Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (19), 7103-15

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

Affiliations

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

Guadalupe Camarero et al. Mol Cell Biol.

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.

Figures

FIG. 1.
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).
FIG. 2.
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-β antibody (red, arrowheads indicate misplaced neurons). (E) Quantification of Tuj1-β-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-β 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.
FIG. 3.
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.
FIG. 4.
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).
FIG. 5.
FIG. 5.
Cortical pathology in B-RAFKIN/KIN embryos. (A and B) Tuj1-β 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-β 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-β-positive cells in the IZ, beneath the SP, is increased in the B-RAFKIN/KIN neocortex (E). Scale bars, 100 μm.
FIG. 6.
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.
FIG. 7.
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).
FIG. 8.
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).
FIG. 9.
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-β 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-β, was quantified. Each assay was done in duplicate from at least four embryos of each genotype. Scale bars, 50 μm (B to E).

Similar articles

See all similar articles

Cited by 8 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback