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Fig. 6. Patterning defects in wild-type cultured trophoblast-ablated
conceptuses. (A) Live view of a typical wild-type E5.5 conceptus
transgenic for an epiblast-specific GFP transgene used for trophoblast
ablation. Note the strictly distal VE thickening (dashed lines) and the
morphologically identifiable embryonic-extraembryonic junction (arrowhead).
(B) Outline of the microsurgery procedure used for trophoblast
ablation: the preplacental trophoblast (PPT) was separated from the
epiblast-visceral endoderm (Epi-VE) by a cut along the
embryonic-extraembryonic junction. This was followed by culture of the Epi-VE
for approximately 30 hours under serum-free conditions. To control for the
accuracy of the ablation, only those Epi-VE whose PPT had no GFP positivity
were used. (C) To further control for the accuracy of ablation, several
PPT and Epi-VE fragments were fixed immediately after ablation and subjected
to in situ hybridization with the EXE-specific Cdx2 probe, to ensure
that any `trophoblast contamination' left on the Epi-VE to be cultured was
minimized. (D-I,K,L) All conceptuses on the left are intact stage E5.5
and those on the right are trophoblast-ablated at the same stage, then
cultured for 30 hours. The expression profile of the indicated gene
transcripts is shown. All are sagittal views (where applicable, posterior is
on the right). Note, in the semithin sections (D), the PS-derived mesenchyme
(m) in the intact, but not in the trophoblast-ablated, conceptus. (J)
Semithin section (left) and whole-mount in situ hybridization with T
(right) of conceptuses trophoblast-ablated at E6.0, then cultured in
serum-free conditions for 24 hours. Scale bars: 100 µm; in E for
E-I,K,L.
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