embryo, Germ cells, Gonads, Sertoli cells, Sex differentiation After formation of the indifferent gonad, the pathways of male and female gonadogenesis diverge sharply and produce either a testis or an ovary. The indifferent gonad is so named because its eventual outcome cannot be predicted by morphological criteria. Under directive of the male genotype, the indifferent gonad becomes a testis. Under directive of the female genotype or in the absence of the male genotype, the indifferent gonad becomes an ovary. Once the decision is made to embark on either the male or female differentiation pathway, the structure of the gonad is specified, as is the structure of the corresponding reproductive tract. Although there is some variance in the literature because different strains and mating regimens have been used, the dates for several critical events of gonadogenesis in the mouse have been established (for a review see McLaren, 1991). The gonadal ridge forms on embryonic day 10.5, primordial germ cells (PGCs) enter the gonadal ridge between days 10.5 and 12, and testicular cords form on day 12.5. The formation of testicular cords is the first histological event that distin0 1996 WILEY-LISS, INC. guishes the future testis and ovary, marking the end of the indifferent stage of gonadogenesis and the onset of sex differentiation. One approach towards a better of understanding of gonadogenesis is to identify specific molecules that are regulated differentially in the male and female during gonad formation. The prototype of these molecules is Mullerian inhibiting substance (MIS), also known as anti-Mullerian hormone. MIS is responsible for the dramatic regression of the Mullerian duct system seen in the male (Joat, 1953).It is the first product known to be secreted by Sertoli cells in the developing testis ("ran and Josso, 19821, and transcripts for MIS are first detected in Sertoli cells of the mouse testis on embryonic day 12.5 (Munsterberg and Lovell-Badge, 1991). Transcripts for MIS do not appear in the ovary until postnatal day 6, well after gonad differentiation. Received April 26,1996 accepted July 30,1996. Address reprint requests to Robert M. DePhilip, Department of Cell Biology, Neurobiology, and Anatomy, 4072 Graves Hall, Ohio State University, Columbus, OH 43210. 536 L.H. LIN AND R.M. DEPHILIP Thus, MIS serves as an example of a gonadal protein-in this case, a differentiation factor-that demonstrates sex-dependent expression. Structural proteins, particularly the intermediate filament proteins, have also been studied to determine whether they exhibit sex-dependent expression during gonadogenesis (Fridmacher et al., 1992; Frojdman et al., 1992, 1993). During an initial, sex-independent phase of gonadal development, the sequential appearance of desmin, vimentin, and cytokeratins 8 and 19 is observed in both sexes. A sex-dependent phase of gonadogenesis follows, and cytokeratin 18 replaces cytokeratin 19 in the testis. Cytokeratin 18 is associated with Sertoli cells as testicular cords form but is not observed a t any time in the ovary (Fridmacher et al., 1992). Given the obvious importance of cell-cell and cellextracellular matrix interactions during gonadogenesis, the sex-dependent expression of cell adhesion molecules has been explored. In the mouse, the neural cell adhesion molecule (NCAM) is expressed by some of the cells lining the developing testicular cords in the male on embryonic days 12.5 and 13.5 but not at later ages. In the female, NCAM reactivity was observed in cells lining clusters of differentiating germ cells between days 12.5 and 18.5. It was suggested that NCAM plays a role in compartmentalization of the developing gonad (Mgller et al., 1991). The expression of the a6 subunit of integrin has also been studied in the developing male (Frijjdman and Pelliniemi, 1994) and female mouse gonad (Frojdman and Pelliniemi, 1995). Here again, localization of the a 6 subunit is consistent with a role in the formation of somatic-germ cell aggregates, and, while there is sex-dependent expression of the a 6 subunit in the tunica albuginea and in the surface epithelium of the male, expression of the a6 subunit is not sex-specific within the gonad itself but is observed in both male and female germ cell aggregates. We are interested in determining the expression pattern and function in the testis of a family of cell adhesion molecules called cadherins (Nose et al., 1987; Kemler, 1992). Cadherins are defined by their unique dependency on calcium ions for adhesive function and for protection from proteolysis (Hyafil et al., 1981; Yoshida-Nor0 et al., 1984). During various developmental events, there is a clear correlation between cadherin expression and adhesive behavior: aggregates of cells express high levels of cadherin, and nonaggregated cells express low levels (Takeichi, 1988). Recently, we have shown that Sertoli cells express placental (P)-cadherin during the first 8 days of postnatal development (Lin and DePhilip, 1996). After day 8, P-cadherin is no longer expressed in Sertoli cells but begins to be expressed in the maturing peritubular cells. The expression of P-cadherin in peritubular cells then continues to adulthood. The association of P-cadherin in early postnatal Sertoli cells raised two questions: (1)what the earliest appearance of P-cadherin in the embryonic testis is and (2) if there is sex-dependent expression of P-cadherin during formation of the testis and ovary. This report provides a morphological comparison of P-cadherin expression in the mouse testis and ovary during embryonic development. Immunohistochemical results show that P-cadherin was expressed in the go- nad of both sexes during the indifferent stage of development. However, after sexual differentiation, P-cadherin continued to be expressed in the testis but was no longer detected in the ovary. In the testis, expression of P-cadherin was spatially and temporally associated with formation of the testicular cords. In addition, we extend the time frame during which epithelial (El-cadherin is associated with germ cells (Wu et al., 1993) and show that E-cadherin is expressed by germ cells of both sexes throughout sexual differentiation. The sustained expression of P-cadherin during testicular development suggests that P-cadherin is regulated by elements of the male determination pathway, a pathway which is currently believed to be initiated by the sexdetermining region of the Y chromosome (Sry) (reviewed by Moore and Grumbach, 1992). The position of P-cadherin expression in this regulatory pathway is discussed. MATERIALS AND METHODS Animals Timed pregnant female ND4 Swiss Webster mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and were maintained on a cycle of 12 h of light and 12 h of darkness with free access to food and water. Pregnant females were killed by cervical dislocation. Embryos were collected at 12 h or 24 h intervals between embryonic days 10.5 and 18.The day on which the copulation plug was identified was taken to be embryonic day 0. Mating was assumed to take place at midnight on the day the plug was identified. Since the breeder pairs were housed together from 4:OO PM to 8:OO AM, there was a possible of error of 2 8 h in the estimated age of the embryos. Only embryos whose estimated age matched their developmental stage, as defined by hind limb development (McLaren and Buehr, 1990), were used. Embryos were collected from the uterus and killed by decapitation with a sharp razor. The sex of the embryos was determined by analysis of chromosomes in amnion cells (Palmer and Burgoyne, 1991). Maintenance and experimental use of animals complied with the NIH Guide for the Care and Use of Laboratory Animals. lmmunohistochemistry and Detection of Alkaline Phosphatase Reactivity Between days 10.5 and 12, the caudal half of the embryo was embedded in O.C.T. Compound (Miles Inc., Elkart, IN). To localize the gonad, continuous cryostat sections (7-10 pm) were cut, and every eighth to tenth section was examined for alkaline phosphatase enzymatic activity on germ cells as described (Chiquoine, 1954),except that a substrate mixture of 400 pM nitro blue tetrazolium and 380 pM 5-bromo-4-chloro-indoyl phosphate in 100 mM Tris-C1, pH 9.5, mM NaC1,5 mM MgCl,, and 1.5 mM CaC1, was used. Between days 12.5 and 18, the gonad was dissected from the embryo and embedded. To localize P-cadherin, the procedure described previously was used (Lin and DePhilip, 19961, except that the concentration of PCD-1 primary antibody (Nose and Takeichi, 1986) was increased to 20 pg/ml. The concentration of the ECCD-2 primary antibody for E-cadherin (Nagafuchi et al., 1987) was also 20 pg/ml. The MGH-4 rabbit antiserum against MIS (Ueno et al., 1989) was used at a dilution of 12300. To P-CADHERIN EXPRESSION I N MOUSE GONADOGENESIS localize the antibodies against cadherins, a 1:30 dilution of rhodamine-conjugated, goat anti-rat IgG antiserum was used as described (Lin and DePhilip, 1996).To localize MGH-4, sections were processed with the WCTASTAINB Elite ABC Kit using instructions supplied with the kit. PCD-1 and ECCD-2 were purchased from Zymed Laboratories (South San Francisco, CA), and the MGH-4 antiserum was obtained from Dr. Patricia K. Donahoe (Massachusetts General Hospital, Boston, MA). To localize alkaline phosphatase enzyme reactivity and E-cadherin immunoreactivity on the same section, the immunofluorescence procedure was performed first, and the section was photographed. The coverslip was then removed, and the section was processed for alkaline phosphatase reactivity and rephotographed. 537 collected from near the longitudinal midpoint of the gonadal ridge in order to eliminate ambiguities due to comparisons between different levels of the gonads. The results shown in Figure 1 demonstrate that the expression patterns of P-cadherin in the male and female indifferent gonad are similar and cannot be used to distinguish the future testis and ovary. P-Cadherin Expression in the Testis and Ovary During Morphological Sex Differentiation The first sign of morphological sex differentiation in the mouse occurs around embryonic day 12.5, when testicular cords define the testis. A second feature of testis formation is development of the tunica albuginea in the region adjacent to the surface epithelium. The ovary remains morphologically undifferentiated and demonstrates neither testicular cords nor a developing RESULTS tunica (Brambell, 1927). We compared the immunoreP-Cadherin Expression in the Gonadal Ridge During the activity of P-cadherin in the testis and ovary on cryIndifferent Stage of Development ostat sections prepared on embryonic days 12,12.5,13, The gonadal ridge is the precursor to the gonad and and 14. This comparison is presented in Figure 2. On can be recognized in the mouse on embryonic day 10.5 day 12, P-cadherin immunoreactivity in the testis was (Rugh, 1968). We used immunofluorescence micros- localized to aggregates of cells forming the gonadal copy to localize P-cadherin in the gonadal ridge in cry- cords and was absent from the region just deep to the ostat sections prepared from lo-, 11-,and 11.5-day-old surface epithelium-the site of the future tunica albuembryos. The sex of the embryos at these early ages ginea (Fig. 2A). In contrast, P-cadherin reactivity in was determined by the presence or absence of Barr bod- the ovary on day 12 remained more uniformly distribies in amniotic cells. The position of the gonadal ridge uted throughout the gonad (Fig. 2B). In both the testis was defined by the presence of alkaline phosphatase and ovary on day 12, P-cadherin reactivity was associreactive germ cells that migrate into this region be- ated with the surface epithelium and with epithelial tween days 10.5 and 12 (McLaren, 1991). Sections se- cells of the mesonephric tubules. As the testis continlected for P-cadherin localization were always between ues to develop on days 12.5, 13, and 14, the testicular sections containing germ cells. cords became progressively more organized and continOn embryonic day 10, P-cadherin was associated ued to express P-cadherin (Fig. 2C,E,G). However, the with the surface epithelium of the gonadal ridge and appearance of P-cadherin in the ovary during this pewith several layers of cells deep to the surface epithe- riod was strikingly different (Fig. 2D,F,H). While the lium (Fig. 1).These P-cadherin-reactive subepithelial reactivity of P-cadherin within the ovary on day 12 was cells were located in an area bordered by the surface easily detected and uniformly distributed, reactivity on epithelium, the attachment of the dorsal mesentery, day 12.5 fell dramatically. The fluorescent signal on and the dorsal aorta and were anterior to the P-cad- day 13 was also low, and by day 14 the only P-cadherin-negative mesonephros. There was no difference herin-reactive cells associated with the ovary were the in either the location or the intensity of P-cadherin epithelial cells on the surface of the organ and in the reactivity in the male and female gonadal ridge on day mesonephric tubules. Throughout this period, epithe10 (Fig. lB,C). P-cadherin reactivity was also ex- lial cells of the mesonephric tubules and the Wolffian pressed by cells of the coelomic epithelium covering the duct were P-cadherin-positive in both the male and the body wall and the abdominal organs and by epithelial female. cells of the Wolffian duct. By day 11,an increase in the Localization of P-Cadherin and of MIS on Consecutive number of cells had caused the gonadal ridge to proSections of the Testis During Gonadogenesis trude slightly into the coelomic cavity, and P-cadherin reactivity was localized to this protruding ridge (Fig. In previous work, we have shown that Sertoli cells 1D). As on day 10, the reactivity of P-cadherin in the express P-cadherin on postnatal days 1, 3, and 8 (Lin male and female gonadal ridge on day 11was indistin- and DePhilip, 1996). This observation and the associguishable (Fig. lE,F). In both sexes, the coelomic epi- ation of P-cadherin with developing cords presented in thelium and the epithelial cells of the Wolffian duct Figure 2 raised the question of whether Sertoli cells expressed P-cadherin on day 11. Beginning on day were responsible for the expression of P-cadherin in the 11.5, continued cellular expansion in the indifferent prenatal testis. MIS is an early differentiation marker gonad made the distinction between it and the meso- for Sertoli cells (Blanchard and Josso, 1974; Tran and nephros more apparent (Fig. lG,H). P-cadherin reac- Josso, 1982), and we compared the localization of tivity was uniformly distributed throughout the ex- P-cadherin with that of MIS in consecutive sections of panded cellular mass of both the male and female the testis on embryonic days 11.5 and 12.5 and fetal indifferent gonad. Cells of the coelomic epithelium and day 18 (Fig. 3). As in Figure 1,P-cadherin reactivity on the Wolffian duct continued to be P-cadherin-reactive. day 11.5 was uniformly expressed throughout the testis Mesenchymal cells of the mesonephros did not demon- (Fig. 3A). However, sections of the testis on day 11.5 strate P-cadherin reactivity a t any developmental age did not express MIS (Fig. 3B). On day 12.5, a large studied here. All sections presented in Figure 1 were number of cells in the developing cords expressed both Fig. 1. The localization of P-cadherin in the gonadal ridge of male and female embryos during the indifferent stage of development. P-cadherin was localized using indirect immunofluorescence in transverse cryostat sections of male (C,D,E,G) and female (A,B,F,H) embryos on days 10 (A,B,C), 11(D,E,F),and 11.5 (G,H).The section from the day 10 female embryo is shown at low magnification in A for orientation and a t higher magnification in B. Similarly, the section from the day 11male embryo is shown a t low magnification in D and at higher magnification in E. The position of the gonadal ridge is indicated with a bracket. bw, body wall; da, dorsal aorta; dm, dorsal mesentery; m, mesonephros; Wd, Wolffian duct. A,D: x 90. B,C,E,F: x 190.G , H x 200. Fig. 2. The localization of P-cadherin in the testis and ovary during the period of sexual dxerentiation. P-cadherin was localized using indirect immunofluorescence in transverse cryostat sections of testis (A,C,E,G) and ovary (B,D,F,H) on embryonic days 12 (A,B), 12.5 (C,D),13 (E,F),and 14 (G,H). c, testicular cords; mt, mesonephric tubules; se, surface epithelium of the gonad; t, region of the developing tunica albuginea. A-E,G: x 195. F: x 245.H: x 225. 540 L.H. LIN AND R.M. DEPHILIP P-cadherin and MIS (Fig. 3C,D). By day 18, the testicular cords are well formed, and germ cells and Sertoli cells can be distinguished from e&h other on the basis of differing size and contour: germ cells are larger and round, while Sertoli cells are smaller and pleomorphic, filling all spaces within the cords not occupied by germ cells. On day 18, P-cadherin reactivity outlined the smaller Sertoli cells, while the larger germ cells occupied spaces within the cords that displayed no fluorescent signal (Fig. 3E). The localization of MIS on an adjacent section matched that of P-cadherin: MIS reactivity was associated with the smaller Sertoli cells situated a t the periphery of the cords but was not associated with germ cells that occupied the cord center (Fig. 3F). Throughout this series, P-cadherin was associated with the surface epithelium of the gonad and with epithelial cells of the mesonephric tubules, while MIS was not expressed at either of these locations. Colocalization of E-Cadherin and Alkaline Phosphatase Reactivities in the Testis and Ovary on Embryonic Day 12.5 The results in Figures 2 and 3 demonstrate the differential expression of P-cadherin during male and female gonadogenesis. In other work, germ cells have been shown to express E-cadherin in the mouse testis on postnatal day 8 (Wu et al., 1993). It was important to determine whether germ cells in the embryonic testis also express E-cadherin and whether there was differential expression of E-cadherin in the testis and ovary that with P-cadherin might contribute to gonadal differentiation. Alkaline phosphatase is a reliable marker for germ cells (Chiquoine, 1954), and we compared the localization of alkaline phosphatase enzyme reactivity and E-cadherin immunoreactivity in the male and female gonad on embryonic day 12.5. At this age, there is an obvious difference in the expression of P-cadherin in the sexes: testicular cords express P-cadherin, while somatic cells surrounding clusters of germ cells in the ovary express relatively low levels of P-cadherin (Fig. 2C,D). Figure 4 demonstrates that all germ cells express both alkaline phosphatase reactivity and E-cadherin immunoreactivity in both the testis and the ovary on day 12.5. In the testis, alkaline phosphatase and E-cadherin were localized to germ cells in the forming testicular cords (Fig. 4A,B). In the ovary, alkaline phosphatase and E-cadherin were associated with the loose aggregates of germ cells that comprise the ovary a t this age (Fig. 4C,D). This one-to-one correlation between alkaline phosphatase reactivity and E-cadherin immunoreactivity of germ cells was also seen in both sexes on day 12 and day 14 (not shown). The low level of alkaline phosphatase reactivity exhibited by the surface epithelium of the testis is of unknown significance and has been observed by others (Paranko and Pelliniemi, 1992). The expression of E-cadherin by epithelial cells of the mesonephric tubules served as an internal, positive control for E-cadherin immunoreactivity. It is noteworthy that the surface epithelium of the testis and the ovary did not express E-cadherin as might be expected for epithelial cells. These results demonstrate that, unlike P-cadherin, E-cadherin is not differentially expressed in the developing testis and ovary. Comparison of E-Cadherin and P-Cadherin in the Prenatal Testis Finally, we compared E-cadherin and P-cadherin reactivities in consecutive sections of the developing testis in order to better demonstrate how these cadherins are localized with respect to each other during testicular cord formation. This comparison was made on three different days and covered the period of testicular cord formation. On embryonic day 11.5, E-cadherin was localized to the aggregating germ cells within the forming gonad, while P-cadherin was expressed uniformly by the more numerous somatic cells in the testis (Fig. 5A,B). On day 14, the segregation of E-cadherin-expressing germ cells within the cords had been completed (Fig. 5C). Similarly, P-cadherin reactivity within the testis became restricted to the cords and was not detected in the interstitial spaces (Fig. 5D). The outline of the E-cadherin-reactive aggregates of germ cells displayed a lobular border that is consistent with the rounded surface of germ cells. The P-cadherin reactivity associated with cords occupied all spaces between the germ cells and between the germ cells and the perimeter of the cords. The relative localization of E- and P-cadherin was most evident on day 18 (Fig. 5E,F). Here, E-cadherin outlined the larger germ cells, while P-cadherin was associated with the smaller Sertoli cells that occupied the base of the cords and filled spaces between germ cells in the center of the cords. The nuclei and cytoplasm of germ cells were P-cadherin-negative and created negative images in the P-cadherin-stained sections (Fig. 5F). Throughout this series, the surface epithelium of the testis was P-cadherin-positive and E-cadherin-negative. DISCUSSION Immunohistochemistry was used here to define two periods of P-cadherin expression during gonadogenesis. During an initial period that coincided with the indifferent stage of gonadogenesis (embryonic day 10 to day 11.5), P-cadherin was expressed in both the male and female gonad in a similar pattern and at a similar level. In a subsequent period that coincided with sex differentiation (embryonic day 12 and later), P-cadherin was associated with testicular cords, while its expression in the ovary fell to undetectable levels. The simplest explanation for these observations is that P-cadherin expression is retained in the male, where it participates in cell adhesion events that contribute to cord formation. The localization of P-cadherin to small, basal cells in the developing testicular cords suggested that it was a product of Sertoli cells. This suggestion was confirmed by demonstrating that P-cadherin was localized to the surface of cells that expressed MIS, an early marker for Sertoli cells in the testis. The first appearance of MIS in the developing mouse testis in our experiments was consistent with previous reports of MIS expression in that MIS was not expressed until testicular cords formed (Tran et al., 1977).Thus, MIS appears in Sertoli cells as P-cadherin becomes associated with Sertoli cells in testicular cords. We had shown earlier that P-cadherin immunoreactivity disappears from Sertoli cells on postnatal day 8 (Lin and DePhilip, 19961, which is around the time that the level of MIS in Ser- P-CADHERIN EXPRESSION IN MOUSE GONADOGENESIS 541 Fig. 3.The localization of P-cadherin and MIS in the testis during gonadogenesis. Cryostat sections of the developing testis were collected on embryonic days 11.5 (A,B) and 12.5 (C,D) and fetal day 18 (E,F).P-cadherin was localized using indirect immunofluorescence (A,C,E),and MIS was localized on consecutive sections using indirect immunohistochemistry (B,D,F). The position of the gonadal ridge (gr) is indicated with a bracket in A and B. Examples of germ cells (g), which are P-cadherin-negative and MIS-negative, are indicated in E and F,respectively. c, testicular cords; mt, mesonephnc tubules. A-D: x 195.E , F x 390. toli cells drops below detectable levels (Taketo et al., 1993). It is interesting that the appearance of MIS and P-cadherin coincide, as does the disappearance of these two molecules from the testis, postnatally. In addition to its well-known, regressive effect on the Mullerian duct of the male, MIS may have a positive influence on testis formation (Vigier et al., 1987). It will be important to determine whether MIS is a positive, autocrine regulator of P-cadherin expression in Sertoli cells or whether MIS and P-cadherin are regulated coordinately as the testis forms (see below). The differential expression of P-cadherin in the testis and ovary during sexual morphogenesis should be compared to the uniform expression of E-cadherin at this time. E-cadherin was expressed by germ cells in both the testis and the ovary before and after sex differentiation. The observation that E-cadherin appears on female germ cells implies that it plays a minor role in cord formation in the male, and the fact that testicular cords can form in the absence of germ cells (Merchant, 1975) is consistent with this interpretation. However, this view that E-cadherin does not play a role in tes- 542 L.H. LIN AND R.M. DEPHILIP Fig. 4. Colmalizationof E-cadherin immunoreactivity and alkaline phosphatase reactivity on germ cells in the developing testis and ovary on embryonic day 12.5. Alkaline phosphatase reactivity was demonstrated in cryostat sections of the testis (A) and ovary (C)on day 12.5. E-cadherin immunoreactivity was localized in the same sections of testis (B) and ovary (D). Note the one-to-one correlation be- tween alkaline phosphatase reactivity and E-cadherin immunoreactivity on germ cells and the absence of E-cadherin reactivity on the surface epithelium (se) of both the testis and ovary (B,D). The epithelial cells of the mesonephrictubules (mt) displayed E-cadherinimmunoreadivity in both sexes.A-D: X 190. ticular cord formation because it is also expressed in will occupy a core position, and the less adhesive cells the ovary may be misleading. The E-cadherin on fe- take up an outer position. Though results supporting male germ cells, while immunoreactive, may not be this model were obtained using two populations of cells functional. There are several factors, most importantly expressing different levels of the same cadherin moleassociation with cytoplasmic molecules called catenins cule, the model would also accommodate two popula(Ozawa et al., 1990), which modulate the adhesiveness tions of cells with different adhesiveness due to expresof cadherins. Therefore, E-cadherin on male germ cells sion of different cadherin molecules. During testicular may promote cell-cell adhesion and insure the inclu- cord formation, one would predict that the E-cadherinsion of germ cells in the forming testicular cords. expressing germ cells are more adhesive than the E-cadherin in the ovary, though immunoreactive, may P-cadherin-expressing Sertoli cells. The arrangement be nonadhesive and allow the dispersion of female of P-cadherin and E-cadherin demonstrated here suggests a mechanism for cord formation that can be germ cells into individual follicles. The segregation of different embryonic cell popula- tested experimentally by using function-blocking antitions into discrete structures is often associated with bodies. The switch in P-cadherin localization from its homothe appearance of different cadherin molecules on specific groups of cells (Takeichi, 1990). On day 18 of ges- geneous distribution in the indifferent gonad to its astation, E-cadherin-expressing germ cells were clus- sociation within cords in the testis parallels the decitered within a mantle of P-cadherin-expressing Sertoli sion of the indifferent gonad to become a testis. This cells. This arrangement calls to mind the “sphere developmental switch is controlled by a gene in the within a sphere” configuration that is predicted to re- sex-determining region of the mouse Y chromosome sult when adhesive forces are maximized between two called €ry (Cubbay et al., 1990). The Sry gene encodes groups of cells that have adhesion molecules on their a transcription factor that initiates a cascade of events surface (Steinberg and Takeichi, 1994). It has been and produces the male phenotype (Harley et al., 1992). demonstrated that when two groups of cells with dif- In the female, the expression of P-cadherin in the inferent adhesiveness are mixed, the more adhesive cells different gonad must be regulated by an autosomal or P-CADHERIN EXPRESSION IN MOUSE GONADOGENESIS 543 Fig. 5. The localization of E- and P-cadherin in the testis before and after testicular cord formation. The localization of E-cadherin (A,C,E) and P-cadherin (B,D,F) wm compared in consecutive sections of the testis on embryonic days 11.5 (A,B) and 14 (C,D) and fetal day 18 (E,F). The arrows in C and E indicate E-cadherin reactivity on the rounded surface of germ cells. Asterisks in F indicate examples of germ cells that are P-cadherin-negative. The surface epithelium of the gonad did not react with E-cadherin at any age examined here, and its position in A is indicated by a dotted line. The surface epithelium (se) of the testis and the epithelial cells of the mesonephric tubules (mt) were P-cadherin-positive a t all ages examined here. A-D: x 245. E , F x 390. X-linked gene. It may also be true that the early expression of P-cadherin in the male is regulated by autosomal or X-linked genes. However, at the point of sex differentiation, the expression of P-cadherin in the male is retained in Sertoli cells and may be regulated by the Sry gene. The sequence of the immediate 5' upstream region of the P-cadherin gene has been determined (Faraldo and Cano, 1993),and initial studies have shown that the regulation of the promoter is likely to be very complex (Hatta and Takeichi, 1994).It is intriguing that a DNA-binding motif (CCT'M'GA) for the mouse Sry gene protein (Nasrin et al., 1991),appears in the 5' flanking region of both the mouse P-cadherin gene and the mouse MIS gene (Shen et al., 1994). This observation, together with the coincident expression of P-cadherin and MIS during male gonadogenesis, raises the possibility that both molecules may be regulated by the Sry gene. Elucidation of the regula- 544 L.H. LIN AND R.M. DEPHILIP t o r y mechanism for the sex-independent and the sex- dependent expression of P-cadherin will provide important insight into the overall process of gonadogenesis. LITERATURE CITED Blanchard, M.-G., and N. 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