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. 2017 Apr 17;13(4):e1006729. doi: 10.1371/journal.pgen.1006729

Fig 3. col-Tv enhancer analysis.

Fig 3

(A-E) T1-T3 region of the Drosophila VNC and a zoom-in on the NB5-6 at stage 14, stained for Dpn, lbe(K)-GFP and β-gal to show the activity of the col-Tv-CRM-lacZ fragment under (A) control conditions and (B-E) in different mutant backgrounds for col upstream activators in NB5-6 i.e., Antp, hth, cas and lbe. Reporter expression under control of the col-Tv-CRM shows reduced expression in all mutant backgrounds. (F) Quantification of β-gal levels from the col-Tv-CRM in NB5-6 at stage 14 in different mutant backgrounds, shows that all mutant backgrounds affect the col-Tv-CRM activity and β-gal levels are reduced significantly compared to each control (*** p≤0.0001, * p≤0.01, n≥30 NBs, Students t-test, +/- SEM). (G-L) Staining for β-gal and Dpn of the NB5-6 at stage14, to assess the col-Tv-CRM activity of (G) the col-Tv-CRM wild type fragment and (H-L) the col-Tv-CRM with mutated potential binding sites for the col upstream activators (indicated by Δ). (L) ΔHCEH denotes a col-Tv-CRM fragment with all potential binding sites mutated (Δ Hox, Cas, Exd, Hth). (M) Quantification of the β-gal levels in the NB5-6 shows that all col-Tv-CRM fragments with mutated binding sites for col upstream activators, show a significant decrease in activity compared to control levels. (*** p≤0.0001, ** p≤0.001, * p≤0.05, n≥30 NBs, Students t-test, +/- SEM). (N) Schematic representation of the col genetic locus together with the position of the col-Tv-CRM fragment. Color coded dots represent potential transcription factor binding sites (TFBS) found in the col-Tv-CRM fragment, together with the conversion pattern, which was used to mutated the potential binding sites. Chr2:attP40 denotes the landing sites at which all, col-CRM as well as col-Tv-CRM ΔTFBS constructs are landed in the fly genome. Genotypes: (A, G) col-Tv-CRM/lbe(K)-GFP. (B) col-Tv-CRM/lbe(K)-GFP; Antp12/Antp25. (C) col-Tv-CRM/lbe(K)-GFP; casΔ1/casΔ3. (D) col-Tv-CRM/lbe(K)-GFP; hthDf7637/hth5E04. (E) col-Tv-CRM/lbe(K)-GFP; lbe12C5/lbeDf. (H) col-Tv-CRM-ΔHox/lbe(K)-GFP. (I) col-Tv-CRM-Δcas/lbe(K)-GFP. (J) col-Tv-CRM-Δexd/lbe(K)-GFP. (K) col-Tv-CRM-Δhth/lbe(K)-GFP. (L) col-Tv-CRM-ΔHCEH/lbe(K)-GFP.