PMC

Elevated and protracted expression of Brn-2 in nerves of Oct-6^βgeo/ΔSCE mice. (A) EMSA with whole-cell extracts from sciatic nerves of P4 Oct-6^βgeo/ΔSCE mice and a radiolabeled octamer probe reveal a strong complex that migrates at the same position as Brn-2 and Oct-6 DNA complexes. This complex is supershifted with antibodies against Brn-2, unmasking the low residual expression of Oct-6 from the ΔSCE allele. Preimmune serum affects none of these complexes. (B) Western blot analysis confirms that Brn-2 protein levels are elevated in the nerves of Oct-6^βgeo/ΔSCE vs. Oct-6^ΔSCE/+ nerves at P4. Tubulin served as a loading control. (C) Semiquantitative RT–PCR using RNA extracted from newborn sciatic nerves shows the higher Brn-2 mRNA steady-state levels in Oct-6^βgeo/ΔSCE mice than in Oct-6^ΔSCE/+ mice. Cyclophilin mRNA served as a control. (D) Brn-2 is expressed in the nucleus of Schwann cells in the nerves of P4 Oct-6^βgeo/ΔSCE mice. Nerves were dissected and teased into single fibers and incubated with antibodies against mouse Brn-2 (α-Brn-2) in green and Neurofilament medium chain (NFM) in red. (E) Expression of Brn-2 and Oct-6 was examined in developing nerves of Oct-6^ΔSCE/+ and Oct-6^βgeo/ΔSCE animals at E17 and P1–P32. Amounts of nerve extract loaded were normalized for acetylated α-tubulin. The build-up of P-zero immunoreactivity over time illustrates the progression of myelination in both genotypes.

(A) The floxed Brn-2 locus is effectively recombined by the Cre recombinase in peripheral nerve. DNA was extracted from adult sciatic nerve of wild-type (wt), Brn-2^wt/flox, Brn-2^flox/flox, and Brn-2^flox/floxCre (Brn-2^flox/flox mice transgenic for the DhhCre transgene) animals, digested with BamHI, and subjected to Southern blot analysis with probe B. Probe B detects fragments of 7.3 kb (wild-type allele), 8.3 kb (targeted allele), and 14 kb (recombined allele). The nonrecombined band present in lane 4 results from DNA derived from cells that do not express the DhhCre transgene, such as nerve sheath cells. (B) The recombination of the Brn-2 allele in Schwann cells is complete, as Brn-2^flox/floxCre nerves do not express Brn-2 atP4 (lanes 1,2). Complete deletion of Brn-2 does not affect Oct-6 expression, as Oct-6 expression levels are the same in nerves of Oct-6^ΔSCE/+ and Brn-2^flox/floxCre animals (cf. lanes 3 and 4). (C) Deletion of Brn-2 in Schwann cells does not affect the morphological maturation of the nerve. The overall morphology appears very similar in cross sections of nerves from both genotypes. Resin sections (1 μm) of P4 sciatic nerves of Brn-2^flox/flox (panel a) and Brn-2^flox/flox/Cre (panel b) animals were stained with ppd. Bar, 10 μm (applies to both micrographs).

Schwann cell-specific deletion of Brn-2 in an Oct-6^βgeo/ΔSCE background results in a severe hypomyelination phenotype. (A) Representative sections (1 μm; ppd-stained) are shown from sciatic nerves at three developmental time points (P16, P56, and P120). In Oct-6^ΔSCE/+ mice, most myelin-competent axons are actively being myelinated by P16 (panel a). At P56 and P120, these nerves are fully matured (panel d). This pattern contrasts with that observed in Oct-6^βgeo/ΔSCE animals, which exhibit a strong delay in the differentiation of Schwann cells (; ; ). At P16, a majority of Schwann cells is arrested at the promyelin stage, with only the largest axons myelinating (panel b). By P56, most if not all myelinating Schwann cells have elaborated a myelin sheath (panels e,h; P120). Deletion of Brn-2 in this genetic background results in a dramatic increase in the severity of the phenotype. At P16, essentially all myelinating Schwann cells are arrested at the promyelin stage of differentiation (panel c). Even at P56 and P120, myelination is largely abnormal, with large numbers of cells at the promyelin stage (panel f). Those axons that are myelinated have very thin myelin sheaths. Bar: panels a–f, 20 μm. (B) Quantification of delayed myelination in genotypes presented in A.

Brn-2 is expressed in chick and mouse sciatic nerves. (A) Developmental expression of a novel octamer-binding complex was examined by EMSA using freeze-thaw extracts from chicken nerves at different stages of development [embryonal stages E14, E17, and E20 and postnatal days 3 (P3) and 20 (P20)]. Whole-cell extracts of COS cells expressing chicken Oct-6 (Oct-6) served as a control (). Free probe is not shown, but all experiments were performed in probe excess. (B) Using E17 chick embryonic nerve, extracts identify Brn-2 in complex X, as antibodies directed against the C-terminal part of mouse Brn-2 specifically affect complex X, whereas chicken Oct-6 antibodies (α-Oct-6) affect the Oct-6 complex but not complex X. Preimmune serum (Pre) does not affect either complex. (C) Brn-2 is expressed in mouse nerves. Incubation of Oct-6-specific antibodies with P4 mouse whole-nerve extracts and an octamer probe results in the formation of a ternary complex (supershift Oct-6 complex; ssOct-6) and unmasks another complex that is specifically supershifted (ssBrn-2) with mouse Brn-2 antibodies (α-Brn-2). When both antibodies are added all complexes are shifted, demonstrating that no other complexes comigrate with the Oct-6/DNA and Brn-2/DNA complexes. (D) Brn-2 is expressed in the Schwann cell lineage. Whole-cell extracts from cultured rat Schwann cells grown in the presence of 20 μM forskolin for 36 h were incubated with the octamer probe in presence of the indicated sera. As in C, Oct-6- and Brn-2-specific antibodies identify two comigrating Oct-6 and Brn-2 protein DNA complexes.

HA-Oct-6 and HA-Brn-2, but not HA-Brn-5, can rescue the developmental delay phenotype of Oct-6^βgeo/ΔSCE mutant mice. (A) Schematic representation of the constructs used to generate mice transgenic for HA-Oct-6 (SCE Oct-6), HA-Brn-2 (SCE Brn-2), and HA-Brn-5 (SCE Brn-5). Two restriction sites used in the generation of these constructs are indicated [NotI (N) and SwaI (Sw)]. The SCE is indicated as a gray box and the triple HA-tag as a yellow box. The intron-less Oct-6 gene is shown as a thick black line. The ORF of Oct-6 is in light green, that of Brn-2 is in pink, and that of Brn-5 is in orange, with their POU-specific domain in dark blue and their POU-homeodomain in light blue. (B) Oct-6^βgeo/ΔSCE mutant mice that express HA-Oct-6 or HA-Brn-2 show high levels of P-zero protein expression in sciatic nerve. Western blot analysis of P4 sciatic nerve extracts from wild-type (wt; lane 1), Oct-6^βgeo/ΔSCE (lane 2), Oct-6^βgeo/ΔSCE/SCE Oct-6 (lane 3), Oct-6^βgeo/ΔSCE/SCE Brn-2 (lane 4), and Oct-6^βgeo/ΔSCE/SCE Brn-5 (lane 5) animals. Levels of HA-tagged proteins are assessed with α-HA antibodies. The amount of protein loaded per lane is estimated by the intensities of the α-tubulin immunoreactive band. (C) Comparison of the morphology of cross sections through P4 sciatic nerves of Oct-6^ΔSCE/+ (panel a), Oct-6^βgeo/ΔSCE (panel b), Oct-6^βgeo/ΔSCE/SCE Oct-6 (panel c), Oct-6^βgeo/ΔSCE/SCE Brn-2 (panel d), and Oct-6^βgeo/ΔSCE/SCE Brn-5 (panel e) animals. Plastic-embedded osmicated nerves were sectioned at 1 μm and stained with ppd. Myelin is strongly stained by this compound and appears as dark rings in cross sections. Bar, 20 μm. (D) Quantification of the promyelin–myelinating transition in nerves of animals in C.

(A) The targeting strategy to generate an inducible deletion allele of Brn-2. The structure of the wild-type Brn-2 locus is shown at the top. The Brn-2 single exon gene is represented in olive green with the POU domain in light green. The Southern blot probes A and B are indicated as horizontal black bars. The positions of restriction enzyme cutting sites are shown for BamHI (B), HindIII (H), EcoRI (E) and ScaI (S). In the targeting construct (targeting vector), the neomycin (NEO) expression cassette (purple box) is flanked by FRT sites (vertical red bars) followed by a 3′ LoxP site (blue triangle) and an eGFP reporter gene (dark green box). The orange box represents the counter-selection cassette, containing the thymidine kinase gene (TK). The targeted allele (the floxed allele) obtained after homologous recombination is shown below the targeting construct. Cre-mediated recombination removes Brn-2 sequences and generates the recombined allele (bottom). (B) Schwann cell-specific recombination is achieved through expression of the Cre recombinase under control of Dhh gene regulatory sequences. The 19-kb construct containing the entire Dhh gene with its three exons (in green) is shown. The Cre recombinase ORF (indicated in red) is preceded by a nuclear localization peptide sequence and cloned in-frame with the Dhh ORF. Restrictions sites for NotI (N) and BamHI (B) are indicated. Expression of the Cre recombinase was examined in crossing with the ROSA26lacz (R26R) reporter mouse. (Panel a) Whole-mount staining of E12.5 embryo reveals expression in the PNS, the snout, and part of the vasculature. (Panel b) Paraffin sections of a stained E14.5 embryo demonstrate the strong expression in immature Schwann cells that populate the peripheral nerves. (Panel c) Strong expression is also observed in the seminiferous tubules of the testis, where Dhh is expressed in the Sertoli cells.