Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.

A dominant molecular explanation for neural induction is the `default model', which proposes that the ectoderm is pre-programmed towards a neural fate, but is normally inhibited by endogenous BMPs. Although there is strong evidence favouring this in Xenopus, data from other organisms suggest more complexity, including an involvement of FGF and modulation of Wnt. However, it is generally believed that these additional signals also act by inhibiting BMPs. We have investigated whether BMP inhibition is necessary and/or sufficient for neural induction. In the chick, misexpression of BMP4 in the prospective neural plate inhibits the expression of definitive neural markers (Sox2 and late Sox3), but does not affect the early expression of Sox3, suggesting that BMP inhibition is required only as a late step during neural induction. Inhibition of BMP signalling by the potent antagonist Smad6, either alone or together with a dominant-negative BMP receptor, Chordin and/or Noggin in competent epiblast is not sufficient to induce expression of Sox2 directly, even in combination with FGF2,FGF3, FGF4 or FGF8 and/or antagonists of Wnt signalling. These results strongly suggest that BMP inhibition is not sufficient for neural induction in the chick embryo. To test this in Xenopus, Smad6 mRNA was injected into the A4 blastomere (which reliably contributes to epidermis but not to neural plate or its border) at the 32-cell stage: expression of neural markers (Sox3 and NCAM) is not induced. We propose that neural induction involves additional signalling events that remain to be identified.

The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.

The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm we show for the first time that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula stage dependent on signals arising from the dorsolateral mesoderm; and second, a maintenance step at the neurula stage dependent on signals from tissues adjacent to the neural crest. By performing tissue recombination experiments and using specific inhibitors of different inductive signals, we show that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula to BMP activation at neurula stages is further supported by the dynamic expression of BMP4 and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The differential requirements of BMP activity allow us to propose an explanation for apparently discrepant results between chick and frog experiments. The demonstration that Wnt signals are required for neural crest induction by mesoderm solves an additional longstanding controversy. Finally, our results emphasise the importance of considering the order of exposure to signals during an inductive event.

There is growing evidence that contact inhibition of locomotion (CIL) is essential for morphogenesis and its failure is thought to be responsible for cancer invasion; however, the molecular bases of this phenomenon are poorly understood. Here we investigate the role of the polarity protein Par3 in CIL during migration of the neural crest, a highly migratory mesenchymal cell type. In epithelial cells, Par3 is localised to the cell-cell adhesion complex and is important in the definition of apicobasal polarity, but the localisation and function of Par3 in mesenchymal cells are not well characterised. We show in Xenopus and zebrafish that Par3 is localised to the cell-cell contact in neural crest cells and is essential for CIL. We demonstrate that the dynamics of microtubules are different in different parts of the cell, with an increase in microtubule catastrophe at the collision site during CIL. Par3 loss-of-function affects neural crest migration by reducing microtubule catastrophe at the site of cell-cell contact and abrogating CIL. Furthermore, Par3 promotes microtubule catastrophe by inhibiting the Rac-GEF Trio, as double inhibition of Par3 and Trio restores microtubule catastrophe at the cell contact and rescues CIL and neural crest migration. Our results demonstrate a novel role of Par3 during neural crest migration, which is likely to be conserved in other processes that involve CIL such as cancer invasion or cell dispersion.