Fig. 1

Expression of Sox2 in chick hindbrain boundaries. A Representative flat-mounted views of hindbrains of 15HH (a,d,g), 16-17HH (b,e,h) and 18HH (c,f,i) chick embryos immunostained for Sox2 (n = 10 for each group). White arrows indicate hindbrain boundaries (HBs). Higher magnification of r3/4 boundary areas, respectively (d–f). Views of confocal-generated 3D models, with yellow arrows indicating HBs and blue arrows indicating rhombomeres (g–i). B Representative transverse section of a boundary (a–c) and rhombomere (d–f) region from 18HH hindbrain, stained with Sox2 and DAPI (n = 5). C Quantification of Sox2⁺ cells in rhombomeres vs. boundaries in 15HH, 16-17HH and 18HH hindbrains (n = 5 embryos for each stage). P value obtained by using t test. D Protocol for marker quantification shows dissociation of the hindbrain (15/18HH) into single cells, immunostaining and analysis by flow cytometry (a). Quantification of Sox2⁺ cells as percentage out of total gated cells in 15HH and 18HH hindbrains (b). Representative flow cytometry plots for this experiment (c). E Illustration of the confinement of Sox2⁺ cells to HBs during maturation from 15HH to 18HH. Arrows indicate the hindbrain in the whole embryo. r = rhombomere, b = boundary, Sec = secondary Ab. only, prop = proportion. Scale bars = 100 μm