KHDC3 affects the lineage differentiation of early embryo by regulating the differential distribution of YAP signal

Hydatidiform mole (HM) is characterized by abnormal fetal tissue loss and placental villous trophoblastic hyperplasia, indicating aberrant early embryonic cell fate differentiation. Clinical observations linking KH Domain Containing 3 (KHDC3) mutations to complete hydatidiform mole (CHM) suggest a potentially unforeseen role for KHDC3 in early embryonic development. Single-cell transcriptome analysis of early embryos revealed notable heterogeneity in KHDC3 expression levels from the 8-cell to blastocyst stage. Initial investigations indicated that varying KHDC3 expression levels at the 8-cell stage could influence the Yes-associated protein (YAP) signaling pathway. Knocking down KHDC3 in individual blastomeres at the 2-cell stage resulted in reduced blastocyst formation rates. Khdc3-knockdown (Khdc3-KD) embryos exhibited disruptions in embryonic cell lineage differentiation, characterized by decreased expression of the inner cell mass (ICM) marker organic cation/carnitine transporter 4 (OCT4) and diminished differential expression of the trophectoderm (TE) marker caudal type homeobox 2 (CDX2). The nuclear entry of phosphorylated YAP in outer cells is pivotal for inducing lineage differentiation. Intriguingly, in Khdc3-KD morulae, a significant reduction in the nuclear entry of phosphorylated YAP in outer cells was observed. Moreover, KHDC3 knockdown simultaneously impaired cortical actin cap formation during the morula stage. Forces generated at the apical cortex during this process segregate cells into inner and outer positions within the embryo, thereby influencing ICM versus TE fate specification. Our research underscores the role of KHDC3 in modulating the YAP signaling pathway to establish distinct inner and outer cell distributions during the morula stage, consequently influencing early embryonic lineage differentiation. Nevertheless, the precise mechanism by which KHDC3 influences the YAP signaling pathway via the actin cytoskeleton remains to be fully elucidated. Given the severity of recurrent miscarriages associated with KHDC3 mutations, elucidating the distinctive role of KHDC3 in early embryonic development warrants further investigation.