Evidence for cross-regulatory relationships between genes in different clusters.

<p>A: mRNA expression levels of the indicated genes in Notch activated cells relative to controls (log₂) in untreated (grey) and cycloheximide treated (black) cells at the times indicated. Cells were exposed to cycloheximide for 60 min prior to Notch activation at 0 min as well as during the timecourse. B,C: Expression of Hairy in the wing imaginal disc pouch from control (B, <i>ptc-Gal4 ; UAS-lacZ</i>) and HLHmβ-VP16 (C, <i>ptc-Gal4</i> ; <i>UAS-HLHmβ-VP16</i>), arrows indicate the stripe of HLHmβ-VP16 expression where Hairy is induced. D,E: Expression of Hairy in muscle progenitors (brackets) from control wing imaginal discs (D, <i>1151-Gal4</i>; <i>UAS-lacZ</i>) and from those expressing HLHmβ (E, <i>1151-Gal4</i>; <i>UAS-HLHmβ</i>). Hairy is reduced in the muscle progenitors (brackets) but not in neighbouring epithelial cells (asterisks). F,G: Quantification of expression levels of Hairy (F) and <i>edl-GFP</i> (G) in muscle progenitors expressing β-galactosidase (con), HLHmβ (mβ) or HLHmβ-VP16 (mβ-VP16). Average pixel intensities from a defined region within the expression domain were measured using ImageJ and normalized relative to background levels from a comparable region in the same discs, >5 discs per genotype. Error bars indicate standard error of the mean. Asterisks indicate that results are significantly different from control (p≤0.05; using an unpaired, 2-tailed student T-test). H: Fold change of the indicated mRNAs in cells treated with dsRNA against <i>hairy</i>, <i>edl</i> or <i>btn</i> in comparison to controls (no RNAi). RNA levels were reduced by 65%, 71% and 61% for <i>hairy</i>, <i>edl</i> and <i>btn</i> respectively. These experiments were performed in the basal state (no Notch activation). Bars represent the average of three biological replicates and error bars indicate standard error of the mean. I: Log₂ fold changes in mRNA levels of <i>hairy</i> (<i>h</i>, brown) <i>hibris</i> (<i>hbs</i>, green) and <i>HLHm</i>β (<i>m</i>β, blue) from the microarray study. Scale for <i>hbs</i> and <i>h</i> is indicated by left axis and for <i>m</i>β, which had larger fold changes, by right axis. J: Summary model of the feed-forward regulatory relationships, dotted line indicates that the direct regulation of <i>hbs</i> (cluster 3) by Notch signaling has not been directly tested here, although <i>hbs</i> and other genes in cluster 3 exhibit Su(H) binding, which implies that at least some undergo direct Notch regulation.