Delayed development of nervous system in mice homozygous for disrupted microtubule-associated protein 1B (MAP1B) gene

Abstract

Microtubule-associated protein 1B (MAP1B), one of the microtubule-associated proteins (MAPs), is a major component of the neuronal cytoskeleton. It is expressed at high levels in immature neurons during growth of their axons, which indicates that it plays a crucial role in neuronal morphogenesis and neurite extension. To better define the role of MAP1B in vivo, we have used gene targeting to disrupt the murine MAP1B gene. Heterozygotes of our MAP1B disruption exhibit no overt abnormalities in their development and behavior, while homozygotes showed a slightly decreased brain weight and delayed nervous system development. Our data indicate that while MAP1B is not essential for survival, it is essential for normal time course development of the murine nervous system. These conclusions are very different from those of a previous MAP1B gene-targeting study (Edelmann, W., M. Zervas, P. Costello, L. Roback, I. Fischer, A. Hammarback, N. Cowan, P. Davis, B. Wainer, and R. Kucherlapati. 1996. Proc. Natl. Acad. Sci. USA. 93: 1270-1275). In this previous effort, homozygotes died before reaching 8-d embryos, while heterozygotes showed severely abnormal phenotypes in their nervous systems. Because the gene targeting event in these mice produced a gene encoding a 571-amino acid truncated product of MAP1B, it seems likely that the phenotypes seen arise from the truncated MAP1B product acting in a dominant-negative fashion, rather than a loss of MAP1B function.

Figure 1

Targeted disruption of the MAP1B gene in ES cells and mice. (A) Targeting strategy: Wild-type allele containing the first coding exon of the MAP1B gene, replacement-type targeting vector, and targeted allele after homologous recombination are shown. P1, primer 1; P2, primer 2; P3, primer 3; B, BamHI; E, EcoRI; H, HindIII. Bar, 1 kb. (B) Genotype of F2 mice of R21 line. Tail DNA was isolated and analyzed by Southern blotting using the 5′-flanking probe (A, probe) after digestion with BamHI. Lane 1, MAP1B−/− mouse; lane 2, MAP1B+/− mouse; lane 3, MAP1B+/+ mouse. WT, wild-type band; HR, homologous recombinant band.

Figure 2

Comparison of our gene disruption strategy with that used by Edelmann et al. (1996). (A) MAP1B polyprotein. 3d2 and YXY, the regions to which two rabbit anti-MAP1B antisera bind, are shown. LCBS, light chain binding site; MTBD, microtubule-binding domain; CTIR, COOH-terminal imperfect repeats; LC1, light chain 1; AA, amino acids. (B) Predicted truncated product of the gene as disrupted by Edelmann et al. (1996). (C) Predicted truncated product of the gene as disrupted by us.

Figure 3

Immunoblot analysis of crude extracts of brain with the anti-MAP1B antiserum 3d2 (A), with the anti-MAP1B mAb 1B6 (B), with the anti-MAP2 mAb HM2 (C), with the anti-tau mAb tau-1 (D), and with the anti-MAP1A mAb 1D1 (E). (A and B) Lane 1, MAP1B+/+ brain at postnatal day 4; lane 2, MAP1B−/− mutant brain at postnatal day 4; lane 3, MAP1B+/+ brain at postnatal week 8; lane 4: MAP-1B−/− brain at postnatal week 8. NF-M, neurofilament M. (C–E) Lane 1, MAP1B+/+ brain at postnatal week 8; lane 2, MAP-1B−/− brain at postnatal week 8.

Figure 4

(A and B) Immunofluorescence micrographs of adult cerebellum. Sagittal cryosections of cerebellar vermis of MAP1B+/+ (A) and −/− (B) mice were stained with anti-MAP1A mAb 1D1. (C and D) Paraffin sections of retinae of MAP1B+/+ (C) and −/− (D) mice stained with hematoxylin-eosin are shown. There are no morphological differences between the MAP1B+/+ and −/− tissues.

Figure 5

(A and C) Schematic illustrations of optic nerve at postnatal day 8 (A) and postnatal week 8–14 (C), showing levels A, B, C, and D, at which ultrathin sections were taken for EM analysis. Each level was determined by the distance from the optic chiasm. (B and D) Quantitative comparison of the numbers of myelinated axons per unit area in juvenile (B) and adult (D) optic nerves of MAP1B+/+ and −/− mice. Solid bars represent each value (per 10² μm²) for the optic nerve of a MAP1B+/+ mouse. Open bars represent each value (per 10² μm²) for the optic nerve of a MAP1B−/− mouse. PD, postnatal days; PW, postnatal weeks.

Figure 6

Electron micrographs showing representative areas of cross sections of optic nerves of MAP1B+/+ (A) and −/− (B) mice at postnatal day 8. Axons in the MAP1B−/− optic nerve (B) are less myelinated than those in the MAP1B+/+ optic nerve (A). Bar, 2 μm.

Figure 7

Quantitative comparison of the numbers of myelinated axons (per 10² μm²) in juvenile (A) and adult (B) anterior pyramidal tract axons of MAP1B+/+ and −/− spinal cords. Solid bars represent each value (per 10² μm²) for the spinal cord of a MAP1B+/+ mouse. Open bars represent each value (per 10² μm²) for the spinal cord of a MAP1B−/− mouse. PD, postnatal days; PW, postnatal weeks.

Figure 8

Electron micrographs showing representative areas of cross sections of optic nerves of MAP1B+/+ (A) and −/− (B) mice at postnatal week 14. There are no differences in the number of myelinated axons per unit area, diameter of axons, thickness of myelin sheaths, and density of MTs. Bar, 500 nm.

Figure 9

Quantitative comparison of the axonal diameters in juvenile (A) and adult (B) optic nerves of MAP1B+/+ and −/− mice. Solid bars represent mean values ± SD (μm) for axons in a MAP1B+/+ mouse. Open bars represent mean values ± SD (μm) for axons in a MAP1B −/− mouse. *Different from value for controls at P < 0.0005, ** at P < 10⁻⁸, and *** at P < 10⁻¹¹. Student's t test was used to determine the significance of the differences. Levels A, B, C, and D are determined in the same way as described in Fig. 5 legend. PD, postnatal days; PW, postnatal weeks. n, the number of axons examined.

Figure 9

Quantitative comparison of the axonal diameters in juvenile (A) and adult (B) optic nerves of MAP1B+/+ and −/− mice. Solid bars represent mean values ± SD (μm) for axons in a MAP1B+/+ mouse. Open bars represent mean values ± SD (μm) for axons in a MAP1B −/− mouse. *Different from value for controls at P < 0.0005, ** at P < 10⁻⁸, and *** at P < 10⁻¹¹. Student's t test was used to determine the significance of the differences. Levels A, B, C, and D are determined in the same way as described in Fig. 5 legend. PD, postnatal days; PW, postnatal weeks. n, the number of axons examined.