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Molecular and Cellular Biology, October 2006, p. 7103-7115, Vol. 26, No. 19
0270-7306/06/$08.00+0     doi:10.1128/MCB.00424-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Guadalupe Camarero,1,{dagger} Oleg Yu Tyrsin,1,{dagger} Chaomei Xiang,1,{dagger} Verena Pfeiffer,1 Sandra Pleiser,1 Stefan Wiese,2 Rudolf Götz,1 and Ulf R. Rapp1*

Institut für Medizinische Strahlenkunde und Zellforschung, Bayerische Julius-Maximilians-Universität, Versbacher-Str. 5, D-97078 Würzburg, Germany,1 Institut für Klinische Neurobiologie, Bayerische Julius-Maximilians-Universität, Josef Schneider-Str. 11, D-97078 Würzburg, Germany2

Received 10 March 2006/ Returned for modification 4 May 2006/ Accepted 3 July 2006


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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-RAFKIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAFKIN/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.


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INTRODUCTION
 
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, 48-50).

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- and C-RAF functions but raise the possibility of cooperation between A-RAF and either B- or C-RAF in rescue before midgestation (23, 48). During midgestation, cooperation of C- with B-RAF might be essential for full extracellular signal-regulated kinase (ERK) activation and for survival activity (45, 48). No specific role has yet been assigned to B-RAF in mouse development after midgestation, when the majority of brain development occurs and when neurons express much higher levels of B-RAF than other cell types. Also, it is not clear whether B-RAF-mediated protection of endothelial cells from apoptosis is associated with its high MEK kinase activity or involves other pathways (3, 46).

To elucidate the role of B-RAF in brain development and in differentiated neurons, we wanted to obtain mice lacking B-RAF that overcome the E11.5/12.5 lethality. B-RAFKIN/KIN mice were created that express A-RAF under control of endogenous B-RAF promoter, and we expected that neurons would survive beyond the critical midgestation cell death phase. Because we had previously observed that B-RAF overexpression can mediate neurite outgrowth (47) and because B-RAF localizes to axons and dendrites in vivo (25), we wanted to study these aspects of B-RAF function in neuronal differentiation.

A-RAF expression under the B-RAF promoter rescued B-RAF-deficient embryos from endothelial apoptosis and allowed them to survive after midgestation. A fraction of these late-stage embryos survived to adulthood. Histological analysis demonstrated that A-RAF can substitute for B-RAF in mediating growth factor-dependent survival. Moreover, in the developing neocortex, impaired neuronal migration was observed in conjunction with disorganization of neuronal layering.


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MATERIALS AND METHODS
 
Generation of B-RAFKIN/KIN mice. Mice used in these studies were generated and maintained according to protocols approved by the animal care and use committee at University of Würzburg. An 8-kb BamHI genomic fragment of the B-RAF gene containing exon 3 was subcloned in pBluescript KS vector. A 2.5-kb fragment containing human A-RAF cDNA with a hemagglutinin (HA) tag and human growth hormone poly(A) signal was generated by PCR from plasmid pCMV5-humanA-RAF(HA) using forward (5'-CGCGCGCTGCAGTGGGCACCGTCAAAG-3') and reverse (5'-GCGCGCGTCGACTACTGAGTGGACCCAACGC-3') primers and cloned into the B-RAF gene fragment cut with NsiI plus SalI. Then, the 7.5-kb 5' arm was subcloned into pKS TKneoLoxP plasmid at the unique BamI and SalI sites. Thus, A-RAF cDNA was introduced in frame with the third exon of B-RAF at the beginning of its own Ras binding domain. A 1.7-kb 3' arm was amplified via PCR (BamHI site at 5' was introduced in forward primer) and cloned into pKS TKneoLoxP vector containing the 5' arm via BamHI and EcoRI sites. The targeting vector was linearized by NotI and electroporated into CJ7 embryonic stem (ES) cells (129Sv background) and surviving colonies were isolated following G418 and gancyclovir selection. Surviving clones were analyzed for homologous recombination by PCR using primers 5'Neo (5'-GTTGGCGCTACCGGTGGATGTGG-3') and 3'armB-RAF (5'-TGTGTATCGATCTGTCCCGGTACACCATG-3'). All positive clones were then verified by Southern hybridization using external fragments as probes (a 1.3-kb EcoRI/BamHI fragment for the 3' end and a 1.6-kb EcoRI/BamHI fragment for the 5' end). Two positive ES clones were injected into C57BL/6-D2 hybrid blastocysts and transferred into pseudopregnant females. The resulting chimeras were mated to C57BL/6 (B6) females, and agouti F1 129Sv/B6 offspring were genotyped by the same Southern analysis. B-RAFKIN/KIN mice were obtained by crossbreeding B-RAF+/KIN 129Sv/B6 mice or by breeding B-RAF+/KIN 129Sv/B6 with CD-1 mice to obtain B-RAF+/KIN 129Sv:B6:CD-1 (1:1:2) animals and their subsequent crossbreeding. Progeny were genotyped by PCR using MB3-1 and MB-del primers to detect the wild-type (WT) allele (50). The MB-del site is deleted in the targeted allele, which was detected with MB3-1 and A-RAF(SacI) (5'-GGACCTCGACAATGAGCTCCTCGCC-3') primers.

Tissue preparation and immunohistochemical analysis. Whole embryos at E10.5 to E16.5 and brains of E18.5 or postnatal mice were dissected into cold phosphate-buffered saline (PBS) and fixed overnight in fresh buffered 4% paraformaldehyde, and 7-µm paraffin serial sections were prepared. Deparaffinized sections were microwaved in 10 mM sodium citrate buffer (pH 5.5) and washed in PBS. Subsequently, the slides were incubated in peroxidase blocking solution (1.5% H2O2 in PBS) and preincubated for 30 min with blocking serum (5% goat or rabbit serum, 0.1% Triton in PBS). Primary antibodies were diluted in blocking serum and applied overnight at 4°C. The primary antibodies used were anti-caspase-3 activated polyclonal (1:100; Cell Signaling), anti-NeuN monoclonal (1:100; Chemicon), anti-MAP2 monoclonal (1:500; Abcam), anti-Nestin monoclonal (1:5; DSHB, Iowa), anti-Tuj1-ß monoclonal (1:100; Chemicon), anti-Brn-2 rabbit polyclonal (1:800; gift from M. Wegner), and anti-Reelin (1:1,000; Abcam). Immunolabeling was revealed by the indirect immunofluorescence procedure using Texas Red and Alexa-488-conjugated secondary antibodies purchased from Vector Labs and Molecular Probes, respectively, or by the avidin-biotin method. For primary cell culture of cortical neurons, cells from E14.5 mouse cerebral cortices were dissociated and seeded on poly-D-lysine and laminin-coated glass coverslips in neurobasal medium (Invitrogen) containing 500 µM glutamine, 2% B27 supplement (Invitrogen), and penicillin-streptomycin (Invitrogen). After 4 h in culture, cells were fixed and stained with anti-B-RAF antibodies specific for the N terminus (1:300; Santa Cruz).

BrdU labeling, immunodetection, and quantification. For the cell proliferation assay, pregnant females were injected intraperitoneally withbromodeoxyuridine (BrdU) (70 µg/g of body weight). After 2 h of labeling, embryos were removed, processed, and serially sectioned. The brain sections were incubated in 2 N HCl for 30 min at 37°C, neutralized in borate buffer (pH 8.5) for 25 min at room temperature, rinsed twice in PBS, and blocked with 3% nonfat milk for 3 h at room temperature. The sections were then incubated with an anti-BrdU monoclonal antibody (1:100; Molecular Probes) overnight at 4°C and rinsed twice in PBS, followed by incubation with an Alexa-488-conjugated secondary antibody (1:100; Molecular Probes). For each genotype, three embryos were analyzed at the indicated stages, and three or four nonadjacent sections at the level of the olfactory bulb were selected to determine the BrdU labeling index. This index was calculated by dividing the number of BrdU-positive nuclei by the total number of the nuclei identified in units of 200-µm-wide ventricular zone (VZ) in a x40 optical view of mutant and control cortical regions. For migration analysis in vivo, BrdU was injected at E14.5, and E17.5 parasagittal sections at the level of the olfactory bulb were used. At this level, the entire cortical region was divided in two parts: upper, at the level of the cortical plate (CP), and deep, at the level of the intermediate zone (IZ) and subventricular zone. The number of BrdU-labeled cells was counted and the relative distribution (as a percentage) in each layer was calculated.

Western blot analysis. Western blotting was performed from embryonic lysates according to standard methods. Tissue was lysed in ice-cold buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1% NP-40, and 1 mM EDTA) supplemented with a protease inhibitor mixture (Roche). Protein concentration was determined by Bradford assay using bovine serum albumin as a standard. Equal amounts of protein (20 µg) were boiled in Laemmli buffer, separated by 10% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and transferred to a 0.45-µm nitrocellulose membrane. Membranes were blocked in 5% nonfat milk in Tris-buffered saline plus 0.5% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibody. The antibodies used were monoclonal anti-HA tag (anti-12CA5), polyclonal anti-ERK2, anti-B-RAF C terminus (Santa Cruz), and anti-A-RAF C terminus (Santa Cruz), each at a dilution of 1:1,000.

Transwell cell migration assay. Migration of dissociated cerebral cortex neurons from E14.5 embryos was assayed using a Boyden transwell system (5-µm pore size; Corning Costar Co.). Two hundred microliters of serum-free medium containing dissociated cortical neurons (2 x 105 cells/cm2) was added to the upper insert of a transwell in neurobasal medium. Prior to seeding, both sides of the transwell were coated with poly-D-lysine and laminin. In the bottom chamber, 500 µl of neurobasal medium with or without BDNF (10 ng/ml; R&D Systems, Minneapolis, MN) was added. Twenty-four hours after seeding, cells were fixed with paraformaldehyde, and cells attached to the upper side of the membrane were thoroughly scraped off with cotton swabs. Migrated neurons attached to the bottom side of the membranes were visualized with anti-Tuj1-ß fluorescence staining. Cell migration was determined by counting the total number of Tuj1-ß-positive cells on the bottom side of the membranes. Each experiment was done in duplicate; a total of four embryos of each genotype were investigated.

Statistical analysis. Data sets from independent experiments were pooled, and the results were expressed as means and standard deviations. Statistical significance between data sets was assessed by Student's t test and assigned when the P value was <0.05.


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RESULTS
 
B-RAF expression in the embryonic forebrain. B-RAF is highly expressed in postnatal brain, including cerebellum, hippocampus, and cortex (2). To study the function and expression of B-RAF and the related isoform A-RAF during embryonic brain development, we chose the embryonic forebrain. The two classes of neural cells in embryonic brain development, proliferating progenitor cells and postmitotic neurons, are localized in two easily discernible layers. Whereas the progenitor cells proliferate in the ventricular zone, the neurons, after being generated in this germinal zone, move to new positions in the developing neocortex. Starting at E10.5, consecutive waves of radial migration of the immature neurons migrate into the preplate and, by dividing it into the subplate (SP) and molecular layer, form the CP. The process of neuronal migration is an instrumental step in neuronal maturation and shaping of the six neocortical layers that will have formed at the end of mouse embryonic development (20, 29). In humans, severe defects in neuronal migration in the cortex cause lissencephaly, less severe defects lead to heterotypia. Both pathologies can cause mental retardation or epilepsy (29).

Western blot analysis of equal amounts of forebrain tissue extracts showed an up-regulation of both kinases at E16.5 compared to E12.5 and E14.5 (Fig. 1A). This observation may indicate that differentiating/mature neurons require higher levels of both kinases than neural progenitor cells. To substantiate this notion, we immunostained sagittal sections of the neocortex of E10.5 and E14.5 embryos with a B-RAF antibody. Homogeneous B-RAF expression in the whole ventricular zone, consisting mainly of progenitor cells, was observed at E10.5 (Fig. 1B, C). At E14.5, predominant B-RAF expression was seen in the IZ migrating neurons and the newly generated CP neurons (Fig. 1E). In higher-magnification pictures within the IZ, B-RAF protein seemed to be accumulated within the presumed leading process and adjacent cytoplasmic region, both thought to be important subcellular sites for neuronal migration (Fig. 1F, G). To further determine the subcellular distribution of B-RAF expression, cortical neurons were isolated from E14.5 forebrain and cultured on laminin for 4 h, fixed, and stained with an anti-B-RAF antibody. Localization of B-RAF was not only restricted to cell bodies but also occurred in the shaft of neurites in neurons with a single process (Fig. 1H).


Figure 1
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  		FIG. 1. Expression of B-RAF and A-RAF in embryonic forebrain. (A) Western blot analysis of equal amounts of forebrain tissue extracts (20 µg) shows up-regulation of both kinases at E16.5 compared to E12.5 and E14.5. The two bands for B-RAF (arrowheads) correspond to the 97-kDa and 94-kDa isoforms, respectively. Reprobing of the blot with an ERK2 antibody (lower panel) was used as a loading control. (B and C) Immunostaining (red) of sagittal sections with an anti-B-RAF antibody showed homogeneous B-RAF expression in the whole VZ at E10.5 (B). The boxed area in panel B corresponds to the enlarged image in panel C. (D) Note the absence of the B-RAF signal in the cortex in a matching section from a B-RAF-deficient E10.5 embryo. (E) B-RAF staining (red) of sagittal sections at E14.5; predominant B-RAF expression is seen in neurons of the IZ and CP. (F) Higher-magnification of B-RAF-stained (red) migrating neurons of IZ. (G) Merged image of panel F with Nomarski differential contrast. (H) Immunostaining for B-RAF (red) of cortical neurons isolated from E14.5 forebrain and grown for 4 h on a laminin substrate. Arrowhead indicates cell body; arrow shows neurite. (I) Omission of the first antibody yielded no specific staining; nuclei (DAPI) are blue. Scale bars, 200 µm (B), 50 µm (C to E), 10 µm (F and G), 20 µm (H and I).

Disorganization and cell death in the ventricular zone in B-RAF-deficient embryos. B-RAF expression has been shown to be essential for survival of sensory neurons, motoneurons, and neural progenitors (12, 46). To determine the function of B-RAF in the neocortex, we compared cell proliferation and survival in wild-type and B-RAF-deficient mice. To obtain living B-RAF-deficient embryos at E12.5, we crossed the targeted B-RAF allele from the B6 background in two consecutive generations into the outbred CD1 genetic background. To investigate whether neuronal birth is impaired in the absence of B-RAF, we performed immunostaining for Tuj1-ß (ß3-tubulin isotype III), the earliest marker of postmitotic neurons (7, 21), in corresponding serial sagittal sections of B-RAF-deficient and littermate control brains (Fig. 2A to D). Tuj1-ß immunoreactivity was restricted to the preplate at E10.5 and E12.5; only occasional staining could be seen in the ventricular zone (Fig. 2A and C). Instead of forming an organized preplate layer as in the WT littermate control embryos, Tuj1-ß-positive cells were dispersed in the cortex of B-RAF–/– E10.5 embryos (Fig. 2B). This effect was even more obvious at E12.5 (Fig. 2D). A quantitative comparison showed a twofold reduction of the number of Tuj1-ß-positive cells in B-RAF–/– embryos compared with the WT (Fig. 2E). This finding may indicate that, in the absence of B-RAF, fewer cortical neurons are being produced. Alternatively, cell survival may be impaired or both cellular processes might be disturbed. To differentiate among these scenarios, we examined the proliferation of cortical progenitor cells by in vivo labeling with bromodeoxyuridine (2-h pulse) (Fig. 2F to I). At E10.5, there was no significant difference in the number of BrdU-labeled cells in the VZ of B-RAF-deficient embryos compared with WT (Fig. 2J). A slightly reduced cell proliferation rate was observed at E12.5 in B-RAF-deficient embryos (Fig. 2H and I). Interestingly, in B-RAF–/– embryos, most BrdU-positive cells seemed to be confined to the basal half of the VZ (Fig. 2G and I), indicating that their return during G2 toward the apical surface was impaired.


Figure 2
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  		FIG. 2. Defects in differentiation, proliferation, positioning, and survival of neural cells in B-RAF-deficient forebrain. (A to D) In sagittal sections from E10.5 and E12.5 WT and B-RAF-deficient embryos, early neurons were identified by staining with Tuj1-ß antibody (red, arrowheads indicate misplaced neurons). (E) Quantification of Tuj1-ß-positive cells at E10.5. (F to I) Sections of WT (F and H) and B-RAF-deficient (G and I) forebrain after a 2-h pulse in vivo with BrdU, followed by immunohistochemistry (green). (J) Quantification of BrdU-positive cells at E10.5. (K to N) Analysis of mitoses using phosphorylated histone H3 (red) staining of neuroepithelial progenitor cells. Mitotic cells are observed outside the ventricular apical surface in the B-RAF-deficient cortex (L and N) (arrowheads indicate ectopic mitosis). (O) Quantification of phosphorylated histone H3-stained cells. (Q) Apoptotic cells were identified by staining for cleaved caspase-3 (red) in sagittal sections from the E12.5 forebrains of B-RAF-deficient embryos. Note the colocalization (yellow to orange) with the Tuj1-ß staining (green, merged images are shown). (P) Note absence of cell death in an equivalent section from the wild-type cortex. Scale bars, 50 µm.

To investigate the interkinetic nuclear movement in the neocortex, we performed immunostaining for phosphorylated histone H3 (pH3) which is a marker of metaphase of mitosis (15). In WT, pH3-positive cells were mostly restricted to the ventricular apical surface at E10.5 and E12.5 (Fig. 2K and M). In B-RAF–/– embryos, the metaphase cell number at both ages was increased compared to the WT (Fig. 2L, N, and O). In addition, more ectopically located pH3-positive cells (present within the ventricular zone more than three cell diameters from the ventricular surface) were found in B-RAF–/– cortices (Fig. 2 L and N). Quantitative analysis of pH3-stained cells showed that about one-third of them were ectopic in B-RAF-deficient embryos, compared to 10% in WT embryos (Fig. 2O). To further explore the mechanism of the reduced neuronal numbers, we performed double-staining of Tuj1-ß-positive cells with the apoptosis marker activated caspase-3. In contrast to the WT cortex, where no cell death was observed (Fig. 2P), we identified many activated caspase-3-positive cells in the central part of sagittal sections of B-RAF–/– cortices at E12.5 (Fig. 2Q) and E10.5 (data not shown). Some of these dying cells were stained for TuJ-1 (Fig. 2Q), indicating that neurons were undergoing cell death in the B-RAF-deficient forebrain.

Generation of mice carrying A-RAF in the B-RAF gene locus (B-RAFKIN/KIN mice). To elucidate the role of B-RAF in neocortex development after E12.5 without interfering with its role in cell survival, we designed and cloned a knock-in construct that expresses A-RAF under the control of the endogenous B-RAF genomic locus. To achieve this gene replacement, the cDNA encoding human A-RAF was inserted into the B-RAF locus. Since B-RAF has multiple splice forms and exon 3 is present in all of them (2), an in-frame fusion was made in the third exon of B-RAF, at the beginning of the conserved Ras binding domain (Fig. 3A). The resulting chimeric kinase of 738 amino acid residues (B-RAFKIN) combines the unique N-terminal part of B-RAF with the remainder of A-RAF (Fig. 3B). Southern blotting confirmed homologous recombination in seven targeted ES cell clones. The expression of the expected mRNA and the 87-kDa chimeric protein in the selected ES cell clones was confirmed by reverse transcription-PCR and Western blotting (data not shown). The neomycin resistance gene, flanked by loxP sites, did not interfere with the expression of chimeric kinase. Chimeric male mice produced from two independent ES clones transmitted the B-RAFKIN allele to their F1 offspring. The F1 heterozygous mice were obtained at a frequency expected for a recessive allele and displayed no visible phenotype, suggesting that there is no dominant negative function of the chimeric kinase.


Figure 3
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  		FIG. 3. Generation of mice carrying A-RAF in the B-RAF gene locus (B-RAFKIN/KIN mice). (A) Targeting strategy to introduce human A-RAF cDNA into the B-RAF locus. Restriction sites: B, BamHI; E, EcoRI; N, NsiI; X, XhoI. Black bars indicate probes used for Southern hybridization. The positions of the neomycin resistance gene (NEO) and LoxP sites (arrows) are shown. (B) The expected chimeric protein harbors 155 amino acids of the N-terminal part of mouse B-RAF and the remainder of A-RAF. An HA tag was introduced at the C terminus of the chimeric kinase. (C) Western blot analysis of E12.5 and E14.5 forebrain lysates, using an antibody specific for the C terminus of B-RAF, revealed the absence of B-RAF protein in B-RAFKIN/KIN embryos (upper panel). After stripping of the antibody, the membrane was reacted with an antibody specific for the C terminus of A-RAF (middle panel). Reprobing of the blot (lower panel) with an ERK2 antibody was employed as a loading control.

B-RAFKIN/KIN mice survive until adulthood. On the mixed inbred 129Sv/B6 background, B-RAFKIN/KIN embryos survived beyond E12.5. The expected 8-kb and 3-kb DNA fragments were detected in genomic DNA isolated from WT and B-RAFKIN/KIN embryos, respectively, after BamHI digestion and Southern blotting (data not shown). The embryos expressed the predicted 87-kDa chimeric A-RAF (Fig. 3C, middle panel) in the absence of B-RAF (Fig. 3C, upper panel). When an outbred genetic background (CD-1) was introduced by intercrossing of heterozygous 129Sv/B6/CD-1 (1:1:2) mice, even more living embryos were obtained at E13.5, and some mice reached a postnatal age of up to 8 weeks (Table 1). However, only about one-third of the B-RAFKIN/KIN embryos survived beyond the critical threshold E13.5 (Table 1). No B-RAF–/– embryos that did not harbor the B-RAFKIN/KIN allele survived beyond E12.5, when the CD-1 genetic background was introduced, as already observed on the B6 background (50). Size-reduced but normal embryos were found after they had passed through the midgestational barrier between E13.5 and 16.5 (Fig. 4A). All postnatal B-RAFKIN/KIN animals investigated (6 at postnatal day 14 [P14] to P24; 1 at P60) were reduced in body weight about twofold but did not display any visible behavioral or developmental defects (Fig. 4A). Newborn B-RAFKIN/KIN mice appeared normal; all major organs, including the brain, were proportional to body size except for the thymus and spleen, which were reduced 10-fold in weight compared to WT littermates.


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  		TABLE 1. Genotyping data from B-RAFkin/+ intercrosses in 129Sv/C57BL/6/CD-1 (1:1:2) background


Figure 4
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  		FIG. 4. B-RAFKIN/KIN mice are rescued from lethality and endothelial apoptosis. (A) Knock-in of A-RAF into the B-RAF locus rescued embryos from death. Representative pictures of embryos and 3-week-old (P21) mice are shown. The developmental stages and genotypes are indicated. (B to D) Absence of endothelial cell death around the dorsal aorta (da) of WT (B) and B-RAFKIN/KIN (C) E11.5 embryos by immunohistochemistry for activated caspase-3. In contrast, a B-RAF–/– embryo showed many dying endothelial cells (arrows) (D). Scale bars, 1,000 µm (A), 50 µm (B to D).

Endothelial and neuronal cell survival in RAFKIN/KIN mice. No endothelial cell apoptosis was seen in immunohistochemical analysis of sections of B-RAFKIN/KIN embryos using an antibody that specifically detects activated caspase-3 (Fig. 4C). To confirm the rescue from apoptosis in another cell type, we tested the survival of motoneurons in vitro in the presence of neurotrophic factors because we had previously shown that sensory and motoneurons from B-RAF-deficient mice do not respond to neurotrophic factors for their survival (46). Similar numbers of motoneurons were isolated from B-RAFKIN/KIN and WT embryos. After plating, the neurotrophic factors GDNF, BDNF, and CNTF (each 1 ng/ml) were added to motoneurons. After 6 days in culture, in the absence of neurotrophic factors, less than 10% of originally plated cells isolated from WT, B-RAF+/KIN, or B-RAFKIN/KIN mice survived, whereas approximately 50% were supported by any single survival factor (data not shown). There was no difference in survival rate between all three genotypes, demonstrating that B-RAF promoter-driven expression of the chimeric kinase had rescued B-RAF deficiency with respect to growth factor-regulated survival of motoneurons.

B-RAFKIN/KIN mice are impaired in neocortical development. The survival activity of the B-RAFKIN/KIN allele was also evident in the embryonic cortex. Neurogenesis in the neocortex in mice starts at around E10.5 and lasts until E17. Tuj1-ß-positive neurons were present in the preplate of the neocortex at E10.5 (data not shown) and E13.5; no significant differences were found in the thickness and distribution of the Tuj1-ß-positive cell layers in B-RAFKIN/KIN compared to WT littermates at this stage (Fig. 5A and B). Quantitative comparison showed similar numbers of Tuj1-ß cells in B-RAFKIN/KIN and WT embryos (data not shown). The progressive change in the intensity of Tuj1-ß expression defines the degree of maturation of these layers. The higher expression levels of Tuj1-ß in SP and molecular layer, compared to CP, allowed us to determine the thickness of cortical layers in E16.5 B-RAFKIN/KIN and WT embryos (Fig. 5C and E). While the distance between the upper boundary of the SP and the pial surface was reduced nearly twofold (127 µm in B-RAFKIN/KIN embryos compared with 246 µm in the control cortex), the total width was reduced by only 16% (497 µm in B-RAFKIN/KIN embryos compared with 592 µm in the control cortex). In addition, the clear boundary between SP and IZ obvious in WT embryos (Fig. 5C) was not visible between SP and IZ in B-RAFKIN/KIN embryos (Fig. 5E). Instead, many neurons were present in the IZ in B-RAFKIN/KIN embryos, indicating that the cells were impaired in their migration toward the CP (Fig, 5E).


Figure 5
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  		FIG. 5. Cortical pathology in B-RAFKIN/KIN embryos. (A and B) Tuj1-ß immunostaining (red) of sagittal sections labels neurons in the preplate of the WT (A) and mutant B-RAFKIN/KIN (B) E13.5 neocortex. (C to F) Tuj1-ß immunostaining (red) and DAPI (blue) staining in sagittal sections of E16.5 cortices. Note that the CP is thinner in the B-RAFKIN/KIN cortex (E and F) than in the WT cortex (C and D) and that the number of Tuj1-ß-positive cells in the IZ, beneath the SP, is increased in the B-RAFKIN/KIN neocortex (E). Scale bars, 100 µm.

B-RAFKIN/KIN mice show a defect in neuronal maturation and dendrite formation. Histological examination of the postnatal cortex (P19) showed a reduction in the thickness of the somatosensory cortex of B-RAFKIN/KIN mice, specifically in layers II, III, and V, and an increased number of cells in layer IV (Fig. 6A and B). Moreover, the neurons in the layers II and III, identified by NeuN staining, were tightly packed in B-RAFKIN/KIN mice (Fig. 6D) compared to WT littermates (Fig. 6C). Interestingly, the neuronal cell bodies in B-RAFKIN/KIN mice were smaller and almost round in shape (Fig. 6B and D), which rarely occurred in WT pyramidal neurons (Fig. 6A and C).


Figure 6
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  		FIG. 6. Cortical pathology in B-RAFKIN/KIN postnatal cortex. (A and B) Hematoxylin and eosin (H&E) staining of WT and B-RAFKIN/KIN mutant sagittal sections of the P19 cortex. Cell layers I to VI are indicated. (C to F) Immunohistochemistry in sagittal sections for NeuN, a nuclear protein expressed in all mature neurons (C and D) and Brn-2, a marker for pyramidal neurons (E and F). (G and H) MAP2 staining in sagittal cortex sections is indicative of a loss of dendritic fasciculation (arrowheads) in layers II and III of mutant cortex. Scale bars: A to F, 100 µm; G and H, 50 µm.

To further characterize the effects of loss of B-RAF signaling during neocortical development, we examined specific populations of cortical neurons in brain sections of postnatalB-RAFKIN/KIN and control mice by immunostaining. The expression of Brn-2, a POU domain transcription factor expressed in pyramidal projection neurons of layers II, III (Fig. 6E), and V (data not shown) of WT littermate animals, was nearly completely lost in B-RAFKIN/KIN in the somatosensory (Fig. 6F) and visual cortex areas (data not shown), whereas in the more anterior cortex of B-RAFKIN/KIN mice, Brn-2 expression was reduced (data not shown). To assay for the presence of another important feature of layer II to V pyramidal neurons, we determined the dendritic formation by MAP2 staining. We observed a dramatic alteration of the apical cortical dendrites in B-RAFKIN/KIN animals because they appeared unfasciculated and disorganized (Fig. 6H), indicating that B-RAF is required for dendrite maturation. Next, we wanted to determine when the loss of Brn-2 expression or the reduction of Brn-2 expressing cells occurred in B-RAFKIN/KIN mice and what cellular process was involved in this phenomenon.

Reduced proliferation of late progenitor cells in B-RAFKIN/KIN cortex. There was no increase in apoptosis in the B-RAFKIN/KIN cortex from E13.5 to postnatal day 19 (data not shown). In fact, there was even an increase in cell density in SP and IZ, as deduced from 4',6'-diamidino-2-phenylindole (DAPI) and Tuj1-ß staining, in B-RAFKIN/KIN embryos at E16.5 (Fig. 5E and F). Therefore, we could exclude increased cell death as the cause for the apparent loss of Brn-2-positive neurons in the postnatal cortex. We next examined the proliferation of cortical progenitor cells by in vivo labeling with BrdU (Fig. 7A to G). Up to E13.5, there was no significant difference in the number of BrdU-labeled cells in the VZ of B-RAFKIN/KIN embryos compared with WT (Fig. 7A, B, and G). A slightly reduced cell proliferation in the VZ of the B-RAFKIN/KIN neocortex was observed at E14.5 that further decreased at E16.5 (Fig. 7C to G). These results indicate that B-RAF has an essential role in the proliferation of late cortical progenitor cells and that the reduction in cortical cell production could be one cause of the hypoplastic cortex seen in the B-RAFKIN/KIN postnatal brain.


Figure 7
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  		FIG. 7. Reduced cell proliferation in late progenitor cells in B-RAFKIN/KIN neocortex. (A to F) E10.5 to E16.5 sagittal sections of the developing cortex after a 2-h pulse in vivo with BrdU, followed by fluorescent staining (green) with an antibody against BrdU. Note impaired proliferation starting at E14.5 in B-RAFKIN/KIN embryos. The developmental stages and genotypes are indicated. (G) Quantification of BrdU-positive cells. Scale bars, 50 µm (A and B), 100 µm (C to F).

Impaired migration of late-born cortical neurons in B-RAFKIN/KIN mice. E14.5 and E16.5 cortical progenitor cells produce neurons that make up the more superficial layers II to V of the neocortex. Our observation of reduced proliferation of late progenitor cells may explain in part the reduction in the thickness of the adult cortex in B-RAFKIN/KIN embryos. However, the nearly complete lack of Brn-2-positive pyramidal projection neurons in postnatal B-RAFKIN/KIN mice (Fig. 6F) would not be consistent with an only twofold reduction in progenitor proliferation between E14.5 and E16.5. Although we observed no significant reduction in the number of Brn-2-positive cells in the E18.5 neocortex of B-RAFKIN/KIN mice, the position of the Brn-2-positive cells was altered in B-RAFKIN/KIN mice compared to the WT (Fig. 8D and E). In B-RAFKIN/KINmice, they were located in the deeper part of the cortical plate (Fig. 8E), while in the WT they had arrived in layer close to the pia (Fig. 8D), indicative of a migration defect specific for this kind of neurons. To assess cell migration defects of the late-born neurons, we labeled proliferating cells in vivo with BrdU at E14.5 and compared the extent of neuronal migration 3 days later in WT control and B-RAFKIN/KIN embryos. As expected, many BrdU-labeled cells were present in the CP of control embryos (Fig. 8A). In contrast, about threefold fewer labeled cells were seen in the CP in B-RAFKIN/KIN embryos (Fig. 8B and C). Correct neuronal migration requires both radial glial fibers as guiding scaffolds for migrating neurons (34) and guidance cues such as Reelin secreted from Cajal-Retzius neurons that play a key role in neuronal lamination (28, 37). The alignment and density of radial glial fibers, labeled with an antibody against nestin, were not altered in B-RAFKIN/KIN (Fig. 8F to I). Furthermore, neither the number of Cajal-Retzius neurons nor their expression of Reelin, as judged from immunolabeling (Fig. 8H and I), were changed in the cortex of B-RAFKIN/KIN embryos. Thus, the migration defect in the B-RAFKIN/KIN cortex does not seem to be a consequence of a disrupted radial glial fiber system or a loss of Reelin-expressing Cajal-Retzius neurons but is most probably caused by a cell-autonomous defect in the cortical neurons themselves.


Figure 8
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  		FIG. 8. Impaired migration of cortical neurons in B-RAFKIN/KIN mice. (A and B) BrdU immunofluorescence in E17.5 sagittal sections. (C) Quantification of BrdU-positive cells (mean ± standard deviation) showed a change in the ratio of cells present in pia-proximal (CP) and subventricular-proximal (IZ/subventricular zone) neocortex (n = 3; P < 0.001, t test). (D and E) Brn-2 immunostaining of sagittal sections from E18.5 cortex of WT and B-RAFKIN/KIN embryos. Notably, Brn-2-stained cells were present in the central region of the cortex in B-RAFKIN/KIN embryos compared to their location close to the pia in WT embryos. (F to I) Nestin staining in E13.5 and E16.5 of B-RAFKIN/KIN embryos compared with WT showed a comparable number of radial glia cells and their fibers between both genotypes. (J to M) Reelin staining in E13.5 and E16.5 B-RAFKIN/KIN embryos compared with WT revealed similar numbers of Cajal-Retzius neurons and expression of the guidance cue Reelin. The developmental stages and genotypes are indicated. Scale bars, 100 µm (A to E), 50 µm (F to M).

To confirm this hypothesis, we isolated cortical neurons from E14.5 embryos and seeded them in the upper chamber of a Boyden transwell (Fig. 9A) using BDNF as a chemoattractant. The chambers were vertically separated by a pored filter (5 µm). Since BDNF was included only in the bottom chamber and cells were seeded in the upper chamber, cells would migrate across the pores toward the bottom side and the chemotactic factor (Fig. 9A). Migrating neurons were identified by Tuj1-ß immunostaining (Fig. 9B to E). As expected, BDNF treatment significantly enhanced the cell migration of WT cortical neurons (Fig. 9F) In contrast, B-RAFKIN/KIN cortical neurons failed to migrate specifically into the lower well. These findings indicate that B-RAF is required for BDNF-induced cell migration of primary cortical neurons.


Figure 9
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  		FIG. 9. B-RAF is required for BDNF-mediated cortical neuron migration. (A) Schematic representation of Boyden transwell chamber used for the migration assays of dissociated cortex neurons. (B to E) TuJ1-ß immunostaining of cortical neurons that had migrated onto the bottom side of the transwell membrane. Dissociated cortical neurons (E14.5) isolated from WT embryos (B and D) and B-RAFKIN/KIN (C and E) were placed into the upper well of a Boyden chamber in the absence (B and C) or presence (D and E) of 10 ng/ml BDNF in the bottom chamber. (F) Twenty-four hours after seeding, the total number of migrating cells, stained with TuJ1-ß, was quantified. Each assay was done in duplicate from at least four embryos of each genotype. Scale bars, 50 µm (B to E).


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DISCUSSION
 
By use of a knock-in strategy with A-RAF, we have rescued the midgestational lethality of B-RAF-deficient embryos, allowing us to delineate the role of B-RAF-specific function in development of the nervous system. A-RAF, the least effective MEK activator of the RAF kinase family, was expressed under the control of the B-RAF promoter in a mouse with a deletion of B-RAF. A-RAF rescued embryonic lethality in a non-Mendelian fashion by suppression of endothelial cell apoptosis. Rescued B-RAFKIN/KIN mice were indistinguishable from wild-type mice in their capability to promote the survival of cortical neurons and motoneurons. Dendrite formation and axon growth were markedly reduced. In vivo, migration of those cortical neurons present in layers II and III was impaired, and the neurons were retained in layer IV, suggesting a role for B-RAF in neuronal migration.

Apoptosis suppression by A-RAF. The organs of B-RAFKIN/KIN mice appeared normal and proportional to body size, excluding the thymus and spleen, which were reduced 10-fold in weight compared to wild-type littermates, indicating the need for B-RAF-specific function in hematopoiesis. Typing for lymphoid and myeloid lineages suggests deficiencies at the level of early progenitor cells (U. Bommhardt, L. Nitschke, and U. R. Rapp, unpublished). Only recently, the interaction of neural stem cells with their local extracellular microenvironment or niche has been investigated. It has become clear that neural stem cell expansion and differentiation are regulated in vivo and in vitro by environmental factors synthesized in the stem cell niche. In the developing central nervous system, ventricular zone cells produce vascular endothelial growth factor, which attracts vessel growth toward them (4). It has also been shown in coculture experiments that endothelial cells stimulate self-renewal and induce neurogenesis of neural stem cells in vitro (39). Thus, loss of endothelial cells in the niche populated by neural stem cells would be expected to adversely affect proliferation of neural stem cells and their ability to differentiate into neurons. Loss of signaling caused by death of endothelial cells in B-RAF-deficient mouse embryos might contribute to the increased rate of apoptosis of differentiating neurons in the forebrain at E10.5. Besides suppression of apoptosis in endothelial cells by A-RAF in B-RAFKIN/KIN mice, two other neuronal cell types that undergo apoptosis when B-RAF is missing were rescued in B-RAFKIN/KIN animals: cortical neurons and motoneurons. Three possibilities may be envisioned for how A-RAF, expressed from the B-RAF promoter and therefore under physiological regulation, might protect from cell death. First, an A-RAF-specific function in apoptosis suppression could be at play. It was described that the N-terminal part of A-RAF specifically interacts with two mitochondrial proteins, hTOM and hTIM, which are similar to components of mitochondrial outer and inner membrane protein-import receptors from lower organisms. Moreover, it was reported that A-RAF is imported into mitochondria via a system for isoform-specific uptake (52). Rescue of B-RAF-deficient mice from endothelial apoptosis may be related to such mitochondrial activity of chimeric A-RAF. Indeed, our preliminary results demonstrate the ability of recombinant A-RAF from SF9 insect cells to directly phosphorylate proapoptotic BH3-only protein BAD in vitro (unpublished data). Another explanation for the antiapoptotic function of B-RAFKIN kinase may be a cooperation between A-RAF and C-RAF. Early evidence for B-RAF/C-RAF cooperation through heterodimer formation (42, 45, 48) has recently been confirmed in an elegant series of experiments (8). In addition, there is also genetic evidence for A-RAF/C-RAF cooperation (23, 48). Recent studies demonstrated an important role for C-RAF in protection of vascular endothelial cells from apoptosis via its action on two distinct signaling pathways: (i) through vascular endothelial growth factor-mediated ERK activation and (ii) through basic fibroblast growth factor-mediated accumulation of C-RAF at mitochondria instrumental for the antiapoptotic activity of C-RAF (1, 16). A third possible explanation for the antiapoptotic function of chimeric A-RAF which harbors the N-terminal 155 amino acids of B-RAF might be due to the survival function of a B-RAF effector activated by this N-terminal domain. A candidate for this role is PA28{alpha}, a subunit of the 11S regulator of proteasomes, known to specifically interact with B-RAF N terminus (19). The connection between proteasomal activity and apoptosis has been described. Dependent on the cellular background, programmed cell death was induced or inhibited by the proteosomal inhibitor lactacystin (13, 13, 38). Neurons were previously shown by us to specifically require B-RAF function for responsiveness to neurotrophic factors (46). C-RAF could not compensate for the loss of B-RAF. The basis for the different finding with A-RAF may be A-RAF/C-RAF heterodimer formation analogous to B-/C-RAF heterodimers that we have described earlier (45). Such heterodimers might be formed due to the presence of the N-terminal part of B-RAF in the fusion protein overexpressed in the B-RAFKIN/KIN mice and allow the B-RAF function to be partially complemented, e.g., in mitochondria. If this would be the case, A-/C-RAF dimers but not C-RAF homodimers would be predicted to couple neurotrophic receptors to the mitogenic cascade and/or survival signaling at the mitochondria.

Role of B-RAF in neuronal migration. The cortex is formed through the coordinated processes of neurogenesis and migration. Loss of B-RAF led to a severe reduction of Brn-2 expressing pyramidal projection neurons in the upper layers of the postnatal cortex and most dramatically in the somatosensory and visual cortex (Fig. 6F). This observation was puzzling in light of the finding that, at E18.5, the number of Brn-2-positive cells was not reduced (Fig. 8E), although they were misplaced. Conceivably, the "improper" environment distant from the pia might have induced a down-regulation of Brn-2 expression, leading in consequence also to a loss of dendrite formation. In melanoma cells, Brn-2 has been shown to be activated either by B-RAF (10) or by Wnt signaling (9). In cortical neurons, Brn-2 controls in a cell autonomous manner the expression of p35, a regulatory subunit of Cdk5 (22), and the signal transducer Dab (40) and thereby mediates migration of cortical neurons. Two different modes of radial migration have been described for newly generated neurons (26). In somal translocation, neurons move their cell soma toward a leading process that is stably attached to the pial surface. Later on during embryogenesis, when the cortex has grown in size, neurons move by locomotion along radial glial fibers. Based on the timing of the histological defects and misplaced neurons, we conclude that migration by locomotion is impaired in neurons lacking B-RAF. Starting at E14.5, we observed a reduction in proliferation and migration, indicating that these cellular activities are dependent on B-RAF. Four distinct phases have been discerned in locomotion at this time of corticogenesis: initial radial migration, arrest of migration in the subventricular zone, migration back toward the ventricle, and finally, secondary migration to the appropriate layer (27). What step in migration is specifically impaired cannot be concluded from static histological analysis but would require time-lapse imaging of slice cultures. In contrast to neurons, lack of B-RAF in fibroblasts led to an increase in migration, in conjunction with reduced levels of F-actin which have been proposed to act through reduced ROCKII expression and impaired phosphorylation of cofilin (31). We have recently uncovered a role of C-RAF in cell migration (33). This work has identified prohibitin as an essential component of MEK/ERK signaling in epithelial cell adhesion and migration. In the absence of prohibitin, the C-RAF kinase fails to become activated downstream of Ras, so that the cells remain adhered very tightly and epidermal growth factor no longer induces cell migration. Considering that cortical neurons migrate along radial glial fibers and that their migration modes are more complex than those of fibroblast cells, the mechanistic details of how B-RAF controls cortical migration remain to be elucidated. In summary, we have shown that the survival function of B-RAF is replaced by that of A-RAF in several cell systems but A-RAF does not fully substitute for B-RAF in neuronal migration and dendrite formation, indicating a specific requirement for B-RAF in these processes.


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ACKNOWLEDGMENTS
 
We thank A. Gancher, N. Gribanov, D. Heim, M. Mennig, and H. Troll for expert technical assistance and members of our group for their support. We are grateful to I. Girkontaite, K. D. Fischer, and K. Weber for materials and Ester Asan, H. Calderon-Sanchez, I. Varela-Nieto, and Lev M. Fedorov for technical advice. We thank R. McKay (NIH, Bethesda, MD) for providing the nestin antibody via the DSHB (Iowa) and M. Wegner for the kind donation of Brn-2 antibody.

This work was supported by Deutsche Forschungsgemeinschaft SFB 581 TP B5, GK1048, and DAAD grant D/04/39971. G.C. was a postdoctoral fellow supported in part by the Spanish Government (Ministerio de Educación y Cultura).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany. Phone: 49 931 201 45141. Fax: 49 931 201 45835. E-mail: rappur{at}mail.uni-wuerzburg.de. Back

{dagger} These authors contributed equally to this work. Back


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Molecular and Cellular Biology, October 2006, p. 7103-7115, Vol. 26, No. 19
0270-7306/06/$08.00+0     doi:10.1128/MCB.00424-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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