Ednrb is highly expressed in satellite glial cells.

A. Representative whole-mount-stained images of DRG from Lycopersicon Esculentum Lectin (LEL) injected Fabp7CreER::Ai14 mice, labeled for TUJ1 (green), tdTomato (magenta), and LEL (grey). 3D reconstruction of blood vessels via LEL labeling (scale bars, 200 μm). Note that TUJ1 antibody staining is limited by penetration of the antibody in the whole mount.

B. Representative images of sectioned DRG from Lycopersicon Esculentum Lectin (LEL) injected C57BL/6 mice, labeled for TUJ1 (green), Fabp7 (red), and LEL (cyan) (Scale bars, 100 μm).

C. UMAP analysis of adult DRG 10X sequencing data identified 8 cell clusters based on known marker genes.

D. Dot plot analysis showing the average gene expression (color coded) and number of expressing cells (dot size) for the marker genes.

E-H. UMAP overlay for expression of Ednra (E), Ednrb (F), Edn1 (G) and Edn3 (H).

I. Representative RNAScope in situ hybridization images showing Ednrb (red), Fabp7 (cyan) and DAPI (blue) of L4 DRGs from 3-month-old mice (scale bars, 50 μm).

Endothelin B receptor inhibition increases axonal growth in vitro and ex vivo.

A. Representative images showing TUJ1 (black) immunostaining of neurons in DRG cultures (scale bars, 100 μm).

B. Representative image of TUJ1 (green) and FABP7 (magenta) immunostaining of neurons and SGCs in control DRG cultures (scale bars, 50 μm).

C,D. Quantification of axonal radial length (B) and TUJ1+ area (C) per neuron. Different colors indicate biological replicates. N = 246 (Veh; 8 replicates), 318 (BQ788; 8 replicates), 320 (IRL1620; 8 replicates), and 244 (BQ123; 8 replicates). Data presented as mean ± SD.

E. Scheme of drug treatment and DRG explant model.

F. Representative images of DRG explants 7 days after drug treatment, immunostained for TUJ1 (black) (scale bars, 1000 μm).

G. Quantification of radial length of the 35 longest axons from DRG explants from indicated groups N=36 explants from 6 indivual mice (BQ788; 18 replicates, Veh; 18 replicates).

H. Representative images of DRG explants immunostained for TUJ1 (green), FABP7 (magenta), and merged (scale bars, 50 μm).

Bosentan treatment improves axon regeneration after peripheral nerve injury in adult mice.

A. Scheme of drug treatment and peripheral nerve injury model.

B. Quantification of the length of the 10 longest axons in indicated conditions.

C. Quantification of the regeneration index, calculated as the distance along the nerve where the SGC10 intensity is 50% of the SCG10 intensity at crush site.

D. Representative longitudinal sections of sciatic nerves 24h after SNC, immunostained for SCG10, from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SGC10 intensity (scale bars, 200 μm).

E. Quantificaiton of SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N = 5 mice/condition.

F. Scheme of long-term Bosentan treatment.

G. Representative images of hindpaw skin after long-term Bosentan treatment immunostained for PGP9.5 (white) and DAPI (blue) (scale bars, 50 μm).

H. Quantification of intraepidermal nerve fiber density (IENFD) 24 days after sciatic nerve crush from the indicated groups. N=5 mice/condition.

I. Scheme of adult DRG neuronal culture and treatments.

J,L. Representative images showing TUJ1 (black) immunostaining of neurons in DRG cultures (scale bars, 100 μm).

K,M. Quantification of axonal radial length (K) and total TUJ1+ area (L). Different colors represent different biological replicates. N (neuron number) = 177 (vehicle; three biological replicates, naïve mice), 168 (Ambrisentan, three biological replicates, naïve mice), 183 (Bosentan; three biological replicates, naïve mice), 186 (vehicle; three biological replicates, injured mice), 204(Ambrisentan, three biological replicates, injured mice) and 210 (Bosentan; three biological replicates, injured mice), respectively. The data are presented as mean ± SD.

Bosentan treatment rescues aging-dependent neuronal regenerative decline.

A. Scheme of drug treatment and DRG explant model.

B. Representative images of DRG explants 7 days after drug treatment (scale bars, 1000 μm).

C. Quantification of radial length of the 50 longest axons from DRG explants. N=36 explants from 6 indivual mice (BQ788; 18 replicates, Veh; 18 replicates).

D. Representative longitudinal sections of sciatic nerves 3 d after SNC immunostained for SCG10 from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SCG10 intensity (scale bars, 200 μm).

E,F. Quantification of the 10 longest axons in indicated groups (E). Quantificaiton of 50% regenerative index, calculated as the disatnce along the nerve where the SCG10 intensity is 50% of the SCG10 intensity at crush site (F).

G. Quantification of the SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N (mouse number) = 4(adult, vehicle), 3 (Aged, vehicle) and 3 (Bosentan+Age), respectively. The data are presented as mean ± SD.

Aging alters SGC abundance and morphology.

A. UMAP plot of adult and aged snRNA-seq identified 16 cell clusters based on known marker genes.

B. Dot plot analysis showing the average gene expression (color coded) and number of expressing cells (dot size) for the marker genes.

C,D. UMAP plot of DRG cells from adult (C) and aged (D) mice.

E. Bar plot of cell proportions in DRGs of adult and aged mice.

F. Representative TEM images of DRG sections from adult (2M), middle aged (12M) and aged (21M) mice showing neuronal cell bodies and the enveloping SGCs (SGCs are pseudo-colored in red) (scale bars, 5 μm).

G. Quantification of the average width of SGCs sheath per neuron soma.

H. Frequency of neurons soma in TEM images with 0, 1, 2, or 3 SGCs nuclei in 2M, 12M, and 21M old mice.

ETBR inhibition increases the expression of Cx43 in SGCs in adult and aged mice.

A. UMAP overlay for expression of Gja1 in adult and aged mouse DRG.

B. Scheme of drug treatment and peripheral nerve injury model.

C. Quantification of the percentage of the Cx43/FABP7 expression area.

D. Quantification of the average number of Connexin 43 (Cx43) puncta per FABP7+ cell. The ratio of total Cx43 puncta to the number of FABP7+ cells surrounding a TUJ1+ neuron was measured. N(cell number) = 60(adult, uninjured), 62(aged, uninjured), 96(vehicle, adult, SNC), 117(bosentan, adult, SNC), 74(vehicle, aged, SNC) and 74(bosentan, aged, SNC), respectively.

E. Representative immunostaining images showing Connexin 43 (Cx43), FABP7 and TUJ1 in L4 DRGs from the indicated condition (scale bars, 50 μm).

F. Proposed model for the role of ETBR in age-dependent decline in axon regenerative capacity