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Ravanelli and Appel
SUPPLEMENTAL DATA
Movie S1. pMN Progenitors Differentiate as Motor Neurons in the Absence of Self-Renewing Divisions (Related to Figure 1)
Maximum intensity projection of confocal z-stack over time of transplanted olig2:EGFP+actb2:mCherry+ pMN progenitors. Time-lapse images show a lateral view of trunk spinal cord, with dorsal to the top. Basal processes of olig2+ progenitors become axons that exit the spinal cord. Images were captured at 30 minute intervals for 19 hours, beginning at 24 hpf.
Movie S2. pMN Progenitor Tracking (Related to Figure 1)
Movie shows an olig2:EGFP embryo injected with mRNA encoding dsRed2nuc. Main image is focused on the trunk spinal cord from lateral orientation and dorsal to the top. Left and top images are orthogonal projections showing transverse and dorsal orientations. Asterisk tracks one example cell that originated next to the ventricle and moved radially without an accompanying division. Images were captured at 25 minute intervals for 19 hours, beginning at 24 hpf.
Movie S3. Dorsal Origin of New pMN Cells (Related to Figure 3)
Confocal image series of olig2:Kaede over time. Kaede was photoconverted at 24 hpf and allowed to recover to determine the location of new olig2:Kaede expression. Merged images are on top. olig2:Kaedered is in the middle. olig2:Kaedegreen is on bottom.
Time-lapse images show a lateral view of trunk spinal cord, with dorsal to the top. Images were captured at 30 minute intervals for 11 hours, beginning at 24 hpf.
Movie S4. Recruitment of New pMN Progenitors Via Ventral Movement (Related to Figure 3)
Movie shows an olig2:EGFP embryo injected with mRNA encoding dsRed2nuc. Images are focused on the trunk spinal cord from lateral orientation and dorsal to the top. Nuclei of EGFP– (*)cells are tracked continuously. Daughter cells following divisions are also tracked with asterisks. By 39 hpf, numerous cells that originated dorsal to the pMN domain occupied positions within the pMN domain and expressed olig2:EGFP.Images were captured at 25 minute intervals for 19 hours, beginning at 24 hpf. Tracked cells in this movie are highlighted in Figure 3B with “o”.
Movie S5. Ventral Movement of Cells Within the pMN Domain During Progenitor Recruitment (Related to Figure 3)
Time-lapse movie of same embryo and imaging as movie S4, with a different population of cells highlighted for tracking.Movie shows an olig2:EGFP embryo injected with mRNA encoding dsRed2nuc. Images are focused on the trunk spinal cord from lateral orientation and dorsal to the top. Nuclei of EGFP+ (*)cells are tracked continuously. Daughter cells following divisions are also tracked with asterisks. By 39 hpf, numerous cells that originated at more dorsal positions within the pMN domain occupied more ventral positions within the pMN domain.Images were captured at 25 minute intervals for 19 hours, beginning at 24 hpf. Tracked cells in this movie are highlighted in Figure 3B with “+”.
Movie S6. Maintenance of Pard3-GFP Apical Localization During Progenitor Recruitment (Related to Figure 4)
olig2:Kaede embryos were injected with CMV:pard3-GFP DNA and photoconverted repeatedly during imaging to allow visualization of Pard3-GFP (green). Kaedered cells appear white. Orthogonal (yz (left, merge) and xy (right, Pard3-GFP only)) projections of confocal z-stacks of trunk spinal cord imaged from 24 hpf.Arrowheads denote Pard3-GFP apical localization in olig2:Kaedered-negative cell at 24 hpf that moves ventrally over time to become olig2:Kaedered-positive. Images were captured at 25 minute intervals for 24 hours, beginning at 24 hpf.
Movie S7. Loss of Pard3-GFP Apical Localization Concomitant With Dorsolateral Migration of Differentiating Neurons (Related to Figure 4)
olig2:Kaede embryos were injected with CMV:pard3-GFP DNA and photoconverted repeatedly during imaging to allow visualization of Pard3-GFP (green). Kaedered cells appear white. Orthogonal (yz (left) and xy (right)) projections of confocal z-stacks of trunk spinal cord imaged from 24 hpf.Arrowheads denote Pard3-GFP apical localization. olig2:Kaedered-negative cell at 24 hpf that differentiates and moves dorsolaterally over time to become a neuron is outlined by dashed magenta line.Images were captured at 25 minute intervals for 24 hours, beginning at 24 hpf.
Movie S8. Inhibition of Smoothened with Cyclopamine Blocks Ventral Movement and Progenitor Recruitment (Related to Figure 5)
Movie shows an olig2:EGFP embryo injected with mRNA encoding dsRed2nuc. Embryo was treated with 5 uM cyclopamine at 24 hpf and imaged continuously. Images are focused on the trunk spinal cord from lateral orientation and dorsal to the top. Nuclei of EGFP– (*)cells are tracked, and do not move ventrally or initiate expression of olig2:EGFP.Images were captured at 20 minute intervals for 19 hours, beginning at 24 hpf.