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Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation

Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

View larger version (31K): [in a new window]   Fig. 1. Generation of Smad1-deficient mice. (A,B) Targeting strategies. Thick lines represent Southern probes and arrows the direction of transcription. (A) The Smad1Robm1 (m) vector replaces most of the first coding exon (yellow box) and 30 bp of 3' flanking sequence with a PGK-neo cassette. (B) The Smad1RobPC vector introduces an EcoRI (R) and loxP site (green triangle) 2 kb 5' of the first coding exon and a loxP-flanked (blue and red triangles) hygro cassette 1.7 kb 3' of the exon. Germline Cre-mediated excision of the Smad1RobPC allele was used to produce the Smad1Robcn (cn) allele, which retains part of the 5' and 3' loxP site (red/green triangle). (C) Southern blot analysis of EcoRI-digested DNA from m/+ intercross embryos using the 5' external probe. (D) Southern blot analysis of BamHI-digested DNA from cn/+ intercross embryos using the 5' internal probe. (E) Northern blot analysis of RNA from wild-type (+/+), Smad1 heterozygous (m/+, cn/+) or Smad1-deficient (m/m, cn/cn) 9.5 dpc embryos. A Smad1 probe detects a 2.8 kb wild-type and a 2.2 kb mutant band. (F) Western blot analysis of individual 9.5 dpc embryos and as a control, COS cells mock transfected or transfected with an human Smad1 expression construct and probed with an antibody that recognizes the MH2 domain (amino acids 147-258) of Smad1, Smad5 and Smad8. This antibody is predicted to recognize potentially truncated Smad1 proteins produced from either Smad1 allele. A single band (asterisk) of approximately 55 kDa corresponds to full-length Smad1. No intact or truncated products are detected in homozygous embryos. H, HindIII; M, MluI; S, StuI; B, BamHI; P, PstI; R, EcoRI.

View larger version (122K): [in a new window]   Fig. 2. Gastrulation and extra-embryonic defects in Smad1-deficient embryos. (A) At 7.0 dpc, Smad1 mutant (-/-) embryos develop characteristic folds (arrowhead in all panels) in the extra-embryonic region proximal to the primitive streak. (B) A transverse section through the extra-embryonic region of the embryo in A reveals folding of the VE, while more distally (C), all three tissue layers are abnormally twisted. (D) Smad1 mutant embryos at 7.5 dpc demonstrate characteristic folds. (E) A frontal section of the embryo in D showing extensive twisting of the epiblast. (F) Posterior view of early headfold mutant embryo (8.0 dpc) showing lack of a recognizable allantoic bud. (G) In a transverse section through the embryo in F, a small allantoic rudiment is evident. (H) By contrast, a transverse section through an age matched wild-type (+/+) littermate reveals extensive growth of the allantois. (I) By 8.5 dpc, mutant embryos show a normal AP axis, but extra-embryonic tissues are abnormal. (J) In sagittal section, an 8.5 dpc mutant embryo has an erratically folded chorion and lacks an extended allantois. (K) A magnified view of a sagittal section through the posterior folds of the embryo in I demonstrates that the extra-embryonic mesoderm has migrated properly to line the entire exocoelomic surface of the chorionic ectoderm and visceral yolk sac. (L) By contrast, in wild-type littermates an elaborated allantois is evident (arrow), that in sagittal section (M) is fused with the chorion to produce a placenta. (A-D,I-L) anterior is towards the left. al, allantois; am, amnion; ch, chorion; ec, ectoderm; ep, epiblast; epc, ectoplacental cone; me, mesoderm; pl, placenta; ve, visceral endoderm.

View larger version (117K): [in a new window]   Fig. 3. Smad1-/- allantois morphogenesis is abnormal. Three allantois phenotypes are observed in Smad1-deficient embryos at 9.5 dpc. The broken line in A,B represents the plane of section in (A',B'). (A,A') Some embryos develop a balloon-like structure containing pooled blood. (B,B') In other mutant embryos, the allantoic rudiment is compact and forms a dense mesenchyme with blood apparent throughout the structure. Frontal sections of wild-type (C) and mutant (D) embryos. (C) The wild-type allantois has prominent blood vessels and has fused uniformly across the entire surface of the chorionic plate. (D) In a proportion of Smad1-deficient embryos, the allantois fuses peripherally and non-uniformly. ab, allantois blood vessel; al, allantois; am, amnion; bl, blood; cp, chorionic plate; da, dorsal aorta; hg, hindgut diverticulum; va, vitelline artery.

View larger version (107K): [in a new window]   Fig. 4. Disturbed mesoderm formation and migration in Smad1 mutants. Whole-mount in-situ hybridization of wild-type (+/+) and mutant (-/-) embryos. (A) T is normally expressed in the primitive streak and in nascent mesoderm of 7.0 dpc wild-type embryos. (B) In Smad1-/- littermates, T expression is patchy and less intense. (C) A posterior view of a Smad1 mutant embryo showing discontinuous T expression. (D) Fgf8, normally expressed in the nascent mesoderm, streak and at the base of the allantois at 7.0 dpc (left), is similarly expressed in mutant embryos (right), but is distorted, owing to the twisted epiblast. (E) Transverse section of the mutant embryo in D, showing Fgf8-expressing cells within the posterior folds (arrowhead). (F) Fgf3, expressed in the extra-embryonic mesoderm and streak in wild-type embryos is also present throughout the extra-embryonic folds of mutant embryos. (G) A transverse section of the mutant in F. Anterior is towards the left in all panels except C. ch, chorion; ep, epiblast; ve, visceral endoderm.

View larger version (85K): [in a new window]   Fig. 5. Extra-embryonic defects in Smad1-/- embryos. (A) Afp expression in wild-type (left) and mutant (right) 7.5 dpc embryos. Compared with the wild-type littermate, Afp is excluded from the anterior extra-embryonic region of the mutant embryo. (A') A transverse section through the proximal portion of the mutant embryo demonstrates that Afp expression is localized to the ruffled VE (arrowhead). (B) Eomesodermin (eom) expression demarcates the extra-embryonic ectoderm and anterior streak. In Smad1-/- embryos, eom-positive cells in the extra-embryonic region are greatly expanded. (C) BMP4 expression delineates the extra-embryonic ectoderm and mesoderm. In the mutant embryo, BMP4 expression is expanded throughout the extra-embryonic part of the embryo. (D) Sox2 is expressed throughout the embryonic and extra-embryonic ectoderm of 8.0 dpc embryos. Note the canal of tissue that normally extends from the anterior portion of the chorion towards the head of the embryo (arrow). (E) In 8.0 dpc Smad1-/- embryos, Sox2-positive tissue emanates from the posterior portion of the embryo towards the chorion (*). (E') A transverse section through the embryo in (E) demonstrates that the posterior Sox2 projection (*) is a ring of cells. (E'') A section near the EPC shows the erratic folding of the chorion. am, amnion; ec, ectoderm; me, mesoderm; ve, visceral endoderm; ys, yolk sac.

View larger version (169K): [in a new window]   Fig. 6. Expression patterns of Smad1, Smad5 and Smad8. Brightfield and darkfield images of sagittally sectioned embryos at 6.5 (A,D,G), 7.25 (B,E,H) and 8.5 dpc (C,F,I) hybridized using Smad1 (A-C), Smad5 (D-F) or Smad8 (G-I) specific probes. (A,A') At the onset of gastrulation, Smad1 is highly expressed in the VE and early primitive streak. (B,B') At late streak stage, Smad1 is observed in the VE, extra-embryonic and embryonic mesoderm derivatives. as well as the primitive streak. (C,C') By 8.5 dpc, Smad1 is ubiquitously expressed. (D,D') At 6.5 dpc Smad5 is uniformly expressed at low levels throughout the conceptus. (E,E') At late streak stages, Smad5 is expressed throughout the embryonic region and the allantois. (F,F') Smad5 transcripts are found throughout 8.5 dpc embryos. (G,G') At 6.5 dpc a Smad8 hybridization signal cannot be discerned from background. (H,H') At late primitive streak stages, Smad8 is expressed in the VE, posterior primitive streak and extra-embryonic mesoderm derivatives. (I,I') By 8.5 dpc Smad8 is widely expressed. al, allantois; am, amnion; ch, chorion; ec, ectoderm; ep, epiblast; ps, primitive streak; ve, visceral endoderm.

View larger version (126K): [in a new window]   Fig. 7. Colonization of Smad1 mutant embryos by wild-type ES cells fails to rescue gastrulation defects. Chimeras were generated by injecting lacZ-positive wild-type ES cells into host blastocysts collected from Smad1+/- intercrosses. (A,B) Two examples of Smad1-/- embryos significantly colonized by wild-type ES cells. (A,A') The posterior visceral endoderm defect is apparent in whole-mount and transverse section. (B,B') A distal section from a second strongly chimeric embryo illustrates that the twisting of the epiblast is not rescued by the presence of wild-type cells. The arrowhead points to the VE ruffles. Anterior is towards the left in all panels. ep, epiblast; me, mesoderm; ve, visceral endoderm.

View larger version (136K): [in a new window]   Fig. 8. Reduced numbers of PGCs in Smad1, but not Smad2, mutant embryos. (A-C) Dorsal views of alkaline phosphatase (AP)-stained, wild-type and Smad1 mutant hindgut pieces at the 17 somite stage. Arrows indicate PGCs. (A) Wild-type embryo with 87 PGCs. Smad1 homozygous mutants with (B) no PGCs or (C) 14 PGCs. (D) Abundant AP-positive PGCs in an advanced Smad2-/- embryo are mostly present in foci. (E) High-power magnification of a PGC cluster showing that the darkly staining foci contain typical PGCs with a characteristic cytoplasmic spot and darkly staining cell membrane.