Conditional Mutagenesis of erbB2
We generated an erbB2flox
allele in which two loxP
sites flank three exons encoding part of the extracellular domain of the receptor. We chose these exons since their deletion introduces a frameshift mutation, which results in the production of an mRNA that encodes a truncated receptor. A two-step procedure was employed to generate the floxed allele in ES cells (; see also Gu et al. 1993
). The mutant ES cells were used to generate mice that carry the erbB2flox
allele. Mice homozygous for the erbB2flox
allele appeared normal and were fertile. The erbB2flox
allele was crossed into a cre
-deleter strain (Schwenk et al. 1995
). The resulting erbB2Δ
allele ( A) was combined with the previously described genetical-null allele of erbB2
(Britsch et al. 1998
mice die before E11.5 and are phenotypically indistinguishable from erbB2
−/− mice, indicating that Cre-induced recombination of the erbB2flox
allele generates a nonfunctional erbB2
gene encodes a transcription factor expressed in promyelinating and myelinating Schwann cells and, thus, appears from ~E16 in peripheral nerves (Topilko et al. 1994
). Furthermore, Schwann cell precursors that reside in the spinal roots express Krox20
earlier, starting ~E11. To mutate erbB2
, we used a mouse strain in which cre
was introduced into the Krox20
locus by homologous recombination. In this strain, cre
expression faithfully reproduces the expression pattern of the endogenous Krox20
gene; its detailed characterization is reported elsewhere (Voiculescu et al. 2000
). The Krox20-cre
allele was combined with a reporter allele, a lacZ
gene which is expressed upon Cre-induced recombination (Akagi et al. 1997
). In teased preparations of the sciatic and other nerves of such mice, we observed β-galactosidase staining in the majority of cells with elongated nuclei that were spaced at regular intervals along the nerve. In contrast, more closely and irregularly spaced cells were not stained ( and ). Sectioning of nerves, complemented by immunohistochemical analysis of teased fibers using antibodies directed against β-galactosidase and myelin basic protein confirmed the identity of the stained cells as myelinating Schwann cells (, C–E). Southern hybridization was used to quantify recombination of the erbB2flox
allele, and showed that recombination occurred in 40–50% of cells associated with sciatic nerves and spinal roots in animals with the genotype Krox20-cre
/+ ( F). It should be noted that these nerves contain significant numbers of nonmyelinating Schwann cells, connective tissue, blood cells, and vessels in which recombination does not occur. Recombination was also observed in the skin and ear: hair follicles express Krox20
, accounting for the recombination in these tissues (Levi et al. 1996
). No recombination was detected in the spinal cord.
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 (more ...)
ErbB2 Is Required for Correct Myelination of Peripheral Nerves
Conditional mutant animals with the genotype Krox20-cre/+; erbB2flox/− were viable, but displayed various behavioral abnormalities within the first weeks of postnatal life. Alterations included kinked or serpentine tails, gait abnormalities, difficulties in hindlimb movement, and wasting associated with weight loss and premature death (5 out of a group of 37 mutants died within 6 mo). Such behavioral abnormalities were observed in all conditional mutants, but were variable in the time of onset and severity. Control animals, for instance animals with the genotype Krox20-cre/+; erbB2+/−, did not display these behavioral phenotypes.
Examination of the sciatic nerves from Cre-induced mutants showed no gross changes in morphology or histology at P3.5 ( A, compare to B), indicating that the Schwann cell precursor pool in this nerve is established. However, sciatic nerves of mutants at P15 were translucent and thin. Histologically, a strikingly reduced thickness of myelin sheaths was apparent ( and ). Reduced thickness of the myelin sheaths persisted, and was also observed at 6 or 14 mo (, E–J, , arrowheads, and data not shown). Myelin of normal thickness ( E, arrow) was found only in 1.7 ± 1.0% of all myelinated axons in the sciatic nerve. Thus, the Krox20-cre–induced erbB2 mutation reproducibly affected virtually all myelinating Schwann cells. Thin myelin was not observed in control mice with a genotype Krox20-cre/+; erbB2+/−, or Krox20-cre/+; erbB2flox/+, or Krox20+/+; erbB2flox/−, or Krox20+/+; erbB2flox/+. Electron micrographs were used to quantify myelin thickness (). In 6-mo-old animals, the thickness of myelin was reduced two- to threefold. This is reflected in G ratios (ratios of axon diameters to fiber diameters) of 0.83 ± 0.04 in mutant, and 0.68 ± 0.04 in control sciatic nerves (P < 10−6). Hypomyelination in sciatic, sural, saphenous nerves and nerves innervating lower leg muscles were observed in all mutant animals examined (28 animals). In the spinal roots, the phenotype was more dramatic (see below). The thin myelin was due to the presence of fewer wraps of myelin around the axon.
Figure 3 Peripheral nerve histology in mice with conditional erbB2 mutation. Semi-thin sections of peripheral nerves from Krox20-cre/+; erbB2flox/− (A, C, E, G, and I) and control (B, D, F, H, and J) animals. The age of the animals is indicated. (more ...)
Figure 4 Electron microscopy of sciatic nerve branches from conditional erbB2 mutants. Electron microscopic analysis of peripheral nerves from Krox20-cre/+; erbB2flox/− (A and C) and control (B and D) animals at 6 mo of age. Shown are sural nerves (more ...)
In addition, we observed occasionally large-caliber axons lacking myelin that were still surrounded by a Schwann cell basal lamina in the sciatic nerve ( C, arrow and inset). Such Schwann cells frequently ensheathed several axons ( and , arrows). These profiles were more frequent in nerves innervating muscle than in cutaneous sensory nerves. Extensive patches of amyelinated axons were observed in some Cre-induced mutants ( I). The histology of nonmyelinating bundles appeared normal (, asterisks).
In early postnatal stages or in the embryo, a more severe phenotype was observed in dorsal and ventral spinal roots. These nerves display a severe lack of Schwann cells in all animals with Krox20-cre–induced erbB2 mutations at P3.5 and at P15 (10 examined; , , and data not shown). In accordance, myelin was absent in ventral roots, whereas thin myelin sheaths were surrounding a small proportion of axons in dorsal roots. This coincides with the early onset of Krox20-cre expression in these cells, revealing the early requirement for ErbB2 in establishment of the Schwann cell precursor pool. As animals reached adulthood, Schwann cell numbers increased, but the myelin sheaths formed were thin (). Localized patches of amyelinated axons persisted. Moreover, a single perineurium surrounds the entire spinal root in control animals, whereas perineurial sheaths compartmentalized the roots into six to seven branches in mutants (, arrowheads).
Effect of Krox20-cre–induced Mutation of erbB2 on Schwann Cell Number
Figure 5 Histology of spinal nerve roots from conditional erbB2 mutants. Semi-thin sections of ventral roots from Krox20-cre/+; erbB2flox/− (A and C) and control (B and D) animals at P15 (A and B) and adult stages (C and D). Arrowheads (A and C) (more ...)
Biochemical Characteristics and Growth Properties of Schwann Cells in Conditional erbB2 Mutants
We determined the absolute numbers of Schwann cells in sciatic nerves and spinal roots. In the sciatic nerves of animals at all stages tested (P3.5, P15, and 6 mo), Schwann cell numbers were comparable in the control and Cre-induced mutant mice (). In contrast, in dorsal and ventral roots, the numbers of cells were severely reduced. At P3.5, a 10-fold reduction was observed in the ventral, and a 5-fold reduction in the dorsal roots of the mutant mice. This reduction was still apparent at P15, and again more pronounced in ventral than in dorsal roots (). At 6 mo, numbers were similar in the dorsal and ventral roots of control and mutant mice. Thus, compensatory mechanisms exist that allow repopulation of spinal roots with Schwann cells; this repopulation occurs earlier in dorsal than in ventral roots.
Schwann cell proliferation and apoptosis determine the absolute numbers of Schwann cells, and were analyzed using BrdU incorporation and TUNEL labeling, respectively. Compared with the controls, the numbers of BrdU-positive cells were increased in the sciatic nerves of mutants at P3.5 and 5 wk ( A). The numbers of apoptotic nuclei were marginally increased at P3.5 ( B). In adults (age 6 mo), proliferation as well as apoptosis frequencies were below detection limits. Increased cell proliferation was also observed in ventral roots of 5-wk-old animals.
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 (more ...)
To assess the effect of the conditional erbB2 mutation induced by Krox20-cre on Krox20 and SCIP protein levels, sciatic nerve extracts were analyzed by Western blotting ( C). The level of Krox20 protein was similar in the nerves of control and Krox20-cre–induced erbB2 mutants. The level of SCIP protein was slightly increased in mutant nerves at P15, but levels attained baseline in control and mutant nerves at later stages. Levels of myelin protein were reduced in conditional mutants, in accordance with the histology.
Neuropathology in Krox20-cre–induced erbB2 Mutants
To assess whether neuron numbers were altered in Krox20-cre–induced erbB2 mutant mice, we counted axons in the L5 ventral root, which contains mainly motor axons. The numbers of such axons were frequently reduced in mutant animals, amounting to 20% loss on average. From 11 mutant animals, and 6 control animals examined, the number of axons in the mutant was 808 ± 152, compared with 1,020 ± 65 in control nerves (P < 0.002). The numbers of neurons in L5 dorsal root ganglia were similar in the control and mutant animals (not shown).