Figure 1. Mycobacterial Granulomas in Larvae Induce Expression of Epithelial Markers.

(A) Cartoon of granuloma formation depicting macrophage aggregation and morphological rearrangement to an interdigitated, flattened epithelial appearance. (B) M. marinum (cyan) granulomas stained for E-cadherin (magenta) and l-plastin (red) at 4 dpi in larva infected with 250 fluorescent M. marinum. Scale bar – 25 µm. (C) Schematic of gene trap approach used in generating the Gt(ctnna-citrine)^ct3a and Gt(jup-citrine)^ct520azebrafish lines: an exogenous citrine ORF flanked with a splice donor site (SD) and splice acceptor (SA) is introduced into the endogenous ctnna and jup genes. (D) Granulomas were identified by DIC imaging (red circle). Fluorescent imaging demonstrating localization of Gt(ctnna-citrine)^ct3a fusion protein (green) at adherens junctions formed in M. marinum (cyan) larval granulomas at 4 dpi. Scale bar – 50 µm. (E and F) Quantitation of Gt(ctnna-citrine)^ct3a positive granulomas from 1–4 dpi in larval zebrafish infected with 150–250 fluorescent bacteria (FB)/fish M. marinum n = 39, representative of 3 independent experiments. (E) Percentage of fish with at least one Gt(ctnna-citrine)^ct3a positive granuloma by day. (F) Number of Gt(ctnna-citrine)^ct3a positive granulomas per fish. Each dot represents one fish. Bars represent mean ± SEM. (G) Fluorescent images of a 4 dpi larval granuloma showing E-cadherin staining (magenta) colocalized with plakoglobin (yellow) in a granuloma in M. marinum (cyan)-infected zebrafish. Fish were infected with 150–250 FB/fish. Scale bar – 25 µm. Images representative of results from 12 animals. (H) Plakoglobin and E-cadherin fluorescence intensity as a function of distance was measured along the white line in (G), demonstrating colocalization of E-cadherin fluorescence with plakoglobin. See also Figure S1.