09.10.13 1 CHAPTER 13-2, NEURONAL SPECIFICATION AND AXONAL SPECIFICITY ZOO3603C Diagram of a motor neuron Dendrites --- pick up electric impulses from other cells Axon --- extend for several feet Synapses 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. Induction and patterning of a neuron-formation (neurogenic) region. 2. Birth and migration of neurons and glia. 3. Specification of the cell fates. 4. Guidance of axonal growth cones to specific targets. Goodman & Doe list 8 stages of neurogenesis 5. Formation of synaptic connections. 6. Binding of trophic factors for survival and differentiation. 7. Competitive rearrangement of functional synapses. 8. Continued synaptic plasticity during the organism’s lifetime The Generation of Neuronal Diversity 09.10.13 2 Specification of neurons in a hierarchicalmanner 1. Whether a ectodermal cell will become a neuron, a neural crest cell, or epidermis. 2. What type of neuron? Motor neuron, sensory neuron, commissural neuron or some other type? 3. What is a specific target of the neuron? Motor neuron organization and Lim specification in the spinal cord • Column of Terni projects into ventral sympathetic neuron. • Lateral motor column projects into the muscle. In Thoracic If Lim3 is expressed, it projects dorsally into the axial muscle. Motor neuron organization and Lim specification in the spinal cord • Medial motor column projects into axial muscle. • Lateral motor column projects into the limb muscle. In Hindlimb If Lim3 is expressed, it projects dorsally into the axial 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 The targets of these motor neurons are specified before their axons extend into the periphery. At 6 days old embryo, neurons were found at their proper pathway. 09.10.13 3 Pattern Generation in the Nervous System Pattern Generation in the Nervous System Three steps specification of axonal connections 1. Pathway selection  Travel along a route 2. Target selection  Recognize and bind to a set of the cells 3. Address selection  Initial patterns are refined Motor neurons can establish their normal patterns of innervation in the absence of neuronal activity. Independent Independent Several neurons 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 09.10.13 4 Repulsion of dorsal root ganglion growth cones Anterior Posterior In vitro assay on ephrin stripes 10 minutes exposure to ephrin In vivo motor axons migration The growth corn contains Eph receptor and neuropilin receptors that are responsive to ephrin and semaphorin on the posterior sclerotome cells. 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 Growth inhibition No inhibition NT3-responsive neurons + Semaphorin-3 • Netrinsandotherreceptors • Slitandrobo Guidance by diffusible molecules 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 09.10.13 5 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 Floor plate Dorsal Spinal Cord COS cells Outgrowth of commissural axons No Outgrowth Netrins elicit axon outgrowth of commissural neurons Dorsal Spinal Cord COS cells with netrin-1 Dorsal Spinal Cord COS cells with netrin-2 Outgrowth of commissural axons induced by netrin UNC expression and function in axonal guidance 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. 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. Netrins inhibit the outgrowth of trochlear axons from explants of the mouse brain stem Netrins and UNC-6 appear to be chemotactic to some neurons and chemorepulsive to others. Dorsal spinal cord, no inhibition Chick fibroblast cells, no inhibition Chick fibroblast cells with netrin-1, no outgrowth Ventral floor plate, no outgrowth 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 Along the neurons of the longitudinal tracts of the CNS axon scaffold. Antibody to all CNS neurons Slit KO, failing to leave the midline Robo/Slit regulation of midline crossing by neurons Netrin: attractive Slit: repulsive 09.10.13 6 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)09.10.13 7 13.29 Differentiation of a motor neuron synapse with a muscle (Part 2) Address selection by competition