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First published online 23 June 2004
doi: 10.1242/dev.01248

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Differential requirements for Smad4 in TGFß-dependent patterning of the early mouse embryo

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA

View larger version (42K): [in a new window]   Fig. 1. Generation of Smad4CA and Smad4N alleles. (A) Strategy used to flank the first coding exon of Smad4 with loxP sites (red triangles). Targeted clones were identified using a 3' external probe (red line) and an internal probe (blue line). A, ApaI; B, BstZ17I; R, EcoRV; St, StuI; X, XbaI; Xh, XhoI. (B) Southern blot analysis of drug-resistant ES clone DNA digested with BstZ17I and screened using the 3' external probe yields 9 kb wild-type (WT) and 11.2 kb targeted (TA) alleles. (C) Smad4TA/+ ES cell clones were transiently transfected with Cre recombinase, and DNA from resultant clones was digested with XbaI and screened by Southern blot using the internal probe. Rearrangement of the Smad4TA allele generates both Smad4 conditional (Smad4CA) and null (Smad4N) alleles. (D) PCR analysis of DNA samples from wild-type, Smad4N/+, Smad4CA/+ and Smad4CA/CA homozygous mice. PCR primer locations are indicated in A.

View larger version (140K): [in a new window]   Fig. 2. Smad4N/N embryos fail to gastrulate. (A,B) Wild-type (WT) and Smad4N/N embryos (N/N) at E6.5. The wild-type embryo displays normal egg cylinder morphology with prominent epiblast (epi) epithelium. By contrast, the mutant embryo (B) shows retarded growth, with circumferential thickening of the visceral endoderm (ve). (C,D) Sagittal sections of E6.5 wild-type and Smad4N/N embryos within the deciduum. The wild-type embryo has distinct embryonic and extra-embryonic regions and has begun gastrulation, as evidenced by the formation of posterior mesoderm (m). By contrast, Smad4N/N embryos show no mesoderm formation, the visceral endoderm accumulates distally and the epiblast is significantly reduced, with intermingling of extra-embryonic ectoderm (eee). epc, ectoplacental cone. (E-J) Whole-mount in situ hybridization at E6.5 of (E,G,I) wild-type and (F,H,J) Smad4N/N mutant embryos. (E,F) Oct4 is robustly expressed in the epiblast of both mutant and wild-type embryos. (G,I) Bmp4 and eomesodermin (Eomes) expression identifies extra-embryonic ectoderm atop the epiblast. In Smad4N/N embryos, this expression domain is displaced distally into the embryonic region (H,J). (I) Eomes transcripts also mark the nascent primitive streak in wild-type embryos.

View larger version (79K): [in a new window]   Fig. 3. Loss of Smad4 from the epiblast disrupts midline formation but does not perturb mesoderm development. (A-E) Developmental time course of (A-E) wild-type (WT) and (A'-E') Sox2Cre;Smad4CA/N (mut) embryos. (A,A') At early gastrulation (E6.5), wild-type and mutant embryos are indistinguishable. (B,B') By E7.0, mutant embryos have a broadened embryonic region that becomes progressively distorted as development continues. (C,C') At the early headfold stage (E7.75), anterior elaboration of neural folds and invagination of foregut do not occur in the mutant. (D,D',E,E') By E8.5, the mutant embryo has developed within a normal visceral yolk sac (vys). Spherical head fold-like structures appear (hf), a protruding heart (ht)-like structure is observed anteriorly, and somites (so) form, which are fused across the midline. fb, forebrain; nt, neural tube. (F-K) Transverse sections of (F,H,J) wild-type and (G,I,K) E7.5 mutant embryos. (H) Anteriorly, the wild-type embryo shows the normal topological arrangement of the three definitive germ layers: inner anterior ectoderm or epiblast (ep); mesodermal (m) wings; and outer layer of definitive endoderm (de). At the midline, the epiblast directly contacts the axial mesendoderm (ame; red arrow) without an intervening layer of mesoderm. Arrowheads indicate parietal endoderm. (I) In mutant embryos, however, interposing mesoderm (m; red arrow) is observed at the midline, and (J,K) the posterior primitive streak (ps) is broadened compared with the control embryos. (L) Sagittal section of an E8.5 Sox2Cre/+;Smad4CA/N embryo reveals a rudimentary heart (ht), neural tissue approximating a headfold (hf) as well as visceral yolk sac endoderm with associated blood islands (vys + bl). am, amnion. (M-O) Coronal sections of a similarly staged E8.5 mutant embryo at (M) anterior, (N) mid and (O) posterior levels. The neural plate (np) fails to form the neural tube. Somitic (so) and lateral plate mesoderm (lpm) is seen. Posteriorly, stalk-like chorionic tissue (ch) extends from the amnion and is associated with allantois (al). (P) PCR performed on microdissected tissue fragments of embryos of a Smad4CA/CA x Sox2Cre;Smad4N/+ intercross. In embryos expressing the Sox2Cre transgene (lanes 2-4, 7, 8), conversion of the conditional allele to the null allele is seen. The yolk sac, which is derived from the epiblast and extra-embryonic endoderm, shows only partial conversion. The epiblast-derived allantois and headfold tissue show complete conditional-to-null conversion.

View larger version (106K): [in a new window]   Fig. 4. The anterior primitive streak and its derivatives are absent in Smad4 mutant embryos. Whole-mount in situ hybridization of (A,C,E,G,I,K,M,O,Q) control and (B,D,F,H,J,L,N,P,R) Sox2Cre;Smad4CA/N mutants. (A,B) Foxa2, (C,D) Gsc and (E,F) Lim1 are expressed in the anterior primitive streak (APS) in wild-type embryos but are absent in mutants. AVE expression is unaffected. (G-L) T expression between E6.5 and E8.5. (G,H) At E6.5 T expression confirms normal formation of the posterior primitive streak and the initiation of gastrulation. (I,J) By E7.5, T expression marks the fully extended primitive streak, and emerging axial mesoderm (arrow). In mutant embryos, no T-expressing axial mesoderm is observed. (K,L) Consistent with this result, mutant embryos lack midline expression of T in the notochord at E8.5. Posterior T transcripts indicate ongoing gastrulation. (M-P) At E7.5, Nodal and Shh transcripts identify the node, but are absent in mutant embryos. (Q,R) Loss of notochord is further illustrated by the lack of midline Shh expression, although some ectopic staining is observed.

View larger version (109K): [in a new window]   Fig. 5. Definitive endoderm is not formed in Smad4 mutant embryos. (A,B) Hex transcripts mark the anterior visceral endoderm (AVE) and emerging definitive endoderm (DE) at the early primitive streak stage. Smad4 mutant embryos express Hex in the AVE, but not in the ADE. (C-F) A Hex-lacZ transgene identifies a portion of medial DE cells (C, lateral view; D, frontal view) at E7.5. By contrast, (E, lateral view; F, frontal view) no transgene activity is detected in mutant embryos. (G,H) Cerl expression within the DE shows a drastic reduction in distribution and intensity in mutant embryos, while expression in the AVE is retained. (I,J) At E7.5 Foxa2 transcripts mark the axial mesendoderm that extends from the anterior streak to the embryonic/extra-embryonic junction. Smad4 mutant embryos show residual Foxa2 expression with the AVE but lack axial expression. (K,L) Mixl1, a marker of the primitive streak and a putative downstream target of Smad4, is correctly expressed in mutants at the late primitive streak stage. (M-P) lacZ expression in (M,N) control and (O,P) Sox2Cre/+;Smad4CA/N;ROSA26R/+ E7.5 embryos viewed in coronal section. In control embryos, the definitive endoderm layer (arrows) is blue, indicating an epiblast-derived origin. By contrast, this layer is white in mutant embryos, indicating origin from non epiblast-derived visceral endoderm and failure to form DE. Note the absence of midline (m) in mutant embryos. Arrowheads indicate parietal endoderm.

View larger version (108K): [in a new window]   Fig. 6. Anterior neural patterning of Smad4 mutants. Whole-mount in situ hybridization of (A,C,E,G,I) control and (B,D,F,H,J) mutant embryos. (A-D) Otx2 expression marks the anterior epiblast of both wild-type and mutant embryos at E7.5, but by E8.5 normal expression within the fore- and midbrain is confined to a small patch of rostral neuroepithelium in Smad4 mutants. (E,F) Similarly, Six3 expression within the anterior forebrain is reduced in mutant embryos. (G,H) Fgf8 transcripts identify the anterior neural ridge (ANR; red arrows) and midbrain/hindbrain junction (black arrows) in wild-type embryos. ANR expression is lost in Smad4 mutants, while expression probably corresponding to the midbrain/hindbrain junction is observed (arrows). (I,J) Evidence of partial hindbrain formation is demonstrated by Krox20 expression. (I) Control, lateral view; (J) mutant, ventral view.

View larger version (118K): [in a new window]   Fig. 7. Several Bmp-dependent developmental processes occur in the absence of embryonic Smad4. (A-G) Heart formation in Smad4 mutant embryos. Specification of the heart field in control (A) and mutant (B) E7.5 embryos as demonstrated by the cardiac marker Nkx2.5 (A,B, lateral view; A',B', anterior view). (C) Higher magnification view of the anterior region of a mutant embryo at E8.5. (D) Coronal section shows heart tube formation with cardiomyocyte differentiation, which is further confirmed by expression of cardiac alpha actin at E8.5 (E,F; higher magnification of mutant shown in G). (H,I) The chorion (ch) forms a stalk-like structure often associated with epiblast-derived allantoic mesoderm (al), as shown by lacZ expression in Sox2Cre/+;Smad4CA/N;ROSA26R/+ embryos. (J-M) Alkaline phosphatase (AP) staining for identification of primordial germ cells (PGCs) viewed in flattened tissue preparations. (J) At E7.5, PGCs cluster at the base of the allantois in wild-type embryos. (K) In Smad4 mutants, PGC formation is not observed. (L) In wild type at E8.5, PGCs begin their anteriorwards migration and intercalate into the hindgut endoderm. (M) Despite formation of allantois, most Smad4 mutants lack recognizable PGCs at this stage.