Schwann cell multiplication in developing rat unmyelinated nerves — a radioautographic study

The expression of the neuronal type III intermediate filament protein peripherin was examined in the rat embryo during and following neuronogenesis in the spinal cord and the peripheral nervous system. In situ hybridization analysis reveals that peripherin mRNA is found in the mid-gestational rat embryo in ventral and lateral motoneurons in the spinal cord, and in neurons of all peripheral ganglia examined, including spinal, sympathetic, and enteric ganglia. Peripherin mRNA is seen only in post-migratory motoneurons or neuronal cells in aggregating ganglia, indicating that precursor cells do not express peripherin. To examine the expression of the protein, an affinity-purified antibody (anti-per) specific for a bacterially produced peripherin fusion protein was generated. Anti-per specifically recognizes a 58 kDa, cytoskeletal-enriched, nerve growth factor (NGF)-inducible protein of the expected tissue distribution. Immunocytodetection with anti-per shows that the initiation of peripherin protein synthesis is coincident with the morphological differentiation of neurons. In development, peripherin is one constituent of a program of gene expression activated at terminal neuronal differentiation.

Two proximal branches of the rat facial nerve were transected and anastomosed end-to-end within a silicone tube, each of them being exposed to a massive invasion of ascending regenerating axons. The proximal nerves contained extremely large bundles of regenerated fibers, often associated with preexistent “parent fibers.” The bundles showed many signs of rash and disordered cell proliferation and myelination. These included multiple Schwann cells surrounded by a common basement membrane, occurrence of different phases of myelination and even myelination of two axons by one Schwann cell. There was no evidence of mitogenic signals for fibrocytes. This model may be used for studying the mitogenic effect of axons on Schwann cells. It also suggests that so-called “groups of regenerating fibers” in neuropathy are caused by Schwann cell recruitment.

Monoclonal antibodies to stage-specific cell surface antigens of oligodendrocytes have been used to investigate the expression of antigens 05 through 011 in the peripheral nervous system of the mouse by immunohistology. In the adult sciatic nerve antigens 05 through 09 and 011 were diffusely positive. 010 antigen was not detectable in the peripheral nervous system at any age tested. During development antigens 05, 06 and 07 were first detectable at birth in tracts at the proximal part of the sciatic nerve. At day 2 the whole diameter of the nerve was positive for 05 antigen, while antigens 06 and 07 were detectable only in part of the nerve and antigens 08 and 09 were just about to appear. At day 4 antigen 011 was the last to appear. At day 7 all antigens were strongly detectable throughout the nerve. After transection of adult sciatic nerve expression of antigens 05 through 09 and 011 was studied at the proximal and distal ends of the cut. Three days after transection all antigens were fully detectable in the degenerating myelin and its debris. After 15 days residual debris was still distinctly positive, while Schwann cells in the bands of Büngner were antigen-negative. At approximately two weeks a connecting bridge between proximal and distal ends of the cut nerve had developed, but the 0 antigens were not detectable in this bridge until day 21. At day 42 all antigens were again fully detectable in the regenerating nerve. In hypomyelinating mouse mutants no difference to the normal control littermates was seen in staining pattern and intensity for jimpy and shiverer, while quaking showed an increase in staining intensity for 05 through 08 antigens. In trembler antigens 05, 06 and 07, but not 08, 09 and 011 appeared associated with non-myelin-forming Schwann cells, while the few recognizable myelin-forming Schwann cells expressed all antigens. These observations show that we have characterized 4 new monoclonal antibodies as further reagents to look at developmentally distinct steps in myelination of the peripheral nervous system.

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J.3, 3183–3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci.5, 1058–1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA80, 3513–3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 ± 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev.48, 534–569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.

Schwann cell mitosis has been demonstrated in chronically denervated cat tibial nerves re-innervated by axons regenerating from the proximal stump of a coapted peroneal nerve. Thymidine incorporation rose above baseline levels at the axon front, with no detectable increase in more distal regions occupied by denervated Schwann cells. Schwann cells therefore enter S phase upon the arrival of a regenerating axon in vivo as previously described in tissue culture. Intraneural treatment of the denervated distal stump with Mitomycin C prior to re-innervation delayed the subsequent appearance of myelin formation. This supports the notion that axonally stimulated division of Schwann cells is a prerequisite for myelination during nerve regeneration. Axonal advancement was also retarded by drug treatment, possibly because of a reduced level of trophic support provided by the compromised Schwann cells. A comparable absence of myelin and poor re-innervation was found in chemically untreated distal stumps that had been maintained in the denervated state for prolonged periods when Schwann cell columns are known to undergo progressive atrophy. These observations suggest that nerve repair should be delayed for limited periods if efficacious regeneration is desired.

The roles of cyclic AMP and calcium in the transduction of the mitogenic effects of central nervous system axolemma and myelin-enriched fractions on cultured Schwann cells were examined. Cyclic AMP levels were not elevated in axolemma or myelin-stimulated Schwann cells, but were increased when stimulated with cholera toxin, an adenyl cyclase activater. The mitogenicity of axolemma and myelin was markedly reduced by 2.5 mM citrate, a calcium chelator, and 10 uM trifluoroperazine, an inhibitor of calmodulin. Treatment of Schwann cells with several tumor-promoting phorbol esters caused significant enhancement of the mitogenicity of the axolemma and myelin preparations. These data suggest that the mitogenic effects of axolemma and myelin are not mediated by cyclic AMP, but may be mediated by calcium ions.