Figure 6.

Characterization of FP Induction by RA

(A) Quantification of patterned cysts after stimulation with 100, 250, and 750 nM RA for 9, 18, and 24 hr. R1 cysts were analyzed on day 7 by immunostaining for SHH and OLIG2, and the percentage of cysts that were double positive was determined. Nine hours of incubation with 100 nM RA led to the formation of 7.2% ± 3.3% of self-patterned R1 cysts. This number increased significantly when the incubation period was prolonged to 18 hr (28.3% ± 7.6%) or 24 hr (30% ± 2%). The total fraction of patterned cervical cysts at 100 nM RA irrespective of incubation length never reached the same percentage as that obtained with 250 or 750 nM RA. Incubation with 250 or 750 nM RA led to similar results at any time point investigated (9 hr incubation, 25% ± 2.6% at 250 nM RA; 27.1% ± 5.4% at 750 nM RA; 18 hr or 24 hr incubation, around 40% of patterned cysts). These results indicate that optimal cyst patterning is dependent on a certain RA concentration and incubation time.

(B) Characterization of the patterning onset in R1 cysts. Days 4–7 cysts were stained for OLIG2, FOXA2, and SHH after RA application on day 2, and the percentage of R1 cysts that was positive for any combination of the 3 markers, notably FOXA2⁺ SHH⁻ OLIG2⁻; FOXA2⁺ SHH⁺ OLIG2⁻ and FOXA2⁺ SHH⁺ OLIG2⁺, was determined.

(C) Inhibition of FP induction through the administration of the SHH signaling inhibitor cyclopamine. On day 2, 1 and 5 μM cyclopamine was added together with RA for 18, 72, or 120 hr to growing cysts. On day 7, the percentage of patterned cysts based on the coexpression of FOXA2 and SHH was determined. FP induction was almost completely inhibited when cysts were grown for 120 hr in 1 or 5 μM cyclopamine.

Data are represented as mean ± SD (n = 3 independent experiments with 100 cysts counted per experiment). See also Figure S7.