PMC

Brdp heterozygous mice are hyperactive. Wild-type and brdp/+ heterozygous mice (n = 6 for each class) were measured in an open field for 30 min. Heterozygotes covered more distance (A) and were more exploratory (B) than wild-type animals. Data are shown normalized to wild-type ± SEM.

Brdp/brdp mutants have significant cortical malformations. (A, D and G) Wild-type, brdp/+ and brdp/brdp embryos are indistinguishable upon initial dissection at E18.5 except for subtle craniofacial defects in the mutants. (B, E and H) Microdissection of the brain reveals significant phenotypes only in the brdp/brdp mutants including smaller olfactory bulbs (asterisks) and ventriculomegaly. Histological analysis (C and F) in the coronal plane shows no significant differences between wild-type and brdp/+ mice. (I) The brdp/brdp mutant cortical neuroepithelium is thinner than wild-type and the reduction is most pronounced in the caudal and lateral cortices. Agenesis of the CC is also noted in the brdp/brdp mutants (arrow).

Histological analysis of brdp/brdp phenotype. (A–H) Brdp/brdp phenotypes at E18.5 include cortical thinning, loss of striatal tissue and agenesis of the CC (Probst bundles indicated by asterisks; B). A higher magnification view shows wave-like perturbations in the cortical plate (D), a smaller, irregular VZ (D and F) and the loss of striatal tissue (H). (I–N) Sections from more posterior regions of the brain show a more severe phenotype (compare B and J) with the dramatic thinning of the neuroepithelium especially pronounced in the area of the rhinencephalon (J and N) (VZ, ventricular zone; the double asterisk in E and J indicates the choroid plexus).

Brdp defects are visible throughout cortical development. (A–D) Brdp/brdp phenotypes including cortical thinning, loss of striatal tissue, agenesis of the CC and anterior commissure are evident by E16.5 in the anterior forebrain (B). Similar to perinatal stages, the brdp defect increases in severity in more posterior regions (D). At E14.5 (E–H), the loss of basal ganglionic structures is already evident throughout the anterior–posterior axis (E and F anterior to G and H). (I and J) At E11.5, the onset of neurogenesis, the brdp forebrain looks largely normal, suggesting that initial pattering is correct (ac, anterior commissure; bg, basal ganglia; s, striatum).

Brdp heterozygous mice have cortical defects. RNA in situ hybridization for multiple markers of cortical development shows subtle defects in the brdp/+ heterozygous cortex. Each experiment was quantified as shown. Data are presented as the number of cells expressing the marker/ImageJ area, normalized to the wild-type average + SEM. Expression is shown for Tbr1 (A), Pvrl3 (B), Rorb (C), Fezf2 (D), Foxp2 (E) and Gad1 (F). No significant difference was seen in the number of expressing cells for any gene except Gad1, which shows an 11% decrease in heterozygous compared with wild-type (P = 0.037). Pvrl3 is not expressed in significantly fewer cells but is expressed at generally lower levels and over a broader portion of the cortex.

Tubb2b is the gene mutated in brdp mutants. (A) Sequencing of the Tubb2b locus identified a single-nucleotide change (indicated by an asterisk in brdp/brdp mutants resulting in a coding change from an asparagine residue to serine). (B) This N247S mutation is in a hyper-conserved region of the β-tubulin gene family: a GxxNxD domain conserved in all species examined. (C) The structure of the assembled α-tubulin (red structure) and β-tubulin (blue structure) subunits with the N247 residue of the β-subunit shown in yellow and the non-hydrolyzed GTP in green (pdb:1JFF). (D) More detailed view of the residue mutated in brdp mutants. (E–P) Transfection of wild-type Tubb2b-eGFP (E–G, K–M) or a Tubb2b^brdp-eGFP (H–J, N–P) into NIH3T3 cells followed by immunostaining for α-tubulin shows that both wild-type Tubb2b-eGFP and mutant Tubb2b^brdp-eGFP can incorporate into microtubular networks. (K–P) The microtubule network was depolymerized by nocodazole treatment and after a 10 min recovery; both wild-type and mutant monomers are incorporated into actively growing microtubules.

Molecular analysis of cortical development in brdp/brdp mutants reveals multiple defects. (A–D) The number of mitotic cells measured with pHH3 is increased in mutants at E14.5. We also note a significant increase in pHH3-positive cells in the intermediate zone (IZ; arrows in B). (E and F) Phospho-vimentin staining is also increased in mutants. (G–I) Tbr2 immunopositive cells are increased in mutants at E14.5. (J and K) Neuronal differentiation as marked by TuJI (E14.5) expression is reduced in mutants. Radial glial cells look morphologically normal in mutants (L and M). (N–P) Cell death is increased in the mutant cortex at E18.5 (N and O) and in both the striatum and the cortex at E16.5 (P). (Q and R) Neurons labeled with BrdU at E14.5 are able to migrate to the outer cortical plate by E18.5 in both mutant and wild-type. All data are graphed as a normalization to wild-type ± SEM. *P < 0.01, **P < 0.001.