Adult spinal cord radial glia display a unique progenitor phenotype

Abstract

Radial glia (RG) are primarily embryonic neuroglial progenitors that express Brain Lipid Binding Protein (Blbp a.k.a. Fabp7) and Glial Fibrillary Acidic Protein (Gfap). We used these transcripts to demarcate the distribution of spinal cord radial glia (SCRG) and screen for SCRG gene expression in the Allen Spinal Cord Atlas (ASCA). We reveal that neonatal and adult SCRG are anchored in a non-ventricular niche at the spinal cord (SC) pial boundary, and express a "signature" subset of 122 genes, many of which are shared with "classic" neural stem cells (NSCs) of the subventricular zone (SVZ) and SC central canal (CC). A core expressed gene set shared between SCRG and progenitors of the SVZ and CC is particularly enriched in genes associated with human disease. Visualizing SCRG in a Fabp7-EGFP reporter mouse reveals an extensive population of SCRG that extend processes around the SC boundary and inwardly (through) the SC white matter (WM), whose abundance increases in a gradient from cervical to lumbar SC. Confocal analysis of multiple NSC-enriched proteins reveals that postnatal SCRG are a discrete and heterogeneous potential progenitor population that become activated by multiple SC lesions, and that CC progenitors are also more heterogeneous than previously appreciated. Gene ontology analysis highlights potentially unique regulatory pathways that may be further manipulated in SCRG to enhance repair in the context of injury and SC disease.

Figure 1. Radial Glia at the Pial Boundary of the Neonatal and Adult Spinal Cord.

In situ hybridization images from the ASCA show that both neonatal (PND 4) and adult (PND 56) SCRG (arrowheads on soma) express genes commonly associated with embryonic RG. (A, B) Blbp (a.k.a. Fabp7), (C, D) Gfap, (E, F) Glast, (G, H) Sox2 and (I, J) Reln. In all cases, SCRG are prominent in the neonatal SC (A, C, E, G & I), and less abundant in the adult (B, D, F, H & J). The boxed areas correspond to the magnified images (A′–J′) and inserts show high magnification of SCRG. Reln: Reelin. Scale bars: 100 µm.

Figure 2. Spinal Cord Radial Glia Shift their Expression of Functionally Important Genes from Neonatal to Adult.

(A) Venn diagram indicates the shift in SCRG gene expression during maturation. 7/122 SCRG genes are exclusive to neonate, 109/122 are expressed in both the neonatal and adult SCRG, and 6/122 genes are detected only in adult RG. (B–D) ASCA ISH images of genes expressed in SCRG (arrowheads) at both time points. (B) Tnc and Cux2 are present only in the neonatal, (C) Hopx (a.k.a. Hod) and Id3 are present in both neonatal and adult SCRG, while (D) Sema3B and Nes are exclusive to adult SCRG. Hopx: HOP homeobox; Tnc: tenascin C; Cux2: cut-like homeobox 2; Nes: nestin. Scale bars: 100 µm.

Figure 3. Spinal Cord Radial Glia Heterogeneity is Highlighted by the Co- Expression of Neural Stem Cell Proteins in the Neonatal and Adult Spinal Cord.

(A, C, E) In the neonatal (PND 5) SC, confocal imaging shows that BLBP⁺ (green) SCRG extend processes (arrows) from the marginal edge of the SC and through the WM. These cells (A) rarely co-express NES (red), but distinct subpopulations (B) robustly co-express VIM (red) and (E) nuclear SOX2 (red). Boxed areas outline higher magnifications (A′–F′) of the z-stacks, rotated on a 3D plane to highlight the anchored SCRG cytoarchitecure at the pial boundary. (B, D, E) In the adult (PND 75) SC, the BLBP⁺ (green) SCRG are less abundant, but their processes display enhanced expression of (B) NES (red) and (D) VIM (red). (F) SCRG nuclei remain at the sub-pial edge of the SC, where rare subpopulations retain expression of SOX2 (red). (A″–F″) Distribution of progenitor marker expression is depicted in schematics of SC cross-sections, and highlight the shifting cytoarchitecture of the SCRG and CC progenitors of the neonatal and adult SC. Immuno-positive multipolar cells and BV are included. Arrow: SCRG process; double arrow: SCRG; dotted line: marginal edge of SC; BV: blood vessels. Scale bars: 50 µm.

Figure 4. Progenitor Gene Expression in Neonatal and Adult Central Canal.

(A) Venn diagram illustrating the number of genes that overlap (or are unique) in expression between CC, SCRG and SVZ. (B) Schematic of SC cross-section detailing the position of the CC progenitor niche. (C–J) ISH images from the ASCA show the shifts in expression of key neural progenitor genes shared with SCRG in the (C–F) neonatal and (G–J) adult CC: (C, G) Nes, (D, H) Blbp, (E, L) Sox2 and (F, J) Id4. (K–T) Confocal z-stack images of neural progenitor proteins in the CC of (K, L) neonatal and (P, Q) adult wild-type mice, and (M–O) neonatal and (R–T) adult Fabp7 (BLBP)-EGFP transgenic reporter mice (M–O) neonatal and (R–T) adult CC (where all reporter⁺ cells are GFP⁺). In the (K, M, N) neonatal CC, NES⁺ processes emanate dorso-ventrally from the CC, some of which co-express VIM and are more abundant than in the (P, R, S) adult CC. In the (L, M, O) neonatal SC, SOX2⁺ nuclei are clustered within and around (arrowheads in L) the CC some of which co-express BLBP or GFP (green), but (Q, R, T) in the adult, become tightly restricted to the ependymal and subependymal layers. (O) Few PCNA⁺ (red) cells are localized around the CC in both the neonatal and (T) the adult, some of which are also GFP⁺ (double arrowheads). (P) Boxed area corresponds to magnified inset (P′). CC: Central canal; arrowhead: soma; arrow: process; double arrowhead: triple-positive cell; dotted line: marginal edge of SC; VIM: Vimentin; BV: blood vessel. Scale bars: 50 µm.

Figure 5. Radial Glia Are Distributed in an Increasing Cervical-to-Lumbar Gradient in the Spinal Cord of the Adult Fabp7-EGFP Reporter Mouse.

(A) Schematic of SC cross-section detailing the position of the dorsal, lateral and ventral areas assayed in B–F, with arrowheads marking the anatomical boundaries for quantification. (B) Expression of eGFP was detectable, but enhanced by a GFP antibody, that confirms GFP co-localization with both rabbit and mouse anti-BLBP antibodies. (B–F) Confocal z-stack images of (C) neonatal (PND 5; cervical level) SC and (B, D–F) adult SC (P56; cervical, thoracic and lumbar levels) Fabp7-EGFP mice. (C) In the neonate, GFP⁺ SCRG are abundant all around the SC. (D–F) In the adult SC GFP illuminates the cytoarchitecture of the processes and soma of pial RG (double arrow) and reveals that (G, H) GFP⁺ SCRG significantly increase in frequency from cervical to thoracic and lumbar levels (p<0.05), and also shift in their relative distribution in dorsal, lateral and ventral SC at different levels. Boxed areas outline magnified regions (C′, D′–F′) of the z-stack. Arrowhead: soma; arrow: process; double arrow: SCRG; dotted line: marginal edge of SC. Scale bars: 50 µm.

Figure 6. An Extensive Scaffold of Fabp7-EGFP-Expressing Spinal Cord Radial Glia Retain Progenitor-like Morphology and Gene Expression.

(A–L) Confocal imaging of the SC of (A–C) neonatal (PND 5; cervical level) and (D–L) adult (P56; lumbar level) Fabp7-EGFP mice has allowed us to reveal a more extensive population of SCRG than detected by immunofluorescence (A–C) BLBP⁺ (green) SCRG processes (arrows) begin at the marginal edge of the SC and extend throughout the WM and up to the astrocyte-rich (arrowhead) GM. Subpopulations of GFP⁺ SCRG co-express (A, C) SOX2 (blue) and (B) VIM robustly (blue) but rarely (A, B) NES (red). Many nuclei contain (C) PCNA (red). (D–L) Although less abundant than in the neonatal SC, the (D–L) GFP⁺ (green) processes of adult SCRG also demonstrate enhanced expression of (E, H, K) VIM (blue) and (D, E, G, H, J, K) NES (red). Their nuclei remain at the sub-pial edge of SC and small subpopulations retain expression of (D, F, G, I, J, L) SOX2 (blue) and (F, I, L) PCNA (red). Boxed areas outline magnified regions (G′–I′, L′) of the z-stack that were rotated and tilted on a 3D plane to best highlight the anchored cell soma and processes at the pial boundary. (G″–I″) Gray scale images of a single SCRG cell (asterisk in G′–I′). Arrowhead: astrocyte; arrow: process; double arrowhead: triple-positive cell; double arrow: SCRG; open arrowhead: cell on SCRG process; dotted line: marginal edge of SC; BV: blood vessels. Scale bars: 50 µm.

Figure 7. Spinal Cord Radial Glia Expand and Transform Morphologically During the Response to SC Compression and Experimental Autoimmune Encephalomyelitis.

Confocal z-stack images of an adult SC reveal similar responses by SCRG and CC progenitors 14 days after a (A) dorsal column compression (red arrowhead), where analysis was performed both (B, B′, E, H, K) at the injury core and (C, D, F, G, I, J) peri-lesion area (≤250 µm rostral or caudal from the site of injury). (A′) Schematic of SC cross-section detailing the position of (B′–M) images (B–B′) After dorsal column compression BLBP⁺/GFAP⁺ SCRG appear disorganized but their processes are readily detected. In the peri-lesion area, SCRG processes appear hypertrophied and strongly express (C–E) NES (green) and VIM (red). Nuclei containing (F–H) SOX2 or (I–K) PCNA are more abundant after lesion in all locations - at the pial boundary, arrayed along BLBP⁺ (green) processes and (G, J) at the CC. (L–M) Analysis of expression following the induction of EAE showed a similar profile of SCRG hypertrophy and CC expansion. CC: Central Canal; arrow: process; double arrowhead: double-positive cell; double arrow: SCRG; open arrowhead: cell on SCRG process; dotted line: marginal edge of SC Scale bars: 100 µm (A), and 50 µm (A′–M).