QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Uy, G. D. Articles by Gardner, R. L. Search for Related Content PubMed PubMed Citation Articles by Uy, G. D. Articles by Gardner, R. L.

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

¹ Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford, OX1 3PS, UK
² Department of Anatomy, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706, USA

View larger version (24K): [in a new window]   Fig. 1. ExE/ChE microdissection. ExRs were microdissected from the conceptus and extra-embryonic visceral endoderm and extra-embryonic mesoderm were removed by trypsin/pancreatin treatment. Proximal and distal are as indicated. ExEs and ChEs were microdissected into fractions perpendicular to the proximodistal axis with a glass needle. Individual ExE/ChE fractions were explanted into separate TSC culture wells following dissociation with pronase, while EPC/ExE transition tissues were explanted as whole, loosely dissociated masses.

View larger version (137K): [in a new window]   Fig. 2. Non-coherent growth of ExE. Isolated day 7 ExE several minutes after cell A was injected with flourescein-dextran-lysine. Note that cell B, though clearly not adjacent to A, has become about as strongly labelled as A and must therefore be its sister. The more weakly labelled intervening cell, C, is presumably a product of the division immediately preceding the one yielding A and B. Scale bar: 50 µm.

View larger version (18K): [in a new window]   Fig. 3. TSC colonies and cell death from dissociated whole ExE and ChE throughout post-implantation development. (A) TSC colonies derived from dissociated whole ExE/ChEs throughout stages in post-implantation development. The mean frequency of TSC colonies was determined as the number of TSC colonies/number of tissues dissociated. The range of TSC colonies obtained from individual tissues is indicated in square brackets. (B) The mean percentage of instantaneous cell death in ExE/ChEs after dissociation, as determined by the Trypan Blue exclusion test. The percentage of Trypan Blue-positive cells was calculated as a function of total cells. A significant increase in cell death was observed between late-streak ChEs and 4 s.p. ChEs at P<0.05. However, there was no significant difference between early-streak ExEs and late-streak ChEs at P>0.05. Error bars indicate s.e.m.

View larger version (53K): [in a new window]   Fig. 4. Analysis of TSC potential in the 9 s.p. ChE. (A) RT-PCR analysis of TSC markers in late-streak and 9 s.p. ExRs. ß-actin is a positive control. The sizes of the PCR products are indicated on the left. (B) Photomicrograph of a 40 XX chromosome spread from a representative cell of the polyclonal TSC line, derived from a single 9 s.p. ChE after approximately 2 months in culture, used for chimaeric analysis. Numbers represent specific chromosomes. Scale bar: 10 µm. (C) Photomicrograph of a late-headfold stage TSC chimaera produced by blastocyst injection of the 9 s.p. TSC line in B. Arrows indicate contribution of blue staining ROSA26 +/– TSCs to the ChE and EPC. Scale bar: 500 µm.

View larger version (138K): [in a new window]   Fig. 5. Photomicrograph of 4 s.p. allantoic distal tips in co-culture with TSC colonies. After 9 days in TSC culture, TSC colonies (arrows) from a line derived from a wild-type early-streak ExE continued to proliferate on, and in close proximity to, blue ROSA26 +/– allantoic tissue. Scale bar: 200 µm.

View larger version (125K): [in a new window]   Fig. 6. Distribution of mitotic cells in the developing ChE. (A,B) Representative photomicrographs of sagittally sectioned, mitotically arrested ChEs. Examples of mitotic cells are indicated by arrows. Note that mitotic cells were located throughout the ChE, but lay predominantly apically in the chorionic plate in the early-headfold ChE. (A) Early-headfold ChE that has not yet undergone fusion. (B) 5 s.p. ChE that has undergone fusion. Arrow in the ChE median box indicates an exceptional mitotic cell. Scale bar: 25 µm.

View larger version (8K): [in a new window]   Fig. 7. Distribution of TSC potential in the 6 s.p. ChE. TSC colonies derived from the ChE median and ChE perimeter of 6 s.p. ChEs (n=20). Range of TSC colonies obtained from each region of individual tissues are indicated in square brackets. There was a significant difference between the ChE median and perimeter at P<0.001. One exceptional ChE produced TSC colonies greater in number than three times the mean in each ChE region: ChE median, 28 TSC colonies; ChE perimeter, 69 TSC colonies.

View larger version (105K): [in a new window]   Fig. 8. Scoring mitotic figures in unfused and fused chorionic ectoplacental junctions for data in Table 5. Brightfield photomicrographs of a late-headfold stage exposed to colcemid for 2 hours and prepared for histochemistry (see Materials and Methods). Arrows in B-D indicate examples of mitotic figures in metaphase. (A) The chorionic area scored for mitotic figures is indicated between the ‘brackets’. The three arrows point to the region of ChE shown in B-D. (B) ChE median of A showing the fused chorionic area beneath the broken lines and bordered on the left by a unbroken black line. (C) Unfused ChE perimeter to the left of the unbroken black line of B, the extent of which is enclosed by a ‘bracket’. (D) Unfused ChE perimeter to the right of the fused ChE median, the extent of which is also enclosed by a ‘bracket’. Abbreviations: ac, amniotic cavity; am, amnion; EPCav, ectoplacental cavity; EPC, ectoplacental cone; x, exocoelomic cavity; ys, yolk sac. Scale bar: 100 µm for A; 25 µm for B-D.