No evidence of involvement of E-cadherin in cell fate specification or the segregation of Epi and PrE in mouse blastocysts

Abstract

During preimplantation mouse development stages, emerging pluripotent epiblast (Epi) and extraembryonic primitive endoderm (PrE) cells are first distributed in the blastocyst in a "salt-and-pepper" manner before they segregate into separate layers. As a result of segregation, PrE cells become localised on the surface of the inner cell mass (ICM), and the Epi is enclosed by the PrE on one side and by the trophectoderm on the other. During later development, a subpopulation of PrE cells migrates away from the ICM and forms the parietal endoderm (PE), while cells remaining in contact with the Epi form the visceral endoderm (VE). Here, we asked: what are the mechanisms mediating Epi and PrE cell segregation and the subsequent VE vs PE specification? Differences in cell adhesion have been proposed; however, we demonstrate that the levels of plasma membrane-bound E-cadherin (CDH1, cadherin 1) in Epi and PrE cells only differ after the segregation of these lineages within the ICM. Moreover, manipulating E-cadherin levels did not affect lineage specification or segregation, thus failing to confirm its role during these processes. Rather, we report changes in E-cadherin localisation during later PrE-to-PE transition which are accompanied by the presence of Vimentin and Twist, supporting the hypothesis that an epithelial-to-mesenchymal transition process occurs in the mouse peri-implantation blastocyst.

Fig 1. E-cadherin localisation and distribution in E3.5–E5.5 embryos.

(A) Immunofluorescence for E-cadherin in embryos at E3.5 (n = 5, a–a”‘), E3.75 (n = 9, b–b”‘), E4.5 (n = 10, c–c”‘), E4.75 (n = 7, d–d”‘) and E5.5 (n = 8, e–e”‘). PrE cells/precursors are GFP-positive (Pdgfrα^H2B-GFP). The arrow denotes a parietal endoderm cell lacking E-cadherin. Ex–extraembryonic part, Em–embryonic part. (B) Scheme for measuring fluorescent signal to determine the relative levels of E-cadherin in E3.5, E3.75 and E4.5 embryos. Red lines indicate measurement of fluorescent intensity between two Epi and two PrE cells. (C) Estimated levels of E-cadherin in PrE cells relative to Epi cells in E3.5, E3.75 and E4.5 embryos; 95% confidence intervals are plotted for the ratios.

Fig 2. Downregulation of E-cadherin by microinjection of dsRNA.

(A) Immunostaining for E-cadherin 24 hours after dsEcad injection. Stars are used to mark the progeny of injected blastomeres. (B) mRNA levels for E-cadherin and N-cadherin in the progeny of cells injected with dsEcad and in the progeny of non-injected cells from the same embryos. C) Plotted are 95% confidence intervals for the ratio of E-cadherin level between non-injected and injected cells. (D) Immunostaining for N-cadherin in embryos injected with dsEcad.

Fig 3. Relative levels of cadherin gene expression and immunostaining of EMT proteins.

(A) Relative levels of mRNA for Gata4, Gata6, E-cadherin, N-cadherin, P-cadherin, R-cadherin, VE-cadherin, OB-cadherin and T-cadherin in Epi and PrE populations of E3.5 and E4.5 embryos. (B) Immunostaining for Vimentin in E3.5, E4.5 and E5.5 embryos. (C) Immunostaining for Twist (a–a”“) in the E4.5 blastocyst.

Fig 4. Immunostaining for N-cadherin in mouse embryos.

Immunostaining shows a lack of N-cadherin in E3.5, E4.5 and E5.5 mouse embryos.