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 +/+, T^C/+ and T^C/T^C 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 T^C/+ (b) allantoises, but was detected in mesothelial and several underlying cell layers. By contrast, all of the cells in the T^C/T^C 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 T^C/T^C (b,d) allantoises. (a) Vcam1 localized to distal mesothelial and core cells (arrowheads) of +/+ allantoises at 5-s, but Vcam1 was not detected in T^C/T^C (b) allantoises, although it was detected in the developing T^C/T^C 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) T^C/T^C 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 T^C/T^C 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 T^C/T^C specimens. Error bars represent s.e.m. in D and E; P-values are reported above each bar. Abbreviations as in Fig. 1.