At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

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the midbrain and they are involved in multiple important brain functions. However, the mechanisms of GABAergic neurogenesis in the midbrain are not well understood. Previously, it has been shown that Ascl1 is required for development of GABAergic neurons in the midbrain. Here we have characterized in more detail the role of Ascl1 in the development of distinct subpopulations of midbrain GABAergic neurons. In Ascl1-null mutants GABAergic neurons are completely absent in the dorsal part of the midbrain.Ventrally part of the cells differentiate but the amount of GABAergic neurons is reduced.Tracing back to the origin of these defects we show that Ascl1 is regulating differently both GABAergic neurogenesis and neuronal subtype specification in distinct subpopulations of the ventral midbrain. We are currently conducting additional studies to further specify the ventral GABAergic populations that are affected in the Ascl1 mutants and to examine the downstream differentiation factors involved in the GABAergic neurogenesis.Results towards understanding the molecular and developmental mechanisms regulated by Ascl1 will be presented. Noggin is one of the secreted antagonists of Bone Morphogenetic Proteins. Interference with the function of Noggin in mice results in a series of patterning and developmental defects, like an increased chondrogenesis, a deregulated ossification, exencephaly and a degeneration of the tail. Additionally, muscles at 18.5 dpc are characterized by a reduction in size and an abnormal organization of nuclei in the myofibres. To determine the molecular basis of this phenotype we carried analysis of muscles from Noggin / mice at different stages of development. We show that the absence of noggin results in the increased BMP signaling in the muscle tissue and induction of BMP target genes associated with inhibition of myogenesis. These data therefore demonstrate that during limb development a balance between BMP and Noggin is required to maintain muscle development. The enteric nervous system (ENS) comprises of a network of interconnected ganglia that control its motility, secretion and microcirculation. This composition of a ''homogeneous-looking'' neuronal population makes the tracing and study of individual classes of neurons a daunting task. Therefore, the development of tools that will allow the identification and study of individual enteric neurons and their processes is fundamental for uncovering the logic that governs enteric neuronal networks. Ret, a receptor tyrosine kinase has been designated as a critical molecule in ENS development. Despite extensive analysis of the developmental role of this signalling pathway, key questions relating to the invivo role of Ret in neuronal differentiation, connectivity and gangliogenesis remain unanswered. We will be reporting on the use of a novel approach, called Mosaic Analysis with Double Markers (MADM) which we have adopted for our combined analysis of morphology and genetics of individual enteric neural crest cells/neurons. This approach requires the s...