Fig. 7. Fever-associated heart defects following remote activation of TRPV1 in neural crest cells.

(A) GCaMP6 fluorescence in primary chick neural crest cells electroporated with TRPV1^WT (blue lines) or TRPV1^FeRIC (red lines) in the absence or presence of the TRPV1 inhibitor SB366971 (gray and black lines) following RF (gray box) then 1μM capsaicin (bar). Bar graph shows cumulative responses representing 3–4 separate experiments with 22–138 cells/group analyzed. (B) mCherry⁺ neural crest streams migrating to the pharyngeal arches (white arrows) in HH14 control or TRPV1^FeRIC electroporated embryos. 6 independent experiments. (C) Whole-mounted hearts from RF^NegTRPV1^FeRIC embryo and RF^10minTRPV1^FeRIC embryo with arrows noting the position of the aorta (Ao) and pulmonary trunk (P) with respect to the right ventricle (RV). 5 independent experiments were performed. See Supplemental Movie 1 for an MRI reconstruction of a RF^10minTRPV 1^FeRIC-induced DORV heart defect in chick. (D) Percent of embryos with histologically confirmed conotruncal heart defects within indicated groups at HH36. (E) Representative histological sections through the aorta and pulmonary trunk at the level of the coronary artery (*) in Control RF, RF^10minTRPV 1^WT, RF^10minTRPV1^FeRIC, and RF^10minTRPV^FeRIC with SB366791 pretreatment. The double arrowheads highlight the luminal areas of the aorta and pulmonary trunk estimated using the Cavalieri probe. Bar = 200μm. (F) Graph of the Cavalieri probe estimates of cross sectional areas through the aorta at the level of the coronary artery in the indicated treatment groups (CE=0.03). (G) Graph of the Cavalieri probe estimates of the crosssectional areas through the pulmonary trunk distal to the semilunar valve in the indicated treatment groups (CE=0.03). Significance was determined using unpaired t-test (A), Fisher exact test (D) or one-way ANOVA followed by Bonferroni’s multiple comparisons test (F,G). *P<0.02, **P<0.005, ***P<0.0001. The number of biological replicates is indicated by n in the graphs in (F) and (G).