Single-Cell RNA Sequencing Reveals Regulatory Mechanism for Trophoblast Cell-Fate Divergence in Human Peri-Implantation Embryo

Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after embryonic implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development remains elusive. In this study, we modeled human embryo peri-implantation development from blastocyst to early post-implantation stages by using an in vitro coculture system, and profiled the transcriptome of individual trophoblast cells from these embryos. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast into syncytiotrophoblast. The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development.

a set of genes that tend to be coexpressed at a certain development stage ( Figure 3A). 128 By relating module expression to development day, we found these eight modules 129 collectively represent three genetic networks that were specifically upregulated at day  To further identify the genes that might play a critical regulatory role in these 147 three genetic networks, we identified 240 hub genes based on the WGCNA measure 148 of intramodular gene connectivity (kME). Hub genes are genes that centrally located within a gene module (kME > 0.8, P<0.05) and have co-expression relationship with 150 many other genes, therefore could have critical regulatory functions. We found that 151 many hub genes are related to critical placental function.  together contain all cells from day 9, day 10, and a few cells from earlier days. By 166 examining the expression of previously defined sublineages marker genes, we found 167 that EVT or ST markers highly expressed in Cluster 2 or 5, whereas Cluster 4 168 co-express CT and EVT marker genes similar to Cluster 1 ( Figure 4C). We then 169 identified genes that were specifically expressed in Cluster 2, 4 and 5. We found that 170 many ST marker genes, such as HSD3B1, CYP19A1, SDC1, ERVW-1 (Syncytin-1), 171 ERVV-1, CGA and CGB, were specifically highly expressed in Cluster 5. CT markers, 172 such as ITGA6 and FZD5, were specifically expressed in Cluster 4. A few EVT 173 marker genes such as MMP2 were specifically highly expressed in Cluster 2 ( Figure   174 4D). Taken together, these results suggest that Cluster 5, Cluster 4 and Cluster 2 175 represent ST, CT and EVT respectively. The rest clusters consist of trophoblasts from 176 day 6 through day 8, and express CT markers. These results indicated that these 177 clusters represent multipotent trophoblasts that have not committed to differentiation.
We then tried to determine when EVT and ST were established in embryos. We 180 found that ST first appears in cocultured embryos at day 7, even though at a very low 181 percentage (1 out of 60 cells). ST cells then become more abundant at day 8 (16 out of 182 168 cells) ( Figure 4E). Immunostaining showed that hCGβ positive cells can be 183 detected as early as day 7, and become more abundant at day 8 ( Figure 4F). These 184 results suggested that ST cells first occur after day 7, and become more abundant after 185 day 8. Similarly, we found EVT cells were absent in all of the day 7 embryos but 186 appear at day 8, indicating EVT were generated after day 7 ( Figure 4E). Taken

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The ST is an important trophoblast sublineage that forms the primary barrier 193 between maternal and fetal circulation and synthesize hormones vital for pregnancy.

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The ST is derived from multipotent trophoblasts within TE and CT. Previous studies 195 using mature placentas and cell lines have demonstrated that many regulatory factors 196 and pathways have been reported to be linked with the human ST formation [18][19][20][21].

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However, these results must be interpret carefully, because these in vitro 198 differentiation models may not perfectly recapuliate the mechanism for trophoblast 199 differentiation in vivo.    The generation of ST can be characterized by cell fusion and ST marker gene expression [20,22]. In control JEG-3 cells that were not treated by 8-Br-cAMP, the 241 cell fusion ratio was less than 1%, and ST marker hCGβ expression was almost 242 undetectable, indicating ST generation before treatment is minimal ( Figure 5A   Trophoblasts undergo magnificent morphological movement and cellular changes 303 after implantation. In this study, by profiling over 500 single cells in 19 embryos 304 generated using a coculture system, we reconstruct the transcriptome dynamics of 305 trophoblasts through blastocyst to early post-implantation stages (Figure 7). Our 306 study complements previous studies that use scRNA-seq to profile trophoblasts and 307 other cell types within mature placenta [4][5][6][7].    Single-cell RNA seq data were first trimmed with TrimGalore! using following 373 parameters "-q 20 --phred33 --gzip --length 30 --paired" to remove adaptor sequences 374 and low-quality bases. The trimmed data was then aligned to human reference 375 genome hg38 using STAR v2.6.0 in the pair-end mode with default parameters. The 376 number of reads mapped to each gene was counted using featureCounts v1.6.2, using 377 Gencode hg38 gene annotation. Customized R scripts and published R packages, 378 including Seurat, were used in subsequent analysis. normalized and quality-controlled using Seurat. The lineage identity for each cell was 388 then determined using a previous strategy reported before [35]. Briefly, for each cell, 389 a "TE score", an "EPI score" and a "PE score" were computed using AddModuleScore function implemented in Seurat package, based on its expression of 391 previously identified markers for each lineage respectively. The cell lineage was then 392 defined as the lineage that had the highest score. To identify maintained trophoblast 393 markers, we started with previously identified TE marker genes for pre-implantation 394 embryos, and excluded genes that were lowly expressed in trophoblasts at any stage 395 between day-6 to day-10 (mean FPKM < 10 over all trophoblasts on each day).     Table S1: Primers used for qRT-PCR 712 Figure S1: Scatterplot of TE-lineage markers identified previously and in our study.