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Bone morphogenetic proteins negatively control oligodendrocyte precursor specification in the chick spinal cord

Centre de Biologie du Développement, UMR 5547 CNRS/UPS, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France

View larger version (79K): [in a new window]   Fig. 1. BMP4 inhibits the development of oligodendrocytes and motor neurones in medial neural plate cultures. Medial neural plate explants were dissected from E1.5 embryos (stage 10), and grown in vitro in the absence (A,D,G) or in the presence (B,E,H) of 0.6 nM BMP4. Oligodendrocytes were stained after 7 days in vitro (div) by O4 antibody (A,B) and after 11 div by OL-1 antibody (D,E), which labels more mature oligodendrocytes. Motoneurones and floor-plate cells were stained after 2 div (G,H) with anti-Islet 1 antibody (red) and by anti-HNF3ß antibody (green). Note that BMP treatment strongly inhibited the development of oligodendrocytes and motoneurones but had no effect on the development of floor plate cells. (C,F,I) The mean number of cells of the oligodendrocyte lineage (C-F) and of motoneurones (I) was determined in control and treated conditions. Scale bars represent the mean±s.e.m. of three to five independent experiments.

View larger version (82K): [in a new window]   Fig. 2. BMP4 inhibits the development of oligodendrocytes in cultures of ventral neural tube explants, with no obvious stimulation of astrocyte differentiation. E1.5 medial neural plate explants (MNPs; A,B,E,F), initially grown for 3 days (final age 4.5 days), and ventral neural tube explants (VNT; C,D) dissected at E4-4.5 were left untreated (A,C,E) or treated (B,D,F) with 0.6 nM BMP4. After 4 and 7 days of treatment of MNP and VNT explants, respectively, cultures were stained with O4 antibody (A-D) or GFAp antibody (E,F). Note the drastic reduction in the number of O4+ cells (quantified in Table 1) in BMP treated explants (B,D). By contrast, the density of GFAp-positive cells is not increased upon BMP treatment (E,F); see text for quantification.

View larger version (157K): [in a new window]   Fig. 3. Grafts of BMP2-producing cells obliterate the development of oligodendrocytes in the spinal cord in vivo. Such grafts, however, neither block SHH production nor affect the development of cells of the astrocyte lineage. Pellets of BMP2-overexpressing Q2bn cells or control cells were implanted at E3-E3.5 between neural tube and somites and embryos were fixed at E7-7.5. (A) Section outside the manipulated region in an embryo grafted with BMP2-producing cells. Ventricular O4-positive cells are found in normal position within the ventral ventricular zone, close to the floor plate (fp). (B,C) Embryo grafted with control-Q2bn cells. (B) Dark field/fluorescence view showing the position of DiI-labelled grafted cells (red). (C) Confocal image of the field framed in B, showing O4 immunostaining. Note the normal development of O4-positive cells in the ventral ventricular zone. (D-F) Sections in embryos grafted with BMP2-producing cells. (D) At the level of the graft, shown in red in E, O4-positive cells are totally absent. In particular, they are not found in the ventricular region above the floor plate (fp). (F) Section at the level of the graft in another embryo. There are no O4-positive neuroepithelial cells on the grafted side (right hand side of the micrograph), but some are discernible on the contralateral side (arrowhead). (G-I) Sections stained with anti-Shh antibody. (G) Control section. Floor-plate cells display Shh immunoreactivity. (H,I) Section in an embryo grafted with BMP2-producing cells. (H) Low-power view showing the large mass of grafted cells (red) flanking the lateral wall of the neural tube. (I) Enlargement of the area boxed in H. Distribution and intensity of Shh immunoreactivity in the floor plate appear comparable with those seen in control sections. (J) Control section stained with anti-GLAST antibody. GLAST is expressed at the plasma membrane of neuroepithelial cells and floor plate cells (fp). Dispersed staining is also found in radial fibres and dispersed cells within the mantle layer. (K) Section in an embryo grafted with BMP2-producing cells, stained with O4 antibody. Grafted cells appear in red on the left-hand side of the micrograph. Note that the ventricular region lacks O4-positive cells. A few are discernible in the marginal layer on the contralateral side. (L) Enlargement of the area boxed in K. The section has been counterstained with anti-GLAST antibody. Staining is comparable with that seen in control sections, both in the neuroepithelium and in the floor plate (fp).

View larger version (106K): [in a new window]   Fig. 4. BMP4 inhibits oligodendrocyte development without significantly affecting astrocyte development. E4 spinal cord flat mounts were organ cultured in control conditions (A,C,F) or treated (B,D,G) with 0.6 nM BMP4. In all micrographs, broken lines delineate the floor plate area. (A,B) Transverse sections from spinal cords grown for 2 days, stained with O4 antibody. In control spinal cords, oligodendrocyte precursors are found in the ventral neuroepithelium, close to the floor plate (arrows in A). BMP4 treatment alone totally abolishes the emergence of O4-positive cells (B). (C-G) Transverse sections from spinal cords grown for 8 days and stained with antibodies directed against GFAp (C,D) or PLP/DM20 (F,G). In control sections, numerous astrocytes (C) and oligodendrocytes (F) are observed, with higher density at the lateral edge of the flat mount. BMP4 treatment alone abolishes oligodendrocyte development (G) but does not appear to modify the pattern of expression of GFAp (D). (E) Quantification of the area covered by GFAp immunoreactive material in control or BMP4 treated spinal cord flat mounts, expressed in percent of total section surface.

View larger version (113K): [in a new window]   Fig. 5. OLPs arise from the intermediate and dorsal regions of the neuroepithelium after dorsal spinal cord ablation. The dorsalmost region of the spinal cord in E3 embryos was excised in ovo or, in control experiments, was simply opened without removing dorsal tissue. In both cases, this led to the formation of a spina bifida. Operated embryos were fixed at E7-7.5. Transverse sections were stained with O4 antibody. (A) Left hemisection from a control spinal bifida embryo (dorsal is towards the top). The spinal cord is widely opened and the ventricular surface is exposed dorsally. O4 antibody delineates a ventricular focus of OLPs of normal extension (bracket), close to the floor plate (fp). (B) Right hemisection from a dorsal spinal cord excision. The ventricular focus of O4-positive cells (bracket) is enlarged compared with that found in the control embryo. (C) Section in another embryo with dorsal spinal cord excision. The position of the sulcus limitans is denoted by the broken line on the left side. Ectopic O4-positive cells (arrows) are found away from the original focus of oligodendrogenesis (bracket), evenly spaced along the neuroepithelium up to the sulcus limitans. (D-F) Section in an embryo with dorsal spinal cord excision, at a level where the spinal cord remained closed. (D) The broken lines indicate the level of the sulcus limitans. Note the important development of O4-positive cells in the marginal zone. (E,F) Enlargements of the areas boxed in D. Ectopic O4-positive cells (arrowheads) develop dorsally to the sulcus limitans (E) and in the intermediate neuroepithelium (F). Note one O4-positive cell that seems to have migrated away from the dorsal neuroepithelium (arrow in E).

View larger version (170K): [in a new window]   Fig. 6. Grafts of noggin-producing cells cause ectopic OLPs development in the neuroepithelium. Pellets of noggin-overexpressing CHO cells were implanted at E3 between neural tube and somites. Embryos were fixed at E7-7.5 and transverse sections were stained with O4 antibody. (A) Control section showing the normal appearance of O4-positive OLPs in the ventral ventricular zone. (B) Section at the level of the graft in an operated embryo. Note the dorsal enlargement of the O4-positive ventral ventricular zone on the grafted side (bracket), while the contralateral side displays normal development of O4-positive cells. (C,D) Section at the level of graft in another operated embryo. (C) Darkfield/fluorescence view. CHO cells (red) are found dispersed all along the neural tube. (D) Confocal image at higher magnification of the area boxed in C. The O4+ ventricular zone is enlarged on the operated side compared with the contralateral side (brackets). Note also the presence of an ectopic neuroepithelial O4-positive cell located dorsal to the normal level of origin of OLPs (asterisks in C and D).

View larger version (56K): [in a new window]   Fig. 7. BMP4 abolishes Shh-mediated induction of oligodendrocytes. Ventral neuroepithelial explants were dissected from E5 embryos, before oligodendrocyte induction has occurred. They were grown for 2 days or 7 days in the absence or presence of Shh, with or without BMP4, and stained with O4 antibody (A-C) or with anti-PLP/DM20 antibody (E-G). (A-C) When isolated at E5, the ventral neuroepithelium is unable to generate oligodendrocytes, as shown by absence of expression of O4 antigen (A). In the presence of 12 nM Shh, such explants generate numerous O4-positive cells within 2 days (B). However, explants treated simultaneously with 12 nM Shh and 0.6 nM BMP4 failed to generate O4-positive cells (C). (D) Quantification of the number of O4-positive cells developing in ventral neuroepithelial explants. Bars represent the mean±s.e.m. of at least 12 separate explants. Oligodendrocyte production is maximal with 12 nM Shh. Note that BMP4 totally blocks the Shh-mediated induction of oligodendrocyte formation, irrespective of Shh concentration. (E-G) Confirming the above results, E5 ventral neuroepithelial explants do not generate PLP/DM20-positive oligodendrocytes after a week in culture (E). Shh treatment induces the development of PLP/DM20-positive cells, which display typical oligodendrocyte morphology (F). Again, SHH/BMP double treatment results in the absence of oligodendrocyte development (G).

View larger version (20K): [in a new window]   Fig. 8. BMP4 effect on E6 neuroepithelial explants. Ventral neuroepithelial explants were dissected from E6 embryos, i.e. just after oligodendrocyte induction has occurred. They were grown for 2 days in the absence (A) or presence (B) of 0.6 nM BMP4. (A) Control explant. E6 ventral neuroepithelial explants generate numerous O4-positive cells. (B) Upon BMP4 treatment, explants generate reduced numbers of O4-positive cells. (C) Quantification of the number of O4-positive cells developing in ventral neuroepithelial explants. Bars represent the mean±s.e.m. of at least 17 separate explants. At this stage, BMP4 treatment does not totally block O4-positive cell production.

View larger version (89K): [in a new window]   Fig. 9. BMP4-mediated inhibition of oligodendrocyte development is accompanied, in vitro as in vivo, by strong repression of Olig2 but does not depend on inhibition of Nkx2.2 expression. (A-D) Ventral neuroepithelial explants were dissected from E5 embryos, before oligodendrocyte induction has occurred. They were grown for 2 days in the absence or presence of 12 nM Shh, with or without 0.6 nM BMP4, and double-stained with Olig2 antiserum (green) and anti-Nkx2.2 monoclonal antibody (red). (A) Control explant displaying small numbers of Olig2 and Nkx2.2-positive nuclei. Only few nuclei are double-labelled. (B) BMP4 treatment alone inhibits Olig2 expression but does not significantly affect Nkx2.2 expression. (C) Shh treatment, which induces oligodendrocytes, also enhances the expression of Olig2 and Nkx2.2. Note that most nuclei are double labelled. (D) Co-treatment with Shh and BMP4, which abolishes Shh-mediated oligodendrocyte induction, also strongly inhibits Olig2 expression but does not affect Nkx2.2 expression. Note, in particular, Nkx2.2-positive nuclei in neuroepithelial cells forming the side of the explant (arrowhead). (E) Quantification of the number of Nkx2.2-positive and Olig2-positive nuclei per explant (20-33 explants per experiment). Bars represent the mean±s.e.m. of at least three independent experiments. Significance of the differences between treated and control conditions, assessed by Student's t-test: *, P<0.01; **, P<0.001. (F-K) Grafts of BMP2-overexpressing Q2bn cells were implanted at E3-E3.5 between neural tube and somites and embryos were fixed at E7-7.5. Transverse spinal cord sections were double stained with Olig2 antiserum (green) and Nkx2.2 antibody (red). (F-H) Control section. (F) Olig2-positive neuroepithelial cell nuclei are found several cell diameters above the floor plate. (G) Nkx2.2-positive nuclei are located more ventral in the neuroepithelium. (H) The overlay shows co-expression of the transcription factors in some nuclei (yellow), indicating that their respective domains of expression overlap at this stage. (I-K) Section at the level of the graft from an embryo implanted with BMP2-Q2bn cells. (I) Expression of Olig2 is totally abolished on the grafted side. (J) By contrast, Nkx2.2 expression appears not to be affected by the implanted BMP2-producing cells. (H) The overlay shows co-expression of the transcription factors in some nuclei on the contralateral side.

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