A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool

Abstract

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.

Figure 2

Tissue specificity of Krox20-cre–induced mutations. (A–E) Nerves from reporter lacZ mice (see Akagi et al. 1997) that also contain a Krox20-cre allele were stained for β-galactosidase or β-galactosidase activity. (A and B) Teased nerves stained with X-gal (A) and DAPI (B). (C) X-gal–stained nerves were also osmicated, sectioned, and counterstained with neutral red and eosin. The myelin and axon of an individual nerve fiber stained dark and light red, respectively. (D and E) Teased nerve fiber triple stained with antibodies against myelin basic protein (D, red), β-galactosidase (D, green), and DAPI (E, blue). Arrows point towards β-gal–positive Schwann cells; arrowheads point towards other cells and nuclei that do not stain; perineurium (p) and capillaries (cp). (F) Southern hybridization analysis of DNA digested with BamHI and EcoRV. Lane 1, erbB2 ^Δ/+; lane 2, wild type; lanes 9 and 10, erbB2^flox/+; all other lanes contain DNA obtained from tissues of mice with a genotype Krox20-cre/+; erbB2^flox/+. Lane 3, liver; lane 4, muscle; lane 5, ear; lane 6, lung; lane 7, skin; lane 8, spleen; lanes 11 and 12, sciatic nerves; lanes 13 and 14 spinal roots; lane 15, spinal cord; and lane 16, ovaries. Densitometric analysis showed that recombination occurred in 40–50% of cells associated with peripheral nerves. Bars: (B) 50 μm; (C and E) 20 μm.

Figure 3

Peripheral nerve histology in mice with conditional erbB2 mutation. Semi-thin sections of peripheral nerves from Krox20-cre/+; erbB2^flox/− (A, C, E, G, and I) and control (B, D, F, H, and J) animals. The age of the animals is indicated. Shown are the tibial (A–D) and sural (E and F) branches of the sciatic nerve and nerves innervating lower leg muscles (G–J). Arrowheads (C, E, G, and I) point towards axons with abnormally thin myelin sheaths. The arrow in E points towards rare axons with myelin of normal thickness; arrows in G and I point towards large diameter axons that are not myelinated. Bar, 20 μm.

Figure 4

Electron microscopy of sciatic nerve branches from conditional erbB2 mutants. Electron microscopic analysis of peripheral nerves from Krox20-cre/+; erbB2^flox/− (A and C) and control (B and D) animals at 6 mo of age. Shown are sural nerves (A and B) and nerves innervating lower leg muscles (C and D). Arrowheads (A and C) point towards abnormally thin myelin sheaths in nerves from mutant mice; the arrow in C indicates a large diameter axon surrounded by a basal lamina, which is also shown enlarged in the inset. Asterisks denote bundles of nonmyelinated small diameter axons. Bars, 1 μm.

Figure 5

Histology of spinal nerve roots from conditional erbB2 mutants. Semi-thin sections of ventral roots from Krox20-cre/+; erbB2^flox/− (A and C) and control (B and D) animals at P15 (A and B) and adult stages (C and D). Arrowheads (A and C) point towards the abnormal perineuria observed in mutant animals. Note that in mutant animals, the extreme lack of Schwann cells observed at P15 is compensated at later stages, although myelin sheaths remain abnormally thin. Bar, 20 μm.

Figure 6

Growth and biochemical properties of Schwann cells in conditional erbB2 mutants. Fractions of nuclei, which were positive for BrdU (A) or TUNEL (B) in sciatic nerves or in the ventral roots, were determined from control or Krox20-cre/+; erbB2^flox/− mice at the indicated ages. Data are shown as mean ± SD; *P < 0.17; **P < 0.01. (C) Western analysis of extracts from sciatic nerves using antibodies directed against Krox20, SCIP, P₀, and PMP22. Ages of control (C) and Krox20-cre–induced mutant (M) animals are indicated. Asterisk denotes lanes loaded with extracts from COS cells transfected with Krox20 or SCIP expression plasmids.