TCFAP2C Is Required for Transcriptional Regulation of Tight Junction Biogenesis and Cavity Formation During Blastocyst Development.

In human ART as many as 80% of preimplantation embryos fail to develop to the blastocyst stage. However, the underlying causes of this failure are not well defined. Utilizing a mouse model we are examining the transcriptional mechanisms that govern cellular processes associated with blastocyst formation (i.e., tight junction biogenesis and fluid transport). Transcription factor TCFAP2C is widely expressed during mouse preimplantation development and becomes enriched in the trophoblast lineage during blastocyst formation. Knockout of TCFAP2C via conventional approaches results in embryonic lethality at around 7.5 days post coitum with defects in the trophoblastic tissue. However, the role of TCFAP2C in blastocyst formation has not been explored. We employed an RNAi approach to deplete both maternally and zygotically expressed TCFAP2C. One-cell embryos were injected with either Tcfap2c or control siRNA and cultured for 4 days. Microinjection of Tcfap2c siRNA resulted in a ~90% reduction in Tcfap2c mRNA and complete loss of protein as assayed by immunocytochemistry (ICC). TCFAP2C knockdown (KD) and control embryos developed at similar rates to the morula stage (p>0.05), however, only a small percentage of TCFAP2C KD morulae formed blastocysts (14.51% vs. 89.32%;p<0.001). These results suggest that TCFAP2C may regulate key genes required for blastocyst formation. To address this we conducted (1) transcriptome analysis to identify genes upregulated during blastocyst formation and (2) a literature search on genes required for blastocyst formation. These lists were compared with a published TCFAP2C ChIP-Chip dataset in trophoblast stem (TS) cells and analyzed for TCFAP2C binding motifs using TRANSFAC. Accordingly, we found that a large number of genes upregulated during blastocyst formation contained putative TCFAP2C motifs and were enriched for TCFAP2C binding in TS cells. Interestingly, many of these genes play important roles in tight junction assembly, cell polarity, and fluid transport during blastocyst formation. To determine whether these genes are deregulated in TCFAP2C KD embryos we conducted real-time qPCR and ICC analysis at the morula stage. Genes important for tight junction biogenesis (e.g., Tjp2, Cldn4, Cldn7), cell polarity (e.g., Pard6b), and fluid transport (e.g., Atp1b1, Aqp3) were downregulated in TCFAP2C KD morulae (p<0.05). To test whether TCFAP2C directly activates these genes via binding to their promoter regions, we employed Cdx2-inducible ES cells that differentiate into TS cells. Chromatin immunoprecipitation (ChIP) analysis showed that during differentiation TCFAP2C was recruited to the promoters of Tjp2, Cldn4, Cldn7, Pard6b, and Aqp3 and this was coupled with increased mRNA expression. Finally, the function of tight junction complexes in TCFAP2C KD embryos and control embryos was tested by subjecting them to a FITC-dextran permeability assay. TCFAP2C KD embryos exhibited increased permeability to FITC-dextran suggesting that tight junction complexes were not properly formed (p<0.05). In summary, our results show that TCFAP2C regulates a diverse set of genes necessary for blastocyst formation. To our knowledge this is the first study to demonstrate the function of a single transcription factor in controlling tight junction assembly, polarity, and fluid transport in blastocysts. Future, studies will investigate whether TCFAP2C is implicated in human fertility. This work was supported by NIH R01GM095347 to JGK.