Failure of spinal cord oligodendrocyte development in mice lacking neuregulin - PubMed (original) (raw)

Failure of spinal cord oligodendrocyte development in mice lacking neuregulin

T Vartanian et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/-) in a temporal sequence of developmental events that replicates that observed in vivo. However, in spinal cord explants derived from mice lacking neuregulin (NRG -/-), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG -/- mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG -/- explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.

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Figures

Figure 1

Figure 1

Oligodendrocytes fail to develop in spinal cord explants from neuregulin knock-out mice. Timed pregnancies from NRG +/− female and NRG +/− male matings were carried to ≈9.5 dpc, at which time females were killed, and embryos were removed. Each embryo was assigned a code number used to blind investigators from the genotyping. Genotyping was carried out by PCR on DNA extracted from the head of each embryo. Spinal cords were excised from embryos, cut transversely into segments, plated onto poly-

l

-lysine- and laminin-coated cover glasses, and cultured for 9 days in DMEM supplemented with 1% fetal bovine serum, N2 additives, and 10 ng/ml platelet-derived growth factor-AA. Cultures were stained with mAb O4 as described (32). Genotyping was unblinded after assessment of oligodendrocyte numbers and photography of explant cultures. (A and B) Micrographs of explants from NRG +/− embryos. (C and D) Micrographs of explants from NRG −/− embryos. O4 staining images are shown on the Left adjacent to the corresponding phase-contrast images on the Right. There are no identifiable O4+ cells in explants from NRG −/− embryos (the fluorescence within the explant is background not cellular staining). In contrast, explants from NRG +/− embryos have numerous O4+ oligodendrocytes. NRG −/− explants do, however, have an extensive neuritic outgrowth and growth of cells other than oligodendrocytes as seen in the phase-contrast images.

Figure 2

Figure 2

Exogenous recombinant neuregulin will rescue oligodendrocyte development from NRG −/− explant cultures. Spinal cord explant cultures were generated from litters ≈9.5 dpc and genotyped as described in the legend to Fig. 1. Spinal cords initially were divided into six to eight segments. Segments that did not obviously contain both ventral and dorsal spinal cord were discarded. Explants from a single embryo received either 0.1% BSA in PBS as a control or 1 nM neuregulin (epidermal-growth-factor-like β1 domain) at the time of plating. Explant cultures were then allowed to grow for an additional 8 days, equivalent to ≈17 dpc, and processed for O4 staining as described in the legend to Fig. 1. (C–F) Micrographs of explant cultures from NRG −/− embryos. (A and B) Micrographs of explant cultures from a wild-type littermate. The NRG −/− explants (C and D) as well as the wild-type explants (A and B) received 0.1% BSA in PBS. The explants shown in E and F received 1 nM neuregulin for the entire in vitro period. O4 staining images are shown on the Right adjacent to the corresponding phase-contrast images on the Left. The addition of neuregulin to NRG −/− explants restores oligodendrocyte development, and cells are indistinguishable from those in wild-type explants.

Figure 3

Figure 3

Neutralizing neuregulin activity inhibits the formation of oligodendrocytes in wild-type spinal cord explant cultures. IgB4 is a chimeric protein consisting of the extracellular domain of erbB4 (ligand-binding domain) and the Fc portion of human IgG1. Spinal cord explants were generated from E9.5 wild-type mice and cultured for 2.5 days in standard growth medium then 9 days in the presence of human Fc fragment as a control (a–d) or IgB4 (e–h). At day 11, cultures were surface stained with mAb O4 and O1 combined to identify immature and mature oligodendrocytes, fixed, and, in some cases, double-labeled with antineurofilament. (a, c, e, g) Combined O4 and O1 staining. b, d, and f are Nomarski images corresponding to a, c, and e, respectively. h shows the neurofilament staining corresponding to g. In cultures treated with IgB4, there are few or no oligodendrocytes identified. In contrast, wild-type explant cultures treated with the control buffer containing human Fc fragment had abundant numbers of oligodendrocytes.

Figure 4

Figure 4

Neuregulin is present in developing mouse spinal cord within motor neurons and the VVZ. E14 mouse embryos were emersion fixed in 4% paraformaldehyde, cryoprotected, and mounted in TissueTec, and 20-μm frozen transverse sections were mounted on glass slides. Sections were stained with a rabbit polyclonal antiserum that recognizes the “a” cytoplasmic domain of neuregulin, detected by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antiserum, and visualized by epifluorescence. Neuregulin is present within motor neurons (MN), dorsal root ganglion neurons (DRG), and in the VVZ itself. CC = central canal.

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