Overlapping expression of GAD and VGAT revealed by double in situ hybridization. Anterior is to the left and whole mount scale bars are 50 μm. A-C: Lateral views of a 16-hr neurula. A: GAD expression is detectable in three spots of the neural tube. B: Double-labeled embryo viewed by epifluorescence showing five pairs of VGAT-positive neurons. C: Merger of A and B showing the colocalization of GAD and VGAT in three anterior pairs of neurons. D,E: Lateral views of a 18-hr neurula. D: Double-labeled embryo. The dark brown staining shows co-expression of GAD and VGAT, while the red staining shows only VGAT expression. E: Magnification of the double-labeled embryo in F showing VGAT-positive neurons in epifluorescence. F,H: Double-labeled 22-hr neurula. F: Magnification showing GAD and VGAT co-expression (dark brown) and VGAT expression (red). The arrow indicates the first pigment spot. G: Magnification of the bracketed region in F.