Specifying neurons and circuits for limb motor control Alana I. Mendelsohn Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2016 ©2016 Alana I. Mendelsohn All rights reserved Abstract Specifying neurons and circuits for limb motor control Alana I. Mendelsohn The emergence of limbs in vertebrates represents a significant evolutionary innovation. Limbs facilitate diverse motor behaviors, yet require spinal networks that can coordinate the activities of many individual muscles within the limb. Here I describe several efforts to characterize the specification of spinal motor neurons and assembly of spinal circuits in higher vertebrates. I discuss the formation of selective presynaptic sensory inputs to motor pools, a process which has long been thought to occur in an activity-independent manner. I demonstrate an as yet unappreciated role of activity-dependent refinement in patterning the set of sensory-motor connections that link motor pools with synergist function. I also explore the genetic specification of motor pools that project to defined muscle targets. I show that the motor pools that control digits engage distinct developmental genetic programs which reflect underlying differences in Hox and retinoic acid signaling. The divergent mechanisms underlying the specification of digit- innervating motor neurons may reflect the unique status of digit control in the evolution of motor behavior. Table of contents List of figures .......................................................................................................................... v 1 Motor pools underlie limb-based motor behavior.............................................................. 1 Organization of motor neuron subtypes in tetrapods .............................................................. 2 Coordination of motor pool output during locomotion........................................................... 7 Organization of sensory inputs to motor pools ....................................................................... 9 Sensory-motor circuit assembly ............................................................................................ 14 Activity-dependent refinement in sensory-motor circuit development ................................ 18 Specification of motor pools to distinct muscle targets ........................................................ 20 Motor system evolution for coordinated behavior ................................................................ 28 Common mechanisms underlie limb and spinal patterning .................................................. 33 Distinguishing aspects of digit development ........................................................................ 36 2 Proprioception organized by limb biomechanics ............................................................. 39 2.1 Introduction .................................................................................................................... 39 2.2 Results ............................................................................................................................ 40 Defining patterns of heteronymous sensory-motor connectivity .......................................... 40 Functional validation of heteronymous connection specificity and density ......................... 46 2.3 Discussion ...................................................................................................................... 51 3 Activity regulates the incidence of heteronymous connections....................................... 54 3.1 Introduction .................................................................................................................... 54 3.2 Results ............................................................................................................................ 55 Motor pool position is unlikely to determine patterns of heteronymous connectivity ......... 55 Tetanus toxin expression in proprioceptors blocks sensory-motor transmission ................. 56 i Increased incidence of heteronymous sensory-motor connections in PvTeNT mice ............... 64 Sensory-motor spike timing may explain synaptic refinement............................................. 69 3.3 Discussion ...................................................................................................................... 73 Sensory afferent activity and the refinement of synaptic density ......................................... 73 Limitations of sensory blockade in shaping patterns of sensory-motor connectivity ........... 76 4 Genetic profiling of digit-innervating motor neurons ..................................................... 78 4.1 Introduction .................................................................................................................... 78 4.2 Results ............................................................................................................................ 78 Genetic profiling of biomechanically distinct motor pools .................................................. 78 Confirmation of specific markers of intrinsic hand and foot motor neurons ........................ 85 4.3 Discussion ...................................................................................................................... 93 Gene expression within the intrinsic digit-innervating motor cohort ................................... 93 Functional relevance of genes identified in digit-innervating motor neurons ...................... 96 Linking intrinsic hand and foot motor neuron specification ................................................. 97 5 Divergent signaling pathways in digit-innervating motor neurons ................................ 99 5.1 Introduction .................................................................................................................... 99 Retinoic acid in motor neuron specification ......................................................................... 99 Mechanisms of retinoid acid signaling ............................................................................... 102 BMP signaling .................................................................................................................... 105 5.2 Results .......................................................................................................................... 108 Digit-innervating motor neurons do not make retinoic acid ............................................... 108 Intrinsic hand and foot motor neurons engage limb-dependent BMP signaling ................ 116 5.3 Discussion .................................................................................................................... 123 Intrinsic and extrinsic factors in patterning digit-innervating motor neurons .................... 123 Do digit-innervating motor neurons engage in retinoid signaling? .................................... 126 ii 6 Hox and retinoid contribution to digit motor neuron identity ..................................... 130 6.1 Introduction .................................................................................................................. 130 Retinoic acid and Hox signaling are interdependent .......................................................... 130 Hox genes coordinate the specification of motor neuron subtypes..................................... 132 6.2 Results .......................................................................................................................... 136 Hoxc8 and Hoxc9 coexpression is sufficient to induce digit-innervating motor neurons at brachial levels ..................................................................................................................... 136 Retinoid induction disrupts digit-innervating motor neurons ............................................. 145 6.3 Discussion .................................................................................................................... 153 A model of Hox and retinoid specification of brachial digit-innervating motor neurons .. 153 Additional mechanisms involved in digit-innervating motor neuron development ........... 158 7 General discussion ............................................................................................................ 163 7.1 Hierarchical mechanisms in sensory-motor circuit assembly ...................................... 164 7.2 Divergent mechanisms in fine motor circuit assembly ................................................ 168 7.3 Future directions ........................................................................................................... 171 Synaptic specificity in sensory-motor circuits .................................................................... 171 Motor system neurobiology in evolutionary context .......................................................... 173 Spinal interneuron and descending circuits for fine motor control..................................... 178 7.4 Conclusions .................................................................................................................. 187 8 Experimental procedures ................................................................................................. 189 Mouse strains ...................................................................................................................... 189 Immunohistochemistry ......................................................................................................