E-cadherin and β-catenin were co-expressed in undifferentiated spermatogonia
A subpopulation of undifferentiated spermatogonia, SPCs, was identified as PLZF+ cells residing in the first layer in seminiferous tubules and found to regulate SPCs fate (Fig. 1A). Since the expression of E-cadherin and β-catenin could be detected in the same population (Fig. 1B and C), we postulated that both molecules might be involved in modulating SPCs fate. To study the role of E-cadherin, SPCs were purified using THY1.2+ MACS from neonatal mouse testis, and grape-like clones were observed after 2 passages on MEF feeder layers (Fig. 1D), which were able to be stably maintained in vitro for more than 30 passages [26]. The expression of SPC markers, including Plzf, Cdh1, Gfra1 and Id4, was examined to characterize their identities using RT-PCR (Fig. 1D). Moreover, IF staining against PLZF, E-cadherin, β-catenin, Axin2 and ZO-2 further confirmed that SPCs were notably enriched (Additional file 1: Fig.S1 A–E). Subsequently, co-IF staining demonstrated an overlap of E-cadherin+ and PLZF+/β-catenin+ populations (Fig. 1E and F). In all, these observations confirmed a co-expression pattern of E-cadherin and β-catenin both in vivo and in vitro, demonstrating that E-cadherin in combination of canonical Wnt signaling pathway might play a role in SPCs.
Differentiation markers up-regulated after disturbing E-cadherin expression in SPCs
To better understand the role of E-cadherin, RNAi was employed to disturb the expression of E-cadherin in SPCs. IF staining revealed the efficient decrease of E-cadherin expression in primary SPCs (Fig. 1J–L), compared to scramble siRNA group (Fig. 1G–I). Although neither obvious morphological change (data not shown) nor difference in the number of SPCs was observed 72 h post-transfection (Fig. 1M), the expression of SPC markers was altered (Fig. 1N, O). Reduced PLZF and GFRA1 along with the increased differentiation marker c-Kit suggested that the disturbance of E-cadherin might jeopardize the undifferentiated state of SPCs under in vitro culture. The binding of E-cadherin to β-catenin was further confirmed in SPCs by co-IP (Fig. 1P). Though E-cadherin knockout maintained the capacities of SPC homing and spermatogenesis [14], our observations revealed a possible influence of E-cadherin on SPCs fate. Therefore, a better understanding of the regulatory mechanism of E-cadherin in SPCs could be greatly valued, especially its interaction with β-catenin.
Conditional knockout of E-cadherin in germline promoted differentiation at protein levels
To further characterize the effect of E-cadherin deficiency on SPCs fate, LoxP-Cre system was employed to conditionally knockout E-cadherin in mouse SPCs (Fig. 2A). Germline-specific E-cadherin knockout mice (E-cadherinL/L;Ddx4-Cre+) were generated by mating E-cadherin floxed females with E-cadherinL/+;Ddx4-Cre+ males (Fig. 2B). Testes from 3-month E-cadherinL/L;Ddx4-Cre+ males were harvested for histological analysis, and germ cells at different differentiation stages could be easily distinguished (Fig. 2C–F), indicating that E-cadherin deficiency neither affected seminiferous tubule structure nor disturbed spermatogenesis. However, when evaluating the expression of undifferentiated spermatogonia marker PLZF using IHC, we noticed that the number of PLZF+ cells in E-cadherinL/L;Ddx4-Cre+ testis remarkably decreased compared to control group (Fig. 2G–I), and the number of differentiating population represented by c-Kit staining intensively increased in E-cadherin deficient tubules (Fig. 2J–L). These observations demonstrated that E-cadherin might inhibit SPCs differentiation. To obtain a better understanding, testes from E-cadherinL/L and E-cadherinL/L;Ddx4-Cre+ littermates were collected for evaluating the expression of self-renewal and differentiation markers using Western blot. Consistently, E-cadherin deficient testes expressed decreasing level of SPC markers such as GFRA1, PLZF and ITGA6, and increasing expression level of differentiation marker c-Kit compared to control group, respectively (Fig. 2M and N). Noteworthy, expression of AXIN2 and GSK3-β was up-regulated (Fig. 2M and N), suggesting that E-cadherin deficiency possibly enhanced the activation of β-catenin degradation complex. Meanwhile, the expression of PCNA, BAX and BCL-2 was not affected in E-cadherin deficient group (Fig. 2M and N), implying that E-cadherin loss promoted differentiation, but not proliferation or apoptosis, in testis.
Subsequently, we explored the impact of E-cadherin deficiency on β-catenin’s translocation into nucleus. IF staining of β-catenin in E-cadherinL/L and E-cadherinL/L;Ddx4-Cre+ testes revealed limited nuclear distribution of β-catenin, while no remarkable difference observed between wild type and E-cadherin deficient SPCs (Fig. 2O and P). Likewise, strong β-catenin signal was restricted to cytoplasm of purified SPCs (Fig. 2Q), we postulated that β-catenin was mainly distributed in cytoplasm in both genotypes. Collectively, E-cadherin could play an important role in SPCs differentiation by modulating both differentiation and SPC marker expression.
The impact of E-cadherin deficiency on the fates of β-catenin in SPCs
A decreased β-catenin expression in E-cadherin knockout SPCs compared with WT controls might be detected because of two possibilities: 1. E-cadherin knockout led to reduced expression of β-catenin; 2. E-cadherin knockout enhanced the degradation of β-catenin. To test these hypotheses, we first compared the expression of β-catenin at mRNA level in SPCs from both genotypes, and noticed an attenuated β-catenin expression in E-cadherin knockout SPCs (Fig. 3A). Subsequently, different phosphorylated forms of β-catenin were determined. A β-catenin antibody targeting phosphorylation at Ser33/Ser37/Tyr41 was used to examine the degradation of β-catenin, and the declined phosphorylation implied that E-cadherin knockout reduced the degradation of β-catenin in SPCs (Fig. 3B and C). Interestingly, phosphorylation at Ser675 representing a transcriptional active form of β-catenin, was declined in E-cadherin knockout SPCs as well (Fig. 3B and C), indicating that E-cadherin deficiency down-regulated the transcriptional activity of β-catenin. Furthermore, a sustained effect on β-catenin expression and phosphorylation was observed when E-cadherin got deleted in cultured SPCs with CRISPR/Cas9 (Fig. 3D, E). The expression of PCNA and BAX was not changed, while expression of anti-apoptosis protein BCL-2 was down-regulated. Thus, we proposed that E-cadherin might play a role in anti-apoptosis, since the ratio of BCL-2/BAX reduced after E-cadherin loss. Considering that E-cadherin deletion increased Axin2 and GSK-3β expression in testes (Fig. 2M, N), we concluded that E-cadherin knockout down-regulated β-catenin expression, resulting in a reduced transcriptional activity of β-catenin.
CDH22 co-regulates β-catenin with E-cadherin in SPCs
In addition to E-cadherin, we were also interested in other types of cadherins expressed in SPCs, particularly CDH22, a key signal molecule regarding SPCs fate [27]. In rats, Cdh22 encodes two splicing proteins. The shorter one lacking catenin binding domain is associated with SSCs self-renewal through interacting with JAK-STAT and PI3K-AKT signaling pathways, while the longer one contains catenin binding domain [27]. Notably, Cdh22 in mouse ovary only encodes the latter one, which interacts with β-catenin to regulate female germline stem cells (FGSCs) self-renewal [28]. Here, we wondered whether CDH22 could compensate for E-cadherin loss in SPCs. As shown in Fig. 3F, CDH22 was detected in SPCs residing in basal membrane and freshly isolated SPCs. Western blot results revealed that only one band was detected in mouse SPCs (Fig. 3G), which was consistent with that of mouse FGSCs. Subsequently, we disturbed Cdh22 expression in SPCs and confirmed the reduced phosphorylation levels of S33/S37/T41 and S675 of β-catenin (Fig. 3H, I), and decreased expression of anti-apoptotic protein BCL-2 (Fig. 3H, I), indicating that CDH22 was positively correlated with transcription activity of β-catenin and anti-apoptosis capacity in SPCs, similar to E-cadherin. Also, simultaneous transfection of Cdh1 and Cdh22 siRNA into SPCs aggravated the decline of β-catenin expression (Fig. 3J and K), suggesting that CDH22 might regulate β-catenin expression in SPCs along with E-cadherin in a synergistic manner. More importantly, the binding of CDH22 and β-catenin was confirmed using co-IP (Fig. 3L), indicating a direct interaction between CDH22 and β-catenin in SPCs. Based on these observations, we postulated that β-catenin could be a critical intermediate molecule interacting with CDH1 and CDH22 to regulate SPCs fate.
Identification of ß-catenin co-regulatory factors in SPCs
Due to lack of DNA binding domain, β-catenin needs to bind to TCF family including LEF1, TCF1, TCF3 and TCF4 in mouse and human to regulate target gene expression [21]. Using RT-PCR, Lef1, Tcf3 and Tcf4 mRNA were detected in SPCs (Fig. 4A). Interestingly, LEF1 and TCF3 were restricted to SPCs residing in the basal membrane, while TCF4 was broadly distributed in undifferentiated spermatogonia, differentiating spermatogonia and mature spermatocytes (Fig. 4B). IF staining confirmed the expression of LEF1, TCF3 and TCF4 in purified SPCs (Fig. 4C–E), and co-IP assays demonstrated the binding of β-catenin to LEF1 and TCF3, but not TCF4 in SPCs (Fig. 4F). As shown in Fig. 4G, β-catenin knockdown in SPCs showed no impact on the expression of LEF1, TCF3 and TCF4. On the other hand, though decreased expression of LEF1 led to no significant change in β-catenin expression, a down-regulation of PLZF was observed (Fig. 4H). Considering β-catenin combined with LEF1 regulates Plzf expression in innate memory-like CD8 thymocytes [29], a similar regulatory pattern might exist in SPCs to maintain the undifferentiated state. Also, we hypothesized that β-catenin displaced the suppressor TCF3 from self-renewal associated genes (such as Plzf). Conversely, knockdown of Tcf3 caused up-regulation of PLZF (Fig. 4I), suggesting opposite roles of LEF1 and TCF3 in regulating SPCs fate by cooperating with β-catenin. Our current finding raises up a question that whether β-catenin displaces its suppressor TCF3 from binding to self-renewal associated genes (such as Plzf) to maintain SPCs undifferentiated state, which requires further investigation.
Validation of co-regulatory role of HDAC4 with PLZF in SPCs
In addition to TCF family, β-catenin is able to cooperate with other co-factors as well, such as SOX1, SOX2 and KLF4 [21]. Among them, we were specifically interested in HDAC family, known as pivotal partners in regulating gene expression [30], especially in germline [31]. Consequently, we wondered if HDAC was able to directly bind to β-catenin as a cooperator. Indeed, purified SPCs expressed Hdac1-9 mRNA (Fig. 5A), and HDAC4 was predominately expressed in SPCs residing in the basal membrane of seminiferous tubules (Fig. 5B). In purified SPCs, HDAC4 signal was highly overlapped with PLZF in the nucleus (Fig. 5C–G) demonstrating the co-localization of HDAC4 and PLZF. Subsequently, co-IP assay revealed the binding of β-catenin and HDAC4 in SPCs, as well as STAT3 (Fig. 5H), another key transcription factor for SSCs self-renewal and differentiation via cooperation with the β-catenin/TCF4 complex [32]. To understand the interaction between β-catenin and HDAC4, β-catenin knockdown was performed in SPCs, resulting in a slightly increased expression of c-Kit and BCL-2, as well as decreased GFRA1, PLZF, Cyclin D1, HDAC4 and BAX (Fig. 5I and J), implying differentiation was enhanced, but proliferation and apoptosis were declined in SPCs. This observation is consistent with a previous study showing that hyper-proliferation is accompanied with enhanced apoptosis in Wnt hyper-active gonocytes [18]. Meanwhile, RNAi assay was employed to reveal the role of HDAC4 in SPCs, and the results showed that the growth condition of SPCs transfected with Hdac4 siRNA was not remarkably altered during 48 h post transfection compared with control (data not shown), but the expression of PLZF was suppressed, and the expression of differentiation markers including c-Kit, STRA8 and SOHLH2, were up-regulated (Fig. 5K and L). Notably, Hdac4 loss led to down-regulation of PCNA and AXIN2, without affecting apoptosis (Fig. 5K and L), suggesting that HDAC4 is more likely a regulator to maintain SPCs self-renewal, and is probably associated with canonical Wnt signal pathway. Collectively, these observations indicated a positive correlation between HDAC4 and β-catenin expression in SPCs, which might synergistically regulate SPCs differentiation, proliferation or apoptosis. Thus, we proposed that β-catenin combined with HDAC4 in SPCs to maintain the undifferentiation state and proliferation capacity.
Similarly, STAT3 might be involved in the regulation of differentiation and proliferation in SPCs, since disturbance of Stat3 also led to decreasing PLZF and PCNA, and increasing STRA8 (Fig. 5M and N). Moreover, STAT3 in SPCs seems to maintain β-catenin activity, since Stat3 loss caused decreased AXIN2 (Fig. 5M and N). The increased value of BCL-2/BAX implied that Stat3 loss strengthened the anti-apoptosis capacity in SPCs, further confirming that the positive correlation of proliferation and apoptosis in SPCs. Overall, these observations suggested that HDAC4 and STAT3 could be potential collaborators of β-catenin that synergically regulated SPCs fate.
HDAC4 directly repressed c-Kit expression through deacetylation in SPCs
Since HDAC family members could cooperate with transcription factors [25], we investigated whether HDAC4 bound to differentiation suppressor PLZF in SPCs. Co-IP assay confirmed the binding of HDAC4 to PLZF in SPCs (Fig. 6A), suggesting HDAC4 might be a co-suppressor of PLZF in the inhibition of SPCs differentiation. Considering that HDAC4 also bound to β-catenin (Fig. 5H), we checked whether HDAC4 could form a complex with β-catenin and PLZF in SPCs. Co-IP showed no direct binding between β-catenin and PLZF (Fig. 6B), suggesting that HDAC4 might bind to β-catenin and PLZF separately. Although knockdown of β-catenin or Hdac4 led to SPCs differentiation (Fig. 5I–L), it was not clear how the β-catenin-HDAC4 complex involved in this biological process, nor the role of HDAC4-PLZF complex. As a type of ubiquitous deacetylase, HDAC family members generally bind to target gene to repress gene expression through modulating its acetylation level [33], and our previous work revealed that PLZF could repress SPCs differentiation via direct binding to the promoter regions of c-Kit and Stra8 [7]. Thus, dual luciferase report assay was performed to test whether HDAC4 could regulate c-Kit or Stra8 expression through directly binding to their promoter regions. The c-Kit promoter region from − 1846 bp to − 6 bp was subcloned into pGL3 basic plasmid, and then transfected into HEK 293T cells with the recombinant HDAC4 and/or PLZF overexpression plasmids. As shown in Fig. 6C, the relative luciferase activity was remarkably declined in pGL3-c-Kit when co-transfected with Plzf, and further decreased in Hdac4 co-transfected group. Surprisingly, simultaneous co-transfection of Plzf and Hdac4 overexpression plasmids showed no further suppression of c-Kit activity compared to Hdac4 transfected group, which may probably due to a more significant inhibitory effect of HDAC4 on c-Kit than PLZF. Similarly, the inhibition effect was also observed on Stra8 (Fig. 6D), further confirmed the co-regulatory mechanism of HDAC4 on SPCs differentiation. Considering that HDAC4 overexpression demonstrated more efficient suppression of c-Kit and Stra8 than PLZF, we hypothesized that the deacetylation level might be dependent on the gene transcription activity. Therefore, we measured acetylation levels of c-Kit and STRA8 using acetylation lysine immunoprecipitation coupled with Western blot analysis against c-Kit or STRA8. The acetylation levels of c-Kit and STRA8 in Hdac4 knockdown group remarkably increased compared to that of control group (Fig. 6E). Thus, we postulated that HDAC4 synergically suppressed SPCs differentiation with PLZF through direct binding to differentiation associated genes (such as c-Kit and Stra8) and regulating acetylation (Fig. 6F).
Collectively, a putative regulatory pattern of E-cadherin on SPCs fate through β-catenin and HDAC4 is summarized (Fig. 6G). E-cadherin plays structural and signaling roles in SPCs. Proliferation, differentiation and apoptosis are inhibited since SPCs are attached in the niche by E-cadherin. Under the physiological condition, β-catenin is dynamically balanced among three statuses: binding to cadherins anchored at cell membrane, residing in cytoplasm and ultimately going into degradation by APC, or translocation into nucleus for transcriptional activity. In the nucleus of SPCs, β-catenin interacts with TCF/LEF, HDAC4 or STAT3 to inhibit differentiation and regulate proliferation. Based on our observations, deficiency of E-cadherin reduces cellular contents of β-catenin and its phosphorylation level, resulting in less β-catenin for degradation and nuclear localization. Consequently, the downstream targets associated with undifferentiation state of SPCs are disturbed. Meanwhile, the synergetic effect on inhibition of differentiation genes with HDAC4 or STAT3 is attenuated, to further promote SPCs turning to differentiation state.