A novel marker for terminal Schwann cells, homocysteine-responsive ER-resident protein, as isolated by a single cell PCR-differential display

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Abstract

Terminal Schwann cells (TSCs) that cover motor neuron terminals are known to play important roles in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. We have established a method of selectively and efficiently collecting TSCs so that cDNA analysis can be done properly. The expression of 1–2% of whole mRNAs was compared between myelinating Schwann cells (MSCs) and TSCs, and it turned out that approximately one-third of the bands could be categorized as cell-type-specific bands. TSCs thus constitute a distinct entity from the viewpoint of gene expression. As one of the cDNA clones belonging to TSC-specific bands was identified homocysteine-responsive ER-resident protein (Herp), and in situ hybridization confirmed that Herp mRNA is expressed in TSCs on motor nerve terminals but not in MSCs, both in developing and adult rats. In conclusion, we have been able to identify Herp as a novel molecular marker for TSCs.

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Materials and methods

Animals. Adult and developing Wistar rats (12-week-old female and 14-day-old male) were purchased from a breeder (SLC, Japan), 1 week prior to experiments, and they were maintained in a temperature- and humidity-controlled animal facility with a 12:12-h light:dark cycle. All the animals were treated and/or handled in accordance with the “Recommendations for Handling of Laboratory Animals for Biomedical Research,” compiled by the Committee on the Safety and Ethical Handling Regulations for

Screening of genes marking TSCs

To identify genes specifically expressed in TSCs, we performed a PCR-based DD screening of two types of Schwann cells, viz., TSCs and MSCs. Herein, we applied a method of uni-directional synthesis and amplification of enriched full-length cDNA to RNA purified from a single cell, and we could obtain total cDNA fraction as moderately strong smears ranging from 0.2 to 4.0 kb with scattering several bright bands. Representative data are shown in Fig. 1A.

To check the quality and the origin of the

Discussion

We have identified a gene called Herp, as a novel molecular marker for terminal Schwann cells, by applying a single cell PCR-DD.

Myelinating Schwann cells (MSCs) are known to contribute to axonal outgrowth and guidance in the regeneration process after nerve injury. On the other hand, terminal Schwann cells (TSCs) appear to play critical roles in maintaining the structure and function of neuromuscular junctions (NMJs) as well as in the repair process. Whereas molecular characteristics of MSCs

Acknowledgements

This work was supported in part by Grant-in-Aid for Scientific Research (B) (to S.F.) and Grant-in-Aid for Young Scientists (A) (to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by research grants from National Cardiovascular Center of Japan (to S.F.).

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