Cortical Migration Defects in Mice Expressing A-RAF from the B-RAF Locus

Abstract

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.The founding member of the RAF family of protein serine/ threonine kinases was discovered as the oncogene of mouse sarcoma virus 3611 (35). In vertebrate species, three RAF genes (A-RAF, B-RAF, and C-RAF) have been identified (5,14). B-RAF-deficient mice die between embryonic day 11.5 (E11.5) and E12.5 due to vascular hemorrhaging caused by increased apoptosis of endothelial cells (50). These animals also suffer from neuronal cell death (46) and a range of other defects that arise as a consequence of a significant disruption to ERK activation in these cells (48). Our earlier work further established that B-RAF is the major MEK activator in vivo and that C-RAF is required for normal B-RAF function (48). Targeted disruption of A-RAF or C-RAF genes demonstrated that their functions are not fully redundant with B-RAF, since null mutations for each gene resulted in distinct phenotypes (18,24,30,(48)(49)(50).Knock-in experiments support an important role of C-RAF in apoptosis suppression (6,18,53). The presence of multiple interaction partners of RAF that have been implicated in the control of apoptosis (36) and genetic experiments (18,24,48) raise the possibility that modulation of C-RAF kinase activity in survival depends on interaction with a different set of proteins, including Bcl-2 and Bag1 (11,12,43,44). A-RAF, the least well-characterized member of the family, appears to have the lowest specific activity for MEK (32, 51), although it clearly functions as a transforming gene and activates the mitogenic cascade when overexpressed in an activated form (17, 41). Moreover, like B-and C-RAF, A-RAF activation is coupled to stimulation of growth factor receptors such as nerve growth factor and epidermal growth factor receptors and expression of activated variants of all three isozymes causes differentiation and neurite formation in PC12 pheochromocytoma cells (47).Before determination of differentiated cell lineages in midgestation, C-RAF alone can fully compensate B-RAF function and vice versa (18,24,49,50). Double knockout experiments demonstrate that A-RAF alone cannot compensate B-...

Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum

“…In the hippocampus, migration defects can lead to cell dispersal [43]. As BRaf has been implicated to play a role in neuronal migration in the embryonic cortex [44], we inspected hippocampal sections of cKO mice at P21 but did not observe cell dispersal in the dentate gyrus (Figure 1D, E). In order to exclude sex-related differences, quantification of the granule cell layer volume and all other subsequent experiments were performed exclusively with male littermates.…”

“…After appropriate labelling periods, mice were anaesthetized with Ketanest®/Rompun® and perfused transcardially with saline, followed by 4% paraformaldehyde (PFA) in PBS, pH7.4. Brains were dissected and postfixed in 4% PFA/PBS overnight at 4°C and subsequently embedded in paraffin and serially sectioned as described [44]. The left part of the brain was serially cut into a spate of ten 10 µm sagittal sections always using the first slide for Nissl staining.…”

“…Immunohistochemical analysis was performed as described [44] . Antigen demasking was performed by carefully boiling the sections in a microwave oven in 10 mM citrate buffer pH6.0.…”

B-raf Alternative Splicing Is Dispensable for Development but Required for Learning and Memory Associated with the Hippocampus in the Adult Mouse

“…However, conditional ablation of B -raf in neural precursors resulted in severe neurological defects caused by central nervous system (CNS) dysmyelination, leading to death three weeks after birth [8]. These specific B-Raf functions were confirmed by the fact that changing the B-Raf kinase domain with that of A-Raf in knockin experiments was not sufficient to fully rescue the phenotype observed in B-Raf null animals [9]. These findings are likely related to the high levels of B-Raf expression in the nervous system that could not be compensated by other Raf proteins.…”

EphA/ephrin A reverse signaling promotes the migration of cortical interneurons from the medial ganglionic eminence

“…2D). These assays have widely been used to study the response of various neuronal cell types, including cortical interneurons, to guidance cues such as neurotrophins and ephrins (Santiago and Erickson, 2002;Camarero et al, 2006;Perrinjaquet et al, 2011). Thus, dissociated cells from the MGE of E14.5 embryos were placed in the upper compartment of a Boyden chamber and exposed to pre-clustered EphA4-Fc.…”