pubmed.ncbi.nlm.nih.gov

nkx2.2a promotes specification and differentiation of a myelinating subset of oligodendrocyte lineage cells in zebrafish - PubMed

nkx2.2a promotes specification and differentiation of a myelinating subset of oligodendrocyte lineage cells in zebrafish

Sarah Kucenas et al. Neuron Glia Biol. 2008 May.

Abstract

During development, multipotent neural precursors give rise to oligodendrocyte progenitor cells (OPCs), which migrate and divide to produce additional OPCs. Near the end of embryogenesis and during postnatal stages, many OPCs stop dividing and differentiate as myelinating oligodendrocytes, whereas others persist as nonmyelinating cells. Investigations of oligodendrocyte development in mice indicated that the Nkx2.2 transcription factor both limits the number of OPCs that are formed and subsequently promotes their differentiation, raising the possibility that Nkx2.2 plays a key role in determining myelinating versus nonmyelinating fate. We used in vivo time-lapse imaging and loss-of-function experiments in zebrafish to further explore formation and differentiation of oligodendrocyte lineage cells. Our data show that newly specified OPCs are heterogeneous with respect to gene expression and fate. Whereas some OPCs express the nkx2.2a gene and differentiate as oligodendrocytes, others that do not express nkx2.2a mostly remain as nonmyelinating OPCs. Similarly to mouse, loss of nkx2.2a function results in excess OPCs and delayed oligodendrocyte differentiation. Notably, excess OPCs are formed as a consequence of prolonged OPC production from neural precursor cells. We conclude that Nkx2.2 promotes timely specification and differentiation of myelinating oligodendrocyte lineage cells from species representing different vertebrate taxa.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. nkx2.2a and olig2 RNA expression patterns in spinal cord

All panels show transverse sections with dorsal to the top. (A–D) At 24 and 36 hpf (A,B) nkx2.2a expression is restricted to the p3 precursor domain (brackets). At 48 and 72 hpf (C,D) the nkx2.2a ventral expression domain (brackets) expands dorsally and nkx2.2a+ cells appear in dorsal spinal cord near the pial surface (arrows). (E–H) olig2 expression marks the pMN precursor domain. Scale bar: (in H), 20 μm.

Fig. 2
Fig. 2. nkx2.2a and olig2 expression define two distinct classes of spinal cord OPCs

All panels show images of 10 μm transverse sections, dorsal to the top, of Tg(nkx2.2a:megfp);Tg(olig2:dsred2) embryos and larvae. (A) At 24 hpf, cells that express nkx2.2a, revealed by EGFP fluorescence (blue) and olig2, revealed by DsRed2 fluorescence (red) occupy distinct domains within the ventral spinal cord. (B) By 48 hpf, EGFP expression expands dorsally and some cells express both EGFP and DsRed2 (arrows). (C–E) Sections labeled with anti-Sox10 antibody (blue) to reveal OPCs. Sox10+ OPCs are both nkx2.2a+ olig2+ (arrows) and nkx2.2a− olig2+ (arrowheads). Scale bar: (in E), 20 μm.

Fig. 3
Fig. 3. In vivo, time-lapse imaging reveals two classes of spinal cord OPCs

All panels show images from the side of the spinal cord with dorsal up. Numbers in lower right corners denote time elapsed from the first frame. (A) Frames captured from a 24 h time-lapse sequence (Supplemental Video 1) of a Tg(nkx2.2a:megfp);Tg(sox10(7.2):mrfp) embryo beginning at 52 hpf. Cells with OPC morphologies and behaviors are nkx2.2a+ sox10+ (yellow, arrowheads) and nkx2.2a− sox10+ (red, outlined arrowheads). (B) Frames captured from a 56 h time-lapse sequence (Supplemental Video 2) of a Tg(nkx2.2a:megfp);Tg(olig2:dsred2) embryo beginning at 60 hpf. OPCs are nkx2.2a+ olig2+ (red outlined by green membrane, arrowheads) and nkx2.2a− olig2+ (red cytoplasm, outlined arrowheads). Scale bars: 10 μm.

Fig. 4
Fig. 4. OPCs are born as nkx2.2a+ and nkx2.2a− cells

All panels show images from the side of the spinal cord with dorsal up. Frames were captured from a 24 h time-lapse sequence (Supplemental Video 3) of a Tg(nkx2.2a:megfp);Tg(sox10(7.2):mrfp) embryo beginning at 40 hpf. (A) Arrows indicate a green nkx2.2a+ cell that begins to turn yellow from co-expression of sox10. This OPC remained in ventral spinal cord whereas a neighboring nkx2.2a+ sox10+ OPC migrated dorsally (arrowheads). (B) Frames captured from the same time-lapse sequence showing a nkx2.2a+ sox10+ OPC (yellow, solid arrow) and a nkx2.2a− sox10+ OPC (red, outlined arrow). White asterisks mark ectopic expression of the sox10 reporter gene in interneurons. Scale bar: (in A), 10 μm.

Fig. 5
Fig. 5. Loss of nkx2.2a function causes a transient deficit of OPCs

(A–D) Transverse sections, dorsal to the top, of Tg(nkx2.2a:megfp);Tg(olig2:dsred2) embryos and larvae labeled with a Sox10 antibody. Solid arrows mark nkx2.2a+ olig2+ OPCs and outlined arrows indicate nkx2.2a− olig2+ OPCs. At 48 nkx2.2a MO-injected embryos (B) have fewer nkx2.2a+ OPCs and fewer total OPCs than control embryos (A). By 72 hpf, nkx2.2a MO-injected larvae (D) have more nkx2.2a− OPCs and more total OPCs than control larvae (C). (E) Quantification of total OPC number revealing a deficit at 48 hpf and excess at 72 hpf. (F) Quantification of OPC subclasses at 48 hpf revealing a deficit of nkx2.2a− olig2+ OPCs and small excess of nkx2.2a+ olig2+ OPCs in nkx2.2a MO-injected embryos compared to wild-type (WT) controls. (G) Quantification of OPC subclasses at 72 hpf revealing equal numbers of nkx2.2a− olig2+ OPCs and excess nkx2.2a+ olig2+ OPCs in nkx2.2a MO-injected embryos compared to WT. Error bars represent standard error of the mean. P values represent comparisons between MO-injected animals and corresponding controls. Statistical significance was determined using the paired t-test. Scale bar: (in D), 20 μm.

Fig. 6
Fig. 6. nkx2.2a promotes oligodendrocyte differentiation

(A–E) Transverse sections of larvae processed for in situ RNA hybridization with dorsal to the top. Arrows mark differentiating oligodendrocytes. At 3 dpf, nkx2.2a MO-injected larvae have more plp/dm20+ cells (B) but fewer mbp+ cells (F) than corresponding controls (A,E). At 4 dpf, nkx2.2a MO-injected larvae have comparable number of plp/dm20+ cells (D) but fewer mbp+ cells (H) than corresponding controls (C,G). (I) Quantification of plp/dm20+ cells. The difference between WT and MO-injected larvae at 4 dpf is not statistically significant. (J) Quantification of mbp+ cells. Error bars represent standard error of the mean. P values represent comparisons between MO-injected animals and corresponding controls. Statistical significance was determined using the paired t-test. Scale bar: (in H), 20 μm.

Fig. 7
Fig. 7. Loss of nkx2.2a function extends the proliferative period of olig2+ precursors

(A–F) Ten micrometer transverse sections, dorsal to the top, of Tg(olig2:egfp) embryos and larvae treated with BrdU and labeled with anti-Sox10 (blue) and anti-BrdU (red) antibodies. olig2 expression is revealed by EGFP fluorescence (green). Wild-type control and nkx2.2a MO-injected embryos have similar numbers of total BrdU+ cells and olig2+ BrdU+ cells at 48 hpf (A,B). By 60 hpf, control larvae (C) have many fewer BrdU+ cells than at 48 hpf but nkx2.2a MO-injected larvae (D) have substantial numbers of olig2+ BrdU+ cells (arrowheads). By 72 hpf, control larvae (E) have few olig2+ cells that incorporate BrdU whereas olig2+ cells in nkx2.2a MO-injected larvae (F) continue to incorporate BrdU. Solid and outlined arrows mark BrdU− and BrdU+ OPCs, respectively. (G) Quantification of olig2+ BrdU+ precursor cells. The difference between 48 hpf wild-type (WT) control and MO-injected embryos is not significant. (H) Quantification of Sox10+ BrdU+ OPCs in 72 hpf larvae. The difference is not statistically significant. Error bars represent standard error of the mean. P values represent comparisons between MO-injected animals and corresponding controls. Statistical significance was determined using the paired t-test. Scale bar: (in F), 20 μm.

Fig. 8
Fig. 8. Loss of nkx2.2a function extends the period of OPC production and dorsal migration

Panels show frames captured from a time-lapse sequence, beginning at 72 hpf, of a Tg(sox10(7.2):mrfp) larvae injected with nkx2.2a MO. Images are shown from the side with dorsal up. Numbers in upper right corners indicate time elapsed from beginning of sequence. Arrows mark OPCs initiating dorsal migration. Asterisks mark migrating OPCs in subsequent frame.

Similar articles

Cited by

References

    1. Barth KA, Wilson SW. Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development. 1995;121:1755–1768. - PubMed
    1. Baumann N, Pham-Dinh D. Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiological Review. 2001;81:871–927. - PubMed
    1. Bradel EJ, Prince FP. Cultured neonatal rat oligodendrocytes elaborate myelin membrane in the absence of neurons. Journal of Neuroscience Research. 1983;9:381–392. - PubMed
    1. Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000;101:435–445. - PubMed
    1. Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, Rubenstein JL, et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature. 1999;398:622–627. - PubMed

Publication types

MeSH terms

Substances