1. Developmental Biology
  2. Neuroscience

Spinal cord precursors utilize neural crest cell mechanisms to generate hybrid peripheral myelinating glia

  1. Laura Fontenas
  2. Sarah Kucenas  Is a corresponding author
  1. Department of Biology, University of Virginia, United States
https://doi.org/10.7554/eLife.64267
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Cite this article
  1. Laura Fontenas
  2. Sarah Kucenas
(2021)
Spinal cord precursors utilize neural crest cell mechanisms to generate hybrid peripheral myelinating glia
eLife 10:e64267.
https://doi.org/10.7554/eLife.64267
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10 figures, 7 videos, 2 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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MEP glia are hybrid, centrally derived, peripheral myelinating glia.

(A) Timeline of MEP glial development. MEP glia (purple) are specified in the ventral spinal cord (gray) after 36 hpf, exit through motor exit points at ~50 hpf, divide, and migrate to eventually initiate myelination of motor root axons (pink) starting at 72 hpf. mn: motorneuron, LFP: lateral floor plate. (B–F) Lateral views of the motor exit point showing (B) a nkx2.2a+/foxd3+ MEP glia (outlined arrowhead) exiting the spinal cord at the motor exit point at 48 hr post-fertilization (hpf); (C) an olig2+/foxd3+ MEP glia (outlined arrowhead) along motor nerve root axons at 55 hpf; (D) sox10+/nkx2.2a+ MEP glia (outlined arrowheads) and sox10-/nkx2.2a+ perineurial glia (arrow) at 72 hpf; (E) sox10+/nkx2.2a+ MEP glial sheaths (outlined arrowheads) and sox10-/nkx2.2a+ perineurial cells at 4 days post-fertilization dp (arrowhead) and (F) foxd3+ MEP glia making mbp+ myelin sheaths (outlined arrowheads) at 4 dpf. Insets show single z-plane images. (G) In olig2:egfp and nkx2.2a:nls-egfp larvae at 55 hpf, olig2+ pMN domain cells (teal), OPCs (gray), and MEP glia (purple) are labeled, as are nkx2.2a+ LFP cells (yellow), OPCs (gray) and MEP glia (purple). These images were used for fluorescence intensity measurement. (H) Violin plot of mean intensity of GFP fluorescence of olig2:egfp+ cells at 55 hpf (OPCs: 54914 ± 1674 arbitrary units (A.U.), MEP glia: 7149 ± 394, pMN domain cells: 29934 ± 1481). (I) Violin plot of mean intensity of GFP fluorescence of nkx2.2a:nls- egfp cells at 55 hpf (OPCs: 22981 ± 2013, MEP glia: 28610 ± 1624, lateral floor plate cells: 24049 ± 1602). (H–I) (n = 28 MEP glia, n = 28 OPCs and n = 28 neural tube cells from seven embryos). Asterisks denote the dorsal root ganglion (DRG) and yellow dashed lines denote the edge of the spinal cord. Scale bar (B–G) 20 µm.

Figure 1—source data 1

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Figure 1—figure supplement 1
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Development of nkx2.2a+MEP glia.

(A) Lateral view of the trunk of a foxd3:mcherry;nkx2.2a:mcerulean embryo showing nkx2.2a+/foxd3+ MEP glia (outlined arrowheads) and nkx2.2a+/foxd3- perineurial glia (arrowheads) at MEP TZs at 50 hpf. (B) Percentage of MEP glia versus perineurial glia that exit the neural tube first; n = 52 motor nerves. (C) Lateral view of a motor exit point in an olig2:dsred; nkx2.2a:megfp embryo showing nkx2.2a+/olig2+ MEP glia dividing (outlined arrowheads) at 55 hpf. (D) In situ hybridization of olig2+ cells in the spinal cord at 48 and 72 hpf. Dashed lines denote the edge of the spinal cord and asterisks denote the DRG. Scale bar (A) 25 µm, (C) 20 µm, (D) 10 µm.

Figure 1—figure supplement 1—source data 1

Source data for Figure 1—figure supplement 1 .

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Figure 1—figure supplement 2
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Fluorescence intensity over the timecourse of MEP glial development.

(A) Single z-plane confocal images of the motor exit points showing foxd3+/nkx2.2a+ MEP glia (arrowheads) at 48, 50, 55,and 72 hpf in foxd3:mcherry;nkx2.2a:nls-egfp embryos. (B) Single z-plane confocal images of the motor exit points showing foxd3+/olig2 + MEP glia (arrowheads) at 48, 50, 55 hpf and foxd3+/olig2- MEP glia (arrowheads) at 72 hpf in foxd3:mcherry;olig2:egfp embryos. (C) Relative GFP fluorescence intensity of MEP glial cells. Mean ± SEM of nkx2.2a fluorescence levels at 48hpf: 1 ± 0.05; 50hpf: 0.92 ± 0.06, p=0.53; 55hpf: 0.79 ± 0.06, p=0.03; 72hpf: 0.76 ± 0.05, p=0.02. Mean ± SEM of olig2 fluorescence levels at 48hpf: 1 ± 0.04; 50hpf: 0.62 ± 0.03, p<0.0001; 55hpf: 0.47 ± 0.03, p<0.0001; 72hpf: 0.36 ± 0.02, p<0.0001. (A–B) Scale bar, 20 μm.

Figure 1—figure supplement 2—source data 1

Source data for Figure 1—figure supplement 2 .

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Figure 1—figure supplement 3
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Transverse sections of the neural tube of olig2:egfp;nkx2.2a:nls-mcherry embryos at 24, 30, 36, 48, and 72 hpf showing olig2+/nkx2.2a+ cells (outlined arrowheads).

All sections were collected along the yolk extension. White dashed lines show the dorsal edge of the lateral floor plate and yellow dashed lines denote the edge of the spinal cord. Scale bar, 25 µm.

Figure 2 with 1 supplement
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Ventral neural tube radial glial precursors give rise to MEP glia.

(A) Transverse section of a foxd3:mcherry;olig2:egfp embryo at 48 hpf showing a foxd3+/olig2+ MEP glial cell (outlined arrowhead) ventral to the olig2 pMN domain in the spinal cord. (B) Transverse section of a foxd3:mcherry;nkx2.2a:mcerulean embryo at 48 hpf showing a foxd3+/nkx2.2a+ MEP glial cell (outlined arrowhead). (C) Transverse section of a foxd3:mcherry;nkx2.2a:mcerulean;olig2:egfp embryo at 48 hpf showing a foxd3+/nkx2.2a+/olig2+ triple positive MEP glial cell (outlined arrowhead) in the p3 domain of the neural tube, just ventral to the pMN domain. (D) Transverse section of a nkx2.2a:nls-egfp embryo showing nkx2.2a+/GFAP+ radial glia (outlined arrowhead) at 48 hpf (top panel) and 72 hpf (bottom panel). Yellow dashed lines outline the edge of the spinal cord. Top right corner white boxes show higher magnification of bottom white boxes. Scale bar, (A–D) 10 µm.

Figure 2—figure supplement 1
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Lateral view of the trunk of a foxd3:mcherry;olig2:egfp embryo showing one foxd3+ MEP glial cell (outlined arrowhead) at the MEP TZ at 48 hpf.

Yellow dashed lines denote the edge of the spinal cord and asterisks denote the DRG. Scale bar, 10 µm.

Figure 3 with 2 supplements
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MEP glia require foxd3 to exit the spinal cord.

(A) MEP TZ in a foxd3:mcherry;olig2:egfp larvae showing foxd3+ MEP glia (white outlined arrowhead) along the motor root of a foxd3+/- control larva and stalled in the spinal cord of a foxd3-/- larva (white arrowhead) at 3 dpf. Note the presence of peripheral OPCs (yellow outlined arrowheads) along motor nerve axons in a foxd3-/- sibling lacking peripheral MEP glia. (B) Bright-field images of foxd3+/- and foxd3-/- siblings at 3 dpf reveal no developmental delay in foxd3-/- larvae. (C) Percentage of motor nerves with MEP glia in foxd3+/- (n = 150 nerves from 10 larvae) and foxd3-/- siblings (n = 270 nerves from 27 larvae) at 3 dpf. (D) Mean ± SEM of peripheral OPCs in olig2:egfp;foxd3:mcherry;foxd3+/- (0.07 ± 0.07, n = 15 larvae) and foxd3-/- (14.07 ± 1.2, n = 27 larvae) larvae at 3 dpf; p<0.0001. (E) Transverse sections of the spinal cord in foxd3:mcherry;foxd3+/- and foxd3-/- larvae showing MEP glia (outlined arrowheads) at the motor nerve root in a control sibling and MEP glia (arrowheads) in the lateral floor plate in a foxd3 mutant at 72 hpf. (F) Immunohistochemistry showing Sox10+/foxd3+ MEP glia (white outlined arrowheads) along motor nerve root axons in a foxd3+/- control embryo, and a Sox10+/foxd3+ MEP glial cell (white arrowhead) stalled in the spinal cord of a foxd3-/- larvae at 3 dpf. Yellow outlined arrowheads show OPCs in the spinal cord in a control larva and along peripheral axons in a foxd3-/- larva. Asterisks denote the DRG in control larvae. Note the absence of DRG in foxd3 mutants. (G) Dot plot of markers first detected in MEP glia, in percent. (H) Control and foxd3 mRNA-injected nkx2.2a:nls-egfp;foxd3:mcherry embryos showing a nkx2.2a+/foxd3+ MEP glia (outlined arrowhead) in the PNS in a control embryo and nkx2.2a+/foxd3+ MEP glia both in the CNS (arrowhead) and PNS (outlined arrowhead) in the injected embryo, at 50 hpf. (I), Mean ± SEM of additional CNS-located MEP glia indicates 0.08 ± 0.03 MEP glia in control embryos (n = 76 hemi-segments from 10 embryos), and 0.54 ± 0.07 MEP glia in foxd3 mRNA injected embryos (n = 78 hemi-segments from 10 embryos) at 50 hpf; p<0.0001. Asterisks denote the DRG and yellow dashed lines denote the edge of the spinal cord. Scale bar, (A, F) 25 µm, (B) 0.5 mm, (E) 10 µm, and (H) 20 µm.

Figure 3—source data 1

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Figure 3—figure supplement 1
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MEP glia do not exit the spinal cord infoxd3mutant larvae.

(A) In situ hybridization showing wif1+ MEP glia (yellow outlined arrowheads) along the motor nerve root in a foxd3+/- control larva and wif1+ MEP glia (black arrowheads) in the spinal cord of foxd3-/- larva at 3 dpf. (B) Lateral view of a foxd3:mcherry; olig2:egfp;foxd3-/- larva at 3 dpf showing a foxd3+ MEP glia in the CNS (white arrowhead) and olig2+ OPCs in the PNS (yellow outlined arrowheads). Dashed box shows single z-plane confocal image of a HuC immunohistostaining showing olig2+/HuC+ motorneurons and a foxd3+/HuC- MEP glial cell (white arrowhead). Scale bar, (A–B) 10 µm.

Figure 3—figure supplement 2
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Schematic of the zebrafish neural tube showing foxd3-dependent MEP glial (purple) delamination from the lateral floor plate (LFP), mirroring foxd3-dependent neural crest cell (red) migration from the dorsal neural tube.
Figure 4
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Matrix metalloproteinases participate in MEP glial exit.

(A) Lateral view of the zebrafish trunk showing a nkx2.2a+/foxd3+ MEP glial cell (outlined arrowhead) delaminate from the lateral floor plate and exit the spinal cord in a control embryo and nkx2.2a+/foxd3+ MEP glia unable to exit the spinal cord (white arrowhead) in an embryo treated with 100 µM GM6001 from 36 to 48 hpf. (B) Percentage of MEP glia in the spinal cord, absent, or present along motor nerves at 55 hpf in DMSO control (n = 68 nerves from six embryos) and GM6001- treated embryos (n = 68 nerves from eight embryos). (C) Percentage of MEP glia in the spinal cord, absent, or present along motor nerves at 72 hpf in DMSO control (n = 70 nerves from seven larvae) and GM6001-treated larvae (n = 70 nerves from seven larvae). (D) Transverse sections of foxd3:mcherry;nkx2.2a:megfp spinal cords at 72 hpf showing nkx2.2a+/foxd3+ MEP glia in the PNS (outlined arrowheads) in DMSO control and nkx2.2a+/foxd3+ MEP glia in the lateral floor plate (arrowhead) in GM6001-treated larvae. Yellow dashed lines outline the edge of the spinal cord. (E) In situ hybridizations showing mmp17b in the spinal cord of foxd3+/- and foxd3-/- siblings and adamts3 expression in the spinal cord of foxd3+/- and foxd3-/- siblings and foxd3 mRNA injected embryos at 48 hpf. Scale bar, (A, D, E) 10 µm.

Figure 4—source data 1

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Figure 5 with 1 supplement
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Clonal analysis reveals that neural tube precursors give rise to one MEP glial progenitor per motor exit point.

(A) Lateral view of Sox10 immunohistochemistry in a nkx2.2a:cre;ubi:zebrabow larvae showing Cre recombinase driven recombination (blue and green) in nkx2.2a+ cells at 4 dpf. Floor plate cells (bracket) and Sox10+ MEP glia along two distinct nerves (white and yellow outlined arrowheads respectively) labeled. (B) Percentage of MEP glia present along a single motor nerve sharing a common (96.67%) vs distinct neural tube precursor (3.33%); n = 214 MEP glia and n = 60 motor nerves in 20 animals. (C) Ternary plot showing the intensity profile of 14 MEP glia present on three adjacent motor nerves (white and yellow symbols in C correspond to white and yellow outlined arrowheads in A) for RFP, YFP and CFP, in percent. (D) Mean ± SEM of MEP glial cell divisions per motor nerve (2.19 ± 0.18) between 48 and 72 hpf; n = 27 nerves in 12 larvae. (E) DMSO control and aphidicolin/hydroxyurea (HUA)-treated nkx2.2a:nls-egfp;foxd3:mcherry larvae showing nkx2.2a+/ foxd3+ MEP glia (outlined arrowheads) along motor nerve root axons at 72 hpf. (F) Mean ± SEM of MEP glia along motor nerve roots at 72 hpf indicating 2.82 ± 0.12 MEP glia in control larvae (n = 79 hemi-segments from 10 larvae), and 0.90 ± 0.04 MEP glia in aphidicolin/HUA treated larvae (n = 89 hemi-segments from 10 larvae; p<0.0001). (G) Lateral view of a foxd3:mcherry;sox2:egfp larvae at 55 hpf showing foxd3+/sox2+ MEP glia (white outlined arrowhead) and DRG (asterisk). (H) Singe z plane transverse section of a BLBP immunostaining in foxd3:mcherry;nkx2.2a:mcerulean larvae showing BLBP+/foxd3+/nkx2.2a+ MEP glia (outlined arrowheads) outside the spinal cord along the motor nerve root at 55 hpf. Asterisks denote the DRG and yellow dashed lines denote the edge of the spinal cord. Scale bar, (A) 25 µm, (E, H) 20 µm, (G) 10 µm.

Figure 5—source data 1

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Figure 5—figure supplement 1
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MEP glial proliferation.

(A) Experimental design for MEP glial EdU incorporation assay. (B) Lateral view of a nkx2.2a:nls-eGFP larva showing EdU+/GFP+/Sox10+ MEP glia at 72 hpf (outlined arrowheads). (C) Percentage of EdU+ MEP glia per motor nerve at 72 hpf; n = 89 MEP glia in 10 larvae. (D) Distribution of the number of MEP glia per motor nerve at 4 dpf; n = 214 MEP glia and. n = 60 motor nerves in 20 larvae. (E) Lateral view of a sox2:egfp larvae at 55 hpf showing sox2+ MEP glia (white outlined arrowhead). Yellow dashed lines denote the edge of the spinal cord. Scale bar, (B) 20 µm, (E) 10 µm.

Figure 5—figure supplement 1—source data 1

Source data for Figure 5—figure supplement 1 .

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Figure 6 with 2 supplements
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MEP glial development is axonal dependent.

(A) Lateral view of sox10:eos;nbt:dsred WT control and plexinA3-/- siblings at 3 dpf showing MEP glia along both motor nerve axons (outlined arrowheads) and ectopic motor nerve axons (arrowheads). Brackets denote ectopic motor exit points. (B) Lateral views of sox10:tagrfp;hb9:yfp-ntr larvae from 48 to 72 hpf, treated with either 2% DMSO or 20 mM metronidazole(MTZ)/2% DMSO from 9 to 72 hpf, showing sox10+ MEP glia (white outlined arrowheads) exit the spinal cord and migrate onto healthy motor axons in the control embryo. In the MTZ-treated larvae, MEP glia did not exit the spinal cord. Asterisks denote the DRG and yellow dashed lines denote the edge of the spinal cord. Scale bar, (A) 25 µm, (B) 20 µm.

Figure 6—figure supplement 1
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Ectopic motor axons are associated with ectopic MEP glia in plexinA3 mutant larvae.

(A) Lateral view of three somites of photoconverted sox10:eos;plexinA3 siblings at 72 hpf, showing green, unphotoconverted MEP glia along three motor nerves (white outlined arrowheads) in plexinA3+/+ and -/- larvae, and along ectopic motor nerves (white arrowheads) in plexinA3-/- larvae. (B) Lateral view of one somite of photoconverted sox10:eos;plexinA3 siblings showing green, unphotoconverted MEP glia along motor axons (white outlined arrowheads) and ectopic motor axons (white arrowhead), and red, photoconverted neural crest cells (shown in magenta) at 72 hpf. Yellow dashed lines denote the edge of the spinal cord and asterisks denote the DRG. Scale bar (A–B) 25 μm.

Figure 6—figure supplement 2
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MEP glia do not exit the spinal cord in the absence of motor axons.

(A) In situ hybridization showing wif1+ MEP glia (outlined arrowheads) along the motor nerve root in a DMSO-treated hb9:yfp-ntr larva and wif1+ MEP glia (arrowheads) in the spinal cord of a MTZ-treated hb9:yfp-ntr larva at 3 dpf. (B) Lateral views of foxd3:mcherry;hb9:yfp-ntr larvae at 50 and 72 hpf, treated with either 2% DMSO or 20 mM metronidazole (MTZ)/2% DMSO from 9 to 72 hpf, showing foxd3+ MEP glia (outlined arrowheads) along motor axons in the control larvae. In the MTZ-treated larvae, foxd3+ MEP glia did not exit the spinal cord. Yellow dashed lines denote the edge of the spinal cord and asterisks denote the DRG. Scale bar, (A) 10 µm, (B) 20 µm.

Figure 7 with 1 supplement
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Neuregulin 1 type III drives MEP glial directed migration.

(A) Motor exit point of sox10:eos WT and nrg1 -/- siblings photoconverted at 48 hpf and imaged at 3 dpf showing the presence of MEP glia (outlined arrowheads) and Schwann cells (SCs) (arrowhead) in a control larva and the presence of an oligodendrocyte membrane extension (pink arrowhead) in a nrg1 -/- larva that lacks MEP glia and SCs. Yellow dashed lines denote the edge of the spinal cord. (B) In situ hybridization showing wif1+ MEP glia along motor nerve root axons in a WT control larva (outlined arrowheads, n = 24 larvae) and the absence of wif1+ MEP glia along motor nerve root axons in a nrg1 mutant larva at 3 dpf (n = 16 larvae). Arrowheads indicate the presence of wif1+ cells near the lateral line nerve in a nrg1 mutant larva at 3 dpf. (C) In situ hybridization showing wif1+ MEP glia along motor nerve root axons in a WT control larva (outlined arrowheads, n = 20 larvae) and the absence of wif1+ MEP glia along motor nerve root axons in an erbb3b mutant larva at 3 dpf (n = 14 larvae). Arrowheads indicate the presence of wif1+ cells near the lateral line nerve in an erbb3b mutant larva at 3 dpf. Scale bar (A–C), 10 μm.

Figure 7—figure supplement 1
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MEP glia are absent along the motor root in nrg1 mutant larvae.

(A) In situ hybridization showing plp1a+ oligodendrocytes in the spinal cord (outlined arrowheads) in nrg1+/+ larvae and in both the CNS (outlined arrowheads) and the PNS (arrowhead) in nrg1-/- larvae at 3 dpf. (B) Dorsal view of the zebrafish trunk showing wif1+ MEP glia in WT and nrg1-/- larvae at 3 dpf. wif1+ MEP glia are located at the edge of the spinal cord (white outlined arrowheads) in WT larvae while their location is lateral (white arrowheads), close to the skin, in nrg1-/- larvae. Yellow brackets denote the width of the spinal cord. White dashed lines denote the trunk. Scale bar, (A) 10 µm, (B) 250 µm.

Figure 8 with 2 supplements
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Neuregulin 1 type III drives MEP glial development.

(A) In situ hybridization showing wif1+ MEP glia (white outlined arrowheads) along motor nerve roots in WT larvae (n = 30 larvae) at 3 dpf and both inside (green outlined arrowheads) and outside (white outlined arrowheads) the spinal cord in neuroD:gal4;UAS:hNrg1 larvae (n = 30 larvae). Zrf1 immunostaining shows GFAP+ radial glia and denotes the outline of the spinal cord. (B) Lateral view of 3 dpf nkx2.2a:nls-egfp;foxd3:mcherry control and neuroD:gal4;UAS:hNrg1 larvae showing nkx2.2a+/foxd3+ MEP glia (outlined arrowheads) along motor nerve root axons. MEP glia are found in both the PNS (outlined arrowheads) and in the ventral spinal cord (arrowheads) in neuroD:gal4;UAS:hNrg1 larvae. Asterisks denote the DRG. (C) Mean ± SEM of additional, centrally located MEP glia per hemi-segment in control (0.17 ± 0.11; n = 12 larvae) and neuroD:gal4;UAS:hNrg1 larvae (1.3 ± 0.3; n = 20 larvae) at 3 dpf. (D) PH3 immunostaining on spinal cord transverse sections in neuroD:gal4;nkx2.2a:nls-egfp control larvae and neuroD:gal4;UAS:hNrg1;nkx2.2a:nls-egfp larvae showing PH3+/nkx2.2a+ proliferating radial glia (outline arrowheads). (E) Mean ± SEM of PH3+/nkx2.2a+ cells in the ventral spinal cord per 20 µm section in neuroD:gal4 (0.35 ± 0.12; n = 17 sections from four embryos) and neuroD:gal4;UAS:hNrg1 larvae (1.22 ± 0.3; n = 18 sections from four embryos). (F) Transverse section of a nkx2.2a:megfp larva at 4 dpf showing Sox2+/nkx2.2a+ radial glial precursors. Yellow dashed lines denote the edge of the spinal cord. Scale bar (A,B,D,F), 10 µm.

Figure 8—source data 1

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Figure 8—figure supplement 1
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In situ hybridization showing the absence of Schwann cells in the spinal cord of a control sibling (n = 20 larvae), and the presence of krox20+ Schwann cells (white outlined arrowheads) in the ventral and dorsal spinal cord in a neuroD:gal4;UAS:hNrg1 larva at 4 dpf (n = 20 larvae).

Zrf1 immunostaining shows GFAP+ radial glia and denotes the outline of the spinal cord. Scale bar, 10 µm.

Figure 8—figure supplement 2
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EdU proliferation assay.

(A) Experimental design for MEP glial EdU incorporation assay. (B) Mean ± SEM of number of EdU+ MEP glia per motor nerve in control (1.74 ± 0.15) and neuroD:gal4;UAS:hNrg1 (2.70 ± 0.18) larvae at 72 hpf (p=0.0002). (C) Mean ± SEM of total number of MEP glia per motor nerve in control (2.48 ± 0.12) and neuroD:gal4;UAS:hNrg1 (3.53 ± 0.16) larvae at 72 hpf (p<0.0001). (B– C) n = 58 nerves in 10 control larvae and n = 62 nerves in 12 neuroD:gal4;UAS:hNrg1 larvae.

Figure 8—figure supplement 2—source data 1

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Videos

Video 1
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foxd3+ MEP glia (arrow) exit the spinal cord and migrate onto olig2+ motor axons in foxd3:mcherry;olig2:egfp;foxd3+/- larvae between 48 and 72 hpf.

Images were taken every 15 min and the movie runs at 10 frames per second (fps).

Video 2
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foxd3+ MEP glia are stalled in the spinal cord (arrow) in foxd3:mcherry;olig2:egfp;foxd3-/- larvae imaged between 48 and 72 hpf.

Images were taken every 15 min and the movie runs at 10 fps.

Video 3
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A single nkx2.2a+/foxd3+ MEP glia (arrow) has exited the spinal cord and divides in the PNS in control foxd3:mcherry;nkx2.2a:megfp;nkx2.2a:nls-egfp embryos.

Video on the right is the merge of GFP and mCherry channels. Images were taken every 15 min from 48 to 72 hpf and the movie runs at 10 fps.

Video 4
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An additional nkx2.2a+/foxd3+ MEP glia (arrow) exits the spinal cord in foxd3 mRNA-injected foxd3:mcherry;nkx2.2a:megfp;nkx2.2a:nls-egfp embryos.

Video on the right is the merge of GFP and mCherry channels. Images were taken every 15 min from 50 to 72 hpf and the movie runs at 10 fps.

Video 5
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One foxd3+/nkx2.2a+ MEP glial progenitor delaminates from the lateral floor plate, exits the spinal cord, and divides to give rise to several MEP glia along the motor nerve in a foxd3:mcherry;nkx2.2a:megfp control embryo.

Images were taken every 10 min from 48 to 72 hpf and the movie runs at 10 fps.

Video 6
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One foxd3+/nkx2.2a+ MEP glial progenitor delaminates from the lateral floor plate and stalls at the MEP in the spinal cord in a foxd3:mcherry;nkx2.2a:megfp embryo treated with GM6001 from 36 to 72 hpf.

Images were taken every 10 min from 48 to 72 hpf and the movie runs at 10 fps.

Video 7
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One nkx2.2a+/foxd3+ MEP glial progenitor exits the spinal cord at the MEP transition zone and undergoes cell division in the PNS to generate several MEP glia in a nkx2.2a:nls-egfp/foxd3:mcherry embryo.

Images were taken every 15 min from 50 to 72 hpf and the movie runs at 10 fps.

Tables

Key resources table
Reagent type
(species)
or resource		DesignationSource or referenceIdentifiersAdditional information
Strain
(Danio rerio)		AB*ZIRCRRID:ZFIN_ZDB-GENO-960809-7
Genetic reagent
(Danio rerio)		Tg(nkx2.2a(3.5):Cre;
cmlc2:eGFP)uva42		This paper
Genetic reagent
(Danio rerio)		Tg(nkx2.2a(3.5):
mCerulean3)uva41		This paper
Genetic reagent
(Danio rerio)		Tg(nkx2.2a(3.5):nls-eGFP)uva1This paper
Genetic reagent
(Danio rerio)		Tg(mbp:
eGFP-CAAX)ue2		Almeida et al., 2011RRID:ZFIN_ZDB-ALT-120103-2
Genetic reagent
(Danio rerio)		Gt(foxd3:
mcherry)ct110R		Hochgreb-Hägele and Bronner, 2013RRID:ZFIN_ZDB-ALT-130314-2
Genetic reagent
(Danio rerio)		Tg(XlTubb:
DsRed) zf148		Peri and Nüsslein-Volhard, 2008RRID:ZFIN_ZDB-ALT-081027-2
Genetic reagent
(Danio rerio)		Tg(neuroD1:Gal4;
cmlc2:eGFP)uva22		Fontenas et al., 2019RRID:ZFIN_ZDB-ALT-191209-8
Genetic reagent
(Danio rerio)		Tg(nkx2.2a(3.5):
nls-mCherry)uva2		Zhu et al., 2019RRID:ZFIN_ZDB-ALT-200513-2
Genetic reagent
(Danio rerio)		Tg(olig2:eGFP)vu12Shin et al., 2003RRID:ZFIN_ZDB-ALT-041129-8
Genetic reagent
(Danio rerio)		Tg(olig2:
DsRed2)vu19		Shin et al., 2003RRID:ZFIN_ZDB-FISH-150901–8168
Genetic reagent
(Danio rerio)		Tg(sox10(4.9):Eos)w9McGraw et al., 2012RRID:ZFIN_ZDB-ALT-110721-1
Genetic reagent
(Danio rerio)		Tg(sox10(4.9):
nls-Eos)w18		McGraw et al., 2012RRID:ZFIN_ZDB-ALT-110721-2
Genetic reagent
(Danio rerio)		Tg(sox10(4.9):
TagRFP)uva5		Zhu et al., 2019RRID:ZFIN_ZDB-ALT-200513–7
Genetic reagent
(Danio rerio)		Tg(sox10(7.2):
mRFP)vu234		Kucenas et al., 2008bRRID:ZFIN_ZDB-ALT-080321–3
Genetic reagent
(Danio rerio)		Tg(UAS:hNrg1
type III)st85		Perlin et al., 2011RRID:ZFIN_ZDB-ALT-120221–8
Genetic reagent
(Danio rerio)		Tg(ubi:
Zebrabow-M)a131		Pan et al., 2013RRID:ZFIN_ZDB-ALT-130816–2
Genetic reagent
(Danio rerio)		Tg(2xNRSE-2xMnx1-
Mmu.Fos:KalTA4,5xUAS-
ADV.E1b:GAP-YFP-2A-Eco.
NfsBT41Q/
N71S/F124T)lmc008		Mathias et al., 2014RRID:ZFIN_ZDB-ALT-151021–5
Genetic reagent
(Danio rerio)		Tg(sox2(2.9):
eGFP)uva55		This paper
Genetic reagent
(Danio rerio)		neuregulin1z26Perlin et al., 2011RRID:ZFIN_ZDB-ALT-120308–1
Genetic reagent
(Danio rerio)		plexinA3 p13umalPalaisa and Granato, 2007RRID:ZFIN_ZDB-ALT-071126–1
Genetic reagent
(Danio rerio)		erbb3bst48Lyons et al., 2005RRID:ZFIN_ZDB-ALT-050512–6
Recombinant
DNA reagent		p5E-nkx2.2a(−3.5)Pauls et al., 2007N/A
Recombinant
DNA reagent		pME-mcerulean3Zhu et al., 2019N/A
Recombinant
DNA reagent		p5E-sox2(−2.9)This paper
Recombinant
DNA reagent		pME-nls-eGFPKwan et al., 2007N/A
Recombinant
DNA reagent		p3E-polyAKwan et al., 2007N/A
Recombinant
DNA reagent		pDestTol2CG2Kwan et al., 2007N/A
Recombinant
DNA reagent		pME-creThis paper
Recombinant
DNA reagent		Human adamts3Genomics-onlineCat.
#ABIN3996515
Commercial
assay or kit		pENTR 5´-TOPO
cloning kit		InvitrogenCat. #K59120
Commercial
assay or kit		LR clonase II plusInvitrogenCat. #12538–120
Commercial
assay or kit		Click-it EdU Cell
proliferation kit
for imaging.
Alexa Fluor 647 dye		InvitrogenCat. #C11340
Commercial
assay or kit		mMESSAGE mMACHINE
sp6 transcription kit		FisherCat. #AM1340
Chemical
compound, drug		DAPI fluoromount-GSouthern BiotechCat. #0100–20
Chemical
compound, drug		DIG RNA labeling mixRocheCat.
#11277073910
Chemical
compound, drug		metronidazoleSigmaCat#M1547;
CAS#443-48-1		20 mM
Chemical
compound, drug		GM6001Enzo Life SciencesCat.
#BML-EI300-0001;
CAS# 142880-36-2		100 μM
Chemical
compound, drug		aphidicolinSigmaCat. #A0781;
CAS# 38966-21-1		150 μM
Chemical
compound, drug		hydroxyureaSigmaCat. #H8627;
CAS#127-07-1		20 mM
AntibodyMouse anti-GFAPZIRCCat. #Zrf1 ;
RRID:AB_10013806		1 :1000
AntibodyAnti-digoxigenin-AP,
Fab fragments
from sheep		SigmaCat#11093274910;
RRID:AB_514497		1:5000
AntibodyRabbit anti-sox10Binari et al., 2013N/A1 :5000
Antibodychicken anti-GFPAbcamCat. #ab13970;
RRID:AB_300798		1 :500
AntibodyAlexa Fluor 488
goat anti-chicken		ThermoFisherCat. #A-11039;
RRID:AB_2534096		1:1000
AntibodyAlexa Fluor 647
goat anti-rabbit
IgG(H+L)		ThermoFisherCat. #A-21244;
RRID:AB_2535812		1:1000
AntibodyRabbit anti-BLBPSigmaCat. #ABN14;
RRID:AB_10000325		1:1000
AntibodyRabbit anti-PH3MilliporeCat. #06–570;
RRID:AB_310177		1:2000
AntibodyRabbit anti-sox2AbcamCat. #ab97959;
RRID:AB_2341193		1:500
AntibodyAlexa Fluor 647
goat anti-mouse
IgG(H+L)		ThermoFisherCat. #A-21235;
RRID:AB_2535804		1:1000
Sequence-based
reagent		Cre-FThis paperPCR primers5’-ATGTCCAATCTTCTAACCGT-3’
Sequence-based
reagent		Cre-RThis paperPCR primers5’-TTAGTCTCCATCCTCCAGCA-3’
Sequence-based
reagent		Foxd3-FThis paperPCR primers5’-CAGGGATCCATGA
CCCTGTCTGGAGGCA-3’
Sequence-based
reagent		Foxd3-RThis paperPCR primers5’-GAACTCGAGTCATTGA
GAAGGCCATTTCGATA-3’
Sequence-based
reagent		Sox2-FThis paperPCR primers5’-GTGAGTAACTTTT
GGGTGTGCGG-3’
Sequence-based
reagent		Sox2-RThis paperPCR primers5’-TTAAACCGATTTTC
TCGAAAGTCTAC-3’
Sequence-based
reagent		Mmp17b-FThis paperPCR primers5’-GGGAAGTGCTG
TGGATGTTT-3’
Sequence-based
reagent		Mmp17b-RThis paperPCR primers5’-TAATACGACTCACTATAGATG
AAACTCGAGCAGTGTTGG-3’
Sequence-based
reagent		Adamts3-FThis paperPCR primers5’-TCCTGGGGCT
AGACATGTGA-3’
Sequence-based
reagent		Adamts3-RThis paperPCR primers5’-TAATACGACTCACTATAGAGC
GCACAGTACGGATTTGA-3’
SoftwareImageJ/FijiImageJ.nih.govRRID:SCR_003070
SoftwarePrism 9GraphPad softwaresRRID:SCR_002798
SoftwareMetamorphMolecular DevicesRRID:SCR_002368
SoftwareImaris 9.6Oxford InstrumentsRRID:SCR_007370
SoftwareRStudioRStudioRRID:SCR_000432
Table 1
Strains and transgenic lines.

Picture description: Cross section of the spinal cord showing Sox2+ neural precursors (magenta), nkx2.2a+ precursors (cyan) and Sox10+ glia (yellow) at 4 days post-fertilization.

Full nameabbreviationReference
Tg(mbp:eGFP-CAAX)ue2mbp:egfp-caaxAlmeida et al., 2011
Gt(foxd3:mcherry)ct110Rfoxd3:mcherryHochgreb-Hägele and Bronner, 2013
Tg(XlTubb:DsRed) zf148nbt:dsredPeri and Nüsslein-Volhard, 2008
Tg(neuroD1:Gal4; cmlc2:eGFP)uva22neuroD:Gal4Fontenas et al., 2019
Tg(nkx2.2a:meGFP)vu17nkx2.2a:megfpKucenas et al., 2008b
Tg(nkx2.2a(3.5):Cre; cmlc2:eGFP)uva42nkx2.2a:creThis paper
Tg(nkx2.2a(3.5):mCerulean3)uva41nkx2.2a:mceruleanThis paper
Tg(nkx2.2a(3.5):nls-eGFP)uva1nkx2.2a:nls-egfpThis paper
Tg(nkx2.2a(3.5):nls-mCherry)uva2nkx2.2a:nls-mcherryZhu et al., 2019
Tg(olig2:eGFP)vu12olig2:egfpShin et al., 2003
Tg(olig2:DsRed2)vu19olig2:dsredShin et al., 2003
Tg(sox10(4.9):Eos)w9sox10:eosMcGraw et al., 2012
Tg(sox10(4.9):nls-Eos)w18sox10:nls-eosMcGraw et al., 2012
Tg(sox10(4.9):TagRFP)uva5sox10:tagrfpZhu et al., 2019
Tg(sox10(7.2):mRFP)vu234sox10:mrfpKucenas et al., 2008b
Tg(UAS:hNrg1 type III)UAS:hNrg1Perlin et al., 2011
Tg(ubi:Zebrabow-M)a131ubi:zebrabowPan et al., 2013
Tg(2xNRSE-2xMnx1-Mmu.Fos:KalTA4,5xUAS-ADV.E1b:GAP-YFP-2A-Eco.NfsBT41Q/N71S/F124T)lmc008hb9:yfp-ntrMathias et al., 2014
Tg(sox2(2.9):eGFP)uva55sox2:egfpThis paper
AB*wildtype
neuregulin1z26nrg1Perlin et al., 2011
plexinA3 p13umal (sidetracked)plexinA3Palaisa and Granato, 2007
erbb3bst48erbb3bLyons et al., 2005

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  1. Laura Fontenas
  2. Sarah Kucenas
(2021)
Spinal cord precursors utilize neural crest cell mechanisms to generate hybrid peripheral myelinating glia
eLife 10:e64267.
https://doi.org/10.7554/eLife.64267