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Sonic hedgehog-dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFR signaling

Nathalie Spassky¹, Katharina Heydon¹, Arnaud Mangatal¹, Alexandar Jankovski², Christelle Olivier¹, Françoise Queraud-Lesaux¹, Cécile Goujet-Zalc¹, Jean Léon Thomas^* and Bernard Zalc^*,

¹ Laboratoire de Biologie des Interactions Neurones/Glie, INSERM U-495, Université Pierre et Marie Curie, IFR des Neurosciences, Hôpital de la Salpêtrière, 75651 Paris Cedex 13, France
² Neuromorphologie: Développement Evolution, INSERM U-106, Université Pierre et Marie Curie, IFR des Neurosciences, Hôpital de la Salpêtrière, 75651 Paris Cedex 13, France
^* These authors contributed equally to this work

View larger version (87K): [in a new window]   Fig. 1. Grafted postnatal SVZ of the lateral ventricles and embryonic olfactory bulb (OB) generate oligodendrocytes in the corpus callosum. (A-E) Fragments from the SVZ of the lateral ventricles of ß2nZ3'1/1.9 mbp-lacZ bigenic mice were transplanted into the SVZ of wild-type mice, as schematized in A,B (A, donor; B, host). (C) Three weeks later, transplanted mice were analyzed after X-gal staining of frozen coronal sections. The presence of ß-gal-expressing cells in the SVZ of a transplanted animal indicates the site of the graft. (D,E) Mbp+ myelinating oligodendrocytes are detectable in the corpus callosum (D), whereas none is seen in the OB (E). In myelinating oligodendrocytes from the donor, the product of X-gal reaction stained the cell body and diffuses along the myelinated fibers. By contrast, ß2nZ3'1+ interneurons, which are X-gal positive only in the nucleus, are numerous in the OB (E), illustrating that they have migrated from the graft into the OB. (F-I) Fragments from E17.5 OB from 1.9 mbp-lacZ transgenic embryos were transplanted into the SVZ of the lateral ventricles of P3-P5 wild type, as schematized in F,G (F, donor; G, host). Three weeks later, transplanted mice were analyzed after X-gal staining of frozen coronal sections of the corpus callosum (H) and the OB (I). Mbp+ myelinating oligodendrocytes are seen in the corpus callosum but none is detected in the OB.

View larger version (21K): [in a new window]   Fig. 2. The development of oligodendrocytes in E9.5- and E10.5-derived telencephalic explants is not restricted to caudal telencephalon. Telencephalon of embryos at E9.5 or E10.5 was carefully split into caudal (CT), rostral (RT) and dorsal (DT) territories. (A) The plane of dissection of an E9.5 embryo used to separate the caudal, rostral and dorsal telencephalon. Explants were cultivated for various periods of time before being analyzed after immunolabeling with O4 mAb. (B) At E9.5, none of the telencephalic explants contained O4+ cells after 11 days in vitro (equivalent to E20). After 13 days in vitro (equivalent to P2), 47% and 56% of caudal and rostral telencephalic explants, respectively, contained O4+ cells. By contrast, more than 85% of dorsal explants were negative for O4+ cells at 13 days in vitro. (C) At E10.5, both caudal and rostral territories were able to generate O4+ cells after 10 or 12 days in vitro (P2). The data represent the mean proportion of explants in each category±s.e.m. The number of explants analyzed in each category is indicated above the bars. White bars, fewer than 10 O4+ cells per explant; hatched bars, 10 to 100 O4+ cells per explant; black bars, more than 100 O4+ cells per explant. di, diencephalon; epa, entopeduncular area; mge, medial ganglionic eminence; os, optic stalk.

View larger version (125K): [in a new window]   Fig. 3. Cells in caudal (A,D), rostral (B,E) and dorsal (C,F) E9.5-derived telencephalic explants stained with O4 mAb at 13 days in vitro. (A,B) Note that the morphology of O4+ cells in caudal and rostral explants is identical. Inset in B is a rostral culture stained at 15 DIV with O1 mAb, showing that cells generated in rostral explants are also O1+ and therefore differentiated oligodendrocytes. (C) The vast majority of explants derived from the dorsal telencephalon did not generate O4+ cells after 13 days in vitro. This inability was, however, only transient, and O4+ cells were observed at 19 days in vitro (inset in C). (A-C) O4 labeling; (D-F) corresponding interferential contrast images. Scale bar: 35 µm; 50 µm in the inset.

View larger version (92K): [in a new window]   Fig. 4. Low levels of Shh are detected in the rostral telencephalon. (A) Sagittal cryosection of E10.5 mouse forebrain hybridized with Shh digoxigenin-labeled antisense cRNA probe. The most rostral signal for Shh is in the anterior entopeduncular area (AEP), caudal to the medial ganglionic eminence (MGE). No hybridization signal for Shh is detected in the rostral (RT) and dorsal (DT) telencephalon. AHy, anterior hypothalamus; ChPl, choroid plexus; OR, optic recess. (B) mRNA was extracted from rostral (RT), caudal (CT) and dorsal (DT) explants derived from E9.5 and E10.5 telencephalon at the time of isolation (lane 0) or after 5 (lane 5) and 10 (lane 10) days in vitro and subjected to RT-PCR with primers specific for Shh. In contrast to data obtained by in situ hybridization, a definite positive signal for Shh is amplified from E9.5- and E10.5-derived rostral territory explants at the time of isolation. In dorsal explants, Shh is detected only after 10 or 5 days in vitro for E9.5- or E10.5-derived explants, respectively. Scale bar: 220 µm.

View larger version (170K): [in a new window]   Fig. 5. Development of oligodendrocytes in rostral telencephalon depends on Sonic hedgehog signaling. Explants derived from E10.5 rostral telencephalon were analyzed by double immunostaining with O4 (A,B) and TuJ1 (C,D) mAbs at 12 days in vitro. In control experiments, a large number of O4+ (A) and TuJ1+ (C) cells developed. When similar explants were grown in the presence of a function blocking anti-Shh antibody for 12 days, no O4+ cells were detected (B); however, TuJ1+ cells still developed (D), although less efficiently than in control (C). Scale bar: 130 µm.

View larger version (171K): [in a new window]   Fig. 6. Developmental expression in the OB of oligodendrocyte lineage early markers. (A-F) Adjacent sagittal cryosections of E14.5 (A,D), E16.5 (B,E) and coronal cryosections of P3 (C,F) mouse OB were hybridized with either Olig1/2 (A-C) or Pdgfra (D-F) DIG-antisense riboprobes. (G-K) Sagittal (G-J) and coronal (K) vibratome sections of E14.5 (G,H), E17.5 (I,J) or P3 (K) plp-sh ble-lacZ OBs were treated for histoenzymatic detection of ß-gal activity (bluo-gal). Higher magnifications show that ß-gal+ cells have a unipolar morphology at E14.5 (H), and are often bipolar at E17.5 (J). At all ages examined, ß-gal+ cells are present in the ventricular layer. By contrast, only occasional Olig1/2- and Pdgfra-expressing cells are seen in the ventricular layer at E16.5, and none at E14.5 or P3. Scale bar: 260 µm in A-G,I,K; 80 µm in H; 20 µm in J.

View larger version (90K): [in a new window]   Fig. 7. Plp-expressing cells in the OB express the AN2/NG2 marker of oligodendrocyte precursors. Coronal cryosections of E17.5 plp-sh ble-lacZ OB were double labeled with bluo-gal (A) or X-gal (B) and AN2 polyclonal antibody revealed with peroxidase-conjugated secondary antibody and DAB substrate. (A) Double-labeled ß-gal+/AN2+ cells, some of which (in the upper right corner) have the typical bipolar morphology of oligodendrocyte precursor. (B) In the subventricular layer (the limit of the ventricle (V) is indicated by a broken line), ß-gal+/AN2– cells (arrows) are Plp+ cells, which are probably less advanced in the oligodendrocyte lineage than ß-gal+/AN2+ cells. Note that AN2+ endothelial cells (with a brown precipitate diffusely distributed in the cell body, arrowheads) are ß-gal negative. Scale bar: 13 µm.

View larger version (27K): [in a new window]   Fig. 8. Blocking PDGFR tyrosine kinase do not interfere with development of Plp+ progenitors cells. Rostral (A) or caudal (B,C) telencephalon from E12.5 OF1 wild-type (A,B), or plp-sh ble-lacZ transgenic (C) mouse were dissociated, seeded in 96 wells dishes and cultivated in BS medium supplemented with 1% FCS. After 2 days in vitro, either STI571 (50-500 nM) or PDGF-AA (10 ng/ml) was added. After 13 (A,B) or 12 (C) days in vitro, cultures were immunolabeled with O4 mAb alone (A,B), or O4 mAb and anti-ß-galactosidase Ab (C), and immunopositive cells were counted. In C, the white columns represent the O4+ cells and the hatched columns the ß-gal+ cells. Each column represents the mean±s.e.m. of three separate experiments representing 14 to 24 different cultures (A,B) or two separate experiments representing eight to 19 different cultures (C) (***P<0.0001; * P<0.05, Student’s t-test).