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Retinoic acid decreases intracellular K+ in pectoral fin buds via transcriptional activation of the calcineurin inhibitor rcan2. (A, B) FLIM images of developing fin buds from embryos treated for 6 h with DSMO-treated (A) or with 200 nM RA-treated (B) 32 hpf embryos. (C) FLIM measurements from cells in developing buds at 32 hpf of the indicated treatment groups. (D) FLIM measurements of cells in the ectoderm (Ecto) or the mesenchyme (Mesen) of buds at 32 hpf treated either with DMSO or RA. (E) In situ images of dorsal view (a) and lateral view (b) for kcnk5b expression in 48 hpf embryos. Pectoral fin buds (pfb), ectoderm (e), and mesenchyme (m). (F) Representative lateral view images of pectoral fin buds of in situ experiments for kcnk5b expression at 32 hpf (a), 56 hpf (b), and 72 hpf (c). (G) Representative lateral views of pectoral fin buds after in situ experiments for rcan2 expression at 32 hpf (a), 34 hpf (b), 48 hpf (c), and 56 hpf (d) embryos. (H) qRT-PCR of rcan2 from isolated pectoral fin buds of 48–52 hpf embryos treated with DMSO or 200 nM RA for 6 h. (I) In situ for rcan2 expression in fin buds of 48 hpf embryos after 6 h DMSO treatment. Ectoderm is indicated by (e), and mesenchyme is indicated by (m). (J) In situ images for rcan2 expression in fin buds of 48 hpf embryo after 6 h 200 nM RA treatment. Ectoderm (e), mesenchyme (m). (K, L) Pectoral fin buds of heat-shocked non-transgenic embryo (K) or heat-shocked transgenic hsp70:rcan2-mCherry sibling (L). (M) Measured fin buds were standardized to the area of an eye in the same embryo. (N) FLIM measurements of intracellular K+ in pectoral fin bud cells from heat-shocked non-transgenic and mCherry controls and transgenic rcan2-mCherry-expressing siblings. (O–R) Representative fin buds of embryos expressing empty sgRNA vector “EV” (O), rcan2 sgRNA “KO” (P), mCherry mRNA with rcan2 sgRNA “KO+mCherry” (Q), mutated rescue rcan2 mRNA with rcan2 sgRNA “KO+rcan2” (R). (S) Ratios of fin bud areas to eye areas of embryos of the indicated experimental groups. (T) FLIM measurements of intracellular K+ in the pectoral fin bud cells in control and CRISPR-Cas9 knockout of rcan2 in embryos. Experiments were repeated 3 or more time (N ≥ 3). For FLIM, we measured 2 or 3 locations in each tissue of 1 fin bud per embryo. Each measured value is represented as a data point (C, D, N, T). For the fin bud size measurements, we measured 1 fin bud per embryo (M) and 2 fin buds per embryo (S). For the qRT-PCR experiments, we collected 80 fin bud samples per isolation. Three or more isolations were measured. Each isolation was measured in duplicate or triplicate. Each isolation is represented as a data point. Each in situ repeat contained 6–12 embryos per replicate. P values represent statistical analysis by Student’s two-tailed t test. P values >0.05 are designated as “not significant” (NS). Scale bars represent 50 μm (E–G, I–L, O–R). Numerical data used in this figure are included in S3 Data. FLIM, Fluorescence Lifetime Microscopy; hpf, hours post fertilization; RA, retinoic acid.
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