Figure 5. Generation and Application of Inducible Reporter Knockin Mice (iLumiFluor) for Developing Methods to Target Lingual and Oropharyngeal Epithelia.
(A) General strategy for testing the efficacy of intralingual (i.l.) tamoxifen (TAM) administration to activate the estrogen receptor Cre recombinase fusion (KRT14-CreERtam) and induce eGFP-NanoLuc (GpNLuc LumiFluor) transgene expression within the tongue following Cre-mediated recombination and excision of the STOP cassette.
(B) Schematic of transverse and sagittal views highlighting key lingual anatomic features including the oropharynx, dorsal tongue, and ventral tongue. TAM was delivered by i.l. injection to the posterior, dorsal-lateral tongue to minimize dorsal tongue swelling and disruption to eating or drinking.
(C) Targeted induction of bioluminescent signal in adult tongues. Bioluminescent imaging (BLI) of iLumiFluor and control littermate mice injected either 2× or 3× i.l. versus 5× i.p. with TAM. Live animal, non-invasive BLI was performed temporally and signal levels graphed relative to baseline. Representative images for both live, non-invasive BLI and end-point resected tongues 16 days post-TAM are presented (n = 3, ***p < 0.0001).
(D) Immunofluorescence analysis reveals mosaic reporter expression throughout the adult tongue. Representative sections of adult tongues fixed in paraformaldehyde (PFA) and embedded, and sections were stained for epithelial basement membrane (Integrin-β4; red), LumiFluor (eGFP; green), and nuclei (DAPI; blue). Tile scan of 3× i.l. TAM (top). Comparison of TAM treatment regimen effects at various anatomic locations in iLumiFluor and control littermate mice (bottom). Tile scan of magnification at 20× with 1.25 optical zoom.
Data presented in (C) is shown as mean ± SEM of a representative experiment (n = 3 experiments). Two-way ANOVA with Bonferroni’s post hoc tests were used to determine significance. Related to Figure S6.