{"id":488711,"date":"2024-01-16T19:00:00","date_gmt":"2024-01-17T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/vgll1-cooperates-with-tead4-to-control-human-trophectoderm-lineage-specification-nature-communications\/"},"modified":"2024-01-17T14:55:31","modified_gmt":"2024-01-17T19:55:31","slug":"vgll1-cooperates-with-tead4-to-control-human-trophectoderm-lineage-specification-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/vgll1-cooperates-with-tead4-to-control-human-trophectoderm-lineage-specification-nature-communications\/","title":{"rendered":"VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
We produced TELCs from human na\u00efve H9 embryonic stem cells (ESCs) generated with our newly established 4CL medium33<\/a><\/sup> using a previously reported TELC conversion protocol26<\/a><\/sup> (Fig. 1a<\/a>). Flattened epithelial-like cells with a TE-like morphology were observed at day 5 (Fig. 1b<\/a>). Downregulation of classical (e.g<\/i>., NANOG<\/i> and OCT4<\/i>) and na\u00efve (e.g<\/i>., KLF17<\/i> and DPPA3<\/i>) pluripotency genes, expression of na\u00efve\/TE shared genes such as TEAD4<\/i> and TFAP2C<\/i>, and upregulation of TE-enriched genes like GATA3<\/i> and ENPEP<\/i> were validated by immunofluorescence microscopy and\/or bulk RNA-sequencing (RNA-seq) (Fig. 1c, d<\/a> and Supplementary Fig. 1a<\/a>). To assess the fidelity with which TELCs derived from 4CL ESCs reflect preimplantation embryo TE cells, we performed droplet-based single-cell RNA sequencing (scRNA-seq)39<\/a><\/sup> at day 5 of TELC induction (TELC-D5) from 4CL ESCs and integrated them with previously reported scRNA-seq datasets of 4CL na\u00efve ESCs33<\/a><\/sup>, human preimplantation40<\/a>,41<\/a><\/sup> and postimplantation embryo cells42<\/a><\/sup>. Uniform manifold approximation and projection (UMAP) representation showed that TELC-D5 cells clustered closely with human preimplantation embryo TE cells and their gene expression pattern was comparable (Fig. 1e, f<\/a>). Pearson correlation analysis among the subclusters within TELC-D5 cells showed that they are highly correlated (correlation score > 0.9) except for two subclusters (cluster 7 and 8, 2.08 % of total TELC-D5 cells); cluster 7 also expressed SCT related genes (Supplementary Fig. 1b-d<\/a>). These results suggest that 4CL ESC-derived TELC-D5 cells are relatively homogenous and resemble the human preimplantation embryo TE. We then determined the differentially expressed genes (DEGs) between na\u00efve ESCs and TELC-D5 cells, and found VGLL1<\/i> as the most significantly upregulated gene in TELCs (Fig. 1g<\/a>). In contrast, YAP1<\/i> and TEAD4<\/i> were only moderately upregulated in TELC-D5 cells. We confirmed the exclusivity of VGLL1<\/i> expression in TELCs by immunofluorescence microscopy, real-time quantitative PCR (RT-qPCR) and Western blotting (Fig. 1c, h, i<\/a>). Western blotting also validated the downregulation of the pluripotency genes NANOG<\/i>, OCT4<\/i> and SOX2<\/i> (Fig. 1i<\/a>). Likewise, it confirmed that YAP and TEAD4 are slightly upregulated in TELC-D5 cells compared with na\u00efve ESCs.<\/p>\n a<\/b> Schematic depicting the generation of TELC-D5 from 4CL H9 ESCs with TE medium and the generation of TSCs from TELC-D5 cells with TSC medium. VGLL1<\/i>\/TEAD4<\/i> knockdown (KD) and multiple types of sequencing were performed as indicated. b<\/b> Phase contrast images of 4CL H9 ESCs and TELCs at the indicated time points. Scale bar, 40\u2009\u00b5m. Representative of three independent experiments. c<\/b> Immunostaining images for pluripotency (NANOG and KLF17), na\u00efve\/TE shared (TFAP2C and TEAD4), and TE-enriched (GATA3 and VGLL1) genes in 4CL H9 ESCs (upper panels; scale bar, 50\u2009\u00b5m) and TELC-D5 cells (lower panels; scale bar, 100\u2009\u00b5m). Nuclei were counterstained with DAPI (blue). Representative of three independent experiments. d<\/b> Heatmap showing the expression of pluripotency and TE genes in bulk RNA-seq for the indicated conditions. Example genes for each cluster are shown in the boxes. n<\/i>\u2009=\u20092 biological replicates. e<\/b> UMAP comparing the human embryonic day 4 (E4) to E14 stages with 4CL H9 ESCs and TELC-D5 cells. All reference datasets used in this study are summarized in Methods. f<\/b> Bubble plot showing the frequency of expression and scaled average expression of representative genes in, 4CL H9 ESCs, TELC-D5 cells and human embryo E4-E14 datasets. TE, trophectoderm; CTB, cytotrophoblast; EVT, extravillous trophoblast; SCT, syncytiotrophoblast. g<\/b> Volcano plot showing DEGs between 4CL H9 na\u00efve ESCs and TELC-D5 cells. DEGs higher in TELC-D5 cells (log2<\/sub>(fold change)\u2009> 1) are shown in red. P<\/i> value was calculated using the Wald test and adjusted for multiple testing using the Benjamini-Hochberg correction. h<\/b> RT-qPCR showing the expression of VGLL1<\/i> in 4CL H9 ESCs and TELC-D5 cells. Data are presented as the mean\u2009\u00b1\u2009standard error of the mean (SEM) of the fold-change compared to naive ESCs. n<\/i>\u2009=\u20093 biological replicates. P<\/i> value was calculated using a two-tailed unpaired Student\u2019s t<\/i>-test, ***P<\/i>\u2009<\u20090.001. i<\/b> Western blotting analysis for the indicated proteins in 4CL H9 ESCs, TELC-D5 cells, and TSCs derived from TELC-D5 cells. Representative of three independent experiments.<\/p>\n<\/div>\n<\/div>\n The high levels of VGLL1<\/i> in TELCs derived from naive ESCs prompted us to study its expression kinetics during early in vivo development. The human early blastocyst corresponding to embryonic day 5 (E5) is comprised of cells belonging to the earliest stage of TE and ICM lineage. We investigated the identities and gene expression patterns of human E5 cells from a previously reported scRNA-seq dataset40<\/a><\/sup>. UMAP segregated the TE and ICM cells in addition to pre-lineage cells in E5 cells (Fig. 2a<\/a>). Classical and naive pluripotency genes were highly expressed in pre-lineage and ICM cells but downregulated in TE cells (Supplementary Fig. 1e<\/a>). TE-related transcription factors like GATA3<\/i>, GATA2<\/i> and CDX2<\/i> were upregulated in TE cells compared with pre-lineage or ICM cells (Fig. 2b<\/a>, Supplementary Fig. 1e<\/a>). VGLL1<\/i> was highly and specifically expressed in TE cells. This is relevant because context-dependent highly expressed genes often function as key regulators43<\/a><\/sup>. Unlike mouse, genes like ELF5<\/i> and EOMES<\/i> were only marginally expressed at this stage (Supplementary Fig. 1e<\/a>), which is consistent with previous report44<\/a><\/sup>. To better understand the dynamic changes of gene expression during the human TE and ICM lineage bifurcation, we performed pseudotime analysis of the scRNA-seq data using Monocle 245<\/a>,46<\/a>,47<\/a><\/sup> (Fig. 2c<\/a>). As expected, pluripotency genes such as OCT4<\/i> and SOX2<\/i> increased in the pre-lineage to ICM branch and were downregulated in the pre-lineage to TE branch (Supplementary Fig. 1f<\/a>). In contrast, naive\/TE shared genes like TEAD4<\/i>, YAP1<\/i> and TFAP2C<\/i> remained at similar expression levels along the pseudotime or were moderately upregulated, whereas TE-enriched genes like GATA3<\/i>, GATA2<\/i> and CDX2<\/i> were progressively upregulated (Fig. 2d<\/a>, Supplementary Fig. 1f<\/a>). Further supporting a role in TE specification, VGLL1<\/i> showed a dramatic upregulation at the onset of the TE branch (Fig. 2d<\/a>). The quick induction and highly selective expression of VGLL1<\/i> in TE were further confirmed by using another human early embryo scRNA-seq dataset41<\/a><\/sup> (Supplementary Fig. 1g-j<\/a>).<\/p>\n a<\/b> UMAP visualization showing the different cell types in the human blastocyst at E540<\/a><\/sup>. Dots are colored by cell type. Cell types were annotated by cluster-specific gene expression patterns. b<\/b> Violin plot showing the log-normalized expression of TE genes in different cell types of the human blastocyst at E5. c<\/b> Left panel: the trajectory of the different cell types in the human blastocyst at E5 reveals two branches: pre-lineage to the TE branch and pre-lineage to the ICM branch. Right panel: colors from dark blue to light blue indicate progression through the pseudotime. d<\/b> Expression patterns of TE genes along the pseudotime trajectory of cells in the human blastocyst at E5. e<\/b> UMAP visualization showing the different cell types in the monkey blastocyst at E6-E948<\/a><\/sup>. Dots are colored by cell type. Cell types were annotated by cluster-specific gene expression patterns. f<\/b> Violin plot showing the log-normalized expression of TE genes in different cell types of the monkey blastocyst at E6-E9. g<\/b> Left panel: the trajectory of the different cell types in the monkey blastocyst (E6-E9) reveals two branches: pre-lineage to the TE branch and pre-lineage to the EPI branch. Right panel: colors from dark blue to light blue indicate progression through the pseudotime. h<\/b> Expression patterns of TE genes along the pseudotime trajectory of cells in the monkey blastocyst at E6-E9. i<\/b> UMAP visualization showing the different cell types at the mouse 16-cell stage49<\/a><\/sup>. Dots are colored by cell type. Cell types were annotated by cluster-specific gene expression patterns. j<\/b> Violin plot showing the log-normalized expression of TE genes in different cell types at the mouse 16-cell stage. k<\/b> Left panel: the trajectory of the different cell types at the mouse 16-cell stage reveals two branches: pre-lineage to the TE branch and pre-lineage to the ICM branch. Right panel: colors from dark blue to light blue indicate progression through the pseudotime. l<\/b> Expression patterns of TE genes along the pseudotime trajectory of cells at the mouse 16-cell stage.<\/p>\n<\/div>\n<\/div>\n In addition, we investigated the cell types and gene expression pattern of early Macaca fascicularis<\/i> monkey embryo using a reported scRNA-seq dataset48<\/a><\/sup> and found that, similar to human, VGLL1<\/i> is highly expressed in a TE-specific manner (Fig. 2e, f<\/a>). Pseudotime analysis also showed that VGLL1<\/i> is strongly induced since the beginning of TE lineage specification (Fig. 2g, h<\/a>). In contrast, scRNA-seq of early mouse embryo development49<\/a><\/sup> showed that Vgll1<\/i> is marginally expressed and minimally upregulated at the onset of mouse TE induction in the 16-cell stage (Fig. 2i-l<\/a>), which is consistent with previous report that VGLL1 is not involved in mouse trophoblast specification34<\/a><\/sup>. These results indicate that VGLL1 may function as a TE induction regulator in primates.<\/p>\n To explore whether VGLL1<\/i> has a functional role in human TE specification, we depleted it using short-hairpin RNAs (shRNAs; shVGLL1<\/i>\u22122 and \u22124) during the 4CL naive PSC to TELC induction. Knockdown efficiency was confirmed by RT-qPCR and Western blotting (Fig. 3a, b<\/a>). Notably, we observed that self-renewal (cell proliferation) during the conversion was severely compromised in shVGLL1<\/i> transduced cells compared to the shLuc<\/i> control, and this was also accompanied by a lack of TE-like morphology (Fig. 3c, d<\/a>). We then performed bulk RNA-seq for shLuc<\/i> and shVGLL1<\/i> transduced TELC-D5 cells. Consistent with the reduced proliferation and lack of TE-like morphology, cell-cycle\/self-renewal genes such as MKI67<\/i>, CDK1<\/i>\/3<\/i>\/7<\/i> and CDC42<\/i>, and TE genes like GATA3, ENPEP, NR2F2<\/i> and TEAD1,<\/i> were substantially downregulated in shVGLL1<\/i> transduced cells compared to control shLuc<\/i> (Fig. 3e<\/a>). There was relatively less downregulation of pluripotency genes in shVGLL1<\/i> cells compared to shLuc<\/i> control (Fig. 3e<\/a>). These results were validated by RT-qPCR (Fig. 3f<\/a>). In agreement, gene ontology (GO) analysis for the downregulated genes with shVGLL1<\/i> showed enrichment of terms related to cell cycle DNA replication, cell cycle checkpoint, in utero<\/i> embryonic development and placenta development (Fig. 3g<\/a>). Likewise, upregulated genes in shVGLL1<\/i> transduced cells compared to control shLuc<\/i> were related to development and cell fate commitment (Fig. 3h<\/a>). To further validate the function of VGLL1<\/i> in TELC induction, we generated VGLL1<\/i> knockout (KO) clones (C45 and C68) using CRISPR-Cas9 technology with H9 ESCs (Fig. 3i, j<\/a>). Consistent with the shRNA-mediated VGLL1<\/i> knockdown, both VGLL1<\/i> KO clones, compared to wild-type (WT) clones, showed reduced TE gene expression determined by RNA-seq as well as compromised cell proliferation confirmed by phase contrast imaging and cell counting (Fig. 3k-m<\/a>). The downregulated genes in VGLL1<\/i> KO cells were associated with in utero<\/i> embryonic development, placenta development and positive regulation of cell cycle determined by GO enrichment analysis (Fig. 3n<\/a>).<\/p>\n a<\/b> RT-qPCR showing the VGLL1<\/i> knockdown efficiency for 4CL H9 ESCs transduced with shVGLL1\u2212<\/i>2 or shVGLL1\u2212<\/i>4 compared to the shLuc<\/i> control at day 5 of TELC differentiation. Data are presented as the mean\u2009\u00b1\u2009SEM, n\u2009=\u20093 biological replicates. P<\/i> value was calculated using a two-tailed unpaired Student\u2019s t-<\/i>test, ***P<\/i>\u2009<\u20090.001. b<\/b> Western blotting analysis of the indicated proteins for 4CL H9 ESCs transduced with shVGLL1\u2212<\/i>2 or shVGLL1\u2212<\/i>4 compared to the shLuc<\/i> control at day 5 of TELC differentiation. Representative of three independent experiments. c<\/b> Representative phase contrast images of 4CL H9 ESCs transduced with shLuc<\/i> (control) or shVGLL1<\/i> (2 and 4) at day 5 of TELC differentiation. Scale bar, 100\u2009\u00b5m. Representative of three independent experiments. d<\/b> Analysis of cell numbers for 4CL H9 ESCs transduced with shLuc<\/i>, shVGLL1<\/i>\u22122 or shVGLL1<\/i>\u22124 throughout the TELC induction time course. Data are presented as the mean\u2009\u00b1\u2009SEM. n\u2009=\u20093 biological replicates. P<\/i> value was calculated using a two-tailed unpaired Student\u2019s t<\/i>-test, ***P<\/i>\u2009<\u20090.001, **P<\/i>\u2009<\u20090.01. e<\/b> Heatmap showing the expression of pluripotency, cell cycle and TE genes in 4CL H9 ESC-derived cells from the indicated conditions. Example genes are shown for each cluster in the boxes. n\u2009=\u20092 biological replicates. f<\/b> RT-qPCR showing the expression of TE-related genes for 4CL H9 ESCs transduced with shVGLL1<\/i>\u22122 and shVGLL1<\/i>\u22124 compared to the shLuc<\/i> control at day 5 of TELC differentiation. Data are presented as the mean\u2009\u00b1\u2009SEM. n\u2009=\u20093 biological replicates. P<\/i> value was calculated using a two-tailed unpaired Student\u2019s t<\/i>-test, ***P<\/i>\u2009<\u20090.001, **P<\/i>\u2009<\u20090.01, *P<\/i>\u2009<\u20090.05. g<\/b> Enriched GO terms for downregulated genes in 4CL H9 ESCs transduced with shVGLL1<\/i> compared to shLuc<\/i> control at day 5 of TELC differentiation. P<\/i> value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. h<\/b> Enriched GO terms for upregulated genes in 4CL H9 ESCs transduced with shVGLL1<\/i> compared to shLuc<\/i> control at day 5 of TELC differentiation. P<\/i> value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. i<\/b> TA cloning followed by Sanger sequencing results showing homozygous deletion for VGLL1<\/i>-knockout clones [clone 45 (C45) and clone 68 (C68)]. WT: wild-type. j<\/b> Western blotting analysis of VGLL1 expression in H9 WT and VGLL1<\/i>-knockout clones at day 5 of TELC induction. Representative of three independent experiments. k<\/b> Representative phase contrast images of H9 WT and VGLL1<\/i>-knockout clones at day 5 of TELC induction. Scale bar, 100\u2009\u00b5m. Representative of three independent experiments. l<\/b> Analysis of cell numbers for H9 WT and VGLL1<\/i>-knockout clones at day 5 of TELC induction. Data are presented as the mean\u2009\u00b1\u2009SEM. n\u2009=\u20093 biological replicates. P<\/i> value was calculated using a two-tailed unpaired Student\u2019s t<\/i>-test, ***P<\/i>\u2009<\u20090.001. m<\/b> Heatmap showing the expression of pluripotency, cell cycle and TE genes in bulk RNA-seq of H9 VGLL1<\/i>-knockout clones compared to WT at day 5 of TELC induction. Example genes are shown for each cluster in the boxes. n\u2009=\u20092 biological replicates. n<\/b> Enriched GO terms for downregulated genes in H9 VGLL1<\/i>-knockout clones compared to WT at day 5 of TELC induction. P<\/i> value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction.<\/p>\n<\/div>\n<\/div>\n Trophoblast lineage differentiation potential may vary with cell lines and starting naive PSC states17<\/a><\/sup>. We thus performed VGLL1<\/i> knockdown in another human induced pluripotent stem cell (iPSC) line, UH1050<\/a>,51<\/a><\/sup>, and tested other well-established human naive PSC culture conditions: PXGL and HENSM<\/a><\/div>\n
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\n VGLL1 regulates TELC generation from human na<\/b>ive PSCs<\/b>
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