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First published online 15 February 2006
doi: 10.1242/dev.02277

Development 133, 1059-1068 (2006)
Published by The Company of Biologists 2006
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Ets2 is necessary in trophoblast for normal embryonic anteroposterior axis development

Pantelis Georgiades1,* and Janet Rossant1,2,{dagger},{ddagger}

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
2 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.


Figure 1
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  		Fig. 1. Ets2 expression profile and morphological classification of Ets2db1/Ets2db1 conceptuses. (A-E) Ets2 expression from pre-implantation (E3.5) to early head-fold stage (E7.75). Note that Ets2 is not expressed in the polar trophectoderm prior to implantation, but its expression is trophoblast specific from E5.0-E6.75, and by the early head-fold stage it is also expressed in the PS (asterisk in E). (F-H) Conceptuses on the left of each image are whole-mount views; those on the right are sagittal semithin sections of the same type of embryo. Type-I (H) and type-II (G) Ets2 homozygous mutants tend to be smaller than their wild-type (F) littermates. Type I mutants also have a short PPT, as judged by the position of the embryonic-extraembryonic junction (small arrows), and an abnormally thickened anterior-distal VE (dashed lines), and show absence of mesenchymal (m) cells. Whole-mount images in A-D, and in F-H are of the same magnification. Sections in F-H are of the same magnification with the exception that the right image in H is an enlargement of the image to its left. Scale bars: 100 µm.

 

Figure 2
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  		Fig. 2. Trophoblast defects in type I mutants. (A-Q) All conceptuses on the left of each image are either wild type or heterozygous for the Ets2 mutation, and those on the right are either type I or type II homozygous mutant littermates, showing the expression profile (either single- or double-probe in situ hybridization) of the indicated gene transcripts. All are sagittal views and, where applicable, posterior is to the right. The morphologically identifiable embryonic-extraembryonic junction is denoted by arrowheads. (A-E,J,N,P,Q) E6.75 control conceptuses and their type I mutant littermates, with the exception of P, where a type II mutant is also included. For A and J, the image on the right is a magnification of the image to its left. (F,G) E7.75 control conceptuses and their type I mutant littermates. The chorion is marked by an asterisk. (H,I) E6.75 control conceptuses and their type II mutant littermates. (K-M,O) E5.5 control conceptuses and their homozygous Ets2db1/Ets2db1 mutant littermates with a reduced trophoblast region (prospective type I mutants). Conceptuses in K are semithin sections. Scale bars: 100 µm. All images without scale bars have the same magnification as A.

 

Figure 3
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  		Fig. 3. Epiblast defects in type I mutants. (A-L) All conceptuses on the left of each image are either wild type or heterozygous for the Ets2 mutation, and those on the right are either type I or type II homozygous mutant littermates, showing the expression profile (either single- or double-probe in situ hybridization) of the indicated gene transcripts. All are sagittal views and, where applicable, posterior is to the right. The morphologically identifiable embryonic-extraembryonic junction is denoted by arrowheads. (A,C,F,G,I) E6.75 control conceptuses and their type I mutant littermates, with the exception of I, where a type II mutant is also included. (H) E6.75 control conceptuses and their type II mutant littermates. (B,D,E,K) E7.75 control conceptuses and their type I mutant littermates. (J,L) E8.5 control conceptuses and their type I mutant littermates. Scale bars: 100 µm. All images without scale bars have the same magnification as A.

 

Figure 4
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  		Fig. 4. VE defects in type-I mutants. (A-M) Sagittal views, posterior to the right. The morphologically identifiable embryonic-extraembryonic junction is denoted by arrowheads and the VE area below each arrow denotes the VE of the distal tip. Dashed lines represent the DVE/AVE region and asterisks the definitive endoderm. (A-F) Wild-type conceptuses at the stages indicated showing either the Hex or cerberus expression profile. (G,H,J,K,L) All conceptuses on the left of each image are either E6.75 wild type or heterozygous for the Ets2 mutation, and those on the right are either type I and/or type II homozygous mutant littermates, showing the expression profile (either single- or double-probe in situ hybridization) of the indicated gene transcripts. The image on the right in H is a sagittal section of the image to its left. (I) E7.75 type I mutant (left) and its sagittal section (right), showing expression of Hex. (M) E7.75 control conceptus (left) and its type I mutant littermate (right), showing expression of Otx2. Scale bars: 100 µm; in A for A-D; in E for E-H,J,K.

 

Figure 5
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  		Fig. 5. Type I defects in chimeras generated with 2N ES+/+ cells and 4N Ets2db1/Ets2db1 embryos. All images on the left are bright-field (BF) and GFP views of E7.75 chimeras generated by aggregations of GFP-positive 2N ES+/+ cells with 4N Ets2db1/Ets2db1, 4N Ets2db1/Ets2+ or 4N Ets2+/Ets2+ embryos. All images to the right are whole-mount in situ hybridizations of the conceptuses to their left, showing the expression profile of the genes indicated. (A) 2N ES+/+{leftrightarrow}4N Ets2db1/Ets2+ E7.75 chimera showing normal development that is indistinguishable from its 2N ES+/+{leftrightarrow}4N Ets2+/Ets2+ counterpart (data not shown). (B-D) 2N ES+/+{leftrightarrow}4N Ets2db1/Ets2db1 E7.75 chimeras showing abnormal development with defects identical to those seen in E7.75 type I mutants, as judged morphologically and by T, Hex and Oct4 expression. Scale bars: 100 µm.

 

Figure 6
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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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