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First published online 25 May 2006
doi: 10.1242/dev.02413

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Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning

¹ Ecole Polytechnique Fédérale de Lausanne EPFL-ISREC, Chemin des Boveresses 155, CH-1066, Switzerland.

View larger version (83K): [in a new window]   Fig. 1. Expression of Nodal agonists and antagonists between implantation and E5.5. (A) Nodal is already expressed at E4.5, both in the ICM and PrE of the blastocyst. Furin mRNA is expressed in the PrE lineage until E5.25, before it becomes restricted to the ExE and ExVE (E5.5). Pace4 and Cripto are specifically expressed in the trophectoderm lineage and epiblast, respectively. (B,C) The PrE also expresses the DVE markers Lefty1 (B), Hex and a HexP-GFP reporter transgene (C), whereas Cer1 mRNA is below detectable levels (B). Lefty1 and Hex transcripts are clearly downregulated between E5.0 and 5.25 before they reappear together with Cer1 mRNA in the most distal VE cells at E5.5.

View larger version (89K): [in a new window]   Fig. 2. Early Nodal signaling is required for normal epithelialization of the epiblast and to prevent ectopic expression of extra-embryonic VE markers and Furin in distal regions. (A-D) Whole-mount in-situ hybridization (ISH) of an antisense probe directed against the deleted exon 2 identifies NodallacZ/lacZ null mutants (B) and reveals specific expression of Nodal mRNA in the epiblast and EmVE of wild-type embryos (A). Nodal expression is also detected by ß-galactosidase staining of Nodal+/lacZ heterozygotes (C). In E5.25 NodallacZ/lacZ homozygotes, Nodal expression independent of Nodal activity is detected only in the epiblast (D). (E-H) Immunostaining of E-cadherin (E,F) and laminin (G,H) reveals irregularities in the epithelial organization of mutant epiblasts (F), and detachment of distal embryonic visceral endoderm cells from the laminin-positive basement membrane (arrowhead in H). (I-P) In wild-type and Nodal+/lacZ heterozygous embryos (control), Gata4, Hnf4, Ttr and Furin adopt a restricted expression in the ExVE around E5.5 (I,K,M,O). In sharp contrast, ectopic distal expression of these markers is observed in NodallacZ/lacZ null mutants (J,L,N,P).

View larger version (132K): [in a new window]   Fig. 3. Early Nodal signaling before DVE formation is essential to specify EmVE. In pre-DVE control embryos (A,C,E,G), which have not yet upregulated HexP-GFP expression (E5.25, see Materials and methods for accurate staging of embryos), Lim1, Fgf5, Fgf8 and Bmp2 are specifically expressed in the EmVE. By contrast, all of these markers fail to be induced in Nodal mutants (B,D,F,H). Note that Fgf5 and Fgf8 also show expression in the epiblast of wild-type embryos, which is downregulated in NodallacZ/lacZ null mutants (C,E,D,F).

View larger version (37K): [in a new window]   Fig. 4. The egg cylinder of Nodal mutants does not reach the size that is required for DVE formation. (A) In normal embryos, DVE formation monitored by HexP-GFP expression occurs only when the apex of the egg cylinder has reached a certain distance from the ExE (epiblast length). (B) While the length of the epiblast is difficult to measure in Nodal mutants due to the lack of a sharp epiblast/ExE boundary, a reduction in overall size (conceptus length) is readily apparent. Note that Nodal mutants at E5.5 do not reach the conceptus length that appears to be required for the DVE to be induced.

View larger version (84K): [in a new window]   Fig. 5. Even in the absence of dominant inhibitory signals from ExE, induction of DVE depends on Nodal and proprotein convertase activities. As expected (Rodriguez et al., 2005), embryo explants stripped of ExE at the DVE stage (E5.5) induce Lim1 and HexP-GFP throughout the EmVE endoderm (top row). Purified recombinant Nodal (50 µg/ml) does not prevent ectopic induction of DVE markers (second row). By contrast, blocking endogenous Nodal activity through inhibition of its signaling receptors (+SB) or proprotein convertases (+CMK) prevents ectopic induction of both HexP-GFP and Lim1. Processed Nodal (50 µg/ml) administred to the outer (apical) membrane of the visceral endoderm is not sufficient to ectopically induce HexP-GFP and Lim1, whereas processed Activin (50 µg/ml) is (bottom rows). Note that the thickness of the VE is significantly increased upon inhibition of Nodal signaling compared with untreated controls or explants treated with Activin. SB, SB-431542 (10 µmol/l); CMK, dec-RVKR-CMK (25 µmol/l).

View larger version (104K): [in a new window]   Fig. 6. Nodal mutants prematurely downregulate several determinants of pluripotency during implantation. (A) Whereas expression of Fgf4 and Sox2 is still unaffected, Oct4, Nanog, Foxd3 and Cripto are already downregulated or silenced in Nodal mutants between E5.0 and 5.5. (B) By contrast, molecular markers of the ExE, such as Pace4 and Bmp4, are not affected at this stage. (C) Epiblast explants of post-DVE embryos (E5.75) stripped of ExE and VE fail to maintain expression of Oct4/Pou5f1, unless they are cultured in the presence of recombinant Nodal. Administration of SB-431542 blocked the effect of exogenous Nodal protein, confirming that it is specific, even though in this experiment we used unpurified protein produced in human 293T cells (Beck et al., 2002). (D) Earlier explants isolated from DVE- and pre-DVE-stage embryos without VE did not survived well under the conditions examined (not shown). However, in the presence of VE, they maintained expression of Oct4, Nodal, as well as Furin (top row). By contrast, Oct4 and Nodal were downregulated in explants cultured with inhibitors of endogenous Nodal (SB) or Furin (CMK) activities. Furin remained expressed in the VE, suggesting that these treatments are not toxic.

View larger version (36K): [in a new window]   Fig. 7. Role of early Nodal signaling in overcoming dominant inhibitory signals from the ExE. Nodal initially maintains pluripotent cells in the epiblast (dark red) and must overcome dominant inhibitory signals from the ExE before it can specify DVE. Between E4.5 and 5.0, Nodal signaling (blue) specifies the VE abutting the epiblast to become EmVE and provide an adhesive substratum for the epiblast. Paracrine signals from the ExE (gray) pattern the VE in the extra-embryonic region (ExVE) (Dziadek, 1978), and prevent the conversion of EmVE into DVE (Rodriguez et al., 2005). Therefore, to establish the competence for DVE formation, we propose that EmVE first must promote elongation of the egg cylinder to distance its apex from the ExE. In addition, after overcoming the non-permissive stage, sustained Nodal activity potentiated by the proprotein convertases Furin and Pace4 (Beck et al., 2002) is also essential as an instructive signal, which actively induces DVE-specific genes. In NodallacZ/lacZ null mutants, the entire VE assumes an extra-embryonic character (ExVE) and subsequently fails to form DVE. In addition, the epiblast prematurely downregulates determinants of pluripotency (light red). Whether this is a consequence or a cause of the absence of the EmVE remains to be further investigated.

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