Article Figures & Data
Figures
- Figure 1.
tdTomato is expressed in glial cells of the adult Hb9cre;Ai14 mouse in an increasing rostrocaudal gradient. A, Cartoon depicting the breeding scheme used to generate mice in this study. Hb9cre male sires were mated to Ai14 female dams to produce Hb9cre;Ai14 F1 offspring. B, Gross image of whole spinal cord from a P28 Hb9cre;Ai14 mouse. Black markings and arrowheads indicate the position of C1, T1, and L1 spinal levels. C, Transverse sections of cervical (C5), thoracic (T5), lumbar (L4), and sacral (S4) spinal cord labeled for choline acetyltransferase (ChAT) and tdTomato (tdT). Images are from a P28 Hb9cre;Ai14 mouse. C’, Inset shows tdTomato+ axons in the fasciculus gracilis (fg) but not fasciculus cuneatus (fc) of the cervical spinal cord. C’’, Inset shows small-diameter tdTomato+ neurons in the lumbar dorsal horn. D–F, High-magnification images of sagittal sections from the thoracic spinal cord of a P28 Hb9cre;Ai14 mouse. D, D’, tdTomato+ cells with neuronal morphology. E, E’, tdTomato+ cells with astroglial cell morphology that express glial cell marker Sox9 (arrowheads). F, tdTomato+ cells with oligodendrocyte-like morphology. F’, Some tdTomato+ cells exhibit nuclear expression of Olig2. G–I, Quantification of the number of tdTomato+ (G) neurons, (H) gray matter (GM) astrocytes, and (I) white matter (WM) astrocytes at cervical (C5), thoracic (T5), lumbar (L4), and sacral (S4) spinal segments. J–L, Quantification of the numbers of tdTomato+ (J) neurons, (K) gray matter astrocytes, and (L) white matter astrocytes in the dorsal or ventral halves of each spinal cord tissue section. M, Quantification of the percent of total Sox9+ glial cells that express tdTomato at each spinal level. Detailed descriptions of statistical analyses are provided in Table 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data are mean ± SEM N = 3 per group. Scale bars = 2 mm (B), 250 μm (C), 100 μm (D, F), 50 μm (C’’), and 25 μm (C’, D’, E, E’, F’).
- Figure 2.
tdTomato (tdT) is not expressed in Ai14 spinal cords in the absence of Cre recombinase. Images of sagittal spinal cord sections from cervical (C4–C6; top row) and lumbar (L2–L6; bottom row) spinal cords of adult Ai14 mice. Neurons are labeled with NeuN and astrocyte nuclei are labeled with Sox9. Scale bars = 100 μm.
- Figure 3.
Schwann cells express tdTomato in Hb9cre;Ai14 mice. A, Representative image of tdTomato+ motor axons immunolabeled against neurofilament and synaptic vesicle glycoprotein 2A (2H3-SV2); acetylcholine receptors at the neuromuscular junction are labeled with α-bungarotoxin (α-BTX). B, Some Schwann cells (S100B) at the neuromuscular junction also express tdTomato (arrowheads). C, Quantification of the number of neuromuscular junctions that exhibited tdTomato expression in motor axons only (solid red), or both motor axons and Schwann cells (red checkered bar), in the triangularis sterni (TS), sternomastoid (STM), diaphragm (DIA), extensor hallucis longus (EHL), extensor digitorum longus (EDL), and soleus (SOL). *p < 0.05. All data are mean ± SEM N = 3–4 per group. Scale bars = 20 μm (A) and 10 μm (B).
- Figure 4.
Hb9 expression in embryonic spinal cords. Transverse sections of lumbar spinal cord from (A) E9.5, (B) E10.5, and (C) E11.5 wild-type mouse embryos, with neural progenitor marker Sox2 labeled in green and Hb9 labeled in magenta. Arrowhead indicates emergence of Hb9+ cells at E9.5. Scale bars = 50 μm.
- Figure 5.
tdTomato is expressed in glial cells of the embryonic Hb9cre;Ai14 mouse in an increasing rostrocaudal gradient. A, Fluorescent image of a whole E12.5 Hb9cre;Ai14 embryo. B, Transverse sections of cervical and lumbar spinal cord from an E9.5 Hb9cre;Ai14 embryo. C, C’, Confocal images showing Hb9 immunoreactivity in the nuclei of some cells in the E9.5 lumbar spinal cord. C’, Nuclear Hb9 expression in a few tdT+ cells (arrowheads). D–G, Horizontal sections of lumbar spinal cord from E15.5 Hb9cre;Ai14 embryo. Arrowheads indicate tdT+ cells that express (E) Sox9, (F) Pax7, and (G) Olig2. Scale bars = 1 mm (A), 100 μm (B), 25 μm (C–G), and 5 μm (C’).
- Figure 6.
tdTomato is expressed in neural and glial progenitors in neonatal Hb9cre;Ai14 mice. A, Sagittal section through the nervous system of a P0 Hb9cre;Ai14 mouse. B, tdTomato+ cells with radial glia-like morphology in the spinal cord of a P0 Hb9cre;Ai14 mouse. C, D, tdTomato is expressed in a subset of (C) S100B+ cells and (D) Pax6+ cells in the neonatal spinal cord, indicated with arrowheads. Scale bars = 1 mm (A) and 50 μm (B–D).
- Figure 7.
Cultured neural progenitor cells from Hb9cre;Ai14 embryonic spinal cord give rise to tdTomato+ neurons and glial cells. A, Cartoon shows experimental design for data in panels. B–D, Spinal cords from E12.5 Hb9cre;Ai14 embryos were separated into anterior and posterior halves. Tissue was dissociated, and neural progenitor cells (NPCs) obtained from either anterior or posterior spinal cord were cultured for 10 d in vitro. B, Representative images of tdTomato expression in anterior or posterior NPC cultures after 10 d. C, Expression of neuronal marker NeuN (top row), astroglial marker Sox9 (middle row), and oligodendroglial/motor neuron marker Olig2 (bottom row) in NPC cultures. D–F, Quantification of the percent of (D) total NeuN+, (E) Sox9+, or (F) Olig2+ cells that express tdTomato. All data are mean ± SEM N = 6 per group. ****p < 0.0001. Scale bars = 50 μm (B) and 100 μm (C).
- Figure 8.
Treatment with caudalizing factors promotes gliogenesis and glial tdTomato expression in cultured NPCs. A, Cartoon shows experimental design. Anterior portions of E12.5 Hb9cre;Ai14 spinal cords were dissociated, plated, and treated in culture for 7 d with either vehicle, CHIR 99 021, FGF-8B, GDF-11, or both FGF-8B + GDF-11. B, Images of DAPI staining in cultured cells at 7 DIV. C, Quantification of DAPI+ cell cluster size (μm2). D, Representative images of cells in each treatment group immunolabeled for tdTomato, NeuN, and Sox9. E–I, Quantification of (E) the percentage of all cells that are neurons, (F) the percentage of all cells that are astrocytes, (G) the percentage of all cells that are tdTomato+, (H) the percentage of neurons that express tdTomato, and (I) the percentage of astrocytes that express tdTomato. All data are mean ± SEM N = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Scale bars = 100 μm.
Tables
Antibody Catalog # RRID Dilution Goat anti-ChAT Genetex #GTX82725 AB_11162364 1:400 Chicken anti-GFAP Encor Bio #CPCA-GFAP AB_2109953 1:1500 Mouse anti-Hb9 DSHB #81.5C10 AB_2145209 1:50 Goat anti-mCherry Sicgen #AB0040 AB_2333092 1:2000 Guinea pig anti-NeuN Millipore #ABN90 AB_11205592 1:3000 Mouse anti-neurofilament (NF-M) DSHB #2H3 AB_531793 1:500 Rabbit anti-Olig2 Millipore #AB9610 AB_570666 1:200 Mouse anti-Pax7 DSHB #AB528428 AB_528428 1:50 Rabbit anti-RFP Abcam #62341 AB_945213 1:500 Rabbit anti-S100B Dako (now Agilent: Z031129-2) AB_2315306 1:500 Mouse anti-synaptic vesicle glycoprotein 2A DSHB #SV2 AB_2315387 1:500 Rabbit anti-Sox9 Abcam #185966 AB_2728660 1:2000 Figure Parameter Sample size Statistical test Significance level 1G tdTomato+ neurons in P28 spinal cord N = 3 mice per group Ordinary one-way ANOVA with Tukey’s multiple comparisons test F(3,8) = 90.22, p < 0.0001
C vs T: p = 0.0003
C vs L: p = 0.0001
C vs S: p < 0.0001
T vs S: p < 0.0001
L vs S: p = 0.00021H tdTomato+ gray matter astrocytes in P28 spinal cord N = 3 mice per group Ordinary one-way ANOVA with Tukey’s multiple comparisons test F(3,8) = 81.54, p < 0.0001
C vs T: p = 0.0004
C vs L: p = 0.0004
C vs S: p < 0.0001
T vs S: p = 0.0001
L vs S: p = 0.00021I tdTomato+ white matter astrocytes in P28 spinal cord N = 3 mice per group Ordinary one-way ANOVA with Tukey’s multiple comparisons test F(3,8) = 26.84, p = 0.0002
C vs T: p = 0.0486
C vs L: p = 0.0206
C vs S: p < 0.0001
T vs S: p = 0.0022
L vs S: p = 0.00451J tdTomato+ neurons in P28 spinal cord (dorsal vs ventral) N = 3 mice per group Two-way ANOVA with Sidak’s multiple comparisons test F(1,16) = 133.7, p < 0.0001
(source of variation: dorsal vs ventral)
T dorsal vs T ventral: p < 0.0001
L dorsal vs L ventral: p < 0.0001
S dorsal vs S ventral: p < 0.00011K tdTomato+ gray matter astrocytes in P28 spinal cord (dorsal vs ventral) N = 3 mice per group Two-way ANOVA with Sidak’s multiple comparisons test F(1,16) = 51.32, p < 0.0001
(source of variation: dorsal vs ventral)
T dorsal vs T ventral: p = 0.0004
L dorsal vs L ventral: p = 0.0294
S dorsal vs S ventral: p < 0.00011L tdTomato+ white matter astrocytes in P28 spinal cord (dorsal vs ventral) N = 2–3 mice per group) Two-way ANOVA with Sidak’s multiple comparisons test F(1,14) = 25.83, p = 0.0002
(source of variation: dorsal vs ventral)
S dorsal vs S ventral: p < 0.00011M Percent of all Sox9+ cells that express tdTomato in P28 spinal cord N = 3 mice per group Ordinary one-way ANOVA with Tukey’s multiple comparisons test F(3,8) = 242.9, p < 0.0001
C vs L: p = 0.0055
C vs S: p < 0.0001
T vs L: p = 0.0289
T vs S: p < 0.0001
L vs S: p < 0.00012C tdTomato+ Schwann cells at NMJs in P28 mice N = 3–4 per group
(between 110 and 344 NMJs quantified per individual muscle)Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test F(5,14) = 3.392, p = 0.0324
TS vs SOL: p = 0.0306
TS vs EDL: p = 0.05397D tdTomato+ neurons in vitro N = 12 wells per group Unpaired t test t = 25.15, df = 22
p < 0.00017E tdTomato+ Sox9+ cells in vitro N = 6 wells per group Unpaired t test t = 8.772, df = 10
p < 0.00017F tdTomato+ Olig2+ cells in vitro N = 6 wells per group Unpaired t test t = 8.287, df = 10
p < 0.00018C Cluster size in vitro + drug treatment N = 6 wells per group
(between 153 and 319 clusters quantified per well)Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,1096) = 363.7, p < 0.0001
VEH vs CHIR: p < 0.0001
VEH vs FGF: p < 0.0001
VEH vs FGF+GDF: p < 0.00018E Percent neurons in vitro + drug treatment N = 6 wells per group Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,25) = 8.967, p = 0.0001
VEH vs FGF: p = 0.00038F Percent astrocytes in vitro + drug treatment N = 6 wells per group Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,25) = 36.21, p < 0.0001
VEH vs FGF: p < 0.0001
VEH vs FGF+GDF: p = 0.00278G Percent tdTomato+ cells in vitro + drug treatment N = 6 wells per group Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,25) = 2.502, p = 0.0680
No significant differences8H Percent tdTomato+ neurons in vitro + drug treatment N = 6 wells per group Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,25) = 3.596, p = 0.0190
No significant differences8I Percent tdTomato+ astrocytes in vitro + drug treatment N = 6 wells per group Ordinary one-way ANOVA with Dunnett’s multiple comparisons test F(4,25) = 6.630, p = 0.0009
VEH vs CHIR: p = 0.0244
VEH vs FGF+GDF: p = 0.0002
In this issue
