Respiratory data two weeks post viral PHOX2B shRNA injection. (A) breathing frequency (ƒR,), (B) tidal volume (VT), (C) allometric minute ventilation (VE ALLO), (D) oxygen consumption (VO2 ALLO), (E) convective requirement ratio (VE/VO2 ALLO), (F) hypercapnic ventilatory response (HCVR absolute change in VE ALLO vs. corresponding room air) of baseline (pre-surgery, grey filled box), naïve (black n=8), non-target control shRNA (NT-shRNA, grey n=17) and PHOX2B shRNA (PHOX2B-shRNA, red n=17) rats 2-weeks post injection during room air, hypercapnia 5% and 7.2% CO2. ƒR is equally impaired in all experimental group compared to baseline but no treatment effect was observed (A). VT is significantly impaired following RTN injection in PHOX2B-shRNA group compared to naïve animals (p=0.022) and baseline (p< 0.001) at 7.2% CO2 (B). VE ALLO is increased in naïve rats compared to the other treatment groups (p=0.005) in room air (C). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, “+” indicates arithmetic mean. Bonferroni post-hoc as indicated. Black*, different from naïve; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.

PHOX2B and Nmb expression and total cell count within the RTN area in naïve, NT- shRNA and PHOX2B-shRNA injected rats two weeks post viral shRNA injection. (A) Schematic and representative image of a transverse brainstem section at the level of the RTN (-11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in RTN Nmb+/PHOX2B+ and Nmb+/PHOX2B- neurons in naïve (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view inserts below). Arrowheads indicate absence of PHOX2B protein. Scale bar=400μm (top figures), 150μm (inserts below). (C) The number of total cells (Nmb+/PHOX2B+ plus Nmb+/PHOX2B-) comprising the RTN are reduced in PHOX2B-shRNA rats (n=4) as compared to naïve (n=4) and NT-shRNA (n=4), and in NT-shRNA rats as compared to naïve rats (black#, One-way ANOVA, p<0.001). The number of Nmb+/PHOX2B+ cells are reduced in PHOX2B-shRNA rats as compared to naïve and NT-shRNA (Blue*, One-way ANOVA, p<0 0.001). The number of Nmb+/PHOX2B- cells was unchanged across groups. (D,E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb+/PHOX2B+ (D) and Nmb+/PHOX2B- (E) neurons along the RTN.

PHOX2B and Nmb expression and total cell count within the RTN area in naïve, NT- shRNA and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection. (A) Schematic and representative image of a transverse brainstem section at the level of the RTN (-11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in naïve (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view insert). Arrowheads indicate absence of PHOX2B protein. Scale bar = 400μm (top figures), 150μm (inserts below). (C) The number of total cells (Nmb+/PHOX2B+ + Nmb+/PHOX2B-) comprising the RTN are reduced in PHOX2B-shRNA rats (n=6) as compared to naïve (n=4) (Black#, One-way ANOVA, p=0.0087) but not to NT-shRNA (n=10). The number of Nmb+/PHOX2B+ cells are reduced in PHOX2B-shRNA rats as compared to naïve and NT- shRNA (Blue*, one-way ANOVA, p<0.001). The number of Nmb+/PHOX2B- cells are increased in PHOX2B-shRNA rats as compared to both naïve and NT-shRNA (Green*, one-way ANOVA p<0.001). (D, E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb+/PHOX2B+ (D) and Nmb+/PHOX2B- (E) neurons along the RTN.

Respiratory data following 4 weeks post viral shRNA injection. (A) breathing frequency (ƒR,), (B) tidal volume (VT), (C) allometric minute ventilation (VE ALLO), (D) convective requirement ratio (VE/VO2 ALLO), (E) hypercapnic ventilatory response (HCVR, absolute change in VE ALLO vs. corresponding room air), (F) HCVR at baseline, week 2 and week 4 post-viral injections in naïve (black n=8), non-target control shRNA (NT-shRNA, grey n=10) and PHOX2B-shRNA (PHOX2B- shRNA, red n=6). ƒR is equally impaired in all experimental group compared to baseline but no treatment effect was observed (A). VT is significantly impaired following RTN injection in PHOX2B- shRNA group compared to baseline pre-surgery (p<0.001), naïve rats p<0.001), and NT-shRNA rats (p=0.002) at 7.2% CO2. (B). VE ALLO is impaired in PHOX2B-shRNA rats during exposure to hypercapnia (7.2% CO2) compared to baseline (p=0.0025), naïve rats (p=0.007), and NT-shRNA rats (p=0.002). (C). VE/VO2 ALLO is reduced in PHOX2B-shRNA animals compared to NT-shRNA rats both at 5% (p=0.023) and 7.2% (p=0.004.) CO2 (D). HCVR during 7.2% CO2 is lower in PHOX2B- shRNA rats compared to baseline (p=0.007), naïve rats (p=0.001), and NT-shRNA rats (p=0.016) (E). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, “+” indicates arithmetic mean. Bonferroni post-hoc as indicated. HCVR is significantly impaired only in PHOX2B- shRNA rats 4-weeks post-surgery (One-way ANOVA p=0.007) (F). Black*, different from naïve; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.

PHOX2B knockdown in RTN neurons does not alter TH or Nmb expression but impairs the hypercapnic ventilatory response. (A)

PHOX2B protein (white) and TH (red) expression in C1 neurons of naïve (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats. Magnified view at the bottom. Arrowheads indicate the colocalization of PHOX2B and TH protein in C1 neurones cells. Scale bar = 400μm (top figures), 150μm (bottom figures). (B) No differences are observed in the rostral-caudal distribution of TH+/PHOX2B+ catecholaminergic C1 neurons cells caudal to the RTN between naïve (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rats. (C) PHOX2B protein (white) and Nmb mRNA (green) expression in NT-shRNA rat at the level of NTS and RTN regions. Magnified view on the left. Arrowheads indicate cells with Nmb mRNA expression. Scale bar = 400μm (right figures), 150μm (left figures). (D) Quantification of single cells Nmb mRNA fluorescence intensity along the rostro-caudal extension of RTN in naïve (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rat calculated as average ratio between RTN and NTS cells shows no difference between treatment groups. Data are shown as average cell fluorescence value at different rostro-caudal levels. (D-E) X-Y plot of HCVR during 7.2% CO2 exposure relative to the number of Nmb+/PHOX2B+ (D, slope is different from “0” at p<0.001; r2=0.739) and Nmb+/PHOX2B- (E, p<0.001 for difference between slopes; r2=0.482) in the RTN.

Gpr4 and Task2 mRNA expression within the RTN area in naïve, and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection. (A,D) Nmb (green), Gpr4, Task2 mRNA (red) and PHOX2B protein (grey) expression in RTN neurons in naïve (top) and PHOX2B-shRNA (bottom) rats. Scale bar = 150 μm. Arrowheads indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb+/PHOX2B+ neurons. Asterisks indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb+/PHOX2B- neurons. (B,E) Quantification of single cells Gpr4 and Task2 mRNA fluorescence intensity along the rostro-caudal extension of RTN in naïve (black), NT-shRNA (grey) and PHOX2B- shRNA (red) rats. Black*, different from naïve, Grey*, different from NT-shRNA (C and F) Quantification of single cells Gpr4 and Task2 mRNA fluorescence staining intensity along the rostro-caudal extension of RTN in Nmb+/PHOX2B+ (red filled dot) and Nmb+/PHOX2B- (red empty dot) neurons in PHOX2B-shRNA rats. Data are shown as average cell fluorescence value at different rostro-caudal levels of the RTN. Mean corrected total cell fluorescence (CTCF) value ± SEM combined (naïve, n=4; NT-shRNA, n=10; PHOX2B-shRNA, n=6) (see methods for detail). One- way ANOVA repeated measures.