Promyelinating Schwann cells express Tst-1/SCIP/Oct-6

Abstract

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are transiently arrested at the "promyelinating" stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined beta-galactosidase (beta-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. beta-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenous tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-beta-D-galactopyranoside- (X-gal) and halogenated indolyl-beta-D-galactoside- (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of beta-gal, both in developing and in regenerating nerves. Thus, the expression of beta-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested in tst-1/scip/oct-6-null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.

Fig. 1.

Solution assay of β-gal in developingtst-1/scip/oct-6 +/− sciatic nerves. Theheights of the bars represent the mean RLUs per milligram of protein for P1, P5, P10, P15, P20, P37, and adult mouse sciatic nerves. The RLUs were measured in triplicate for each sample; error bars represent the SEM.

Fig. 2.

Graphical summary of β-gal expression in Schwann cells in tst-1/scip/oct-6 +/− sciatic nerves. At each developmental age (P1, P10, and P30), the heights of thebars show the percentage of Schwann cells of the following morphologies: denervated, bundling, promyelinating > 1, promyelinating, myelinating, and nonmyelinating. The lightly shaded portions of the bars indicate the percentage of β-gal–negative cells, whereas the darkly shaded portions represent the percentage of β-gal–positive Schwann cells (visualized with X-gal and Bluo-gal at P1 and with X-gal at P10 and P30). The fraction over each barshows the number of positive Schwann cells relative to the total number of Schwann cells of that morphology. n, Number of nerves analyzed at each age.

Fig. 3.

Electron microscopy of P1tst-1/scip/oct-6 +/− sciatic nerve stained with Bluo-gal. A, The nuclei of three bundling (b) Schwann cells and one promyelinating (p) Schwann cell. B,C, Higher magnifications of the promyelinating Schwann cell seen in A. Some of the Bluo-gal crystals are indicated (arrowheads); the symbols plusand minus mark the presence or absence, respectively, of crystals in classified cells. To demonstrate the crystals more clearly, we took these photographs from a section that was not counterstained with lead citrate and uranyl acetate. Scale bars: A, 1.0 μm; B, C, 0.5 μm.

Fig. 4.

Electron microscopy of P30tst-1/scip/oct-6 +/− sciatic nerve stained with Bluo-gal. Two Schwann cells, one myelinating (m) and one nonmyelinating (n), are shown. Thearrowhead indicates a Bluo-crystal. The symbols plus and minus mark the presence or absence, respectively, of crystals in classified cells. To demonstrate the crystals more clearly, we took these photographs from a section that was not counterstained with lead citrate and uranyl acetate. Scale bar, 1.0 μm.

Fig. 5.

Solution assay of β-gal in adulttst-1/scip/oct-6 +/− sciatic nerves. Theheights of the bars represent the mean RLUs per milligram of protein from the distal stumps of nerves 1, 4, 8, 12, 24, and 58 d after transection or after crushing. Note that the RLUs of the crushed nerves is higher than that in the transected nerves during the period of ensheathment and myelination (8–24 d after crushing).

Fig. 6.

Electron micrograph of a perineurial-like cell from the distal stump 24 d after crushing. This cell shows the typical features, including a patchy basal lamina (fat arrows), caveoli (arrowheads), and two long processes that ensheathe Schwann cells (s) and axons (a). Scale bar, 1.0 μm.

Fig. 7.

Electron microscopy of lesioned adulttst-1/scip/oct-6 +/− sciatic nerves stained with Bluo-gal. A, A denervated (d) Schwann cell at 12 d after transection.B–D, Promyelinating (p); bundling, promyelinating > 1, and nonmyelinating (b,p>1,n); and myelinating (m) Schwann cells at 12 d after crushing, respectively. Some Bluo-gal crystals are indicated (arrowheads). The symbols plus andminus indicate the presence or absence, respectively, of crystals in classified cells. To demonstrate the crystals more clearly, we took these photographs from sections that were not counterstained with lead citrate and uranyl acetate. Scale bars, 1.0 μm.

Fig. 8.

Graphical summary of β-gal expression by Schwann cells in lesioned adult tst-1/scip/oct-6 +/− sciatic nerves. The bars show the percentage of Schwann cells of each morphology at 8, 12, 24, and 58 d after transection (upper) and after crushing (lower); thelightly and darkly shaded bars indicate the percentage of Bluo-gal–negative and –positive Schwann cells, respectively. The fraction over each barshows the number of positive Schwann cells relative to the total number of Schwann cells of that morphology. n, Number of animals analyzed. The Schwann cell morphologies are as follows:d, denervated; bp>1n, bundling, and promyelinating > 1, and nonmyelinating;p, promyelinating; and m, myelinating.

Fig. 9.

Schematic view of transcription factor expression in developing rodent Schwann cells. This figure summarizes the timing of expression of various transcription factors in developing rodent (mouse and rat) peripheral nerve and relates the expression of these transcription factors to the anatomical relationships of axons and Schwann cells. The thick black lines (e.g., for Krox-20 and Tst-1/SCIP/Oct-6) indicate relatively higher levels of expression than do the thin black lines.