Rbm47 is abundantly expressed in pluripotent stem cells (PSCs) and localizes to both nucleus and cytoplasm
We found that Rbm47 transcript and protein were abundantly expressed in mESCs as compared with differentiated cells such as mouse embryonic fibroblasts (MEF) (Fig. 1B and 1C). To determine the cellular localization of RBM47 protein, we fractionated mouse ESCs into cytoplasmic and nuclear extracts and analyzed them by immunoblotting (Fig. 1D). RBM47 was found to be expressed as both, a nuclear and a cytoplasmic RBP with significant enrichment in the nucleus of mESCs, indicating its discrete roles in nuclear and cytoplasmic RNA metabolism. Similar data were obtained with human iPSC lines and human dermal fibroblasts suggesting closely related functions in human counterparts (Supplementary fig. S1 D, E, and F).
Rbm47 expression profile in specific lineages derived in vitro from mESCs
Early mouse development involves the specification of three lineages at the blastocyst stage. As shown in Fig. 1A, the early blastocyst stage (E3.25) has a fully specified outer layer of cells called trophectoderm (TE), while the inner cell mass (ICM) retains pluripotency. In the late blastocyst stage (E4.5), ICM undergoes lineage specification into the pluripotent epiblast (Epi) and primitive endoderm (PrE). As the blastocyst enters the subsequent stages of development, TE gives rise to the extraembryonic ectoderm (ExE), and the PrE gives rise to visceral endoderm surrounding both the Epi and ExE. Epi gives rise to tri-lineages of the embryo proper. Fortunately, the self-renewing ability of these cells enabled us to derive and maintain stem cell lines from each of these lineages and utilize them to study blastocyst development in vitro . ESCs can be derived from both the ICM and Epi and cultured indefinitely in vitro under defined growth conditions. Currently, protocols are available to convert mESCs into naive endoderm (nEnd) or extraembryonic endoderm (EXEn; similar to PrE) and epiblast-like cells (EpiLCs, similar to Epi) in addition to the traditional germ layer differentiation of embryo proper.
To explore the function of Rbm47 during early embryonic development using mESCs, we subjected mESCs to various differentiation strategies and analyzed Rbm47 expression. First, we differentiated mESCs as embryoid bodies (EBs), which undergo spontaneous differentiation into the three germ layers and mimic post-implantation embryos in vitro. RT-qPCR profiling revealed a 2–3 fold upregulation of Rbm47 expression in differentiating EBs compared with ESCs (Fig. 1E). However, RBM47 protein level was correlated with mRNA only in 3 days-old EBs and gradually decreased during further EB differentiation (Fig. 1F). Expression of Nanog, a core pluripotency marker, significantly reduced both at mRNA and protein levels, served as an indicator for EB differentiation (Fig. 1E and 1F).
Next, to investigate whether Rbm47 undergoes lineage-specific regulation, we converted mESCs into extraembryonic endoderm (cXEN) cells, epiblast-like cells (EpiLCs), definitive endoderm (DE), mesoderm (MES), and neuroectoderm (NE) and profiled its expression both at RNA and protein level (Fig. 1G and 1H). All the cultures were additionally profiled for expression of specific-lineage markers to ensure proper differentiation (Fig. 1H and Supplementary fig. S2). We observed that Rbm47 mRNA was differentially expressed in cXEN and EpiLC, which mimic successive cell-fates of ICM post-implantation. In cXEN cells, Rbm47 mRNA was upregulated nearly two-fold, whereas, in EpiLCs, it was downregulated (Fig. 1G). Interestingly, RBM47 protein level correlated significantly with mRNA level in EpiLC but not in cXEN as protein level was reduced compared to ESCs (Fig. 1H), indicating a possibility of post-transcriptional regulation of Rbm47 in cXEN cells.
To gain insights into Rbm47 expression in the lineages of the embryo proper, we analyzed mRNA and protein levels in mESC-derived DE, MES, and NE. We found that both Rbm47 mRNA and protein levels were upregulated in the DE (Fig. 1G and 1H). In contrast, the expression was compromised significantly in MES and NE compared to ESCs (Fig. 1G and 1H). Together, these results suggest that Rbm47 expression is subjected to lineage-specific gene regulation, with an increase at the protein level in DE and a concurrent decrease in other lineages compared to mESCs.
Rbm47 depletion doesn’t affect in vitro mESC maintenance
We used the RNAi approach for loss-of-function studies to investigate the significance of Rbm47 expression in mESCs. We transduced ESCs with lentivirus that expresses shRNAs targeting either Rbm47 mRNA or lacZ mRNA (non-targeting control) and established stable cell lines (Rbm47 depleted mESCs designated as shRbm47#1 and shRbm47#3; control ESCs as shlacZ in the figures). There was an efficient knockdown of Rbm47 at mRNA (~ 80%) as well as protein levels (~ 50%) (Figs. 2A and 2B). Rbm47-depleted mESCs displayed no apparent change in the undifferentiated state as they expressed similar levels of pluripotency markers as control mESCs as demonstrated by RT-qPCR, western blotting, and immunocytochemistry (Figs. 2E, 2F, and 2G). The cell cycle profile of these mESCs was similar to that of control mESCs (Fig. 2H). In terms of morphology and alkaline phosphatase (ALP) activity, shRbm47#3 mESCs were comparable to control mESCs, whereas shRbm47#1 mESCs showed a slightly diffused morphology with a reduced intensity of ALP staining (Fig. 2C and 2D). However, these cells display a similar pluripotency marker profile as control mESCs. Overall, our data suggest that Rbm47 is not necessary to maintain the pluripotent state of mESCs.
Downregulation of Rbm47 increases primitive endoderm (PrE)-like cells in mESC culture and FGF-ERK pathway inhibition rescues the effect
Since Rbm47 depletion didn't affect the pluripotency and self-renewal of mESCs, we next considered analyzing the expression of differentiation markers specific to PrE, mesendoderm, TE, and neuroectoderm. Previously, it was established that mESC cultures display heterogeneity and comprise a subpopulation of lineage-committed cells [28, 29]. RT-qPCR revealed a significant upregulation of PrE markers Gata6, Gata4, Sox17, Dab2, Pdgfra, and Foxa2 in shRbm47 mESCs as compared with control mESCs (Fig. 3A and 3B). Expression of mesendodermal markers largely remained unchanged while a few neuroectodermal markers (Pax6, Nestin, and Emx1) were marginally downregulated. Most importantly, immunostaining revealed that Rbm47-depleted mESCs contained a higher fraction of GATA4 + PrE-like cells (shRbm47#1: 5.5% \(\pm\) 2.3%; shRbm47#3: 6% \(\pm\) 0.67%) as compared with control mESCs (shlacZ: 2.2% \(\pm\) 0.9%) (Fig. 3C and 3D).
Since ESCs are in vitro models of the ICM, the cells are routinely cultured in a medium supplemented with leukemia inhibitory factor (LIF). However, they can be converted to a hypomethylated ground state of pluripotency (similar to preimplantation ICM) by culturing in a medium containing inhibitors of GSK3ß and MEK1/2 (termed as '2i') . To determine whether a return to the ground state would reverse Rbm47-depletion induced priming towards the PrE lineage, we cultured control and Rbm47-depleted mESCs for two passages in ‘2i’ supplemented ESC medium and analyzed these for PrE marker expression. We indeed observed that culture conditions capturing the ground state were sufficient to reverse the PrE priming in Rbm47-depleted ESCs (Supplementary Fig. 3).
It is well-documented that FGF4-ERK signaling is the central pathway in cell-fate determination of the ICM into NANOG-positive Epi and GATA6-positive PrE at the E4.5 stage of the mouse blastocyst (Fig. 3E). Blocking FGF signaling with inhibitors of FGF receptor (FGFR) and ERK is reported to convert ICM into Epi ; in contrast, overactivation of FGF signaling can transform ICM to PrE . To assess the possible effect of Rbm47 depletion in modulating the FGF-ERK pathway, we treated shRbm47 mESCs with PD0325901 (MEK1/2 inhibitor, MEKi) or PD173074 (Pan-FGFR inhibitor, FGFRi) for 48 h and profiled for expression of PrE markers by RT-qPCR and GATA4 immunostaining. The use of either inhibitor reduced the PrE marker levels significantly, suggesting the FGF-ERK signaling is implicated in increasing PrE-like subpopulation in Rbm47-depleted mESCs (Fig. 3F and 3I). Further, to confirm the activation of this pathway, we quantified the expression of Fgfr1, Fgfr2, and Fgf4 mRNAs in Rbm47-depleted mESCs; however, there was no significant change in the mRNA levels of the FGF receptors and the ligand as compared with control cells (Fig. 3G). Additionally, we did not observe a spike in phospho-ERK1/2 levels in Rbm47-depleted ESCs, a direct measure of FGF signaling (Fig. 3H). Collectively, our findings demonstrate that Rbm47-depleted ESCs displayed upregulated PrE related genes and contained an increased population of GATA4 + cells compared to control ESCs. Culturing these cells in ESC medium supplemented with either 2i, MEKi, or FGFRi could reverse the priming towards PrE, indicating the implication of Rbm47 in regulating the FGF-ERK pathway.
Rbm47 depleted ESCs do not retain a complete multi-lineage differentiation potential
To evaluate the role Rbm47 on differentiation potential of mESCs in vivo, we xenografted shlacZ and shRbm47 mESCs subcutaneously into NSG mice for teratoma formation. Rbm47-depleted ESCs formed significantly smaller teratomas (mean teratoma volume: shRbm47#1 = 251.63 mm3; shRbm47#3 = 324.6 mm3) compared to control ESC-derived teratoma (mean teratoma volume: 2081.36 mm3), indicating that Rbm47 might be necessary for self-renewal during the differentiation of mESCs (Fig. 4A and 4B). However, sectioning and histological observation of these teratomas revealed that Rbm47-depleted mESCs could form structures from all three germ layers despite the drastic gross reduction in teratoma size (Fig. 4C). Next, to assess the multi-lineage differentiation potential of shRbm47 mESCs in vitro, we measured the expression of various markers in six-day serum differentiated monolayer cultures. As shown in Fig. 4D, there was no consistent variation in the transcripts of mesendoderm, but extraembryonic endoderm (ExEn; similar to PrE) markers (Gata6, Gata4, Pdgfra, Foxa2, and Sox17) were consistently upregulated, while neuroectoderm progenitor markers (Nestin, Pax6, and Cdh2) were downregulated in shRbm47 mESCs as compared with control cells. These data indicate that Rbm47 depleted ESCs exhibit a skewed multi-lineage differentiation potential and might have an enhanced tendency for differentiating into ExEn lineage with compromised neuroectodermal and mesendodermal fate.
Lineage-specific differentiation reveals Rbm47 is essential of neuroectoderm and endoderm fate of ESCs
To further probe the effects of Rbm47 depletion on differentiation, we profiled the expression of lineage-specific markers in shRbm47 and control mESCs differentiated into definitive endoderm (DE), mesoderm (MES), and neuroectoderm (NE). As shown in Fig. 1G and 1H, Rbm47 expression undergoes lineage-specific modulation, and the mRNA and protein levels were notably upregulated in wild-type mESC-derived DE. To determine whether Rbm47 is essential for DE formation, we plated control and shRbm47 mESCs to form EBs for 2-days in the serum-free formulation and then directed to DE lineage by supplementing the medium with Activin A. We observed that Rbm47-depleted EBs were significantly smaller and irregularly shaped than the control EBs (Fig. 5A and 5B). RT-qPCR profiling revealed a consistent downregulation of definitive endoderm markers (Sox17, Foxa2, Cdh1, and Hhex) (Fig. 5E), suggesting Rbm47 is necessary for proper differentiation of mESCs to DE.
We next specified Rbm47-depleted ESCs to MES by treating 2-day old EBs with Activin A, VEGF, and BMP4 for two days. A role for Rbm47 in the mesodermal specification was not expected since it was significantly downregulated in wild-type mESC-derived MES (Fig. 1G and 1H) and undetected in mesodermal tissues embryo and adult mouse . However, surprisingly, Rbm47-depleted ESCs not only formed significantly smaller EBs (Fig. 5C and 5D) but also displayed enhanced expression of mesodermal markers such as Mixl1, Kdr, Gsc, Mesp1, and Hand1 (Fig. 5F).
On the other hand, the involvement of Rbm47 in neuroectodermal differentiation was indicated since critical NE progenitor markers (Pax6, Nestin, and Cdh2) were suppressed upon Rbm47 depletion (Fig. 3A and 4D), we expected to be necessary for proper NE differentiation. As expected, we observed a significant decrease in self-renewal of Rbm47-depleted mESCs on day 2 of NE induction with N2B27-high insulin medium, and the effect persisted throughout the duration (day 8) (Fig. 5G). RT-qPCR analysis and immunocytochemistry of NE-differentiated shRbm47 mESCS revealed a significant downregulation of most NE markers, with a clear bias towards ExEn markers as compared with control mESCs (Fig. 5I). Immunostaining of these cultures with NE markers PAX6 and TUBB3 (B-3-Tubulin) and ExEn marker GATA4 correlated with the RT-qPCR data (Fig. 5H). Collectively, our findings from the lineage-specific differentiation models successfully demonstrated that Rbm47 is essential for fine-tuning the cell-fate decisions and lineage specification of mESCs. Rbm47 depletion strongly affects the DE and NE differentiation programs.