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Co-localization of Nkx6.2 and Nkx2.2 homeodomain proteins in differentiated myelinating oligodendrocytes - PubMed

Co-localization of Nkx6.2 and Nkx2.2 homeodomain proteins in differentiated myelinating oligodendrocytes

Jun Cai et al. Glia. 2010 Mar.

Abstract

Recent studies have suggested that Nkx6.2/Gtx and Nkx2.2 homeodomain transcription factors are involved in the regulation of oligodendrocyte maturation and/or myelination which occur predominantly in postnatal stages. However, their cellular specificity in postnatal central nervous system has not been characterized and their dynamic expressional relationship during oligodendrocyte lineage progression has not been determined. Here we report that both Nkx2.2 and Nkx6.2 are selectively expressed in Olig2+ cells of oligodendrocyte lineage in postnatal spinal cords. Although Nkx6.2 is specifically expressed in the APC+ mature oligodendrocytes, Nkx2.2 is initially expressed in differentiating oligodendrocyte precursor cells (OPCs) but quickly down-regulated as OPCs undergo terminal differentiation. Intriguingly, Nkx2.2 expression is up-regulated in mature myelinating oligodendrocytes at later stages. The co-expression of Nkx2.2 and Nkx6.2 transcription factors in myelinating oligodendrocytes suggests their functional interactions in the regulation of myelin sheath formation and/or maintenance.

2009 Wiley-Liss, Inc.

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Figures

Figure 1
Figure 1

Nkx2.2 and Nkx6.2 expression in postnatal mouse spinal cords. Transverse sections from P1 (A, F), P8 (B, G), P15 (C, H), P34 (D, I) and P68 (E, J) wild-type spinal cords were subject to immunofluorescent staining with anti-Nkx2.2 (A-E) or anti-Nkx6.2 (F-J). Only the ventrolateral spinal cord positions were shown. The Nkx6.2+ cells with large nuclei in the grey matter (top-right inset in F) and those with small nuclei in the white matter (bottom-left inset in F) are represented by arrows and arrowheads, respectively. Scale bar: 100µm.

Figure 2
Figure 2

Nkx2.2 and Nkx6.2 expression in postnatal mouse brains. Transverse sections from P15 (A, C) and P30 (B, D) wild-type corpus callosum regions were subject to double immunofluorescent staining with anti-Nkx2.2 and anti-Olig2 (A, B) or anti-Nkx6.2 and anti-Olig2 (C, D). Scale bar: 100 µm.

Figure 3
Figure 3

Non-overlapping of Nkx6.2 and astrocytic markers in postnatal mouse spinal cords. Cross sections from P2 (A and D), P6 (B) and P15 (C and E) wild-type pups were subject to double immunofluorescent staining with anti-Nkx6.2 and anti-GFAP (A-C), anti-Glast (D), or anti-S100B (E). Only the ventrolateral spinal cord positions were shown. Scale bar: 50 µm.

Figure 4
Figure 4

Co-expression of Nkx6.2 with Olig2 and APC in spinal cord oligodendrocytes. Spinal cord sections from P2 (A–C), P4 (J), P8 (K), P13 (D–F), P30 (G–I) and P34 (L) wild-type pups were subject to double immunofluorescent staining with anti-Nkx6.2 and anti-Olig2 (A–I), or anti-Nkx6.2 and anti-APC (J–L). Only the ventrolateral spinal cord positions were shown. The Nkx6.2+++/Olig2+ and Olig2+++/Nkx6.2- single positive cells were represented by arrows and arrowheads, respectively. The percentage of Nkx6.2+ and Nkx6.2- cells in APC+ or Olig2+ population from P1 to P34 were shown in M and N. The Nkx6.2+ cells in C and J and represent the ventral interneurons. The APC+/Nkx6.2- cells were indicated by asterisks. Inset in K showed the APC/Nkx6.2/Dapi triple labeling from the outlined region. Scale bar: 100 µm.

Figure 5
Figure 5

Expression of Nkx2.2 in the oligodendroglial cells of postnatal spinal cords. Spinal cord sections at the thoracic level were prepared from P2 (A, E, I), P4 (F, J), P15 (B, G, K), P34 (C, H, L), and adult (D) wild-type mice. A-D. Immunofluorescence with anti-Nkx2.2 (red) and anti-Olig2 (green). Arrows indicated the Nkx2.2+/Olig2- or weak Olig2+ cells. E-H. Double immunostaining with anti-Nkx2.2 (red) and anti-APC (green). I-L. Double immunolabeling with anti-Nkx6.2 (red) and anti-Nkx2.2 (Green). Arrows in F–H and J–L represented the Nkx2.2+/APC+ and Nkx6.2+/Nkx2.2+ (either strong or weak) cells, respectively. Arrowheads in G and K indicated the Nkx2.2+/APC- or Nkx6.2- cells. The percentage of Nkx2.2+ and Nkx2.2- cells in APC+ or Olig2+ population from P1 to P34 were shown in M and N. Scale bar: 100 µm.

Figure 6
Figure 6

Definition of Nkx2.2 and Nkx6.2 expression by triple immunostaining with stage-specific markers in oligodendrocytes. Spinal cord sections were collected from P4 (A, D-F), P15 (B), and P40 (C) wild-type pups. Only the ventrolateral spinal cord positions were shown. A-C. Immunofluorescent staining with anti-Nkx2.2 (red), anti-Olig1 (green) and Dapi (blue). Arrows delineated the Nkx2.2+/nuclear Olig1+. The Nkx2.2-/cytoplasmic Olig1+ and Nkx2.2+/cytoplasmic Olig1+ cells were represented by arrowheads and asterisks, respectively. D. Triple labeling with anti-Nkx2.2 (red), anti-Sox10 (green), and anti-APC (blue). E. Immunofluorescence with anti-Nkx6.2 (red), anti-Nkx2.2 (green), and anti-APC (blue). F. Triple staining with anti-Nkx6.2 (red), anti-Olig1 (green) and anti-Nkx2.2 (blue). Arrows represented either Nkx2.2+/Sox10+ cells in D or Nkx2.2+/nuclear Olig1+ in F. Open arrows represented Nkx2.2+/Sox10+/APC- cells in D, Nkx6.2-/Nkx2.2+/APC- in E, or Nkx6.2-/Nkx2.2+/nuclear Olig1+ in F. Leis in F delineated the Nkx6.2+/cytoplasmic Olig1+ cells. Scale bar: 50 µm.

Figure 7
Figure 7

Up-regulation of Nkx2.2 in OPCs and pro-oligodendrocytes but down-regulation in mature oligodendrocytes in vitro. Immunofluorescence with anti-Nkx2.2 (red) and A2B5 (green), O4 (green) or anti-MBP (green) were performed in disassociated oligodendrocytes at A2B5 (A, A’), O4 (B, B’), and MBP (C,C’) stages, or co-culture of DRG neurons and oligodendrocytes (D, D’). The percentage of Nkx2.2+ cells oligodendrocyte population at different differentiation stages was summarized in E and E’. ***: p<0.001. Arrows represented either Nkx2.2-/Dapi+ nuclei in A and A’ or Nkx2.2+ /Dapi+ nuclei in D and D’. Arrowheads in B, B’, D, and D’ or stars in C and C’ indicated Nkx2.2+ (weak)/Dapi+ cells.

Figure 8
Figure 8

Delayed expression of Nkx6.2 and APC in Nkx2.2 mutant spinal cords. Cross sections of cervical spinal cord from wild-type (A, C, E) and Nkx2.2 mutant (B, D, F) pups at P4 (A-D) and P6 (E, F) were labeled with anti-APC (A, B) and anti-Nkx6.2 (C-F) antibodies. G. Nkx6.2+ cells per section in P4 and P6 wild-type and Nkx2.2 null mutant spinal cords (n=3). ***: p<0.001.

Figure 9
Figure 9

Schematic illustration of oligodendrocyte development and stage-specific expression of transcription factors during oligodendrogial lineage progression. NPC, neural progenitor cells; OPC, oligodendrocyte progenitor cell; OL, oligodendrocyte; VZ, ventricular zone.

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