Notch1 regulates gonadal development in chicken germ cells
The Notch signaling pathway is highly conserved throughout evolution and is involved in the cell differentiation process [14] through the functions of Notch1, Notch2, Notch3, and Notch4 [15]. Only Notch1 and Notch2 receptors were detected during chicken germ cell differentiation. Notch1 expression was significantly altered during germ cell formation, while Notch2 expression was not (Fig. 1a). Notch1 was strongly expressed in PGCs but only weakly expressed in ESCs and SSCs (Fig. 1a left). These observations suggest that the Notch signaling pathway may only be regulated by Notch1 during germ cell formation in chicken.
To further study the specific functions of the NICD receptor and Notch signaling pathway in chicken germ cell formation, we treated the chicken PGCs and SSCs with the Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) [16, 17] and pcDNA3.0-NICD. DAPT was able to inhibit NICD completely within 24 h but had no significant inhibitory effect on Notch2 (Fig. 1b, Additional file 1: Figure S1C). We injected DAPT and pcDNA3.0-NICD into chicken embryos. We observed that inhibition of NICD expression indeed affects the development of chicken embryos. Specifically, the inhibition of NICD altered the development of the reproductive ridge and resulted in developmental retardation (Fig. 1c, Additional file 1: Figure S1A, B). Inhibition of NICD actually promoted the development of the testes. We found that the seminiferous tubules suffered from hypoplasia in the testicular slices with NICD overexpression (Fig. 1d, Additional file 1: Figure S1D). Taken together, these results indicate that NICD acts as the sole receptor for the Notch signaling pathway and exerts opposite effects on reproductive ridges and testicular development.
CBF-1/RBP-dependent notch signaling regulates downstream gene expression by altering histone acetylation
Classical Notch signaling relies on CBF-1/RBP [18], which is a transcriptional factor that inhibits the expression of downstream genes [19].To explore the transduction of Notch signaling in chicken germ cell differentiation in detail, NICD inhibition and overexpression were performed to inhibit or activate the Notch signal, and downstream signaling molecules were assessed. Expression of mastermind-like (MAML)1, MAML2, and MAML3 as well as expression of the p300/CBP-related factor (PCAF) were significantly upregulated following activation of Notch signaling during the formation of PGCs (Fig. 2a, b). The downstream transcription factor hairy/enhancer of split-1 (HES1) was also upregulated. Expression of the phenomenon, inhibition of Notch1 has the opposite phenomenon (Fig. 2e).
To investigate these effects further, we examined the composition of the CBF-1/RBP complex. We found that activation of Notch signaling resulted in dissociation of co-suppression complexes of CBF-1/RBP, a significant reduction in the enrichment of histone deacetylase (HDAC)1 and HDAC2 in the complex, and formation of co-activated complexes with MAML1 (Fig. 2c). Observation of high expression of PCAF also demonstrated the formation of co-activated complexes (Fig. 2b). The expression of HES1 was promoted by increasing the level of histone acetylation near the transcriptional binding site of HES1, leading to regulation of expression of downstream genes(Fig. 2e).We next transfected DF-1 cells with HES1 and HES5 promoter dual-luciferin reporter vectors and treated the cells with the HDAC inhibitor Trichostatin A (TSA). HES5, a transcription factor downstream of Notch, was not affected by histone acetylation, although both HES1 and HES5 promoters are enriched by RBP as determined by chromatin immunoprecipitation (Additional file 2: Figure S2A, B).Interestingly, with respect to the formation of SSCs, we found that the dissociation process of the CBF-1/RBP co-suppression complex was reversed due to unknown factors. Moreover, the enrichment of HDAC1 and HDAC2 in the co-suppression complex was significantly increased (Fig. 2d), leading to significant reduction in the expression of downstream transcription factors. These results indicate that Notch signaling regulates downstream gene expression by altering histone acetylation levels via dynamic expression of HDAC1 and HDAC2 in CBF-1/RBP complexes.
Notch signaling positively regulates the formation of PGCs, but negatively regulates the formation of SSCs in vivo
We next further explored the specific function of Notch signaling during germ cell formation. To this end, we detected the formation of PGCs (4.5 days) [20] and SSCs (18 days) [21, 22] after activation and inhibition of Notch signaling during chick embryo hatching. With respect to PGCs, we found that the activation of Notch signaling significantly upregulated the expression of Lin28 (8.3427 ± 0.23, p < 0.05) and Blimp1 (12.4213 ± 0.13, p < 0.05), which mark formation of PGCs at 4.5 days. Inhibition of Notch signaling induced the opposite effects on Lin28 and Blimp1 (Fig. 3a). These data indicate that Notch signaling may positively regulate the formation of PGCs. We also analyzed the CVH+ CKIT+ efficiency (which Used to mark PGCs) during the chicken embryo hatching process following manipulation of Notch signaling. Activation of Notch signaling promoted the formation of PGCs in reproductive ridges and the efficiency of CVH+ CKIT+ cells (7.5% ± 0.11, p < 0.05) was significantly higher than that of embryos that did not have activated Notch signaling(control, 6.4% ± 0.26). On the other hand, inhibition of Notch signaling significantly reduced the number of PGCs in the reproductive ridge, and the efficiency of CVH+ CKIT+ cells was only 4.9% ± 0.17 (p < 0.05 compared to control without Notch inhibition (Fig. 3c top). At the same time, the results of periodic acid Schiff (PAS) [23] staining also demonstrated that Notch signaling enhanced the formation of PGCs, although the morphological development of the reproductive ridge was the same in the presence and absence of Notch activation. The number of PGCs was nearly 30 ± 3 in the control group, but was 53 ± 2 following Notch activation. In accord with our previous observations, the number of PGCs formed after inhibition of Notch signaling was only 12 ± 2 (Fig. 3d). These results indicate that Notch signaling positively regulates the formation of PGCs.
The same phenomenon was not observed during the formation of SSCs. We found that the expression of integrin α6 (2.8461 ± 0.31, p < 0.05) and integrin β1 (1.8253 ± 0.44, p < 0.05) were significantly downregulated after Notch signaling activation at 18 days. In contrast, suppression of Notch signaling resulted in upregulation of integrin α6 and integrin β1 (Fig. 3a). We also analyzed the efficiency of integrin α6+ integrin β1+ cell formation following modulation of Notch signaling. Activation of Notch signaling inhibited the formation of SSCs in testicles, and the efficiency of integrin α6+ integrin β1+ cells (12.7% ± 0.08) was significantly lower than that in embryos without Notch activation (control, 14.2% ± 0.33, p < 0.05). Inhibition of Notch signaling, however, significantly promoted the formation of SSCs in the testicles, and the efficiency of integrin α6+ integrin β1+ cells was 16.3% ± 0.16(Fig. 3b, bottom). Therefore, these results underscore the opposing functions of Notch signaling in the formation of PGCs and SSCs.
Notch signaling functions similarly in the Bmp4 model and in vivo
Bone morphogenetic protein (BMP4) is an important endogenous factor for the origin and migration of germ cells [24]; however, this protein not only induces germ cell formation in vitro, but also regulates germ cell differentiation by interacting with Notch1 [25,26,27]. To study the function of Notch signaling in chicken germ cell differentiation in vitro, we established a model of BMP4-induced germ cell formation (Fig. 4a, Additional file 3: Figure S3). In our model, ESCs were treated with different concentrations of BMP4 (0–40 ng/mL), and none of these concentrations caused significant cytotoxicity to ESCs or affected cell proliferation status (Additional file 3: Figure S3).In general, the proliferation of the ESCs treated with the different BMP4 concentrations was similar. During the logarithmic growth phase (2–6 days), the cells continued to proliferate and reached the maximum peak on day 6. When the cells were in the plateau stage, the growth rate was stable for 8–10 days. The cell number stabilized on day 12 and began to decrease after 14 days due to apoptosis. Thus, BMP4 can induce germ cell formation without toxic effects that affect cell proliferation.
Different concentrations of BMP4, however, caused significant changes in cell morphology. A BMP4 concentration of 40 ng/mL resulted in embryoids on day 4, and the number of embryos increased gradually at 6–8 days. Then, the number of germ cells on day 14 was increased, and the number of reproductive cells was increased 12 days after the disintegration of the embryoid bodies (Additional file 4: Figure S4). The change of expression level of each marker also indicated that the concentration of 40 ng/mL was optimal for PGCs and SSCs generation in vitro as assessed by fluorescence activated cell sorting (FACS) (Fig. 4a).
Next, we examined inhibition and overexpression of NICD in the BMP4 induction model in vitro (Additional file 5: Figure S5A–C). Inhibition of NICD expression inhibited PGCs formation and promoted SSCs formation, while overexpression of NICD promoted the formation of PGCs and inhibited the formation of SSCs (Fig. 4b, c, d and Additional file 6: Figure S6). To validate the specific function of Notch signaling in vitro, we examined the expression of reproductive marker genes (Fig. 5a) and analyzed the effect of Notch signaling on germ cell formation before and after activation using FACS (Fig. 5b). The results of the reproductive marker gene expression and the efficiency of PGCs and SSCs generation were consistent with those observed in vitro (Fig. 5a, b). Together, these results demonstrate that Notch signaling plays opposing roles during the formation of PGCs and SSCs in both germ cells: Notch positively regulates PGC formation and negatively regulates SSC formation.
High-throughput sequencing confirms a role for notch in the formation of PGCs and SSCs
To analyze the specific functions of Notch signaling in PGCs and SSCs, we performed transcriptome sequencing of ESCs, PGCs, and SSCs (Additional file 7: Figure S7). The enrichment of differentially expressed genes in Notch signaling was dynamic (Fig. 6a). Specifically, Notch signaling was significantly upregulated during the process of ESC differentiation into PGCs. On the other hand, Notch signaling was downregulated during the process of PGC differentiation into SSCs. Interestingly, during ESC differentiation into SSCs, the Notch signal was enriched and expressed as a suppressive state (Fig. 6b). This indicates that the function of the signal is opposite during the process of PGCs and SSCs formation, which is consistent with the previous experimental results.
With respect to the two types of Notch ligands, Delta-like ligands and Serrate-like ligands, we found that the Delta-like ligands were expressed at their highest levels in PGCs. Conversely, the two Serrate-like ligand subtypes were expressed differently. Specifically, Jagged 1 was expressed at significantly higher levels in PGCs than in ESCs, and levels were decreased slightly in SSCs. The expression of Jagged 2 continued to decrease in all three kinds of cells. The expression of Notch receptors generally increased, with highest expression of Notch1 in PGCs. The expression of HES1 and HES5 was highest in PGCs and lowest in SSCs. Thus, these results illustrate the different functions of Notch signaling in the two germ cell formation processes.
In a previous study, we found that the dissociation of CBF-1/RBP co-suppression complexes was reversed albeit by unknown factors. Thus, we sought to elucidate the responsible factor using the high-throughput sequencing results. Based on the expression of the molecules in the Notch signaling pathway in PGCs and SSCs, we initially mapped the signal transduction mechanism of Notch signaling (Fig. 6c). We found that transducer like enhancer of split 3 (TLE3), TLE4, and C-terminal binding protein 2 (CTBP2) genes regulated the co-suppression CBF-1/RBP complex, and this reversal process is key to the reverse function of Notch signaling in the two types of reproductive stem cells. The expression of CBF-1/RBP complex in SSCs is reversed, which affects the differentiation of PGCs into SSCs.