Our research aim is to understand how neuronal networks are established during development with the long- term goal of determining how this process can be recapitulated to repair circuits damaged after injury or disease. To extend towards its target, an axon must process directional information in the embryonic environment, reach signals at the correct time in development and be competent to interpret the cue in the correct context. However, although the mechanisms that control directionality for axons have been extensively described, it remains unresolved how either the growth rate or competence of growth cones is regulated. We will examine these questions by determining how Bone Morphogenetic Proteins (BMPs) guide commissural (C) axons in the developing spinal cord. In our previous work, we identified a new class of guidance signals: morphogens that also induce specific cell fates. We showed that BMPs, secreted from the roof plate, provide directional signals for C axons by repelling their initial projections away from the dorsal midline. We have now identified two additional critical roles for BMP signaling in C axon guidance: regulating the rate of C axonal outgrowth and directing the responsiveness of C axons to subsequent guidance cues. To investigate these previously unrecognized activities, we will define the mechanism by which BMPs inhibit the rate of C axon outgrowth in Aim 1 and determine the developmental consequences of unregulated axon outgrowth in Aim 2.
In Aim 3, we will assess an integrative mechanism regulated by BMP signaling that permits a C growth cone to change its response to signals over time. By advancing our understanding of the basic mechanisms of axon guidance during development, these studies will facilitate the regeneration of neuronal circuits in the CNS. Approach: We will use convergent in vitro and in vivo methods in these studies, combining biochemical and tissue grafting assays with mouse genetics and chick in ovo electroporation, in the following aims:
Aim 1 : Determine how BMP signaling regulates Limk1/cofilin to control the rate of C axon outgrowth. Hypothesis: BMPs regulate the rate of C axon outgrowth by upregulating Lim kinase 1 (Limk1) in C neurons and thereby inactivating cofilin, a direct regulator of actin polymerization.
Aim 2. Determine the mechanism by which axon extension speed regulates the recognition of guidance cues Hypothesis: The timing of axon outgrowth determines the response of an axon to guidance cues. Guidance errors occur if the rate of C axon outgrowth is increased because either the response of accelerated C growth cones is intrinsically altered to guidance cues, or the extrinsic environment is not in place to guide them.
Aim 3. Determine how BMP signaling regulates the response of C axons to Netrin1 Hypothesis: The activation of BMP signaling in C growth cones directs C axons to respond to Netrin1 as a repellent rather than an attractant. Thus, axons accumulate a history of signaling events that informs future guidance decisions, thereby permitting a few guidance cues to generate multiple distinct axon trajectories.

Public Health Relevance

We are examining the mechanisms by which neurons send out projections, called axons, towards their synaptic targets during embryonic development. We have identified two previously undescribed processes within, or intrinsic to, neurons that control a) the rate at which axons grow and b) how axons regulate their response to signals in the embryonic environment. The identification of intrinsic regulators of axon outgrowth will be a significant advance for studies trying to re-establish axonal circuits after traumatic injury, given that regenerating axons in the central nervous system are unable to overcome the effect of inhibitory signals from the environment.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
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Riddle, Robert D
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University of Southern California
Schools of Arts and Sciences
Los Angeles
United States
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Varadarajan, Supraja G; Butler, Samantha J (2017) Netrin1 establishes multiple boundaries for axon growth in the developing spinal cord. Dev Biol 430:177-187
Varadarajan, Supraja G; Kong, Jennifer H; Phan, Keith D et al. (2017) Netrin1 Produced by Neural Progenitors, Not Floor Plate Cells, Is Required for Axon Guidance in the Spinal Cord. Neuron 94:790-799.e3
Butler, Samantha J; Bronner, Marianne E (2015) From classical to current: analyzing peripheral nervous system and spinal cord lineage and fate. Dev Biol 398:135-46
Kandyba, Eve; Hazen, Virginia M; Kobielak, Agnieszka et al. (2014) Smad1 and 5 but not Smad8 establish stem cell quiescence which is critical to transform the premature hair follicle during morphogenesis toward the postnatal state. Stem Cells 32:534-47
Phan, Keith D; Butler, Samantha J (2013) Bilaterally symmetric populations of chicken dI1 (commissural) axons cross the floor plate independently of each other. PLoS One 8:e62977
Yamauchi, Ken; Varadarajan, Supraja G; Li, Joseph E et al. (2013) Type Ib BMP receptors mediate the rate of commissural axon extension through inhibition of cofilin activity. Development 140:333-42
Gaber, Zachary B; Butler, Samantha J; Novitch, Bennett G (2013) PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors. PLoS Biol 11:e1001676
Hazen, V M; Andrews, M G; Umans, L et al. (2012) BMP receptor-activated Smads confer diverse functions during the development of the dorsal spinal cord. Dev Biol 367:216-27
Hazen, V M; Phan, K D; Hudiburgh, S et al. (2011) Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord. Dev Biol 356:566-75
Phan, Keith Dai; Croteau, Louis-Philippe; Kam, Joseph Wai Keung et al. (2011) Neogenin may functionally substitute for Dcc in chicken. PLoS One 6:e22072

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