a, nkx1-1+ and BWM collagen+ cells (white, double-positive cells from one animal) and nkx1-1 and myoD mutually exclusive expression by FISH (4 animals, 2 experiments) and single-cell (each dot) RNA-seq17. b, Length-to-width ratio (13 control, 17 nkx1-1(RNAi) animals, 3 experiments). Circular-fiber loss after nkx1-1 RNAi by c, IF (3 experiments) and d, TEM (3 animals). Pseudocolored circular (cf), longitudinal (lf), and other fibers (yellow); sm, subepidermal membrane. e, ML-patterning defects after nkx1-1 RNAi (4 experiments). f, Wider anterior poles (2 experiments). g, Ectopic eyes (opsin+), supernumerary gut branches (mat+), wider cephalic-ganglia arc (glutamate decarboxylase+), wider midline (admp+), duplicated midline (ephR1+), and anterior poles (notum+) in 9dpa nkx1-1(RNAi) animals (3 experiments). Cartoon line, measurement site. Animals had 12 dsRNA feedings. Two-tailed Student-t test, mean ± SD, ns: not significant (b,c). Bottom left number: animals with phenotype/total tested. Bars: 10μm (a,c); 1μm (d); 100μm (f,g).