Development of Connectivity and Function of the Mammalian Peripheral Nervous System
Summary: David Ginty's research addresses mechanisms of assembly and function of peripheral nervous system circuits that underlie somatosensation and autonomic control of the body.
The function of the adult nervous system is dependent upon trillions of neural connections. Not only must the organism generate neuronal numbers appropriate for the needs of targets being innervated, but it must also instruct these neurons to extend axons, elaborate dendrites, and generate synapses to establish functional circuits. The goals of my laboratory are to identify key molecular events underlying neuronal growth and survival and to establish the principles governing development of the vertebrate peripheral nervous system (PNS).
Functions and Mechanisms of Neurotrophic Growth Factor Signaling During Development of the PNS
Overproduction of neurons followed by neuronal loss commensurate with circuit requirements is a basic tenet of neural development. What determines whether a neuron lives or dies, and how is neuronal elimination initiated and executed in an expeditious yet precise manner? We found that developmental competition among sympathetic neurons for survival is initiated by target field innervation and retrograde nerve growth factor (NGF) signaling, and it is critically dependent on feedback loops involving sensitization to NGF signaling, paracrine apoptotic signaling, and protection from paracrine apoptotic signals.
More recently, we discovered that retrograde NGF signaling and a balance between the activities of these same prosurvival and prodeath signaling pathways also determine the amount of synaptic connectivity between sympathetic neurons and their presynaptic partners. Thus, target field innervation triggers rapid and robust competition for both survival and synapse formation through long-range retrograde signaling, the initiation of feedback loops, and a balance of the activities of prosurvival/growth and prodeath/disassembly signaling pathways.
Our discovery that the TrkA signaling endosome is a key mediator of retrograde signaling has brought endosomal signaling mechanisms to the forefront of the fields of development and growth factor biology. From our work, a number of key questions have emerged: How is the signaling endosome formed, sorted from other endosomes, and transported to the cell body? How does NGF signaling lead to the production of retrogradely transported TrkA signaling endosomes, whereas neurotrophin-3 (NT3), a vascular (intermediate target)-derived axonal growth factor that also activates TrkA signaling in axons of sympathetic neurons, does not? To address these questions, we have focused on defining the molecular composition of the TrkA signaling endosome. Purification of the signaling endosome was achieved through a combination of cell fractionation, sucrose gradient centrifugation, and affinity purification steps. Purified endosomes were then subjected to proteomic analysis using MALDI-TOF mass spectrometry.
Our analysis revealed dozens of novel protein constituents of the TrkA signaling endosome, including several plausibly involved in sorting, trafficking, and signaling. One intriguing theme that has emerged from our proteomic analysis is that signaling endosomes share an intimate relationship with the actin cytoskeleton, and endosome components modulate actin dynamics. Another fascinating observation is that disease-related proteins implicated in Alzheimer's disease and other degenerative disorders function in endosome dynamics and retrograde signaling.
The Development and Function of Somatosensory Neurons
The first step in the perception of discriminative touch is the detection of indentation, vibration or stretch of the skin, and deflection of hairs by specialized cutaneous mechanosensory end organs. We aim to identify and characterize distinct populations of somatosensory neurons and to elucidate the developmental steps that establish their unique synaptic connections underlying discriminative touch and the perception of form, texture, vibration, and pressure.
We have recently found that a small population of DRG (dorsal root ganglion) neurons expressing the glial cell–derived neurotrophic factor (GDNF) receptor tyrosine kinase Ret immediately following neurogenesis (the "early Ret+" DRG neurons) are the elusive, rapidly adapting (RA) mechanoreceptors, which form Meissner corpuscles, Pacinian corpuscles, and longitudinal lanceolate endings in the skin. The central axonal projections of these RA mechanoreceptors terminate within the spinal cord in lamina III through V and within discrete subdomains of the dorsal column nuclei of the medulla. Moreover, mice lacking Ret signaling components are devoid of Pacinian corpuscles and exhibit a dramatic disruption of RA mechanoreceptor central projections to both the dorsal horn of the spinal cord and the medulla. Thus, the early Ret+ neurons are RA mechanoreceptors and require Ret signaling for the assembly of neural circuits underlying discriminative touch perception. We now aim to use a variety of molecular-genetic, physiological, and behavioral strategies to elucidate the unique functions of RA mechanoreceptors and other subsets of neurons, including slowly adapting mechanoreceptors, during tactile discrimination and object recognition.
Chemical Genetics and Neuronal Growth Factor Signaling in Adult PNS Neurons
To address the adult functions of neurotrophic growth factors that control PNS development, we have developed a powerful chemical-genetic strategy for manipulating the activities of the neurotrophin receptor tyrosine kinases in postnatal mice. This strategy, developed in collaboration with Kevan Shokat (HHMI, University of California, San Francisco) and Pamela England (UCSF), allows for rapid, reversible, and specific inhibition of receptor tyrosine kinases in vivo. We generated mice that harbor knockin (single-codon) mutations within exons encoding the ATP-binding pockets of each of the Trk receptors and other receptor tyrosine kinases. The knockin mutations allow Trk receptor signaling to be inhibited by a small-molecular-weight derivative of the kinase inhibitor PP1, 1NMPP1, which is water-soluble and membrane-permeable. 1NMPP1 potently blocks the mutant but not wild-type kinases. These Trk knockin mice enable rapid and reversible inhibition of neurotrophin signaling in vivo during specific windows of time.
Our recent studies, in collaboration with John Griffin (Johns Hopkins University), show that TrkA signaling in adult mice is critical for maintenance of specific PNS axonal connections with select target tissues in skin and that neurotrophins control regeneration of multiple classes of neurons in the injured PNS. Other related findings show that TrkB signaling is essential for maintenance of motor axons in the limb and the integrity of the neuromuscular junction. A principle that emerges from this research is that growth factor systems that control PNS development also act in the adult to maintain connectivity. We are developing an extension of this technology that will enable both spatial and temporal control of growth factor signaling in vivo. This new strategy will allow us to dissect cell-autonomous functions of trophic factor receptor signals at precise times during development and in adulthood.
Forward Genetics and the Assembly of PNS Circuits
We complement our reverse-genetic and chemical-genetic strategies with murine forward-genetic strategies to identify genes that govern the establishment and maintenance of PNS connectivity. Our goal is to uncover novel ligand-receptor complexes and components of the intracellular machinery that control PNS neuronal survival and axonal extension, branching, and innervation of target fields. In a recessive screen, we are analyzing third-generation progeny of N-ethyl-N-nitrosourea (ENU)-mutagenized C57Bl/6 mice that are crossed with C3H/He mice. Whole-mount neurofilament and LacZ staining assays enable high-throughput visualization of peripheral and central axonal projections of sensory neurons of thousands of embryos. We have used this approach to identify novel mutant alleles of both previously known guidance cue genes, including Sema3A and ErbB3, and a large number of uncharacterized genes, including Sec24b, that control specific steps in neural development and the establishment of PNS connectivity. Some of these genes control early events in the formation of peripheral axonal projections of sensory neurons; others control their central (spinal cord) projections, developmental processes of which we know little.
Thus, our forward-genetic screens and functional characterization of novel PNS axonal extension, guidance, and connectivity genes both complement and extend our studies on growth factor signaling during development of PNS neurons and will define new areas of study for the fields of neural development and function.
This research is also supported by grants from the National Institutes of Health.
As of May 30, 2012