CHAPTER 13 2 NEURONAL SPECIFICATION AND AXONAL SPECIFICITY ZOO3603C Diagram of a motor neuron Synapses Dendrites pick up electric impulses from other cells Axon extend for several feet Connections of axons to a cultured rat hippocampal neuron Red Tubulin indicating dendrites Green Synapsin indicating the synapse In human brain interacting with thousands of other neurons Goodman Doe list 8 stages of neurogenesis 1 2 3 4 Induction and patterning of a neuron formation neurogenic region Birth and migration of neurons and glia Specification of the cell fates Guidance of axonal growth cones to specific targets Goodman Doe list 8 stages of neurogenesis 5 6 7 8 Formation of synaptic connections Binding of trophic factors for survival and differentiation Competitive rearrangement of functional synapses Continued synaptic plasticity during the organism s lifetime The Generation of Neuronal Diversity Specification of neurons in a hierarchical manner 1 2 3 Whether a ectodermal cell will become a neuron a neural crest cell or epidermis What type of neuron Motor neuron sensory neuron commissural neuron or some other type What is a specific target of the neuron Motor neuron organization and Lim specification in the spinal cord In Thoracic If Lim3 is expressed it projects dorsally into the axial muscle Column of Terni projects into ventral sympathetic neuron Lateral motor column projects into the muscle Motor neuron organization and Lim specification in the spinal cord In Hindlimb If Lim3 is expressed it projects dorsally into the axial muscle Medial motor column projects into axial muscle Lateral motor column projects into the limb muscle Lim transcription factors Lim transcription factors probably coordinate the projections of the motor neurons by specifying ephrin A receptors Compensation for small dislocations of axonal initiation position in the chick embryo 7th thoracic to 3rd lumbo sacral segment was reversed in 2 5 day chick embryo Compensation for small dislocations of axonal initiation position in the chick embryo At 6 days old embryo neurons were found at their proper pathway The targets of these motor neurons are specified before their axons extend into the periphery Pattern Generation in the Nervous System Pattern Generation in the Nervous System Three steps specification of axonal connections 1 Pathway selection 2 Independent Target selection 3 Travel along a route Recognize and bind to a set of the cells Independent Address selection Initial patterns are refined Several neurons Motor neurons can establish their normal patterns of innervation in the absence of neuronal activity Cell adhesion and contact guidance by attractive and permissive molecules Growth Cone Migration Outgrowth of sensory neurons Outgrowth of sensory neurons placed on a patterned substrate consisting of parallel stripes of laminin applied to a background of type IV collagen Many of the roads appear to be paved with the glycoprotein laminin Guidance by specific growth cone repulsion Repulsion of dorsal root ganglion growth cones In vivo motor axons migration In vitro assay on ephrin stripes Anterior Posterior The growth corn contains Eph receptor and neuropilin receptors that are responsive to ephrin and semaphorin on the posterior sclerotome cells 10 minutes exposure to ephrin The action of semaphorin 1 in the developing grasshopper limb Semaphorins force turn Semaphorin 3 as a selective inhibitor of axonal projections into the ventral spinal cord Semaphorin 3 as a selective inhibitor of axonal projections into the ventral spinal cord Selective response to semaphorin 3 Mechanoreceptor neurons Semaphorin 3 NT3 responsive neurons Semaphorin 3 Growth inhibition No inhibition Guidance by diffusible molecules Netrins and other receptors Slit and robo Trajectory of the commissural axons in the rat spinal cord Both Shh Netrin 1 synthesized in floor plate Netrin 1 mRNA localize on floor plate Netrins elicit axon outgrowth of commissural neurons Transformed chick fibroblast cells secreting netrins elicit axon outgrowth of commissural neuronsfrom 11 day embryonic rat dorsal spinal cord explants Dorsal Spinal Cord Dorsal Spinal Cord Floor plate COS cells Outgrowth of commissural axons No Outgrowth Netrins elicit axon outgrowth of commissural neurons Dorsal Spinal Cord Dorsal Spinal Cord COS cells with netrin 1 COS cells with netrin 2 Outgrowth of commissural axons induced by netrin UNC expression and function in axonal guidance UNC 6 induces the migrations No UNC 6 causes no migrations in both neurons No UNC 5 causes no migrations in motor neurons No UNC 40 causes no migrations in sensory neurons UNC 5 and UNC 40 are portions of the UNC 6 receptor complex and that UNC 5 can convert a UNC 40 mediated attraction into are repulsion Netrins inhibit the outgrowth of trochlear axons from explants of the mouse brain stem Dorsal spinal cord no inhibition Chick fibroblast cells no inhibition Chick fibroblast cells with netrin 1 no outgrowth Ventral floor plate no outgrowth Netrins and UNC 6 appear to be chemotactic to some neurons and chemorepulsive to others Robo Slit regulation of midline crossing by neurons Part 1 Robo roundabout is the receptor for Slit preventing most neurons from crossing the midline Positive in midline Antibody to all CNS neurons Along the neurons of the longitudinal tracts of the CNS axon scaffold Slit KO failing to leave the midline Robo Slit regulation of midline crossing by neurons Netrin attractive Slit repulsive Robo Slit regulation of midline crossing by neurons Part 3 Netrin attractive Slit repulsive To cross the midline Robo3 block Robo1 13 27 Robo Slit regulation of midline crossing by neurons Part 4 Target selection Embryonic axon from a rat dorsal root ganglion turning in response to a source of NT3 Neurotropins Nerve growth factor NGF Brain derived neurotrophic factor BDNF Neurotrophin 3 Neurotrophin 4 5 Response to a source of NT3 Forming the synapse Activity dependent development 13 29 Differentiation of a motor neuron synapse with a muscle Part 1 13 29 Differentiation of a motor neuron synapse with a muscle Part 2 Address selection by competition One of these axon branches are retracted Myelination by Schwann cells Activity dependent development Differential survival after innervation Neurotrophic factors Effects of NGF and BDNF on axonal outgrowths of different ganglia Over half the neurons die during the normal course of development Neurotropins chemotrophic factors but also regulate the