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Development of Mammalian Peripheral Nervous System Connectivity and Function
Summary: David Ginty is interested in the mechanisms of development and unique functions of neurons in the mammalian peripheral nervous system.
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 proper connectivity. The goals of my laboratory are to identify key molecular events underlying neuronal growth and survival and to establish the principles governing the assembly of mammalian peripheral nervous system (PNS) circuits.
Functions and Mechanisms of Neurotrophic Growth Factor Signaling During Development of the PNS
Upon innervation of target fields, the target-derived growth factor, nerve growth factor (NGF), and its receptor tyrosine kinase TrkA support growth, maturation, and survival of certain subsets of PNS neurons. We recently discovered that retrograde NGF signaling from distal axons to neuronal cell bodies and activation of the transcription factor SRF are essential for axonal growth, branching, and target innervation by embryonic dorsal root ganglia (DRG) sensory neurons. Conditional deletion of the SRF gene in DRG sensory neurons results in defects in extension and arborization of axonal projections into the target field, a phenotype also seen in mice lacking NGF. Consistent with this finding, SRF is necessary and sufficient for NGF-dependent axonal outgrowth in vitro, and NGF is essential for the expression of several SRF-dependent cytoskeletal genes in embryonic DRG neurons. Thus, SRF is a transcriptional mediator of retrograde NGF signaling events that support axonal development and target innervation. Other recent studies have revealed that retrograde NGF signaling also controls the formation of synapses between preganglionic neurons emanating from the spinal cord and postganglionic sympathetic neurons in a manner independent of new gene transcription. Thus, target-derived growth factors signal retrogradely to control transcription-dependent and -independent events underlying circuit formation.
How does retrograde NGF signaling control neuronal survival during the period of developmental competition when approximately half of these neurons die? We found that developmental competition between sympathetic neurons is dependent on a sensitization process initiated by target innervation and mediated by retrograde signaling and a series of transcription-dependent feedback loops. Target-derived NGF promotes expression of its own receptor TrkA in neurons and prolongs TrkA-mediated signal transduction. NGF also promotes expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4). BDNF and NT4 then act through the receptor p75 to kill neighboring neurons with low retrograde NGF–TrkA signaling, while neurons with high NGF–TrkA signaling are protected. In collaboration with Stefan Mihalas and Ernst Niebur (Johns Hopkins University), we used computational modeling to gain insight into the roles of these NGF-dependent gene expression events during competition. Our experimental observations and results from computer simulations support a model in which, prior to target innervation, sympathetic neurons are modestly responsive to NGF because of low TrkA levels. Upon target innervation, neurons acquire NGF, and subtle differences in initial amounts of NGF signaling are amplified through transcription-dependent feedback loops into large cell-autonomous differences in both strength and duration of TrkA signaling; these differences ultimately determine whether a neuron lives or dies. Furthermore, expeditious competition requires target-innervation-dependent expression of punishment cues (BDNF, NT4), susceptibility to punishment (expression of p75), and protection from punishment signals (strong retrograde NGF-TrkA signaling). Thus, retrograde control of gene expression enables neuronal competition that is rapid, robust, and stable, even in a scenario in which all neurons arrive at their target simultaneously and are virtually equivalent in their initial responsiveness to target-derived NGF. These findings highlight a fundamental principle in developmental neurobiology: target field–derived growth factors support the establishment of PNS circuits through retrograde signaling and transcriptional activation of a large set of axonal growth and branching genes, as well as genes that promote survival and neuronal competition.
A current goal of our laboratory is the characterization of the functions of NGF-regulated genes in the control of neuronal maturation, survival, and connectivity. Complementary studies ask how the NGF/TrkA-signaling endosome, which is formed at distal axons, controls retrograde signaling to neuronal cell bodies of sympathetic and sensory neurons. Our proteomic and functional analyses of the molecular components of the retrograde NGF/TrkA endosome are allowing us to delineate the mechanisms by which this signaling vesicle is formed, sorted, trafficked, and functions to control gene expression events that coordinate axonal extension and branching, neuronal competition, and circuit formation.
Neuronal Growth Factors and the Development of Mechanosensory Circuits
Current challenges include the identification and functional characterization of distinct populations of PNS neurons and the elucidation of the developmental steps that establish their unique synaptic connections. Our studies of the glial cell–derived neurotrophic factor (GDNF) family of neuronal growth factors have begun to shed light on the molecular properties and mechanisms of development of specific classes of mechanosensory neurons that mediate discriminative touch and the perception of form, texture, vibration, and pressure.
We have discovered that a small population of DRG neurons expressing the 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. 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 the elusive RA mechanoreceptors and require Ret signaling for the assembly of neural circuits underlying discriminative touch perception. We are now using mouse genetic, physiological, and behavioral strategies to elucidate the unique functions of these RA mechanoreceptors and the related slowly adapting mechanoreceptors during tactile discrimination, object recognition, and the perception of touch.
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.
Using the Trk knockin mice and the chemical-genetic strategy, in collaboration with John Griffin (Johns Hopkins University), we found 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. 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 in 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 several uncharacterized genes that control specific steps in the development of the spinal cord 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, a developmental process that remains poorly understood.
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 define new areas of study for the field of neural development.
This research is also supported by grants from the National Institutes of Health.
Last updated September 17, 2009
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