Figure 4.

Statins perturb tissue growth and SHH signaling. (a) Experimental design to test effect of statins on SHH-organoid development. (b) Four distinct positional domains are identified in day 20 SHH-organoids (400 ng/ml doxycycline). SHH-organoids treated with cyclopamine (1 μM) largely express PAX6, with some GSH2 immunoreactivity, suggesting a near complete inhibition of SHH activity. SHH-organoids treated by AY9944 (1.25μM) fail to induce NKX2.2 and only retain NKX2.1 expression in the immediate vicinity of the organizer, suggesting a strong reduction the range of SHH-signaling activity. SHH organoids treated with Lovastatin (5 μM) exhibit a moderate reduction in NKX2.2 induction, with a concomitant increase in the relative area of the GSH2 expression domain. Summary of drug phenotypes and potential impact on SHH signaling activity is depicted at bottom of panel. (c) Quantification of area of each positional domain, relative to total area. Dots represent individual organoids, Error bars are S.D. One-way ANOVA with Dunnett Test. No drug N=8-14; Cyclopamine N=5-14; AY9944 N=8-16; Lovastatin N=7-13. * P < 0.05; *** P < 0.001, **** P < 0.0001. (d) Lovastatin produces a dose-dependent reduction in the distance at which NKX2.2 is induced from the organizer (red dotted boundary). Insets show NKX2.2 cells in boxed regions. Frequency histograms plot the relative distribution of organizer and NKX2.2 positive cells as a function of distance from the center of the organizer. Dotted lines represent S.E.M. Bar graphs quantify the average distance between the center of NKX2.2 and organizer distributions, illustrated by horizontal grey bars in frequency histograms. Dots represent individual organoids. Error bars are S.D. One-way ANOVA with Dunnett Test. No drug, N=23; Lovastatin (Lova) 2 μM N=18; Lova 5 μM N=24; Lova 20 μM N=23; Simvastatin (Simv) N=12; Atorvastatin (Atorv) N=10 Scale bars: 200 μm (Day 20): 50 μm (Day 6).