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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.
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