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First published online March 24, 2005
doi: 10.1242/10.1242/dev.01777

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A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord

The Wolfson Institute for Biomedical Research and Department of Biology, University College London (UCL), Gower Street, London WC1E 6BT, UK

View larger version (62K): [in a new window]   Fig. 1. Generation of Dbx1-iCre PAC transgenic mice and expression of the transgene. (A) Map of PAC 631-M19 indicating the extent of upstream and downstream genomic regions in the PAC insert. (B) Schematic of the endogenous Dbx1 locus, including intron-exon structure and the relative locations of the BglII and HindIII restriction sites. (C) The targeting vector used in homologous recombination, indicating the positions of the iCre coding sequences, the chloramphenicol resistance cassette (Cmr) and the loxP sites, and the location of the 0.5 kb 3' UTR probe used to hybridize to Southern blots of BglII-digested genomic DNA. The locations of PCR primers used for genotyping are also indicated. (D) Expression of the iCre transgene at E11.5, as revealed by in situ hybridization. Four independent founders had the same pattern of expression at this age. One founder was chosen for further study. (E) Diagram of neuroepithelial precursor domains in the spinal cord, and the reported patterns of Dbx1 and Dbx2 expression at E11.5 (Pierani et al., 2001). FP, floor plate; RP, roof plate.

View larger version (133K): [in a new window]   Fig. 2. Defining the limits of GFP expression in Dbx1-iCrexRosa26-GFP offspring. (A-I) Serial 10-µm sections through the E10.5 upper thoracic spinal cord were subjected to in situ hybridization with various probes (A,B,D,E,H), or immunohistochemistry for GFP or Pax7 (C,F,G,I). In situ hybridization probes are indicated. (G) Double immunolabelling for Pax7 (red) and GFP (green). Brackets (A-C) indicate the approximate limits of the GFP expression domain. Horizontal lines indicate the ventral (D-F) or dorsal (G-I) limits of GFP expression. The ventral limit corresponds approximately to the p2/p1 boundary, and the dorsal limit to the dP5/dP4 boundary. Scale bar: 50 µM.

View larger version (124K): [in a new window]   Fig. 3. Defining the neuronal subtypes derived from the Dbx domain at E12.5. (A) GFP-expressing cells (green) migrating laterally (1), ventro-laterally (2) and ventrally (3) from the Dbx-expressing region. (B-F) Double immunolabelling for (B) GFP (green) and Isl1/2 (red); (C) GFP and Lim3; (D) GFP and Lbx1; (E) GFP and Evx1; (F) GFP and Lim2. Scale bar: 100 µM.

View larger version (160K): [in a new window]   Fig. 4. Radial glia are derived from the Dbx-expressing neuroepithelium. (A,B) GFP expression (green) at E12.5 showing long, fine processes labelled with GFP extending from the ventricular surface to the pial surface (arrows). (C,D) RC2 labelling (red) at E12.5 is seen in many radial glial processes, some of which are also GFP labelled (E,F; arrows). (G-L) Radial glial cell bodies (arrows) appear near the pial surface at E16.5 (G-I) and E18.5 (J-L). Scale bars: in A, 100 µm; in G, 50 µm.

View larger version (108K): [in a new window]   Fig. 5. Oligodendrocytes are generated from Dbx-derived radial glia. (A-D) Radially orientated GFP-positive cells express the oligodendrocyte lineage markers Olig2 (A,C) and Sox10 (B,D) at E16.5 (A,B) and E18.5 (C,D). (E-P) Co-localization of the radial glia marker RC2 with GFP-labelled Olig2-positive cells at E15.5 (E-H) and E16.5 (I-L), or GFP-labelled Sox10-positive cells at E16.5 (M-P). Scale bar: 50 µm.

View larger version (156K): [in a new window]   Fig. 6. Dbx-derived oligodendrocytes at P10. (A) GFP and Sox10 double immunolabelling. (B) Higher magnification confocal view of the area marked in A, showing clear co-expression in a subset of Sox10-positive oligodendrocyte lineage cells. (C) GFP and PDGFRa double immunolabelling. (D) Double immunolabelling of GFP and the differentiated oligodendrocyte marker CC1 (Bhat et al., 1996). Scale bar: 300 µm.

View larger version (38K): [in a new window]   Fig. 7. Shh-independent oligodendrocyte development from Dbx-derived precursors. Dissociated spinal cords from Dbx1-iCrexRosa26-GFP embryos developed Sox10-positive OLPs within 5 days of culture (A). Their development was not inhibited by cyclopamine (B), but was abolished in the presence of PD173074 (C) or both (D). (E) The experiment was quantified by counting the total number of double-labelled GFP-positive/Sox10-positive cells in the cultures. A minimum of 1000 Sox10-positive cells was counted for each condition. Similar data were obtained in two independent experiments.

View larger version (124K): [in a new window]   Fig. 8. Fibrous and protoplasmic astrocytes are derived from the Dbx-expressing neuroepithelium. (A) Co-localization between GFAP (red) and GFP (green) in fibrous astrocytes in the developing white matter of a P10 spinal cord. (B) Confocal microscope view of the area highlighted in A. (C-F) Protoplasmic astrocytes develop from Dbx precursors. (C) All GFP-positive protoplasmic astrocytes co-label with anti-S100ß antibody (red). (D) Protoplasmic astrocytes next to the central canal weakly co-expressed GFAP. (E) GFP and NeuN (red) double labelling in the ventral horn, lying in close proximity to motoneuron cell bodies. (F) Protoplasmic-like astrocytes form bilateral longitudinal columns of cells positioned adjacent to the central canal (arrows). Nuclei are stained with DAPI (blue). Scale bars: in A, 200 µm; in C, 20 µm.

View larger version (32K): [in a new window]   Fig. 9. The origins of oligodendrocytes in the embryonic rodent spinal cord. From E12.5 in the mouse, OLPs are generated from pMN in the ventral VZ. Later, from approximately E16, a subsidiary population emerges from radial glial cells in the Dbx1-expressing domain that spans the dorsoventral midline. The latter population contributes around 5% of all oligodendrocyte lineage cells in the postnatal cord. Our data do not rule out the possibility that there are additional minor sources of oligodendrocytes outside either pMN or the Dbx domain.

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