Figure 1.

ASCs and TPSCs both exhibit robust Ca²⁺ responses to phrenic nerve stimulation (Stim) at E14. A, Left, Spatial map of the SD iu₁₆ values of Ca²⁺ responses in a population of ASCs and TPSCs in the diaphragm of an E14 Wnt1-GCaMP3 mouse imaged in response to 30 s of 40 Hz nerve stimulation; fire CLUT heatmap in SD iu₁₆ and scale bar in micrometers. Note the responses by some but not all ASCs along the secondary phrenic intramuscular branch (arrow) as well as by some but not all TPSCs (arrowhead). Middle, Spatial fluorescence intensity map (in SD iu₁₆) of the same sample in the left panel after treatment with the pan-cholinergic agonist CCh. In contrast to nerve stimulation, CCh induces Ca²⁺ responses in all ASCs and TPSCs. Right, An average fluorescence intensity image generated before application of a stimulus (prestim, in black and white) shows the overall structure of GCaMP3-expressing Schwann cell elements, including the distal end of the primary phrenic trunk (1), the secondary phrenic intramuscular branch (2), as well as several color-coded boxes enclosing ASC or TPSC Ca²⁺ responses that were collected for analysis in the graph to the right. Transient averages of ASC or TPSCs were plotted over time as changes in fluorescence divided by initial fluorescence (ΔF/F₀). B, Left, A prestim image in green of the diaphragm of an E14 Wnt1-GCaMP3 mouse. Middle left, Spatial fluorescence intensity map after 30 s of 40 Hz phrenic nerve stimulation. Note the response of ASCs and TPSCs again. Middle, Enlarged area of inset box in first panel imaged after the diaphragm was immunostained with antibodies against green fluorescent protein (GFP; green; to label GCaMP3-positive Schwann cells) and synaptophysin (SYP; red; to label synaptic vesicle-containing nerves and nerve terminals). Middle right, right, Enlarged regions of inset box in second panel showing α-BTX-labeled nicotinic ACh receptor-enriched postsynaptic apparati in blue together with GFP and synaptophysin staining. Note the presence of GFP-immunoreactive Schwann cells near every synaptophysin-labeled presynaptic nerve ending (right, arrow). At this age, not every α-BTX-labeled postsynaptic ACh receptor (AChR) cluster is innervated (arrowhead).