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First published online 3 August 2006
doi: 10.1242/dev.02497

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Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Erika A. Bosman^1,*, Kirstie A. Lawson^2,, Joke Debruyn¹, Lisette Beek¹, Annick Francis¹, Luc Schoonjans³, Danny Huylebroeck¹ and An Zwijsen^1,

¹ Department of Developmental Biology (VIB7), Flanders Interuniversity Institute for Biotechnology (VIB) and Laboratory of Molecular Biology (Celgen), University of Leuven, B-3000 Leuven, Belgium.
² Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Utrecht, The Netherlands.
³ Thromb-X S.A., Leuven, Belgium.

View larger version (72K): [in a new window]   Fig. 1. Impaired allantois and PGC development in Smad5m1/m1 embryos. (A-C) Allantois dimensions measured in embryos collected from Smad5m1/+ crosses in F2 (C57BL/6J x CBA) background. Results at different developmental stages were expressed as means ± s.e. The numbers under the histogram represent the number of embryos in each group. A numerator indicates the embryos with an allantoic bud. Zero values are not included in the mean. (A) Allantois length (µm). (B) Allantois width (µm) was measured at the base of the allantois (right-left axis). (C) The ratio of the allantois length over the embryonic axis length. (D) Wild-type (5S) embryo with a well-developed head, hindgut entrance (open arrowhead) and heart, and an elongated allantois. The amnion is thin and smooth. (E) Representative stage-matched Smad5m1/m1 (KO) littermate with abnormal body curvature, underdeveloped anterior structures and foregut, vestigial heart, broad base of the allantois and an aggregate on the amnion (*). (F) Number of PGCs detected in embryos of the three Smad5 genotypes at late streak, neural plate and headfold stages. Wilcoxon's summed ranks test was used for statistical analysis (***, P<0.001; **, P<0.01; *, P<0.05), and medians are depicted by a bar. (G,H) Dorsal view of wild-type (28S) and Smad5m1/m1 (26S) mutant embryos dissected free from their yolk sac. The amnion is a thin, smooth membrane that envelops the embryo (G, arrowheads). A characteristic aggregate of cells is present on the mutant amnion (H, arrow). (I) Whole-mount staining for alkaline phosphatase (AP) activity in a mutant amnion (10-11S stage) with an aggregate of cells. A cluster of ectopic AP+ (brown; arrow) PGC-like cells projects from the ectoderm side of the amnion. (J) AP staining of a section through the aggregate of cells on the mutant Smad5m1/m1 amnion shown in H, at the level of the hindbrain. Ectopic PGC-like cells (red) are localized in close association with the amnion ectoderm. Scale bars: 500 µm in G,H; 200 µm in K; 100 µm in C,D,J. Al, allantois; Am, amnion; AmEc, amnion ectoderm; AmMe, amnion mesoderm; EHF, early headfold; He, heart; Hf, headfold; Hg, hindgut; LNP, late neural plate; LS, late streak; NP, neural plate; Nt, neural tissue.

View larger version (97K): [in a new window]   Fig. 2. Epiblast/stem cell and PGC-markers in Smad5m1/m1 embryos. Transverse sister sections through hindgut region (A,E,I,M) and head region (B,F,J,N) of an E8.5 wild-type embryo, and of an E8.5 Smad5m1/m1 littermate with multiple aggregates of cells on the amnion (C,D,G,H,K,L,O,P). The arrow points to the amnion. (A-D) Anti-Oct4 antibody staining demonstrates the presence of PGCs in the hindgut (A; open arrowheads). Oct4+ cells are absent in control (B) and non-thickened mutant amnion (C,D). Ectopic Oct4+ cells can be observed regionally in some aggregates in mutant amnion. Oct4+ cells have an ectoderm oriented localization (C,D). (E-H) PGCs in the hindgut are marked by their fragilis expression (E; open arrowheads). Ectopic fragilis+ cells can be observed within the aggregate of mutant amnion, but not in the non-thickened amnion (G,H). (I-L) Stella is expressed in PGCs in the hindgut (I; open arrowheads). Stella expression was never observed in wild-type (J) or mutant (K,L) amnion, also not in regions where Oct4+, SSEA-1+ and fragilis+ cells are residing (K,L). (M-P) Anti-SSEA-1 antibody staining demonstrates the presence of SSEA-1 in surface ectoderm, visceral endoderm and the neuroectoderm (N). At this stage of development PGCs in the hindgut are SSEA-1 negative (M). There is intense SSEA-1 staining in the mutant amnion ectoderm, and weaker staining can be observed in the aggregates. (Q) Schematic representation of the different regions that can be recognized within the mutant amnion. Scale bars: 100 µm.

View larger version (103K): [in a new window]   Fig. 3. Perturbed homeostasis in the mesoderm and ectoderm component of the amnion in Smad5m1/m1WTlacZ and Smad5m1/m1 embryos. (A,B) Dorsal view of a low-percentage E9.5 Smad5m1/m1WTlacZ chimeric embryo, unstained (A) and stained for ß-galactosidase activity (B). The head is patterned normally and the headfolds and neural tube are closed, but embryonic turning is mildly affected (a twisted tail). The amnion remains locally thickened (white arrow), and red blood cells can be observed within this aggregate of cells (A; open arrowhead). (C-E) Section through low- percentage control Smad5+/m1WTlacZ (chWT) (C) and Smad5m1/m1WTlacZ chimeras (chKO) (D-E). Smad5+/m1 and Smad5m1/m1 ES cell derivatives colonize both the mesoderm and ectoderm of the amnion extensively (E). Aggregates of cells on the amnion are of mixed wild type (blue) and Smad5m1/m1 (purple) origin in a Smad5m1/m1WTlacZ chimera (D). The amnion ectoderm is cuboidal in one part of the aggregate of cells (open arrow). Blood vessels develop always in the mesodermal side of the aggregate. In another Smad5m1/m1WTlacZ embryo the amnion ectoderm is completely of wild-type origin (E). (F) Twist is expressed in amnion mesoderm, including in the clump of cells in Smad5m1/m1 mutant amnion, but Twistneg areas can also be distinguished (dashed circle). (G) Thickened, AP-2+ amnion ectoderm is observed locally in a clump of cells (arrow) in a Smad5m1/m1 mutant amnion. Al, allantois; Am, amnion; AmEc, amnion ectoderm; AmMe, amnion mesoderm; Bv, blood vessel; Ex, extraembryonic region; Fg, foregut; He, heart; Hf, headfold; Nt, neural tissue; Op, optic anlage; So, somite; Ys, yolk sac. Scale bars: 100 µm in A, E-G; 50 µm in B-D.

View larger version (123K): [in a new window]   Fig. 4. Ectopic expression of Bmps in Smad5m1/m1 amnion. (A-F) Ectopic expression of Bmp2 and Bmp4 in mutant amnion at E8.5. Compared with the wild-type littermate (A,B), the expression of Bmp2 is highly elevated in the aggregate of cells (KO, arrow) and the flanking mutant amnion, but not in more remote squamous amnion (arrowhead) (C,D), or in surface ectoderm. Smad5 m1/m1 embryos express much higher levels of Bmp4 throughout the amnion (F), than do controls (E). Bmp4 expression levels appear weaker in mutant surface ectoderm (E,F). Surface ectoderm and amnion ectoderm are often poorly delineated in Smad5 m1/m1 embryos when the aggregate of cells on the amnion is localized anteriorly (dashed circle; C). (G-L) Enhanced expression of Bmp4, but not of Bmp2, in mutant amnion at E7.0. Bmp4 is expressed in wild-type amnion at EB and NP stages, respectively (G,I), but its expression is elevated in the amnion of mutant littermates (H,J). The Bmp2 expression is not yet significantly different in the amnion of wild-type (K) and mutant (L) EB littermates. Short arrows point towards the mesodermal component of the amnion, and are positioned in the exocoelom. Anterior is to the left and posterior to the right. AmMe, amnion mesoderm; He, heart; Nt, neural tube; Ps, primitive streak; Se, surface ectoderm; Ys, yolk sac. Scale bars: 100 µm in B-H,J,L; 50 µm in A,I,K.

View larger version (116K): [in a new window]   Fig. 5. Ectopic Bmp signalling in Smad5m1/m1 amnion. (A-C) Anti-P-ERK1/2 antibody staining can barely be detected in the amnion (arrowhead) at E8.5 (A). It is increased in non-thickened Smad5 mutant amnion but especially elevated in the aggregate of cells (B,C). B and C represent two different Smad5 mutants. (D-F) Anti-phospho-Smad1/5/8 antibody staining can only be detected sporadically in an isolated cell of the amnion (arrowhead) at E8.5 (A). P-Smad1/5/8 is upregulated in non-thickened mutant amnion and within the aggregates of Smad5 mutant amnion. Sections in D-F are sister sections of A-C. (G,H) The Bmp target gene periostin is expressed in the amnion (arrowhead) at E8.5. It is strongly upregulated in the non-thickened Smad5 mutant amnion, although excluded from the aggregate of cells (H). (I,J) The Bmp target gene Msx2 is barely detectable in wild-type amnion (I) but highly expressed in mutant amnion at E8.5 (J). Msx2 expression is primarily confined to the ectodermal component of the amnion (arrowhead) and the aggregate of cells (arrow). The expression of Msx2 in the embryo proper is not altered significantly. AmMe, amnion mesoderm; He, heart; Nt, neural tube; Se, surface ectoderm; Ys, yolk sac. Scale bars: 100 µm.

View larger version (61K): [in a new window]   Fig. 6. Local administration of rBMP4 induces thickening of the amnion in wild-type embryos. (A,B). Lateral view of wild-type embryos that were either control injected (A) or injected with rBMP4 (B) in the exocoelom at the EHF stage (E7.5), and cultured in vitro until the 5S stage. The amnion (arrowhead) developed a local thickening in the rBMP4-injected embryo (dashed circle). Aggregates were always much smaller than in Smad5 mutants, and difficult to see through the yolk sac. (C-F) Sections through control (C) and rBMP4-injected (D-H) embryos. The appearance and size of the amnion thickening (dashed circle) varied significantly between embryos. Thickening of the amnion was only scored when observed in more than five successive sections. The embryos in C-E were injected at the HF stage; D and E are sections from the same embryo, illustrating that amnion thickening can develop at different locations. The embryos in F-H were injected at the NP stage. The embryo in H has an unusually large yolk sac. Al, allantois; Fg, foregut; He, heart; Nt, neural tissue; Ys, yolk sac. Scale bars: 100 µm.

View larger version (26K): [in a new window]   Fig. 7. Model for the role of Smad5 signalling in amnion homeostasis. (A) The squamous, bilayered wild-type amnion at the early somite stage is composed of a mesoderm layer facing the exocoelom that expresses Twist, whereas an epithelial cell layer that expresses AP-2 lines the amniotic cavity. The chimera analysis demonstrates that Smad5 is essential in the mesoderm, where it functions in a non-cell-autonomous fashion, because mutant mesoderm cells can elicit thickening of wild-type amnion ectoderm. Reciprocally, however, we cannot conclude that Smad5 functions also non-cell-autonomously and/or cell autonomously in the ectoderm. (B) At the early somite stage the Smad5m1/m1 mutant amnion displays an altered cellular organization with a zone of primarily thickened mesoderm but also thickened ectoderm. The mutant amnion ectoderm is positive for SSEA-1. The anterior amnion ectoderm remains cuboidal (squares), and is in continuity with anterior surface ectoderm and anterior neural tissue. Robust ectopic expression of Bmp2 and Bmp4 in mutant amnion is associated with increased Smad1/8 and ERK1/2 signalling and expression of periostin, Tbx2, fragilis and Msx2. The differential but overlapping ectopic expression patterns of all these Bmp-target genes suggest that cells in the mutant amnion sense different levels of Bmp signalling. How these different levels of Bmp signalling are established in the mutant amnion remains to be explored. (C) The transformation of the amnion to a complex tissue with different Bmp-sensitive cell types, such as e.g. an Oct4+, AP+, SSEA-1+ and fragilis+ region reminiscent of PGC-competent epiblast, Flk1-expressing endothelial cells and -globin-expressing cells (see Discussion). The ectopic and appreciable numbers of AP+ cells arise suddenly from the 5S stage onwards, which argues strongly for their in situ change in fate. The AP+ cells are localized near the ectodermal component of the aggregate, whereas haematopoietic and endothelial cells are positioned at the exocoelomic side. Our data suggest that Smad5 is the predominant Bmp signal-mediator in the amnion, and controls Bmp expression levels and amnion homeostasis tightly. Upon Smad5 removal the control of Bmp expression is released, which results in an excess of Bmp, in take-over by non-Smad5 pathways and ultimately in gain of Bmp signalling defects.