Characterization of the role of Cripto in trophoblast cells during placental development

Cripto, a member of the Epidermal Growth Factor-Cripto/FRL-1/Cryptic (EGF-CFC1) protein family, plays a crucial role in embryonic development. In addition to embryonic expression, Cripto is expressed in extraembryonic tissues including the various trophoblast cell subtypes spongiotrophoblast and labyrinth sinusoidal giant cells. In human studies, dysregulation of Cripto expression adversely affects placental development and function. Previous findings have reported that Cripto expression is significantly higher in placental pathologies such as placenta accreta and placenta previa compared to normal placentas. Additionally, expression analysis of the EGF-CFC1 gene family demonstrate genetic associations with recurrent pregnancy loss. During mouse embryonic development, the homozygous deletion of Cripto in the inner cell mass is embryonic lethal between days 7.5 and 10.5; however, the deletion of Cripto in the extraembryonic trophectoderm, is yet to be studied. Thus, based on the expression of Cripto in the trophectoderm, and its deregulated expression in pregnancy complications, we hypothesize that Cripto plays an important role in placental development and function. To address that, we generated a Cripto knockout exclusively in mouse TE cells using Tat-Cre recombinant protein. Tat-Cre optimization protocol involving the mating of Cripto floxed females with homozygous mTmG males showed high efficiency of cre-recombination exclusively in TE cells. Following the optimization of the model for knocking out Cripto, homozygous Cripto floxed Tat-Cre-treated embryos were then transferred into uteri of pseudopregnant control mice for continued development. Dissections of the knockout implantation sites at day 10.5 exhibited significantly decreased implantation site size and placental thickness compared to the control sites. Furthermore, hematoxylin and eosin staining revealed positional deviated placentas according to the AM-M axis in the knockouts compared to the controls. Immunofluorescence staining with TE specific markers, placental lactogen and trophoblast specific protein alpha, displayed aberrant cellular organization and differentiation, with increased trophoblast giant cell expansion and diminished spongiotrophoblast layers. In-situ hybridization analysis of the labyrinth syncytiotrophoblasts using GCM-1 showed decreased expression in the knockouts compared to the controls. Dissections of knockout sites at day 8.5 revealed significantly decreased implantation site size and a trending decrease in the viable implantation sites compared to the controls. Addionally, marker analysis using trophoblast specific protein alpha demonstrated a diminished spongiotrophoblast layer at day 8.5 as well, suggesting compromised trophoblast cell differentiation. The findings presented provide deeper insights into Cripto's involvement in placental development. We have shown that Cripto’s deletion in the trophectoderm leads to dysregulated trophoblast cell organization and placental formation during mid-gestation. Therefore, we propose that Cripto, potentially through the Nodal and other signalling pathways, seems to play a role in restraining the growth of the trophoblast giant cell layer while promoting expansion of the labyrinth and spongiotrophoblast compartments