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Background: Mechanisms involved in early patterning of the mammalian gonad as it develops from a bipotential state into a testis or an ovary are as yet not well understood. Sex-specific vascularization is essential in this process, but more specific mechanisms required to, for example, establish interstitial vs. cord compartments in the testis or ovigerous cords in the ovary have not been reported. Adherens junctions (AJs) are known for their roles in morphogenesis; we, therefore, examined expression of AJ components including β-catenin, p120 catenin, and cadherins for possible involvement in sex-specific patterning of the gonad. Results: We show that, at the time of early gonadal sex differentiation, membrane-associated β-catenin and p120 catenin colocalize with cell-specific cadherins in both sex-nonspecific and sex-specific patterns. These expression patterns are consistent with an influence of AJs in overall patterning of the testis vs. ovary through known AJ mechanisms of cell–cell adhesion, cell sorting, and boundary formation. Conclusions: Together these complex and dynamic patterns of AJ component expression precisely mirror patterning of tissues during gonadogenesis and raise the possibility that AJs are essential effectors of patterning within the developing testis and ovary. Developmental Dynamics 241:1782–1798, 2012. © 2012 Wiley Periodicals, Inc.
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- EXPERIMENTAL PROCEDURES
During a remarkably short period of mammalian embryonic development, bipotential gonads become sexually dimorphic as tissue patterning in the testis and ovary diverges. In mice at embryonic day (E) 11.5, the morphologies of male and female gonadal ridges are thought to be comparable; by E12.5, the sex difference is striking. During this short period, two critical and related events in pattern formation occur in the testis: endothelial cells migrate from the mesonephros into the gonad to form male-specific vasculature, and the testis is partitioned into cords (Wilhelm et al., 2007; DeFalco and Capel, 2009). In the ovary, these events do not occur and the overall patterning found at E11.5 is largely maintained at E12.5. Sex-specific vascular patterning is known to be critical to induction of the overall patterning of a testis vs. an ovary (Coveney et al., 2008; Combes et al., 2009). However, in the testis, mechanisms that establish initial compartmentalization between cells of the cords and neighboring interstitial cells, or that organize cells within testis cords, are largely unknown. Similarly, in the ovary, mechanisms that contribute to formation of ovigerous cords—precursors to primordial follicles—are poorly understood (Loffler and Koopman, 2002; Wilhelm et al., 2007).
Many gene products have been reported to influence the molecular choice between development of a testis or an ovary, and expression of these initial, sex-determining genes is generally thought to occur in specific somatic cells, the support cells: Sertoli cells in the testis and pregranulosa cells in the ovary (Fleming and Vilain, 2005; Wilhelm et al., 2007; DeFalco and Capel, 2009). β-Catenin now appears to lie near the center of the sex-determining molecular decision, and is required for full ovarian development (Chassot et al., 2008; Maatouk et al., 2008; Tevosian and Manuylov, 2008). Loss of β-catenin function in XX animals, or gain of function in XY animals, leads to at least partial sex reversal, including changes in vascular and tissue patterning as well as gene expression (Maatouk et al., 2008; Manuylov et al., 2008). On the other hand, loss of β-catenin function in XY animals has shown no apparent effect (Chang et al., 2008; Manuylov et al., 2008; Liu et al., 2009). Together, these data support an effect of β-catenin in the ovary, including maintenance of female-specific tissue patterning. In the development of many tissues, β-catenin is known to participate in at least two distinct molecular mechanisms of action, housed in two subcellular locations, gene transactivation in the nucleus and cell–cell adhesion at the cell membrane (Nelson and Nusse, 2004; Heuberger and Birchmeier, 2010), and one or both of these mechanisms could contribute to sex-specific pattern formation in gonadogenesis.
In its nuclear function, β-catenin (hereafter called “nuclear β-catenin”) is known to act as a co-factor in transactivation of specific subsets of genes, according to tissue and cell type. Canonical Wnt signaling can activate this function by dephosphorylating, or “stabilizing,” β-catenin, thereby preventing its proteasomal degradation (Nelson and Nusse, 2004; Heuberger and Birchmeier, 2010). R-Spondin (Rspo) family members can also activate the nuclear function of β-catenin by enhancing Wnt signaling (Kim et al., 2008). In the context of gonadal development, Wnt4 and Rspo1 are preferentially expressed in the ovary and ablation of either in XX mice results in partial sex reversal (Chassot et al., 2008; Tomizuka et al., 2008). In addition, using a reporter line for Axin2, a transcriptional target of canonical Wnt signaling, β-catenin nuclear function has been detected in the ovary but not testis at stages assayed (E12.5–E14.5; Chassot et al., 2008; Manuylov et al., 2008). It is, therefore, possible that the transactivational function of nuclear β-catenin is involved in patterning during sex-specific gonadal development, although genes expressed downstream of Wnt4/β-catenin have yet to be linked specifically to this process.
These data do not exclude a role for β-catenin at the cell membrane (hereafter termed “membrane β-catenin”) in gonadogenesis. However, membrane β-catenin function has been less extensively studied in gonadal development. Nuclear and membrane mechanisms of β-catenin action can coexist and Wnt signaling—including Wnt4 signaling—can promote β-catenin function in both locations (Hinck et al., 1994; Nelson and Nusse, 2004; Bernard et al., 2008; Heuberger and Birchmeier, 2010).
At cell membranes within many developing tissues, β-catenin takes part in selective cell–cell adhesion as a protein partner in adherens junctions (AJs; Tepass et al., 2002; Gumbiner, 2005; Halbleib and Nelson, 2006). AJs are central to patterning and morphogenesis, where they modulate cell sorting, boundary formation and tissue compartmentalization (Tepass et al., 2002; Steinberg, 2007). AJs are typically among the first adhesion elements found in these tissues, laying the organizational groundwork for other adhesive structures such as tight junctions, desmosomes and gap junctions to follow (Rowlands et al., 2000; Hartsock and Nelson, 2008). The core of the AJ protein complex consists of members of the transmembrane family of cadherins, which preferentially bind like cadherins on neighboring cells. Along with p120 catenin (p120), membrane β-catenin helps bridge the intracellular domain of cadherins to the actin cytoskeleton. While, in some cases, plakoglobin may substitute for β-catenin (Salomon et al., 1997), the co-expression of β-catenin with cadherins at the cell membrane is generally considered an indication that AJs are formed and functional. Selective cell–cell adhesion—the basis of cell sorting—is accomplished through predominantly homophilic association of the extracellular domain of like-cadherin subtypes expressed on specific cell types (Gumbiner, 2005; Halbleib and Nelson, 2006).
Together, these functions make membrane β-catenin, in AJs, a very interesting candidate for involvement in sexually dimorphic patterning of the gonad. Indeed, specifically blocking E-cadherin function interferes with developmental organization of the testis and ovary (Mackay et al., 1999; Bendel-Stenzel et al., 2000; Di Carlo and De Felici, 2000). Furthermore, although colocalization of β-catenin with cadherins at the membrane has not been demonstrated during early gonadal development, several cadherins are expressed in those tissues (Rowlands et al., 2000). Among these, E- and P-cadherin are expressed throughout early gonadogenesis; in both sexes, E-cadherin localizes to germ cells, and P-cadherin to somatic cells—identified as Sertoli cells in males, and unidentified in females (Lin and DePhilip, 1996). Lin and DePhilip proposed that homophilic association between like-cadherin subtypes plays a role in organizing gonadal cells at E12.5, particularly in the testis. However, it was subsequently found that P-cadherin-null mice are fertile (Radice et al., 1997) and that blocking P-cadherin function in cultured E10.5 embryo slices shows no effect on organization of gonadal cells (Bendel-Stenzel et al., 2000). These findings suggest two scenarios: either AJs are not essential in patterning the gonad, or the loss of P-cadherin is compensated in the null model, allowing AJs to still function.
In that mechanisms of tissue patterning in early gonadogenesis are still largely unexplained, that AJs are typically involved in just such functions, that β-catenin is essential in early sex-specific development, and that transcripts of multiple cadherins have been described during gonadogenesis, we hypothesized there could yet be an organizing effect of membrane β-catenin—in AJs—through redundant expression of another cadherin subtype with P-cadherin on somatic cells. Given the dynamic nature of tissue patterning and AJ function, we completed a time course of early AJ component colocalization in the testis compared with ovary. Here, we demonstrate that N-cadherin expression is indeed redundant with the reported expression of P-cadherin at E11.5 and 12.5 (Lin and DePhilip, 1996). This finding could allow us to re-interpret the P-cadherin data to suggest that AJ function, rather than expendable, might be necessary to gonadal patterning.
Together, our data highlight multiple possible roles for membrane β-catenin, in AJs, in patterning of the early testis vs. ovary. We identify cell-specific expression of β-catenin and cadherins that precisely correlates with both sex-specific and sex-nonspecific patterning of the testis and ovary in the early stages of morphological sex differentiation.
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Although much progress has been made in understanding sex differentiation of the mammalian gonad, mechanisms involved in early patterning of cells within the testis and ovary have been elusive. Adherens junctions (AJs) are known to take part in early morphogenesis and patterning of many developing tissues (Tepass et al., 2002; Halbleib and Nelson, 2006; Steinberg, 2007). To determine whether AJs are present early enough to participate in patterning of the developing gonad, we systematically examined the cell-specific expression of key components of AJs in E11.5 and 12.5 mouse gonads. (Findings are summarized in Fig. 6A schematic.)
Figure 6. Schematics of membrane β-catenin and cadherin expression in the embryonic day (E) 11.5 and 12.5 testis and ovary. A: Summary of the data. B: Schematic of a testis cord with proposed sorting by cadherin type and differential total adhesion.
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In brief, at E11.5 we found that expression of AJ components β-catenin, p120, and cadherins reflected both sex-nonspecific and sex-specific patterning of the gonad. Sex-nonspecific expression was found in most cells, where β-catenin and p120 colocalized with either E- or N-cadherin, collectively forming an overall matrix that diminished at the gonad-mesonephros border. Within this matrix, germ cells colocalized β-catenin, p120, and E-cadherin, and tended to self-associate in clusters surrounded by somatic cells that co localized β-catenin, p120, and N-cadherin. The matrix was interrupted by early vascular channels branching off of vasculature beds at the gonad–mesonephric border. Vasculature in both locations, as well as perivascular MAF-positive cells, were membrane β-catenin negative. Sex-specific expression of AJ components was found among support cells. In the testis, Sox9-positive Sertoli cells were β-catenin/N-cadherin-positive and predominantly self-associated. In the ovary, Sprr2-positive pregranulosa cells expressed low or intermittent levels of β-catenin/N-cadherin and tended to be solitary.
At E12.5, sexually dimorphic expression of AJ components paralleled sex-specific patterning of the gonad. In the testis, cells that comprised the partitions between cords—the male-specific vasculature and the perivascular interstitial cells—were negative for membrane β-catenin, p120, and E-/N-cadherins. Within cords, however, all cells were membrane β-catenin/p120/cadherin-positive, and tended to self-associate by cadherin subtype—germ cells with E-cadherin and Sertoli cells with N-cadherin. In the ovary, neither male-like vasculature nor membrane β-catenin-negative interstitial cells partitioned the organ into cords; rather, patterning and expression of AJ components continued much as described at E11.5. When Wnt4 was ablated, membrane β-catenin-negative vascular channels transected both the testis and the ovary by E11.5, partitioning membrane β-catenin-positive cells and giving the appearance of E12.5 testis interstitium and cords.
Other recent studies have shown expression of β-catenin at various stages between E12.5 and E15.5, primarily in the context of its nuclear function (Kimura et al., 2006; Chang et al., 2008; Chassot et al., 2008; Maatouk et al., 2008; Liu et al., 2009; Naillat et al., 2010). At E12.5, the stage that overlaps our work, we found some differences within the literature, and with our findings, specific to membrane β-catenin expression in germ cells. For example, we found membrane β-catenin expression in germ cells to be essentially comparable between the sexes, while another group found β-catenin in the testis to be largely membrane-associated, but in the ovary, they found its expression primarily cytosolic, with some nuclear localization (Chassot et al., 2008). To examine the nuclear function of β-catenin, this group used an antibody raised against the form of β-catenin that is involved in transactivation: dephosphorylated, or stabilized, β-catenin. To examine a possible membrane-associated function of β-catenin, we used an antibody that identifies both its stabilized and unstabilized β-catenin, as both forms can function at the membrane. Comparison of these data raises the possibility that the ratio of stabilized to unstabilized β-catenin at the membrane of germ cells may differ between the sexes. Chassot et al. did find comparable levels of E-cadherin in female and male germ cells at this stage, as did we, and suggested these AJ components contribute to adhesion between germ cells before meiosis.
Our time course of AJ component expression during gonadogenesis correlates well with known mechanisms of AJ function during development of other tissues (Tepass et al., 2002; Steinberg, 2007). First, in AJ-based cell sorting, cells expressing like cadherin subtypes tend to adhere preferentially. In the early testis and ovary, we found that cells largely self-associated—germ with germ and somatic with somatic—according to the cadherin subtype they expressed.
Second, cells expressing differential levels of cadherins/AJs can result in tissue boundary formation and compartmentalization. We found that all vascular/perivascular cell channels were β-catenin/cadherin negative, while cells surrounding these channels were largely β-catenin/cadherin positive. This differential expression of AJ components was seen in the highly vascularized gonad–mesonephros boundary region compared with the body of the gonad. It was also seen between nonvascular and vascular compartments within the gonad, including the boundary between E12.5 testis cords and interstitium, respectively. Of interest, at E11.5 in wild-type animals, somatic cells that did not express markers for support cells were positive for membrane β-catenin, and no partitions subdivided the gonad, while in Wnt4−/− animals, cells of this general description were membrane β-catenin negative, and partitioned both the mutant testis and ovary into cord-like structures reminiscent of the E12.5 testis.
Finally, it has been shown that, in a mixed population of cells, those cells expressing the greatest number of AJs group to the center, and total per cell adhesion strength (to include AJ-based adhesion) successfully predicts such organization (Foty and Steinberg, 2004; Steinberg, 2007). It is inviting to suggest this mechanism plays a role in organizing cells of the E12.5 testis cords, with germ cells grouping toward the center and Sertoli cells at the periphery. While measures of total adhesion strength are beyond the scope of this study, we did note that: (1) β-catenin signal—as a proxy indicator of β-catenin-associated AJs regardless of cadherin subtype—was clearly most intense on germ cells, less so on Sertoli cells, and almost undetectable on surrounding interstitial cells; and (2) strong expression of β-catenin, E-cadherin, and PECAM-1 colocalized at germ cell-germ cell contacts. PECAM-1 is itself a transmembrane cell-cell adhesion molecule that can self-associate, and can also mediate increased cell–cell adhesion of β-catenin-associated AJs (Ilan and Madri, 2003). PECAM-1-null mice are fertile (Duncan et al., 1999) and any PECAM-1-mediated adhesion effect between germ cells would, therefore, be partial. However, convergence of these adhesion molecules, along with considerable cell shape distortion (Lecuit and Lenne, 2007), each correlate with particularly strong adhesion between centrally located germ cells (proposed mechanism outlined in the Fig. 6B schematic).
It is possible that the patterns of AJ expression described here are simply a consequence of other patterning forces. However, several factors argue strongly for active participation by AJs in patterning of the gonad. A body of literature supports a causal role for AJs in early patterning of various tissues, using mechanisms consistent with our data. In the mouse telencephalon, for example, cell sorting and compartment boundary formation are based at least in part on differential expression levels or subtypes of cadherins (Tepass et al., 2002; Steinberg, 2007). AJs are typically involved in establishing initial tissue patterns during development, followed by adhesive molecules that secure more permanent morphological structures (Rowlands et al., 2000; Hartsock and Nelson, 2008), and our data is acquired during such formative stages of gonad patterning. Additionally, previous studies of the gonad have demonstrated specific involvement of E-cadherin in germ cell adhesion and organization during gonadogenesis. When the function of E-cadherin is blocked: (a) in gonad tissue slices, germ cell condensation in the gonad is reduced (Bendel-Stenzel et al., 2000); (b) in dissociated gonadal cells, germ cell re-aggregation and self-organization of cords is blocked (Mackay et al., 1999); and (c) in gonad organ culture, germ cells fail to localize normally (Di Carlo and De Felici, 2000).
This said, multiple molecular mechanisms are likely to act in concert to pattern the gonad. For example, neurotrophic tyrosine receptor kinases have been shown to enhance migration and aggregation between Sertoli cells in culture and in xenographs (Gassei et al., 2008) and may do so in the developing testis, but this mechanism does not address the organizational relationship between Sertoli and germ cells within cords. In both sexes, intercellular bridges between germ cells are made early in development (Braun et al., 1989; Pepling and Spradling, 1998) and may contribute to germ cell clustering. In addition, by E12.5 in the testis, a VEGF/Pdgfr-α signaling interaction appears to promote perivascular cell proliferation that likely contributes to interstitial partitioning (Cool et al., 2011); however, this action on its own does not explain boundary formation between cells of the interstitium and cords.
We propose a model in which AJs contribute to patterning of the gonad through known mechanisms of AJ function (Tepass et al., 2002; Foty and Steinberg, 2004; Halbleib and Nelson, 2006; Steinberg, 2007). At E11.5 in both sexes, AJs between most cells create an overall cell–cell adhesion matrix that establishes the initial architecture of the developing gonad. AJs between cells that lie adjacent to developing, membrane β-catenin-negative vascular/MAF-expressing perivascular cell channels give structure to these channels, in concert with remodeling of the extracellular matrix (Murphy and Gavrilovic, 1999). A reduced number of AJs between cells at the mesonephric border contributes to organ boundary formation. Nonvascular cells within the testis and ovary sort according to homophilic association of like-cadherins, with slight overlap at germ-somatic contact, and with the exception of pregranulosa cells, which bear few or intermittent AJs and in accordance tend not to self-associate. At E12.5 in the testis, cells in the cords are AJ-positive, while vascular/perivascular cells in the interstitium are AJ-negative; this adhesion differential forms compartment boundaries that effectively isolate individual cords. Each cord is now self-contained—essentially untethered from the overall AJ-based adhesion matrix—and germ and Sertoli cells are free to self-organize within cords by cadherin subtype and by relative total adhesion strength. At E12.5 in the ovary, AJs contribute to patterning as described at E11.5.
Several recent studies have together established that β-catenin is required for female sex determination and may in fact be the deciding factor in choosing the female path. Ablation of β-catenin in XX gonads (Manuylov et al., 2008; Liu et al., 2009) and ectopic stabilization in XY gonads (Chang et al., 2008; Maatouk et al., 2008) have clearly shown β-catenin involvement in female sex determination. In addition, there is clear evidence that β-catenin functions in its nuclear, transcriptional role in the ovary at E12.5 and 13.5 (Chassot et al., 2008; Manuylov et al., 2008). We suggest the intriguing possibility that β-catenin may be necessary in both of its roles—nuclear and membrane—during early development of the ovary.
In the testis, on the other hand, relatively few effects have been attributed to the action of β-catenin. Wnt4 ablation results in only a mild phenotype (Vainio et al., 1999; Jeays-Ward et al., 2004), and nuclear β-catenin activity has not been detected (Chassot et al., 2008; Manuylov et al., 2008). Furthermore, conditional ablation of β-catenin from SF-1-expressing cells [primarily Sertoli cells] has shown little effect (Chang et al., 2008; Manuylov et al., 2008; Liu et al., 2009). Together these data have suggested that β-catenin is not essential in testis development. On the other hand, several lines of evidence suggest that an early function of β-catenin cannot be fully dismissed. First, Wnt4 ablation does not eliminate β-catenin expression at the membrane of Sertoli or germ cells (Naillat et al., 2010; and Fig. 5). Second, assays that show a lack of nuclear β-catenin activity in the testis (Chassot et al., 2008; Manuylov et al., 2008) do not rule out a membrane function. Third, the conditional constructs that have been used to ablate β-catenin may not fully operate until after critical testis-specific morphological changes—male-specific vascularization and cord formation—have occurred. The SF-1-cre construct used in studies of sex determination (Manuylov et al., 2008; Liu et al., 2009) is not fully effective until E12.5–E13.5 (Tevosian and Manuylov, 2008), while the AMH-cre construct (Chang et al., 2008) becomes functional after E13. We find wild-type expression of membrane β-catenin, its colocalization with other AJ components, and its strong association with testis patterning already established at E11.5, and suggest its organizational effects in the testis may occur before activation of these conditional knockouts.
As suggested above, during gonadogenesis β-catenin may be required in each of its functional roles, and the literature shows that in tissues where this is the case, regulation of the subcellular localization of β-catenin is both essential and complex (Nelson and Nusse, 2004; Gumbiner, 2005; Perez-Moreno and Fuchs, 2006; Heuberger and Birchmeier, 2010). Indeed, evidence presented here and elsewhere indicate that such regulation may be necessary in the early gonad. In Sertoli cells, for example, we show that β-catenin is expressed at the membrane where it may be involved in male-specific patterning. At the same time, conditional stabilization of β-catenin in Sertoli cells—which likely increases at least its nuclear function in those cells—exacts a high toll: sex reversal (Maatouk et al., 2008). An intriguing possible mechanism in this regard is found in chondrocyte development: Sox9 expression in those cells up-regulates N-cadherin expression, which results in proper adhesion and condensation of these cells (Panda et al., 2001). Sox9 is expressed in both wild-type and Wnt4−/− Sertoli cells (Fig. 5A,B), and could up-regulate N-cadherin in those cells, both enhancing cell–cell adhesion and sequestering β-catenin away from the nucleus.
Regulation of the level of β-catenin may also be critical in the gonad. In germ cells, for example, although β-catenin is required for normal function (Mackay et al., 1999; Bendel-Stenzel et al., 2000; Di Carlo and De Felici, 2000; Liu et al., 2010; Naillat et al., 2010), its levels must be tightly regulated because its overexpression results in defects in germ cell proliferation (Kimura et al., 2006). Of interest, the transmembrane adhesion molecule PECAM-1 has been shown to both sequester β-catenin at the membrane and to enhance cell–cell adhesion by β-catenin-based AJs (Ilan and Madri, 2003). We found PECAM-1 colocalized with β-catenin in germ cells, indicating similar mechanisms could be involved in regulating the functional level of β-catenin in these cells.
AJs have recently emerged in a related but broader capacity than adhesion: they establish links between a variety of signaling pathways, and both regulate and are regulated by these pathways (Halbleib and Nelson, 2006; Lien et al., 2006; Perez-Moreno and Fuchs, 2006). In addition to the dual function of β-catenin as a key component of AJs and a mediator of Wnt signaling (Nelson and Nusse, 2004; Brembeck et al., 2006), other AJ components link to signaling pathways. N-cadherin/catenin, for example, interact with many such pathways (Derycke and Bracke, 2004), including FGF—a pathway that is critical in sex determination (Kim et al., 2006).
Together, our data show that both sex-specific and sex-nonspecific patterning of the early testis and ovary may involve a membrane function of β-catenin, in AJs, through selective cell–cell adhesion, cell sorting, compartmentalization, and boundary formation.