ABSTRACT
Neural cell adhesion molecule close homolog of L1 (CHL1) is a regulator of topographic targeting of thalamic axons to the somatosensory cortex (S1) but little is known about its cooperation with other L1 class molecules. To investigate this, CHL1(-/-)/L1(-/y) double mutant mice were generated and analyzed for thalamocortical axon topography. Double mutants exhibited a striking posterior shift of axons from motor thalamic nuclei to the visual cortex (V1), which was not observed in single mutants. In wild-type (WT) embryos, L1 and CHL1 were coexpressed in the dorsal thalamus (DT) and on fibers along the thalamocortical projection in the ventral telencephalon and cortex. L1 and CHL1 colocalized on growth cones and neurites of cortical and thalamic neurons in culture. Growth cone collapse assays with WT and mutant neurons demonstrated a requirement for L1 and CHL1 in repellent responses to EphrinA5, a guidance factor for thalamic axons. L1 coimmunoprecipitated with the principal EphrinA5 receptors expressed in the DT (EphA3, EphA4, and EphA7), whereas CHL1 associated selectively with EphA7. These results implicate a novel mechanism in which L1 and CHL1 interact with individual EphA receptors and cooperate to guide subpopulations of thalamic axons to distinct neocortical areas essential for thalamocortical connectivity.
Subject(s)
Axons/physiology , Cell Adhesion Molecules/metabolism , Cerebral Cortex/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Neural Pathways/physiology , Thalamus/metabolism , Amino Acids/metabolism , Animals , Axons/drug effects , Axons/ultrastructure , Cell Adhesion Molecules/deficiency , Cells, Cultured , Cerebral Cortex/cytology , Embryo, Mammalian , Ephrin-A5/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Growth Cones/physiology , Humans , Immunoprecipitation/methods , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Cell Adhesion Molecule L1/deficiency , Neurons/cytology , Neurons/drug effects , Neurons/physiology , Receptors, Eph Family/genetics , Receptors, Eph Family/metabolism , Thalamus/cytology , Transfection/methodsABSTRACT
Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin-dependent neurite outgrowth and semaphorin3A (Sema3A)-mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin-radixin-moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane-proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A-induced growth cone collapse and CHL1-dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A-induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.
Subject(s)
Cell Adhesion Molecules/physiology , Cell Membrane/metabolism , DNA-Binding Proteins/metabolism , Growth Cones/drug effects , Neurites/physiology , Neurons/cytology , Neurons/metabolism , Semaphorin-3A/pharmacology , Transcription Factors/metabolism , Animals , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Adhesion Molecules/genetics , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Cerebral Cortex , Cytoskeletal Proteins/metabolism , Embryo, Mammalian , Fibronectins/physiology , Green Fluorescent Proteins/metabolism , Growth Cones/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurites/chemistry , Rats , Transfection/methodsABSTRACT
We report a cooperation between the neural adhesion molecule close homolog of L1 (CHL1) and the semaphorin 3A (Sema3A) receptor, neuropilin 1 (Npn1), important for establishment of area-specific thalamocortical projections. CHL1 deletion in mice selectively disrupted the projection of somatosensory thalamic axons from the ventrobasal (VB) nuclei, causing them to shift caudally and target the visual cortex. At the ventral telencephalon, an intermediate target with graded Sema3A expression, VB axons were caudally shifted in CHL1- embryos and in Npn1(Sema-/-) mutants, in which axons are nonresponsive to Sema3A. CHL1 colocalized with Npn1 on thalamic axons, and associated with Npn1 through a sequence in the CHL1 Ig1 domain that was required for Sema3A-induced growth cone collapse. These results identify a novel function for CHL1 in thalamic axon responsiveness to ventral telencephalic cues, and demonstrate a role for CHL1 and Npn1 in establishment of proper targeting of specific thalamocortical projections.
