Characterization and validation of Tribolium rx-EGFP enhancer trap line.

(A) The Tribolium rx-EGFP enhancer trap was taken from the GEKU screen collection [47] where enhancer traps were generated by piggyBac-mediated transposition. A 3XP3-EGFP-SV40 cassette randomly inserted upstream of the Tc-rx gene in opposite direction (insertion site mapped by [47]). (B) Maximum intensity projections of immunostainings against GFP and Tc-Rx in adult brains of the Tc-rx-EGFP line. The line only marked a subset of Tc-Rx expressing cells. This also applies to the n-dorsal region (Bⁱⁱ). However, all EGFP-expressing cells also expressed Tc-Rx. Coexpression was verified manually. (C) The introduction of the enhancer trap cassette did not visually influence Tc-Rx expression, as domains were highly similar between transgenic Rx-GFP (Bⁱ) and wild-type vermillion-white (v^w, Bⁱⁱ, [90]) animals, as visualized by color-coded maximum intensity projections. Observed qualitative differences in Tc-Rx expression in the transgenic or wildtype condition (N = 3 each) were approximately as large as the differences between the genetic backgrounds. (D) A crop of a maximum intensity projection of cells surrounding the adult protocerebral bridge (yellow arrowhead, Dⁱ) shows the coexpression of GFP (Dⁱⁱ) and Tc-Rx (Dⁱⁱⁱ) in a subset of cells that were subsequently used in this study. (E) An analogous analysis in young pupal brains of cells surrounding the protocerebral bridge (Eⁱ) revealed more EGFP-expressing cells (Eⁱⁱ) with overlap to Tc-Rx cells (Eⁱⁱⁱ) than in the adult (D). (F) Quantification of Rx/GFP double-positive cells in the region of DM1-4 lineages surrounding the protocerebral bridge (yellow rectangle, Fⁱ) revealed that at different developmental stages, the fraction of double-positive cells is different, ranging from approximately 10% to 75%. Scale bars in B and C represent 100 μm, and in D and E, scale bars represent 50 μm. EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; Rx, retinal homeobox protein.

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