Role of the E- to N-cadherin switch in the neural differentiation of embryonic stem cells

Abstract

During early embryonic development in mammals, the initially uniform cells of the naïve epiblast generate the structurally diverse and functionally specific tissues of the adult organism. The naïve epiblast expresses the cell-cell adhesion molecule epithelial (E-) cadherin, which is essential for early embryonic development. During subsequent neural differentiation in the ectoderm, E-cadherin becomes downregulated, and neuronal (N-) cadherin becomes upregulated in a process known as cadherin switching. Premature loss of E-cadherin activity leads to faster, more synchronous neural differentiation in embryonic stem cells by an unknown mechanism (Malaguti et al., 2013). Cadherins are mainly classed as adhesion molecules, but can also modulate cellular signalling by binding various growth factor receptors. This raises the possibility that cadherin switching may modulate signalling during early neural induction, thus affecting differentiation. This thesis investigates the mechanisms by which cadherin switching can bias differentiation decisions during neural induction in mouse embryonic stem (ES) cells . First, the spatio-temporal patterns of cadherins were investigated in the post-implantation embryo and during differentiation of pluripotent cells in vitro. The results of this analysis suggest that cadherin switching is initiated before the loss of pluripotency and co-occurs with lineage priming, consistent with the hypothesis that this process may bias cell fate decisions. Next, ES cells in which the N-cadherin gene was knocked in to the E-cadherin locus (NcKI) were studied. These cells displayed elevated levels of neural marker genes during differentiation, suggesting that forced, premature cadherin switching promotes neural differentiation. To determine whether this effect could be ascribed to the loss of E-cadherin or to the gain of N-cadherin, an inducible N-cadherin overexpressing ESC line was generated. Experiments with this cell line confirmed that N-cadherin alone can promote neural fate. Cadherin switching also occurs at a later developmental stage: during the maturation of bi-potent neuro-mesodermal progenitors (NMPs). Premature induction of N-cadherin was shown to bias immature NMPs towards neural differentiation, similarly to the effect of N-cadherin on pluripotent cells. To establish whether the pro-neural effect of N-cadherin was due to its adhesive function, a quantitative image analysis method was used to measure adhesion defects. Adhesion phenotypes did not correlate with the pro-neural effect observed in NcKI and N-cadherin overexpressing cells, suggesting that adhesion may not be the primary cause for the neural bias of these cells. Finally, the signalling effects of cadherin switching were studied. The loss of E-cadherin in E-cadherin null (EcKO) and NcKI cells was found to correlate with a drop in global and nuclear levels of β-catenin, a central component of the canonical WNT signalling pathway. However, these cells were found to retain WNT responsiveness, suggesting that any neural bias shown during cadherin switching is unlikely to be caused by altered WNT signalling. An mRNA-based signalling pathway analysis showed that cells overexpressing N-cadherin had reduced levels of FGF signalling cascade components at days three and four of neural differentiation. Subsequent experiments in NcKI cells showed that these cells express lower levels of FGF readouts during neural differentiation than WT cells, and that their pro-neural effect can be erased by adding FGF pathway ligands, or replicated by blocking the FGF pathway. These results suggest that the pro-neural effect observed in NcKI cells is caused by dampened FGF responsiveness. Taken together, the results presented in this thesis support a model in which N-cadherin promotes neural differentiation by dampening FGF signalling. This mechanism, rather than being an instructive cue for neural induction, is likely to contribute to the robustness of neural differentiation in the developing embryo.