CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions

Abstract

Coxsackie virus and adenovirus receptor, CXADR (CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.

The Cxadr–Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice

“…In agreement with previous human and porcine studies (Krivega et al, ; Kwon et al, ), the Cxadr transcript was detected during preimplantation, and the transcript levels were highly expressed from the 8‐cell stage onwards. Particularly, the level was 26‐ and 74‐fold at the morula and blastocyst stages, respectively, compared to the 1‐cell zygote (Figure a).…”

“…Moreover, our observation that transcript levels of Cxadr did not change significantly, but Cxadr proteins were localised to nuclei rather than apical regions of the blastocysts led us to examine the existence and expression of different Cxadr forms. As shown in previous studies (B. Choi et al, ; Krivega et al, ), the nuclear form (soluble) was detected in Adam10 KD blastocysts. Moreover, the transcripts of soluble Cxadr were expressed in early, expanded, and hatched blastocysts, but the transmembrane form of Cxadr was relatively abundant in early and hatched blastocysts (Figure S2).…”

“…Moreover, our observation that transcript levels of Cxadr did not change significantly, but Cxadr proteins were localised to nuclei rather than apical regions of the blastocysts led us to examine the existence and expression of different Cxadr forms. As shown in previous studies Krivega et al, 2014), the nuclear form (soluble) was detected in Adam10 KD blastocysts.…”

“…In early mammalian embryos, including humans, mice, and pigs, coxsackievirus and adenovirus receptor (CXADR) is found at the apical contacts of the TE and endometrial epithelium, suggesting that CXADR might be involved in establishing the trophoblast and in further development (B. Choi, Nah, Oh, Gye, & Kim, ; Krivega, Geens, & Van de Velde, ; Kwon et al, ). To investigate the effects of depleting Cxadr on pre‐ and peri‐embryo development, we expand our previous findings (Kwon et al, ; Kwon et al, ) in which porcine CXADR is involved in adherens junction (AJ)/TJ formation and interacts with ADAM10 and TJP1 for TJ assembly during preimplantation development.…”

MINT: A multivariate integrative method to identify reproducible molecular signatures across independent experiments and platforms

“…The two genes selected by MINT on component 1 were Lin28A and CAR which were both found relevant in the literature. Indeed, Lin28A was shown to be highly expressed in ESCs compared to Fibroblasts (Yu et al, 2007;Tsialikas and Romer-Seibert, 2015) and CAR has been associated to pluripotency (Krivega et al, 2014). Finally, despite the high heterogeneity of hiPSC cells included in this study, MINT gave a high accuracy for hESC and hiPSC on independent test sets (93.9% and 77.9% respectively, Figure S6), suggesting that the 15 genes selected by MINT on component 2 have a high potential to explain the differences between those cell types (Table S5).…”