Figure 6: Medaka Block-2 has endogenous enhancer activity.

Enhancer activity of three fragments of the Medaka rnf32 intron was tested by transgenic reporter assay. The tested fragments were 3.9 kb of MACS1, 1.4 kb of Block-2, and the whole intronic fragment encompassing MACS1 and Block-2 (a). GFP reporter expression (green) was monitored in medaka larvae (b–k). The whole intronic fragment drove reporter expression in the posterior pharynx and oesophagus at 2 dpf (b), 3 dpf (c) and 10 dpf (d,e). At 10 dpf, reporter expression was also detected in the dorsal pneumatic duct connecting to the gas bladder (d). Reporter expression driven by the whole intronic fragment (f), MACS1 (g) and Block-2 (h) at 5 dpf. The whole intronic fragment and Block-2 yielded similar signals in the digestive tube (f,h). MACS1 did not drive reproducible expression in medaka larvae (g). Immunohistochemistry for GFP reporter signals driven by the whole intronic fragment at 10 dpf (i–k). Signals were detected in the epithelium of the digestive tube (i–k). The number of larvae exhibiting reproducible reporter expression among the total number of injected eggs is indicated at the bottom in each image (f–h). Scale bars, 500 μm. Galb, gall bladder; Gb, gas bladder; Pd, pneumatic duct.