Effects of <i>bmp4</i> inhibition on markers of differentiated tissues and progenitors.

(A) Following 28 days RNAi of control or bmp4 RNAi, animals were stained for gut marker porcupine and pharynx-associated markers SMU15007112, dd554, foxA, and laminin. Inhibition of bmp4 did not affect porcupine or SMU15007112 expression. bmp4 RNAi caused a subtle change to dd554 expression which kept overall pharynx staining intact but eliminated expression of streams of dd554+ cells located outside of the pharynx (zoom-ins, 6/7 animals had no dd554 expressing cells adjacent to the pharynx, compared with 7/7 control animals displaying this expression). However, inhibition of bmp4 consistently resulted in ectopic non-pharynx expression of foxA and ectopic anterior expression of laminin. 5/8 bmp4(RNAi) animals had relatively less reduction to laminin pharynx staining and less ectopic anterior laminin and 3/8 animals had coincident loss of laminin expression and stronger gain of ectopic anterior expression. N ≥ 7. Scale bars represent 300 μm. (B) Measurements of head, pharynx, and tail regions determined by dd554 expression relative to body region for control and bmp4 RNAi. bmp4 inhibition did not significantly alter AP pharynx scale or position or size of tail and anterior (head) domain as measured with respect to pharynx position. (C) Left: Hoechst-stained animals following 28 days of control or bmp4 RNAi to detect original and ectopic eyes. Right: Quantification of the length from the anterior tip of the eyes (as measured for each pair of eyes along the midline) and normalized to total body size. Top panels show zoom-in of anterior animal regions for control and bmp4(RNAi) animals of eyes. Images are max-projections with z-planes chosen to show only either the original or ectopic bmp4(RNAi) animal eyes as indicated. Compared to the position of control eyes, original eyes in bmp4(RNAi) animals were displaced anteriorly toward the tip of the head while ectopic eyes are positioned at the same relative location as normal eyes in control animals. Scale bars represent 300 um. (B-C) ****p0.05 by 2-tailed t-test (B) or one way ANOVA test on ranks (C). N = 7 animals. Plots shows median values (middle bars) and first-to-third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges and dots are data points from individual animals. (PDF)