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First published online 27 April 2005
doi: 10.1242/dev.01847

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Induction and migration of the anterior visceral endoderm is regulated by the extra-embryonic ectoderm

Tristan A. Rodriguez^1,2,*,¶, Shankar Srinivas^2,3,*,, Melanie P. Clements^2,*,, James C. Smith³ and Rosa S. P. Beddington^2,

¹ Molecular Embryology Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK
² Division of Mammalian Development, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
³ Wellcome Trust/Cancer Research UK Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (73K): [in a new window]   Fig. 1. Removal of the extra-embryonic region leads to ectopic Hex-GFP transgene expression. Bright-field (A,C,E,G) and fluorescence (B,D,F,H) images of 5.5 dpc embryos after overnight culture. (A,B) Control unmanipulated embryo. (C-H) Embryos in which the extra-embryonic region was removed before culture. In control embryos cultured overnight, the expression of the Hex-GFP reporter is restricted to the anterior visceral endoderm (A,B). In embryos where the extra-embryonic region has been removed prior to the migration of the AVE, the expression of the Hex-GFP transgene is observed throughout the visceral endoderm (C-F). However, if the extra-embryonic region was removed after the migration of the AVE, this ectopic expression of the Hex-GFP transgene is not observed (G,H).

View larger version (55K): [in a new window]   Fig. 2. Ectopic Hex-GFP transgene expression is due to de novo expression of the GFP reporter. Representative frames from a movie (supplementary data) of a cultured 5.5 dpc embryo after removal of the extra-embryonic region, showing de novo expression of the Hex-GFP reporter in the visceral endoderm. The time from start of culture is indicated in hours and minutes at the bottom right of each frame. The embryo was imaged every 7 minutes with phase-contrast and fluorescence optics. An overlay of both bright-field and fluorescent images is shown in the top panels and, for clarity, just the fluorescence images below. EGFP fluorescence (green) marks the expression of the Hex-GFP transgene. Arrows in E-H indicate de novo expression of the Hex-GFP reporter.

View larger version (82K): [in a new window]   Fig. 3. Analysis of how the removal of the extra-embryonic region changes the patterning of the visceral endoderm and epiblast. Whole-mount in situ hybridization analysis of 5.5 dpc embryos cultured overnight. (A,C,E,G,I,K,M,O) Control unmanipulated embryos. (B,D,F,H,J,L,N,P,Q,R) Embryos in which the extra-embryonic region was removed before culture. In control embryos Cer1 (A) and Lhx1 (C) are restricted to the anterior visceral endoderm, but in the absence of the extra-embryonic region, both Cer1 (B, n=17/20) and Lhx1 (D, n=7/7) are expressed ectopically throughout the visceral endoderm. Afp expression is observed in the proximal and posterior visceral endoderm in control embryos (E) but is strongly downregulated after removal of the extra-embryonic region (F, n=19/24). Expression of cripto (G) Nodal (I) and T (K) is restricted to the proximal/posterior epiblast in control embryos but is lost after removal of the extra-embryonic region and overnight culture (H, n=14/16; J, n=7/7; and L, n=9/10). Expression of Pou5f1 is observed in the epiblast of both control (M) and manipulated embryos (N, n=11/14), indicating that the downregulation of posterior epiblast markers is a specific effect. No difference is observed in the pattern of Nodal expression in controls (O) and embryos lacking the extra-embryonic region (P, n=6/6) after only 2.5 hours culture. The expression of the anterior neural marker Sox1 is detected only at low levels in embryos lacking the extra-embryonic region after overnight culture (R, n=4 weak and 11 no expression out of 15) but is strongly upregulated throughout the epiblast of these embryos after 42 hours of culture (S, n=7/7).

View larger version (82K): [in a new window]   Fig. 4. Injection of ExE cells inhibits AVE formation and leads to an expansion of posterior epiblast markers. (A) Diagrammatic representation of the approach used to isolate and inject ExE cells adjacent to the AVE of 5.5 dpc embryos. (B-E) Cer1 expression is observed in the AVE of control embryos (B) and of embryos injected with COS-7 cells (E) but is lost in embryos injected with ExE cells (C,D). (F-I) T expression was observed in the posterior epiblast of control embryos (F) but its expression was expanded (G,H) or observed ectopically (I) after the injection of ExE cells.

View larger version (33K): [in a new window]   Fig. 5. Proposed model for the role of the ExE in patterning the pre-gastrulation mouse embryo. Signalling by the epiblast induces the AVE (pink arrows) and inhibitory signals from the ExE restrict this induction to the distal tip of the embryo (inhibitory arrows), The ExE also induces the expression of proximal/posterior markers in the epiblast (orange arrows). In embryos where the ExE has been removed, the epiblast induces AVE markers throughout the visceral endoderm and no expression of proximal/posterior markers is observed in the epiblast.