Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo

Abstract

To define the role of the Raf serine/threonine kinases in nervous system development, we conditionally targeted B-Raf and C-Raf, two of the three known mammalian Raf homologs, using a mouse line expressing Cre recombinase driven by a nestin promoter. Targeting of B-Raf, but not C-Raf, markedly attenuated baseline phosphorylation of Erk in neural tissues and led to growth retardation. Conditional elimination of B-Raf in dorsal root ganglion (DRG) neurons did not interfere with survival, but instead caused marked reduction in expression of the glial cell line-derived neurotrophic factor receptor Ret at postnatal stages, associated with a profound reduction in levels of transcription factor CBF-beta. Elimination of both alleles of Braf, which encodes B-Raf, and one allele of Raf1, which encodes C-Raf, affected DRG neuron maturation as well as proprioceptive axon projection toward the ventral horn in the spinal cord. Finally, conditional elimination of all Braf and Raf1 alleles strongly reduced neurotrophin-dependent axon growth in vitro as well as cutaneous axon terminal arborization in vivo. We conclude that Raf function is crucial for several aspects of DRG neuron development, including differentiation and axon growth.

B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

“…4 C, bottom;Hall et al, 1997;Patel et al, 2000;Xu and Hall, 2007). In addition, we have shown previously that loss of both B-and C-RAF in DRGs does not abrogate the CGRP expression (Zhong et al, 2007). These data together indicate that the restoration of CGRP expression in LSL-kaBraf:nes-Cre:TrkA / :Bax / DRG neurons is not directly caused by the elevation of neuron-intrinsic B-RAF activity, but indirectly through the restoration of cutaneous innervation and subsequent retrograde signaling from skin-derived factors.…”

“…Mouse breeding and genotyping were performed as described previously (Mercer et al, 2005;Zhong et al, 2007). The animal protocol was approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College, and experiments were conducted in accordance with the National Institutes of Health Guide for the Use and Care of Laboratory Animals.…”

“…Western blotting and immunohistochemical staining were performed as described previously (Zhong et al, 2007). An equal amount of total protein was loaded in each lane.…”

“…Bars: (B) 200 µm; (H) 40 µm. (I) The number of branches per nerve trunk was quantitated, blinded to the genotypes, for a defined skin section by counting the total number of branch points observed along each major nerve trunk entering the skin (Zhong et al, 2007). n = 3 for each group.…”

“…We have shown that RAF-MEK signaling robustly promotes axon growth in primary sensory neurons in vitro (Markus et al, 2002). In vivo, conditional gene targeting studies have shown that RAF signaling is necessary for developing sensory neurons to arborize in their target fields in the skin (Zhong et al, 2007). However, it remains unknown whether RAF signaling is sufficient to enable axon growth in vivo or whether concomitant activation of other signaling pathways is necessary to drive longrange axon projections.…”