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First published online November 11, 2004
doi: 10.1242/10.1242/dev.01456

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Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling

Hae-Chul Park^*, Jimann Shin^* and Bruce Appel

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

View larger version (79K): [in a new window]   Fig. 1. Spinal cord olig2+ cells include diverse cell types. (A-H) Transverse sections of Tg[olig2:egfp] embryos. Antibodies indicated on each panel. (A) Twenty-four hpf embryo. Arrows indicate olig2:EGFP+, Isl+ motoneurons. (B) Thirty-six hpf embryo. Arrows and arrowheads mark olig2:EGFP+, Neurolin+ SMNs and olig2:EGFP+, Neurolin- cells, respectively. (C) Forty-eight hpf embryo treated with BrdU from 36 to 48 hpf. Arrows mark olig2:EGFP+, BrdU+ cells (yellow). (D) Three dpf embryo. olig2:EGFP+ cells include Hu+ neurons (arrowheads), dorsally and ventrally migrated Hu- OPCs (asterisks) and Hu- cells with radial morphology (arrows). (E) Three dpf embryo. Arrows and arrowheads mark olig2:EGFP+, zrf-1+ radial cells. Asterisks mark dorsally migrated OPCs. (F) EGFP expression, alone, of section shown in E. (G,H) Twenty-four hpf embryo. Anti-GABA labeling reveals olig2:EGFP+ KA' and putative VeLD interneurons and olig2:EGFP- KA'' interneurons. (I,J) Lateral confocal microscope images of 20 hpf (I) and 24 hpf (J) transgenic embryos showing olig2:EGFP+, GABA+ VeLD interneurons, olig2:EGFP+, GABA+ KA' interneurons and olig2:EGFP-, GABA+ KA'' interneurons. Arrowheads in I mark descending axons of VeLD neurons. Scale bar: 20 µm.

View larger version (67K): [in a new window]   Fig. 2. Clonal analysis of olig2:EGFPP/E neural plate cells. (A) Schematic representation of cell labeling strategy. The grid represents cells of the neural plate. Green boxes are olig2:EGFP+ cells, which, at the time of labeling, overlie the most medial neural plate cells as a consequence of cell movements during early neurulation. (B) Dorsal view of transgenic embryo immediately after labeling. (C-F) Side views, anterior to the left, dorsal up, of 2.5 dpf embryos showing examples of labeled cells, which include PMNs, KA' interneurons and VeLD interneurons. Filled and outlined arrowheads indicate different axon projections. P/E clone numbers correspond to clones in Table 1. Scale bar: 20 µm for B; 40 µm for C-F.

View larger version (63K): [in a new window]   Fig. 3. Clonal analysis of olig2:EGFPD/L neural plate cells. (A) Schematic representation of labeling strategy. (B) Dorsal view of transgenic embryo immediately after labeling. (C-F) Side views, anterior towards the left, dorsal upwards, of 2.5 dpf embryos showing examples of labeled cells, which include SMNs, CiD interneurons, OPCs (asterisks) and cells whose identities could not be determined (question marks). Arrowheads mark some of the axon projections. (D') Combined images of EGFP and rhodamine fluorescence of clone shown in D. The weak EGFP fluorescence of the CiD interneuron is obscured by the bright rhodamine signal. (D'',F') Schematic of clones shown in D,F. (G) Diagram, from a dorsal view, summarizing fate mapping results showing cell fate bias on the medial/ventral (m/v) to lateral/dorsal (l/d) axis of the neural plate/neural tube. Broken line marks midline of neural plate. Scale bars: 20 µm.

View larger version (183K): [in a new window]   Fig. 4. Zebrafish embryos express Hh ligands during the period of oligodendrocyte specification. All panels show transverse sections through trunk spinal cord (sc), with dorsal upwards. (A) Notochord (nc), medial floor plate (mfp) and lateral floor plate (lfp) express shh at 36 hfp. (B) At 48 hpf, medial floor plate and notochord express shh. (C) Medial floor plate also expresses twhh at 36 hpf and (D) 48 hpf. Scale bar: 20 µm.

View larger version (132K): [in a new window]   Fig. 5. Motoneurons and oligodendrocytes have genetically separable requirements for Hh signaling. Top three rows of panels show transverse sections through trunk spinal cord, with dorsal upwards. Bottom row shows lateral views of intact olig2:egfp embryos, with dorsal upwards and anterior towards the left. (A) Wild-type embryos expressed olig2 RNA in the ventral spinal cord (bracket), but the ventralmost cells were olig2-. Broken line indicates the ventral boundary of the spinal cord. (B) olig2:EGFP+ cells of wild-type embryos included Neurolin+ SMNs, OPCs (arrows), Neurolin- cells near ventricle (arrowhead) and a faint GFP+, Neurolin- cell near pial surface (asterisk), which could be a primary motoneuron or VeLD interneuron. (C) sox10+ OPCs in wild-type embryo. (D) Arrows indicate dorsally migrated OPCs in wild-type embryo. Bracket indicates dorsal spinal cord. (E-H) syu-/- embryos injected with twhh MO lacked olig2+ cells (E), secondary motoneurons (F) and OPCs (G,H). (I-L) Wild-type embryos injected with twhh MO expressed olig2 RNA (I) and produced secondary motoneurons (J) and OPCs (K,L). syu-/- embryos expressed olig2 RNA (M), but olig2+ cells were located more ventrally than normal. (M-P) syu-/- embryos had olig2:EGFP+, Neurolin+ secondary motoneurons and olig2:EGFP+, Neurolin- cells (asterisks) (N); however, they did not have OPCs (O,P). (O) sox10+ cells are probably Schwann cells (Dutton et al., 2001), which do not appear in all sections. Scale bar: 20 µm for upper three rows; 40 µm for bottom row.

View larger version (94K): [in a new window]   Fig. 6. Hh signaling is required for oligodendrocyte specification after dorsoventral spinal cord patterning and motoneuron development. All panels in the top five rows show transverse sections, dorsal upwards, of trunk spinal cord. Bottom two panels are side views of whole embryos, dorsal upwards and anterior leftwards. (A-E) Control embryos showing normal expression of various markers. (G-K) Embryos treated with cyclopamine from 6 hpf onwards did not express olig2 (G) or nkx2.2 (H), and expressed iro3 in ventral spinal cord (I), indicating that ventral spinal cord patterning was lost. These embryos did not produce SMNs (J) or OPCs (K). (L-Q) Embryos treated with cyclopamine from 30 hpf onward did not express olig2 by 36 hpf (L) but expressed nkx2.2 (M) and iro3 (N) in their normal patterns. SMNs were produced in normal numbers (O) but OPCs were absent (P,Q). (F) Untreated Tg[olig2:egfp] embryo showing OPCs (arrows) in dorsal spinal cord (brackets). Scale bar: 20 µm for top five rows; 80 µm for F and Q.

View larger version (143K): [in a new window]   Fig. 7. Conditional manipulation of Hh signaling reveals a crucial period for specification of oligodendrocyte precursors. All panels show transverse sections, dorsal side upwards. (A-D) Embryos incubated in cyclopamine from 6-26 hpf. At 26 hpf, these embryos did not express olig2 (A). iro3 expression included the ventralmost spinal cord cells (B; brackets in B,F,J). olig2 expression was not recovered by 36 hpf (C) and no sox10+ OPCs were evident by 48 hpf (D). (E-H) Embryos treated with cyclopamine from 11-26 hpf. olig2 expression was absent by 26 hpf (E) but a small domain of iro3- cells was present in the ventral spinal cord (F). These embryos recovered olig2 expression by 36 hpf, but in an abnormally ventral position (G). At 48 hpf, few sox10+ OPCs had developed (H). (I-L) Embryos treated with cyclopamine from 14-26 hpf. olig2 expression was not maintained until 26 hpf (I) but iro3 expression appeared normal (J, compare with Fig. 6C). olig2 expression was recovered by 36 hpf in ventralmost spinal cord cells (K) but in a larger domain compared with 11-26 hpf treated embryos. These embryos expressed sox10 at 48 hpf, but in an abnormally ventral position (L, compare with Fig. 6E). Scale bar: 20 µm.

View larger version (57K): [in a new window]   Fig. 8. Modulation of Hh signaling can promote OPC specification at the expense of secondary motoneurons. (A,C,E) Transverse sections, dorsal upwards, of Tg[olig2:egfp] embryos labeled with pan-neuronal anti-Hu antibody (red). Asterisks and arrows mark OPCs and radial cells, respectively. (B,D,F) Lateral views of intact Tg[olig2:egfp] embryos, dorsal upwards and anterior leftwards. Brackets indicate dorsal spinal cord. (A,B) Untreated embryos showing normal number and distribution of OPCs. (C,D) Embryos treated with cyclopamine from 11-26 hpf had few OPCs. (E,F) Embryos treated from 14-26 hpf had excess OPCs. (G) Quantification of effects on SMNs and OPCs. Embryos were labeled with anti-Neurolin antibody to reveal SMNs. A total of 20 sections were counted from four embryos for each experiment. Scale bar: 20 µm for top row; 80 µm for bottom row.

View larger version (12K): [in a new window]   Fig. 9. Summary of Hh signaling and ventral spinal cord patterning in zebrafish. Arrows represent Hh ligands expressed by notochord (nc) and floor plate (triangle). The olig2P/E domain (dark green) is formed at neural plate stage and expands to include the olig2D/L domain (light green) by neural keel stage. Together, these comprise the pMN precursor domain. We propose that PMNs, KAs and VeLDs are produced first, from olig2P/E, followed by SMNs and CiDs and, later, OPCs from olig2D/L. Broken lines represent OPC dorsal migration.