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doi: 10.1242/10.1242/dev.00576

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Delta-Notch signaling regulates oligodendrocyte specification

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA

View larger version (116K): [in a new window]   Fig. 1. Delta-Notch is required to maintain the spinal cord precursor pool and for OPC specification. All images show transverse sections, dorsal to the top. (A-C) 1 dpf embryos treated with BrdU and anti-BrdU antibody (pink) to mark S-phase cells and Hoechst (blue) to reveal nuclei. dla-/-;dld-/- (B) and mib-/- (C) embryos had fewer S-phase spinal cord cells than wild type (A). Spinal cords are outlined. (D-F) 1 dpf embryos labeled with anti-phospho-histone-H3 antibody to mark M-phase cells (green) and pan neuronal anti-Hu antibody (red). dla-/-;dld-/- (E) and mib-/- (C) embryos had excess neurons and a deficit of M-phase cells relative to wild type (D). (G-I) 2 dpf embryos probed for sox10 expression, which marks OPCs, by in situ RNA hybridization. Spinal cord cells of dla-/-;dld-/- (H) and mib-/- (I) embryos did not express sox10. (J-L) 3 dpf embryos probed for plp1/dm20 RNA expression, which marks premyelinating oligodendrocytes. dla-/-;dld-/- (K) and mib-/- (L) embryos did not express plp1/dm20. Scale bars: 20 µm.

View larger version (106K): [in a new window]   Fig. 2. Delta-Notch signaling maintains olig2 expression and inhibits ngn1 expression. (A-C) Transverse sections, dorsal up, of 24 hpf embryos. Whereas a discrete group of ventral spinal cord cells expressed olig2 (A), cells that expressed ngn1 were scattered throughout the spinal cord (B). In situ double RNA hybridization revealed that a subset of olig2+ cells (C, red) expressed ngn1 (blue, arrows). (D-G) Dorsal views, anterior left, of 12 hpf embryos. (D,E) Embryos hybridized with probes for olig2 and tlxa. Brackets indicate olig2 expression. tlxa expression marks prospective Rohon-Beard (RB) sensory neurons. In wild-type embryos, olig2 expression was maintained throughout the length of the trunk neural keel (D, arrow). In dla-;dld- mutant embryos, identified by the excess Rohon-Beard phenotype, anterior neural keel cells did not maintain olig2 expression (E). (F,G) Embryos hybridized with probe to reveal ngn1 expression. Brackets indicate region of neural keel that expressed olig2. Excess cells in dla-;dld- mutant embryos expressed ngn1 at high level (G) compared to wild type (F). Scale bar: 20 µm (A-C); 50 µm (D-G).

View larger version (55K): [in a new window]   Fig. 3. Notch1aac expression blocks neurogenesis. Transverse sections, dorsal to top, labeled by immunocytochemistry to reveal Hu proteins. (A) Wild-type control embryo revealing normal distribution of neurons at 48 hpf. (B) 48 hpf transgenic embryo, in which Notch1aac expression was induced at 10, 24 and 36 hpf, had a deficit of neurons. Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 4. Notch1aac increases the number of ventral spinal cord cells that express olig2. Transverse sections, dorsal to top, labeled by olig2 RNA hybridization. (A) Wild-type control embryo revealing normal distribution of olig2+ cells at 26 hpf. (B) 26 hpf transgenic embryo in which Notch1aac was induced at 8 and 24 hpf expressed olig2 similarly to wild type in spinal cord (sc) but expressed olig2 ectopically in somites (so). (C) Wild-type control embryo revealing normal distribution of olig2+ cells at 36 hpf. (D) Excess spinal cord cells expressed olig2 at 36 hpf in transgenic embryo that was heat shocked at 8 and 24 hpf. (E) Quantification of olig2+ cells at 24 and 36 hpf in wild-type (wt) and Notch1aac-expressing embryos. Bars indicate the average number of olig2+ cells per transverse section. Data were obtained from 20 sections from each of five control and five heat-shocked transgenic embryos for each time point. P<0.00000001 by Student's t test. Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 5. Notch1aac promotes OPC specification. Transverse sections, dorsal to top, labeled by sox10 RNA hybridization. (A) 26 hpf wild-type embryo. Spinal cord (sc), somite (so) and notochord (nc) cells did not express sox10. (B) Transgenic embryo in which Notch1aac was induced at 8 and 24 hpf. Somite cells expressed sox10 but spinal cord and notochord cells did not. (C) 52 hpf wild-type embryo hybridized with sox10 probe. sox10+ cells had mostly dispersed from ventral spinal cord (bracket). Neural crest-derived cells on the medial migratory pathway also expressed sox10 (arrow). (D) Transgenic embryo in which Notch1aac was induced at 8, 24 and 36 hpf. Excess ventral spinal cord cells expressed sox10 (bracket) as did presumptive neural crest cells (arrows). (E) Quantification of sox10+ cells at 52 hpf in wild-type (wt) and Notch1aac-expressing embryos. Bars indicate the average number of sox10+ cells per transverse section. Data were obtained from 20 sections from each of 5 control and 5 heat-shocked transgenic embryos. P<0.00000001 by Student's t test. Scale bar: 20 µm.

View larger version (102K): [in a new window]   Fig. 6. Notch1aac inhibits oligodendrocyte differentiation. Side views of 3.5 dpf embryos, dorsal to top. (A) Wild-type embryo showing normal distribution of plp1/dm20+ cells in spinal cord (sc). (B) Transgenic embryo in which Notch1aac was induced at 60 hpf. The number of plp1/dm20+ cells was greatly reduced. Arrows indicate plp1/dm20+ cells in ventral spinal cord. Notochord indicated by nc. Scale bar: 20 µm.

View larger version (12K): [in a new window]   Fig. 7. Model for Delta-Notch regulated specification of pMN precursors for motoneuron and oligodendrocyte fates. Primary motoneurons (pmn) are the first cells to differentiate and activate Notch in neighboring cells via Delta. Notch activation maintains these cells as precursors during the period of primary motoneuron specification. Downregulation of Delta expression in differentiating primary motoneurons results in loss of Notch activity in some precursors, which then adopt secondary motoneuron (smn) fate. These cells upregulate Delta expression, maintaining high Notch activity in remaining precursors, which are specified as OPCs. Maintenance of high Notch activity in OPCs inhibits their differentiation into premyelinated oligodendrocytes (pre oligo). In dla-/-;dld-/- embryos, which have reduced levels of Notch activity, excess pMN precursors are specified as primary motoneurons at the expense of secondary motoneurons and oligodendrocytes. This model does not account for possible lineage relationships between different cell types.