Figure 7. Rotund Zinc-finger protein positively regulates bab2 expression through direct binding to the T13 LAE sequence.

(A) The R32 sequence is required for normal LAE-GFP reporter activation in the proximal-most bab2-expressing cells. GFP expression (green) alone and in combination with Bab2 immunostaining (red) from late L3 leg (D–F and D′–F′, respectively) and antennal (ant) (G–I and G′–I′, respectively) imaginal discs, expressing a given LAE-GFP truncated derivative (left side), are shown. The S5-GFP construct drove wild-type leg (D–D′) and antennal (G–G′) bab2 expression patterns. In contrast, GFP expression in the proximal-most bab2-expressing cells of the R32-truncated derivative S10-GFP was much more weakly detected in leg (E–E′) and not at all in antennal (H–H′) discs (see brackets). The addition of the R32 subsequence T13 to the S10-GFP construct (H3 derivative) was sufficient to increase proximal GFP expression levels (F–F′ and I–I′, see brackets), particularly in antennal tissues. (B) Rn binds in-vitro to R32. The sequence LOGO of the consensus Rn-binding site, including a T-rich track (red bracket), is shown in the upper left side. The LAE sequences included in the four labelled double-stranded DNA probes are indicated beneath, with the same color code. In probes #3–4 the wild-type T6 track was substituted by a linker sequence shown in grey. The position of a consensus G is indicated by a vertical orange line. EMSA experiments are shown on the right. In-vitro translated Rn protein was omitted or added as indicated, and line numbers refer to the four tested probes shown on the left. The positions of non- and Rn-specific shifted DNA-protein complexes are indicated by an asterisk and a horizontal arrow, respectively. Note that Rn bound T13 less strongly than the entire R32 DNA fragment. (C) The R32 sequence is critical for rn-induced LAE-GFP expression in developing limbs. GFP expression (green) alone and in combination with Bab2 immunostaining (red) from late L3 leg (J–K) and antennal (L–M) imaginal discs, over-expressing Rn under the control of the Dll regulatory sequences (Dll-Gal4 plus UAS-Rn constructs), are shown. When Rn was over-expressed throughout the Dll domain, endogenous bab2 and the S5-GFP reporter construct were both up-regulated in nearly all Dll-expressing cells, in either developing leg or antenna. In striking contrast, GFP expression in S10 reporter tissues is not detected, indicating that the R32 sequence is critically required for Rn-mediated S5-GFP expression.