P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer KO revealed discrepancies function, wherein TFAP2C dispensable for blastocyst formation but controls segregation transcription factors mouse embryo. known already an important factor that expressed solely (TE) mouse, both TE epiblast human. Recently, genetic ablation TEAD4 (player HIPPO-pathway) completely abolished which indicates might act before first takes place. In addition, RNA depletion experiments embryos a compensatory upregulation TFAP2A, functional redundancy. Nonetheless, remains how exactly regulates molecular level whether there also implication its isoform TFAP2A. design, size, duration Guide RNAs were designed targeting exon 5 Tfap2c 2 Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes delivered into zygotes via electroporation. Additionally, appropriate non-targeted scramble (inactive crRNA) control groups included. Morphological analysis, immunofluorescence next-generation sequencing (NGS) applied to check gene editing efficiency impact embryonic development segregation. Participants/materials, setting, methods Targeted cultured maximum 4.5 days vitro. They stained different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) SOX17 (hypoblast, PrE) at stages development, such as (E2.5), (E3.5) second (E4.5) Immunostaining was used determine cell number, TE/ICM fraction, marker localization fluorescence intensity. Embryos subjected analysis on-target efficiency. Main results role chance electroporation generated efficiently complete embryos. Of 39 targeted TFAP2C, 38 (97%) them edited. From edited 24 (62%) displayed 100% frameshift mutations, considered KO. until E4.5 still able form blastocysts (13/15, 87%), associated with inferior quality compared groups. Furthermore, TFAP2c-null could hatch herniated multiple places, contrast wild-type blastocysts, typically herniate one full embryos, we observed delayed expression E2.5 (n = 7). At E3.5 5), however, be pertained whereas nuclear disturbed. 6), some primitive endoderm cells presumed ICM (SOX17-positive), no NANOG-positive (EPI) detected. However, TFAP2C-KO erroneous or expression, indicating they adopted another fate irrespective localization. Secondly, TFAP2A (3/5, 60%) exhibited morula arrest (100%; n 3) E4.5. Limitations, reasons caution limited by occurrence mosaicism (more than genotype present embryo) potential off-target editing, will assess silico predicted sites NGS stem cells. The observations study consolidated increasing sample size. Wider implications findings Gene studies enable us unravel interactions are required human preimplantation development. Obtaining novel insights networks TFAP2-transcription family improve our fundamental understanding spatial key factors, crucial successful implantation. Trial registration number Not applicable