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Growth and Survival Signals Controlling Peripheral Nervous System Development
Summary: David Ginty is interested in the molecular control of differentiation, growth, and survival of neurons in the developing vertebrate 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 establish the principles governing development of the vertebrate nervous system.
Retrograde Signaling and Target Field Innervation
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 peripheral nervous system (PNS) neurons. How does NGF signaling control target innervation? We recently discovered that retrograde 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 no deficits in neuronal viability or differentiation but causes dramatic 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.
We now aim to identify and characterize the target genes controlled by SRF and other NGF-regulated transcription factors in sensory neurons. One such NGF-regulated gene encodes the tyrosine kinase Ret, which is the signaling subunit of the GDNF holoreceptor complex. Our analysis shows that NGF-dependent expression of Ret is critical for maturation, hypertrophy, and epidermal penetration by axons of a subset of cutaneous sensory neurons known as nonpeptidergic nociceptors. These findings highlight a fundamental principle in developmental neurobiology: Target fields support neuronal maturation and the establishment of connectivity through retrograde signaling and transcriptional activation of a large set of axonal growth and branching genes. A major current goal of our laboratory is the characterization of the functions of NGF-regulated genes, including SRF target genes, in the control of neuronal maturation, survival, and connectivity.
Retrograde Signaling and Neuronal Survival
How does retrograde NGF signaling control neuronal survival during the period of developmental competition when approximately half of these neurons die? We recently discovered 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), which, through the receptor p75, kill neighboring neurons with low retrograde NGF-TrkA signaling; 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, before 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, which 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.
Other complementary studies address 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 will enable 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.
Chemical Genetics and Neuronal Growth Factor Signaling in Adult Neurons
To address the adult functions of neurotrophic growth factors and their receptors, we have developed a powerful chemical-mouse genetic strategy for manipulating the activities of the neurotrophin receptor tyrosine kinases. 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 recent 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.
Forward Genetics and PNS Axon Development
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 identify novel ligand-receptor complexes and components of the intracellular machinery that control sensory, sympathetic, and motor 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 several uncharacterized genes that control specific steps in the establishment of PNS connectivity. Some of these genes control central (spinal cord) axonal projections of sensory neurons, a developmental process for which we know little.
In collaboration with Alex Kolodkin (HHMI, Johns Hopkins University) and Carlos Lois (Massachusetts Institute of Technology), we are attempting to complement our ENU mutagenesis approach with lentiviral mutagenesis. This lentiviral mutagenesis technique will enable mutagenesis rates that approach saturation and, importantly, it allows for rapid sequence identification of mutated genes responsible for observed phenotypes. Thus, a combination of ENU and lentiviral mutagenesis forward-genetic screens and functional characterization of novel PNS axonal extension, guidance, and connectivity genes both complement and extend our studies on neurotrophin, semaphorin, and growth factor signaling in PNS axons and will 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: August 7, 2008
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