Neuroscience 2005 Abstract
| Presentation Number: | 710.8 |
|---|---|
| Abstract Title: | Key roles for Raf kinases in brain development and neurotrophin-induced axon growth. |
| Authors: |
Zhong, J.*1
; Li, X.1
; Galabova-Kovacs, G.2
; Baccarini, M.2
; Snider, W. D.1
1Neurosci. Center, UNC-CH, Chapel Hill, NC 2Austria, 103 Mason Farm Rd., 27599-7250, |
| Primary Theme and Topics |
Development - Axonal and Dendritic Development -- Axon growth and guidance: Other |
| Session: |
710. Axon Growth and Guidance V Poster |
| Presentation Time: | Tuesday, November 15, 2005 4:00 PM-5:00 PM |
| Location: | Washington Convention Center - Hall A-C, Board # B30 |
| Keywords: | DRG, axon, neurotrophin, Cre |
Raf kinases mediate extracellular signaling via receptor tyrosine kinases. In vitro evidence has implicated Raf kinases in survival and morphological responses to neurotrophins. Because both B-Raf and Raf-1 (C-Raf) null mice die at mid-embryonic stages, the role of Raf signaling in nervous system development has not been defined. We constructed a B-Raf floxed allele with loxP sites flanking the N-terminus of the Ras binding domain. A second B-Raf floxed line, designed to allow truncation of the kinase domain was kindly provided by Alcino Silva (UCLA).
Conditional ablation using a Nestin-Cre deleter showed that B-Raf protein levels were reduced by 90% in the nervous system of young adults. Furthermore, baseline p42/44-ERK phosphorylation in the brain, spinal cord and DRGs was dramatically reduced. B-Raf f/-:NesCre mice on a mixed Bl6/129 background exhibited a severe growth deficit from P15 onwards and exhibited modestly reduced brain size and cortical thinning. Surprisingly, DRG sizes were normal in B-Raf f/-:NesCre mice.
In order to assess the role of Raf-1 in redundant or alternative pathways mediating neurotrophin responses, we generated B-Raf f/f, Raf-1 f/f mice. We then transfected Cre recombinase into E14 DRG neurons from double floxed embryos. To allow recombination and turnover of endogenous Raf proteins, we cultured neurons for 7 days and replated them in the presence of NGF for 48 hours. Cre-transfected neurons exhibited an 80% reduction in axon length compared to GFP-transfected controls (n=6 exps). B-Raf f/f DRG neurons transfected with Cre exhibited no reduction in axon growth.
We conclude that B-Raf function in the nervous system is essential to the normal growth of the animal, but is not required for survival or establishment of peripheral axon projections of neurotrophin dependent neurons. Eliminating both B-Raf and Raf-1 prevents NGF-induced axon growth in vitro. The effect of eliminating both B-Raf and Raf-1 on nervous system development in vivo is under investigation.
Supported by R01 NS31768.
Conditional ablation using a Nestin-Cre deleter showed that B-Raf protein levels were reduced by 90% in the nervous system of young adults. Furthermore, baseline p42/44-ERK phosphorylation in the brain, spinal cord and DRGs was dramatically reduced. B-Raf f/-:NesCre mice on a mixed Bl6/129 background exhibited a severe growth deficit from P15 onwards and exhibited modestly reduced brain size and cortical thinning. Surprisingly, DRG sizes were normal in B-Raf f/-:NesCre mice.
In order to assess the role of Raf-1 in redundant or alternative pathways mediating neurotrophin responses, we generated B-Raf f/f, Raf-1 f/f mice. We then transfected Cre recombinase into E14 DRG neurons from double floxed embryos. To allow recombination and turnover of endogenous Raf proteins, we cultured neurons for 7 days and replated them in the presence of NGF for 48 hours. Cre-transfected neurons exhibited an 80% reduction in axon length compared to GFP-transfected controls (n=6 exps). B-Raf f/f DRG neurons transfected with Cre exhibited no reduction in axon growth.
We conclude that B-Raf function in the nervous system is essential to the normal growth of the animal, but is not required for survival or establishment of peripheral axon projections of neurotrophin dependent neurons. Eliminating both B-Raf and Raf-1 prevents NGF-induced axon growth in vitro. The effect of eliminating both B-Raf and Raf-1 on nervous system development in vivo is under investigation.
Supported by R01 NS31768.
Sample Citation:
[Authors]. [Abstract Title]. Program No. XXX.XX. 2005 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2005. Online.
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