Astrocyte Cell Surface Antigen 2 and Other Potential Cell Surface Markers of Enteric glia in the Mouse Colon

Abstract

Enteric glia regulate gut functions in health and disease through diverse interactions with neurons and immune cells. Intracellular localization of traditional markers of enteric glia such as GFAP, s100b, and Sox10 makes them incompatible for studies that require antigen localization at the cell surface. Thus, new tools are needed for probing the heterogeneous roles of enteric glia at the protein, cell, and functional levels. Here we selected several cell surface antigens including Astrocyte Cell Surface Marker 2 (ACSA2), Cluster of differentiation 9 (CD9), lysophosphatidic acid receptor 1 (LPAR1), and Proteolipid protein 1 (PLP1) as potential markers of enteric glia. We tested their specificity for enteric glia using published single-cell/-nuclei and glia-specific translating mRNA enriched transcriptome datasets, immunolabeling, and flow cytometry. The data show that ACSA2 is a specific marker of mucosal and myenteric glia while other markers are suitable for identifying all subpopulations of enteric glia (LPAR1), glia and immune cells (CD9), or are not suitable for cell-surface labeling (PLP1). These new tools will be useful for future work focused on understanding specific glial functions in health and disease.Summary StatementThis study identifies astrocyte cell surface antigen 2 as a novel marker of myenteric glia in the intestine. This, in combination with other markers identified in this study, could be used for selective targeting of enteric glia.

Keywords: ACSA2; CD9; LPAR1; PLP1; enteric nervous system.

Figure 1.

Characterization of the anti-ACSA2 antibody. Mouse brain slices were co-stained (top row) with ACSA2-APC (magenta), the glial marker GFAP (Green), and the neuronal marker NeuN (blue) or stained with only glial marker GFAP (bottom row). Scale bars, 20 and 40 µm (top and bottom).

Figure 2.

Cell-specific transcript expression of potential surface antigens. Graphs are prepared from published data sets using single nuclei (A-B), single-cell (C), and translating glial mRNA (D) transcriptomics from mouse small (A, C) and large (B, D) intestines. A-B) Single nuclei transcriptome from mouse small (A) and large (B) intestines (Drokhlyansky et al., 2020). Data show normalized expression levels (median ± range) of combined neuronal or glial subpopulations (more detailed summary in Figure S1). *, P < 0.05, ***, P < 0.001, ****, P < 0.0001, multiple Mann-Whitney U tests. C) Single-cell sequencing from adolescent mouse small intestine (Zeisel et al., 2018). Data show normalized expression levels (median ± range) of combined neuronal or glial subpopulations (more detailed summary in Figure S2). *, P < 0.05, **, P < 0.01, multiple Mann-Whitney U tests. D) Ribosomes were isolated from dinitrobenzene sulfonic acid (DNBS) treated glial RiboTag mouse colons (Sox10^CreERT2; Rpl22^fl/fl) and their healthy controls to perform sequencing of the translating glial mRNA (Delvalle et al., 2018). FPKM, fragments per kilobase per million mapped fragments. *, P < 0.05, multiple unpaired t tests.

Figure 3.

Antigen expression of ACSA2, CD9, and LPAR1 in the mouse colon muscularis. Whole mounts of colon longitudinal muscle-myenteric plexus preparations were stained with one of the anti-antigen antibodies (left column, magenta) and one of the glial markers anti-GFAP or anti-s100b (middle column, green). Overlay images are in the right column. Arrows, closed arrowheads, and open arrowheads point to the co-expressed immunoreactivity (ir), only antigen ir, and only glial marker ir, respectively. Note that ACSA2 ir colocalizes with inta- and inter-ganglionic glia, while CD9 and LPAR1 antibodies label intraganglionic and extraganglionic glia. CD9 antibodies also label GFAP- cells outside the myenteric plexus. Scale bar = 50 µm.

Figure 4.

The antigen expression in the mouse colon mucosa. Whole mounts of colon mucosa were stained with one of the anti-antigen antibodies (left column, magenta) and the glial marker anti-GFAP (middle column, green). Overlay images are in the right column. Arrows and closed arrowheads point to the co-expressed ir and only antigen ir, respectively. Note that ACSA2 and LPAR1 ir co-localize with mucosal glia, while CD9 antibody labels GFAP- cells; ACSA2 antibody also labels GFAP- structures due to non-antigen specific reactions (see Figure S4 showing staining with the isotype control). Scale bar = 50 µm.

Figure 5.

Colocalization of CD9 and LPAR1 immunoreactivity with glial and neuronal markers within myenteric ganglia. Whole mounts of colon longitudinal muscle-myenteric plexus preparations were stained with CD9 (top panels) or LPAR1 (bottom panels) antibodies and co-stained with the anti-GFAP glial marker (middle column) and anti-Hu neuronal marker (right column). Overlay images are in the second row of each panel. Optical sections are 5 µm thick. Vertical and horizontal yellow lines in z-projections (large images) mark the optical sections of x- and y-projections placed adjacently at the bottom and the right side of the respective images. Note that CD9 and LPAR1 colocalize with GFAP but not HuC/D. Scale bars, 20 µm.

Figure 6.

Flow cytometry using the anti-ACSA2 antibody. Live mouse longitudinal muscle-myenteric plexus cell suspensions were stained with APC-conjugated anti-ACSA2 antibody while DAPI was used as a viability dye. (A) Gating strategy for cells (left), single events (middle), and live cells (right). (B) APC vs tdTomato fluorescence from the unstained (top) and stained (bottom) cell suspensions derived from WT (left) and Sox10^CreERT2; tdTomato (right) mice. The gating was adjusted using fluorescence minus one controls. (C) Summary graph showing unstained (left, unshaded) and APC-ACSA2 stained (right, shaded) cells from the Sox10^CreERT2; tdTomoto mice that were normalized to the maximal tdTomato+ population. The graph shows three groups of cells: ACSA2+ tdTomato- (blue), ACSA2- tdTomato+ (red), and ASCA-2+ tdTomato+ cells (purple). Note that ACSA2 exclusively labels about 40% of the tdTomato+ cells. N = 4–5 mice.