Fig. 6. Cell arrangement and pMLC distribution at the apical surface of the neural tube. (A) Preparation of flat-mount samples to observe the inner (apical) surface of the neural tube in stage 8-9 chicken embryos. The midbrain areas, marked with dotted squares, were selected for the observations. DM, dorsal midline; VM, ventral midline. In all the images shown below, the dorsal side is at the top and the anterior side is towards the left. (B) Double-immunostaining for ZO-1 and pMLC at the apical surface of the neural tube in stage 8 embryos. Representative cell clusters with rosette-like arrangement, where pMLC is concentrated at the central regions, are encircled; and those with arch-type arrangement are marked by an oval. Arrows indicate examples of linearly arranged pMLC signals. A-P, anteroposterior axis; D-V, dorsoventral axis. Scale bar: 10 µm. (C) The angular distribution of linear pMLC signals on the apical surface of neural tubes. Linear pMLC signals of more than 10 µm (68-89 signals/field) from four different fields from four embryos at stage 8-9 were analyzed for length and orientation. Horizontal and vertical axes represent the anterior-posterior and dorsal-ventral orientations, respectively. Percentages of the signals oriented at the angles between 0° and 30°, 30° and 60°, and 60° and 90° to the anterior-posterior axis were calculated. pMLC signals are more abundant along the dorsoventral axis. (D) Double immunostaining for Shroom3 and ZO-1 or pMLC at the apical surface of the neural tube in stage 9 embryos. ZO-1 colocalizes with Shroom3, whereas pMLC colocalizes only with restricted Shroom3 signals. (E) Effects of Y27632 on the distribution of pMLC and cell morphology. Stage 8(-) embryos (with two or three somites) were incubated in the presence of Y27632 (100 µM) or 1% DMSO (control) in the culture medium for 6 hours prior to fixation, and their neural tubes were double-immunostained for pMLC and ZO-1. Examples of rosette-like and arched cell clusters are marked with a circle and oval, respectively. When rosette is defined as a cluster comprising six cells or more, in which the cells radially converged on a constricted point, cell clusters with such configuration are undetectable in the Y27632-treated sample shown. The bar graphs show the length of apical junctions, apical surface areas and percent of cells with pMLC signals. All the cells in randomly selected fields (105-250 cells/field) were measured. Histograms are the average of three different fields from three embryos, and bars represent the standard deviation. ^*P<0.05; ^**P<0.01 against 1% DMSO-treated embryos. Scale bars: 10 µm.