Extended Data Fig. 5: Cspg4⁺ pericytes wrap around blood vessels and form close contact with CGRP-containing afferent neurons.

a, Colon tissues from Cspg4-DsRed mice were harvested and subjected to whole mount immunofluorescence analysis, using an antibody against CD31 (a marker for blood vessel endothelial cells; magenta). DsRed (cyan) signals were detected directly. DsRed-positive cells surround CD31-positive capillaries in colonic crypts. Maximum intensity projection of the whole stack is presented in Figure 2e. Scale bar is 50 μm.

b, Experiments were carried out as in panel a, except that lymphatic endothelial cells were labeled with an antibody against Lyve-1 (magenta) and DsRed signals were detected using an antibody against DsRed. DsRed-positive cells are clearly separate from lymphatic endothelial cells. Scale bar is 50 μm.

c-f, Experiments were carried out as described in panel a. Enteric glial cells were visualized with GFP fluorescence in examining proteolipid protein 1 (PLP1)-GFP/-Cspg4-dsRed dual reporter mice (panel c). Fibroblasts were labeled using an antibody against platelet-derived growth factor receptor alpha (PGDFRA, magenta, panel d). Myofibroblasts were labeled using an antibody against smooth muscle actin (SMA, magenta, panel e). Mast cells and interstitial cells of Cajal were labeled using an antibody against cKit (magenta, panel f). Scale bar: 10 μm.

g, TcdB induced cell-rounding of cultured human brain vascular pericytes. Scale bar: 25 μm

h, The indicated TcdB mutants were injected into mouse ears via intradermal injection³⁷. Pericytes surrounding ear arterioles were labeled and visualized through DsRed. TcdB and TcdB-FzM induced morphological changes of Cspg4⁺ pericytes surrounding ear arterioles, whereas TcdB-Cspg4M showed no effect. Scale bar, 50 μm. n= 3 mice/group.

i, Experiments were carried out as described in panel h, except that WT mice were utilized and pericytes were detected by immunostaining using an antibody against smooth muscle actin (labeling pericytes). TcdB-FzM disrupted pericytes around ear arterioles, whereas TcdB-Cspg4M has no effect. Scale bar: 50 μm. n= 3 mice/group.

j, Experiments were carried out as in panel a, except that neuronal processes were labeled using an antibody against Tubb3 (magenta, a marker for neuronal processes), showing that neuronal processes are extending alongside DsRed-positive pericytes. Scale bar: 10 μm.

k, Experiments were carried out as in panel a, except that an antibody against CGRP was added to detect CGRP-positive nerve terminals. 3-dimensional reconstruction of images showed that DsRed⁺ pericytes (cyan) surround the vasculature (CD31 endothelial marker; magenta) and contact CGRP-expressing nerve terminals (CGRP; yellow). Scale bar: 10 μm.

l, Immunoblot analysis of cell lysates showed expression of CGRP receptors (CALCRL: calcitonin receptor like receptor; and RAMP1: receptor activity modifying protein 1) in primary cultured human brain vascular pericytes. Two human cell lines, HeLa and U87, were analyzed in parallel as controls, which do not express detectable levels of CGRP receptors. Total protein staining with Coomassie blue was used as a control for protein loading. For gel source data, see Supplementary Figure 1. N = 2 replicates.