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

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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 eduction 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-β. 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.

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Figure 1: Conditional ablation of Braf and Raf1.
Figure 2: Neural tissue-specific ablation of B-Raf.
Figure 3: Loss of B-Raf leads to reduced neural innervation in pituitary gland.
Figure 4: Loss of B-Raf affects postnatal Ret expression, but not survival of DRG neurons.
Figure 5: B-Raf is sufficient to activate the transcription factor complex PEBP2 and induce Ret expression in DRG neurons.
Figure 6: Biochemical and axon projection abnormalities in DRGs missing three Raf alleles (Braff/fRaf1f/+nesCre+) mice at early postnatal stages.
Figure 7: Reduced Erk phosphorylation and axonal growth in embryos after loss of four Raf alleles.
Figure 8: NGF-induced axon growth was reduced in B- and C-Raf deficient DRG neurons.


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The authors wish to thank L. Lei, L. Parada (University of Texas Southwestern), L. Reichardt (University of California San Francisco), T. Jessell (Columbia University) and E. Turner (University of California San Diego) for sharing valuable antibodies, P. Barker (McGill University) for the PC12 cells and K.L. Guan (University of Michigan, Ann Arbor) for the BRAF-ED cDNA. We are grateful to P. Ye and A.J. D'Ercole for blood glucose assays and pituitary gland study. Special thanks are due to the Animal Model Core Facility at University of North Carolina at Chapel Hill for their expert assistance in generating the gene-targeted mice. This work was supported by RO1N0S31768 from the US National Institutes of Health and the Confocal and Multiphoton Microscopy Core Facility supported by National Institute of Neurological Disorders and Stroke center grant NS45892.

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Correspondence to William D Snider.

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Supplementary information

Supplementary Fig. 1

Schematic showing the Braf exon-3 gene targeting construct with chromosomal location of the loxP sites. (PDF 261 kb)

Supplementary Fig. 2

Loss of DRG neuron markers in E12.5 Braf−/− embryos. (PDF 522 kb)

Supplementary Fig. 3

Homeostasis abnormalities in Braff/−nesCre+ mice. (PDF 246 kb)

Supplementary Fig. 4

At P30, the brain size of Braff/−nesCre+ mice is reduced, compared with control littermates'. (PDF 2494 kb)

Supplementary Fig. 5

Profiling of transcription factor activities induced by NGF and B-Raf in PC12 cells. (PDF 4832 kb)

Supplementary Fig. 6

Phenotype in Braff/fRaf1f/+nesCre+ mice. (PDF 3549 kb)

Supplementary Fig. 7

Loss of B- and C-Raf at early embryonic stages impairs NGF-induced axon growth but not neuron survival. (PDF 491 kb)

Supplementary Table 1

List of selected transcription factor complexes activated by NGF or B-Raf 24 h after stimulation. (PDF 136 kb)

Supplementary Video 1

A Braff/−nesCre+ mouse and its Braff/− littermate at P30. The B-Raf conditional null mice are easy to recognize as they are much smaller than their control littermates. Note that mice appear to be hyperactive and clearly have full locomotor activity. This latter observation demonstrates that motor and sensory neurons have survived into adulthood. (MPG 4152 kb)

Supplementary Video 2

A Raf1f/−nesCre+ mouse and its control littermate at the age of P30. The C-Raf conditional nulls have no obvious behavioral phenotype. (MPG 665 kb)

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