Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.037 Byline: Judith J. Eckert (a)(b), Amanda McCallum (a), Andrew Mears (a), Martin G. Rumsby (c), Iain T. Cameron (b), Tom P. Fleming (a) Keywords: Protein kinase C; Mouse embryo; Trophectoderm; Tight junction; ZO-1; Cell contact pattern; Inner cell mass; ZO-2 Abstract: In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKC[delta] and I[paragraph], revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCI[paragraph] activity is involved in regulating ZO-1[alpha]+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis. Author Affiliation: (a) University of Southampton, School of Biological Sciences, Bassett Crescent East, Southampton, SO16 7PX, UK (b) University of Southampton, Developmental Origins of Health and Disease Division, School of Medicine, Coxford Road, Southampton, SO16 5YA, UK (c) University of York, Department of Biology, York, YO1 5DD, UK Article History: Received 19 May 2005; Revised 1 September 2005; Accepted 22 September 2005

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by [Na.sup.+]/[K.sup.+] ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four ([delta], [theta], [iota]/[lambda], [zeta]) PKC isoforms and PKC[micro]/PKD1 showed partial colocalisation with the tight junction marker ZO-1[alpha]+ in TE and all four PKCs ([delta], [theta], [iota]/[lambda], [zeta]) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKC[delta] and [zeta] activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, [Na.sup.+]/[K.sup.+] ATPase [alpha]1 submit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including [Na.sup.+]/[K.sup.+] ATPase. Keywords: Protein kinase C; Mouse embryo; Blastocyst; Trophectoderm; Inner cell mass; Tight junction; ZO-1; [Na.sup.+]/[K.sup.+] ATPase; Cavitation