How Adhesion Forms the Early Mammalian Embryo

Melanie D. White; Nicolas Plachta1    European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia
1 Corresponding author: email address: nicolas.plachta@emblaustralia.org

1 The Mouse Preimplantation Embryo as a Model of Adhesion in Mammalian Development

Most research on adhesion has been performed on cells in tissue culture due to their availability and ease of manipulation. However, it is only during true cellular differentiation within an embryo that the contribution of adhesion to development can be examined directly. The mouse preimplantation embryo provides an ideal system to study adhesion mechanisms that are based exclusively on cell–cell interactions. A glycoprotein membrane, the zona pellucida, encloses the preimplantation embryo so cell–cell adhesion can be studied in the complete absence of extracellular matrix interactions.

Preimplantation development naturally occurs within the oviduct, but it can be recapitulated in vitro without adversely affecting the developmental potential of embryos (Summers & Biggers, 2003). Mouse embryos can be easily removed from the maternal oviducts and cultured in simple media conditions. Under these ex utero conditions, the embryos develop almost as rapidly as they do in utero and if transferred back to the uterus they can implant and continue developing to produce viable offspring.

During the first 2 days of development, the fertilized mouse egg undergoes three cleavage divisions to produce an 8-cell embryo (Fig. 1A). At this stage, the cells are round and visibly indistinguishable. The first major cell morphological changes begin as the 8-cell embryo undergoes compaction. Concomitant with a rise in intercellular adhesion, the cells flatten their membranes against each other, maximizing contact and forming a highly packed mass. This process of increased adhesion and embryo compaction occurs ubiquitously during preimplantation development in different mammalian species and is an absolute requirement for embryo viability. This process is very similar in mouse and humans, making the preimplantation mouse embryo an ideal model to study the role of adhesion in cell shape, position, and fate in early mammalian development. In addition, the cells of the mouse embryo are relatively large, facilitating imaging of subcellular processes. Furthermore, there are many available genetic tools that are applicable in the mouse for manipulation of proteins of interest. Pronuclear microinjection of mRNA or DNA is a well-established technique for expression of exogenous proteins and mouse embryos are resilient enough to withstand this process with high efficiency (Fig. 1B). Moreover, thousands of genetically modified animals are now available carrying targeted endogenous genes or expressing various transgenic constructs.

1.1 Adhesion molecules in the preimplantation mouse embryo

Early studies identified a critical role for calcium-dependent adhesion in embryo compaction, subsequent spatial segregation of the inner cell mass (ICM) and formation of the first differentiated tissue, the trophectoderm (Fleming, Sheth, & Fesenko, 2001). Interfering with adhesion by chelating calcium ions or using antibodies targeting a cell surface glycoprotein decompacted embryos and prevented blastocyst formation (Ducibella & Anderson, 1975; Wales, 1970; Whitten, 1971). In 1981, this cell surface glycoprotein was identified as uvomorulin, now more commonly known as E-cadherin (Hyafil, Babinet, & Jacob, 1981).

Although usually expressed in epithelial cell layers, E-cadherin is also expressed from the very early stages of development. It is initially maternally derived in the oocyte and at the 2-cell stage, de novo E-cadherin zygotic synthesis starts (Vestweber, Gossler, Boller, & Kemler, 1987). Embryos lacking zygotic E-cadherin are preimplantation lethal. They do undergo compaction due to residual maternal E-cadherin but fail to form a blastocyst (Larue, Ohsugi, Hirchenhain, & Kemler, 1994). Using siRNAs to knockdown E-cadherin expression in just half of the embryo prevents those cells from integrating into the compacting embryo (Fig. 2) (Fierro-Gonzalez, White, Silva, & Plachta, 2013). Embryos lacking both maternal and zygotic E-cadherin are unable to compact or form a blastocyst and they appear as loose aggregates of cells (Stephenson, Yamanaka, & Rossant, 2010). Deleting maternal E-cadherin alone delays compaction until the late morula stage but embryos then develop normally due to zygotic E-cadherin expression (De Vries et al., 2004). Adhesion does not develop until the late morula stage in these embryos indicating that although E-cadherin and its binding partners are expressed (Ohsugi et al., 1996; Sefton, Johnson, & Clayton, 1992; Vestweber et al., 1987), they are not required to form functional adhesion complexes at very early stages.

E-cadherin is uniformly distributed in the cell membrane until the 8-cell stage when PKC-α-mediated phosphorylation of β-catenin, a key protein regulating E-cadherin intracellular signaling, is thought to activate the compaction process (Fig. 3; Pauken & Capco, 1999). Compaction can be blocked by inhibition of PKC-α and induced early at the 2- and 4-cell stages by PKC-α activation (Ohsugi, Ohsawa, & Semba, 1993; Winkel, Ferguson, Takeichi, & Nuccitelli, 1990). E-cadherin accumulates basolaterally, forming adherens junctions between cells and connecting to the actin cytoskeleton via catenin proteins (Ozawa, Ringwald, & Kemler, 1990). As adhesion initiates, the actin cytoskeleton is reorganized to define the orientation of the first cellular polarity in the embryo (Stephenson et al., 2010).

After compaction has occurred, tight junctions begin to assemble at apicolateral cell–cell junctions in a process that requires prior activation of E-cadherin-mediated adhesion (Fleming, McConnell, Johnson, & Stevenson, 1989; Ohsugi, Larue, Schwarz, & Kemler, 1997). The timing of tight junction formation is tightly regulated by staggered expression of the constituent proteins from the 8- to 32-cell stage (Sheth et al., 1997). E-cadherin-mediated adhesion may also stabilize tight junction proteins, preventing their turnover once they are assembled at the membrane (Javed, Fleming, Hay, & Citi, 1993). The close intercellular adhesion at tight junctions then forms a permeability seal between...