Sox10 is necessary for oligodendrocyte survival following axon wrapping

Abstract

Cells of the oligodendrocyte lineage, which form the myelinating glia of the vertebrate central nervous system, undergo a stepwise developmental progression entailing specification from neuroepithelial precursors, proliferation, migration to expand and distribute the population, and differentiation to ensheath axons with myelin. Understanding the genetic mechanisms that regulate each of these steps during development is important, because this might lead to therapies to promote remyelination following neural injury or disease. Genetic studies in mice indicated that the Sox10 transcription factor is required during the differentiation stage to promote myelin gene expression. However, whether Sox10 also promotes other features of oligodendroctye differentiation remained unknown. In this study, we used time-lapse imaging to investigate the behavior and fates of oligodendrocyte lineage cells in zebrafish embryos and larvae that lacked Sox10 function. This revealed that the myelinating subset of oligodendrocyte progenitor cells (OPCs) migrates, divides, and wraps axons normally, but then dies. Nonmyelinating oligodendrocyte progenitors divided more frequently, maintaining a normal population size. New oligodendrocytes produced by these progenitors wrapped axons and survived, but did not express myelin genes at high levels. We conclude that, in addition to promoting myelin gene expression, Sox10 function is necessary for the survival of myelinating oligodedrocytes subsequent to axon wrapping but is not required for the survival of nonmyelinating OPCs.

Fig. 1

Loss of sox10 function does not alter oligodendrocyte lineage cell number or distribution. A–H: Lateral views of Tg(olig2:egfp) reporter expression at the level of the trunk spinal cord in wild-type and cls^−/− embryos and larvae. Dorsal is up. Asterisks in (A) indicate examples of dorsally migrated OPCs. Bracket marks the spinal cord (SC). A′ –G′ : Transverse sections, dorsal up, showing Tg(olig2:egfp) reporter expression (green) and Sox10 protein expression (red). Arrows indicate olig2⁺ Sox10⁺ OPCs in wild-type embryos and larvae (A′ –C′) and olig2⁺ Sox10⁻ OPCs in cls^−/− embryos and larvae (E′ –G′). I: Quantification of dorsally migrated olig2⁺ OPCs reveals no difference between wild type and mutant. Error bars represent SEM. Scale bars, 24 μM.

Fig. 2

sox10 function is required for oligodendrocyte differentiation. A–F: Lateral views of spinal cords, dorsal up, showing plp1a, cldnk, and mbp RNA expression in 4.5-dpf larvae. Brackets mark spinal cord (SC) and notochord (NC). G, H: Transverse sections showing MBP expression (red) and anti-acetylated tubulin labeling (blue) at 6 dpf detected by immunocytochemistry. Arrows indicate prominent MBP deposition surrounding Mauthner axons in wild-type larva. I: Quantification of plp1a⁺ and cldnk⁺ cells. Graphs show dorsal, ventral, and total spinal cord cells that express these genes. Error bars represent the SEM. Scale bar, 24 μM.

Fig. 3

OPCs initiate axon wrapping in cls mutant embryos. A–D: Lateral views of spinal cords, dorsal up, of wild-type and cls^−/− embryos and larvae carrying the Tg(nkx2.2a:megfp) reporter to mark the myelinating subset of oligodendrocyte lineage cells. Asterisks mark dorsally migrated OPCs and arrows indicate axon wrapping. OPC number and morphology are similar in wild-type and cls^−/− embryos at 60 hpf (A, C), but mutants have a deficit of oligodendrocytes at 3 dpf (B, D). Transverse (E–G, K–M) and sagital (H–J, N–P) sections of 64 hpf wild-type and cls^−/− spinal cords expressing the Tg(nkx2.2a:megfp) reporter (green) and labeled with anti-acetylated Tubulin (red). OPC processes ensheath axons in both wild-type (E–J) and mutant (K–P) embryos. Scale bars, 24 μM (A–D), 4.5 μM (E–G, K–M), and 9 μM (H–J, N–P).

Fig. 4

Sox10 is required for oligodendrocyte survival following axon wrapping. Frames captured from time-lapse movie of a cls^−/−; Tg(nkx2.2a:megfp) embryo beginning at about 60 hpf. Panels show lateral images of the spinal cord, with dorsal up. Numbers indicate time elapsed since the beginning of the sequence. Large arrow and arrowhead point to the soma of oligodendrocytes, and small arrows and arrowheads indicate initiation of axon wrapping. Oligodendrocytes begin to fragment and die following axon wrapping. Asterisk marks an OPC that migrated into the field of view following oligodendrocyte death. Scale bar, 24 μM.

Fig. 5

Oligodendrocytes undergo apoptotic death in cls mutant embryos. Transverse sections of 64-hpf wild-type (A–C) and cls mutant (D–J) spinal cords expressing the Tg(olig2:egfp) reporter (green) and labeled with antiactivated Caspase-3 antibody (red). Arrows indicate olig2⁺ activated Caspase-3⁺ cells with fragmented appearance. Scale bar, 15 μM.

Fig. 6

nkx2.2a⁻ oligodendrocytes wrap axons in cls^−/− larvae. All images show lateral views of spinal cords, dorsal up, of wild-type and cls^−/− larvae carrying Tg(sox10(7.2):mrfp) and Tg(nkx2.2a:megfp) reporters. In wild type (A–F), most myelinating oligodendrocytes express both nkx2.2 and sox10 reporters (arrowheads). In cls mutants (G–L), some axon wrapping by cells marked by sox10 reporter expression is evident, but these cells appear to express the nkx2.2 reporter at low or nonexistent levels (arrowheads). (M–R) Higher magnification views showing axon wrapping by sox10⁺ nkx2.2a⁺ oligodendrocytes in wild type (M–O; arrowheads) but that in cls mutants oligodendrocytes that wrap axons express only the sox10 reporter (P–R; arrows). Some axons in wild type are also wrapped by oligodendrocytes that express only the sox10 reporter (N,O; arrows). Brackets mark nkx2.2a⁺ axons that descend from the hindbrain through the dorsal longitudinal fasciculus (DLF). Scale bars, 24 μM.

Fig. 7

OPCs divide and wrap axons following oligodendrocyte death in cls^−/− larvae. A: Frames captured from a time-lapse movie of a cls^−/−; Tg(nkx2.2a:megfp);Tg(sox10(7.2):mrfp) larva beginning at 4.5 dpf. Numbers indicate time elapsed since the beginning of the sequence. Panels show lateral views of the spinal cord, with dorsal up. Arrowheads and arrows indicate OPCs. Most OPCs within the field of view divided during the 14-h period of the time-lapse imaging. Dotted box outlines axon wrapping by an oligodendrocyte expressing only the sox10 transgenic reporter. B: Quantification of OPC division. Wild-type and cls^−/− larvae carrying the Tg(olig2:egfp) transgenic reporter were incubated in a BrdU solution for 24 h from 5 to 6 dpf. Graphs show number of BrdU⁺ olig2⁺ dorsal OPCs and BrdU⁺ olig2⁺ pMN precursors. Error bars indicate the SEM. Statistical significance was determined using the unpaired t-test.