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Fig. 4. Loss of T results in loss of the allantoic core but maintenance of the
allantoic mesothelium and chorio-adhesive cells. (A) Cell death in
+/+, TC/+ and TC/TC
whole-mount allantoises assessed by LysoTracker Red (pink signal) at 1-s
(a-e), 3-s (f,g) and 6-s (h-j). Nuclei (blue) were labeled with Hoechst. White
line in each panel outlines the allantois; green bar marks the base of the
allantois (which in panel h lies just beneath the field of view); `ys'
indicates remnants of the yolk sac after dissection to expose the allantois.
Scale bar in j: 100 µm. (B) Immunohistochemical analysis in sections
showing that Bmp4 (brown) was excluded from the dense proximal region of +/+
(a) and TC/+ (b) allantoises, but was detected in
mesothelial and several underlying cell layers. By contrast, all of the cells
in the TC/TC allantois in distal (c) and
proximal (d) regions of a single allantois were Bmp4 positive. Scale bar in d:
100 µm for c,d; 200 µm for a,b. (C) Immunohistochemical detection
of Vcam1 in +/+ (a,c) and TC/TC (b,d)
allantoises. (a) Vcam1 localized to distal mesothelial and core cells
(arrowheads) of +/+ allantoises at 5-s, but Vcam1 was not detected in
TC/TC (b) allantoises, although it was detected
in the developing TC/TC heart (ht, inset in b).
(c) At 16-s (
9.5 dpc), +/+ allantoises united with the chorion (ch) and
contained Vcam1 in the distal mesothelial and core cells (arrowheads). (d)
TC/TC allantoises were not fused with the
chorion, but Vcam1 was observed in the distal-most mesothelium and some core
cells (arrowheads). Black lines distinguish the boundary between the posterior
streak and the allantois in a,b and d. Scale bar in d: 100 µm for a,b,d;
200 µm for c and inset in b. (D) Bar graph of allantoic length in
Vcam1-stained sections reveals that TC/TC
allantoises attain lengths >220 µm (red horizontal line), all of which
contained appropriately localized Vcam1, similar to that shown in C,d.
(E) Bar graph of Vcam1-negative regions in the same histological
sections showing maintenance of the 220 µm Vcam1-negative region in
TC/TC specimens. Error bars represent s.e.m. in
D and E; P-values are reported above each bar. Abbreviations as in
Fig. 1.
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