In the developing embryo, extracellular signals control many aspects of cellular behavior, such as proliferation, growth or cell-cell interactions. A cell within a developing embryo is bombarded by multitudes of signals, all of which must be integrated to produce a coherent and consistent response. While many signals, their cognate receptors and intracellular signal transduction pathways have been identified, how they work in concert remains a very important biological question. This proposal aims to shed light on this issue by studying how signaling pathways intersect in axonal growth cones, which are guided to their targets by multiple molecular cues laid down at decision steps along their routes. In this proposal, we will study the formation of motor neuron and muscle connections in the limbs, and investigate how individual axons can combine information from signals of multiple families of proteins in a 'synergistic' manner, meaning that the combined effect of different cues is stronger than their individual effects added together. This synergy could involve ligand-dependent interactions between the ephrin and Netrin receptor proteins, which are, respectively, required for the limb trajectory of spinal motor axons, and the activation of a common enzyme. To dissect the underlying mechanism of ephrin and Netrin synergy, we propose the following HYPOTHESIS: The synergistic response of motor axonal growth cones to ephrin-B2 and Netrin-1 (e-N) employs molecular mechanisms that are distinct from those mediating the individual response to ephrin-B2 or Netrin-1. This hypothesis will be test through the following specific aims and experimental approaches:SA1: Gain cellular insights into ephrin-Netrin synergy in spinal motor neurons.1.1 Characterize the response of embryonic stem cells derived motor neurons (ES-MNs) to e-N.1.2 Determine the temporal and spatial dissociation of e-N synergy using ES-MN cultures.1.3 Estimate e-N synergy signal amplification using the growth cone collapse assay.1.4 Investigate the ligand-receptor concentration relationship in e-N synergy using ES-MN cultures1.5 Determine genetic interactions underlying e-N synergy using Unc5c, EphB1, EphB2 and EphB3 mutant mice.SA2: Investigate molecular mechanisms of ephrin-Netrin synergy in spinal motor neurons. 2.1 Determine the effect of Src modulation on e-N synergy using the growth cone collapse assay. 2.2 Determine the effect of cAMP modulation on e-N synergy using the growth cone collapse assay. 2.3 Test whether the receptor affinity is increased in e-N synergy. 2.4 Determine EphB signaling pathway phosphorylation levels in e-N synergy.SA3: Investigate EphB2-Unc5c receptor interactions in ephrin-Netrin synergy. 3.1 Structure-function analysis of EphB and Unc5c in e-N synergy 3.2 Detect EphB and Unc5c association in motor axonal growth cones. 3.3 Study the role of direct EphB and Unc5c interactions in synergy. 3.4 Define the interactome of the Unc5c-EphB2 complex.
|Effective start/end date||8/1/19 → 7/31/20|
- motor neuron
- axon guidance
- synergistic integration
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