Extended Data Figure 10. Clonal analysis of ACP neurons.

a, ACP clonal labeling experiment in Ghsr^cre mouse injected with AAV-DIO-eYFP. Left, immunostaining of the soma (dashed outline) in the right Amb of the single eYFP-labeled ACP neuron in this mouse (Clone #2); note it co-stained positive for calbindin (cyan) and negative for BChE (red), confirming ACP identity. Bar, 20 μm. Right, map of ACP neurons (dark blue fill circles) in right Amb (overlay of all sagittal sections of right Amb) showing location of the eYFP-labeled ACP clone (green fill circle). Terminals of the ACP clone (green fibers) were mapped in the heart (left) and lung (right), where it was found to innervate parasympathetic ganglia in both organs (red fill ovals/circles, targeted ganglia). LA, left atrium; RA, right atrium; Ao, aorta; PA, pulmonary artery; PVs, pulmonary veins; SVC, superior vena cava. b, Immunostaining of parasympathetic cardiac ganglion (Ganglion 1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #2. Note innervated neuron stained positive for cholinergic marker VAChT (red) and receives innervation from a cholinergic (red), calbindin-positive (cyan) fiber labeled with eYFP (green). Bar, 10 μm. c, Immunostaining of lung parasympathetic ganglion (Ganglion L1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #2. Note innervated neuron stained positive for neuronal marker Tuj1 (red) and receives innervation from a calbindin-positive (cyan), eYFP-positive (green) fiber. Bar, 10 μm. d, ACP clonal labeling experiment in Calb1^cre mouse injected with limiting dose of AAV-FLEX-GFP. Left, immunostaining of the soma (dashed outline) in the L Amb of the single GFP-labeled ACP neuron in this mouse (Clone #3); note it co-stained positive for calbindin (cyan) and negative for BChE (red), confirming ACP identity. Bar, 20 μm. Right, map of ACP neurons (dark blue fill circles) in left Amb showing location of the GFP-labeled ACP clone (green fill circle, Clone #3). Terminals of the ACP clone (green fibers) were mapped in the heart (left) and lung (right), where it was found to innervate parasympathetic ganglia in both organs (red fill ovals/circles, targeted ganglia). e, Immunostaining of parasympathetic cardiac ganglion (Ganglion 1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #3. Note innervated neuron stained positive for cholinergic marker VAChT (red) and receives innervation from a cholinergic (red), calbindin-positive (cyan) fiber labeled with GFP (green). Bar, 10 μm. f, Immunostaining of lung parasympathetic ganglion (Ganglion RMd1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #3. Note innervated neuron stained positive for neuronal marker Tuj1 (red) and receives innervation from a calbindin-positive (cyan), GFP-positive (green) fiber. Bar, 10 μm.