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First published online 17 March 2004
doi: 10.1242/dev.01072

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Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo

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

View larger version (72K): [in a new window]   Fig. 1. Smad2 and Smad3 are independently regulated and co-expressed in the early embryo. (A-D) Western blot analysis of adult organ and cell lines. (A) Thymus extracts from homozygous Smad3null animals (–/–) lack detectable Smad3 protein. (B) Equivalent Smad2 protein levels are found in wild-type, Smad3+/– and Smad3–/– spleen, thymus and liver extracts. (C) CCE ES cells, STO fibroblasts and spleen express Smad2 protein, whereas KT15 Smad2Robm1 homozygous ES cell lines contain no Smad2. (D) Similar Smad3 levels are observed in thymus, CCE and KT15 Smad2-deficient ES cells, and STO fibroblasts. (E) Semi-quantitative RT-PCR analysis of Smad3, Smad2, Smad4 and hypoxanthine phosphoribosyltransferase (Hprt) expression in blastocysts (E3.5) and gastrulation stage embryos (E6.5 and 7.5). Smad2, Smad3 and Smad4 are co-expressed at all stages examined. (F) Quantitative analysis of Smad2 and Smad3 expression levels by ribonculease protection assay of CCE ES cell, embryo and adult thymus total RNA. Smad2 transcripts are approximately twofold more abundant than Smad3 transcripts in ES cells and E7.5 embryos. Smad3 levels equalize with Smad2 as development progresses, and by E10.5/11.5 the ratio of Smad3:Smad2 transcripts is nearly 1:1. (G-I) Smad3 whole-mount in situ hybridization. (G) Mouse embryos at mid- to late primitive streak stages show low levels of Smad3 expression throughout the embryo. The highest level of expression is seen in the extra-embryonic ectoderm of the posterior amniotic fold (paf) and its later derivative the chorion (ch). The visceral yolk sac endoderm (ve) is negative for Smad3. (H,I) Smad3 expression levels increase within the embryo proper by the early somite stage (E8.0-8.5), and are observed in the midline, node (n) and somites (s).

View larger version (19K): [in a new window]   Fig. 2. Regulation of the Foxh1-dependent (n2)7-luc reporter by Smad2 or Smad3. (A) The constitutively active Alk4 (caAlk4) Nodal receptor with or without mycSmad4 leads to modest upregulation of the (n2)7-luc reporter in Mv1Lu cells. Addition of either FLAG-hSmad3 or FLAG-hSmad2 potentiates reporter activity to levels consistent with the previously demonstrated TGFß-dependent regulation of (n2)7-luc (Saijoh et al., 2000). (B) Western blot analysis of protein extracts from transfected cells confirms appropriate protein production of mycSmad4, FLAG-hSmad3 and FLAG-hSmad2 proteins.

View larger version (106K): [in a new window]   Fig. 3. Abnormalities in Smad2+/–;Smad3–/– mutant embryos. Whole-mount views of E9.5 (A) wild-type (WT), (B) Smad2+/–;Smad3+/– and (D,G) Smad2+/–;Smad3–/– littermate embryos. (B) Smad2+/–;Smad3+/– embryo with wild-type (A) morphology. (C) Transverse section of B. A moderately affected Smad2+/–;Smad3–/– mutant embryo (D) completes turning, has reiterated somites (s) and an otic vesicle (ot), but lacks anterior-most neural structures, including forebrain and optic vesicle (op), and displays a mispatterned, enlarged heart (ht) within an expanded pericardium (pc). (E,F) Transverse sections of embryo in D reveal the presence of a gut tube along the length of the embryo, as well as a thickened anterior tissue mass probably resulting from fusion of the first branchial arches (ba). (G) A more severely affected mutant embryo fails to complete the turning sequence and to close the ventral body wall. Heart development is severely compromised, and anterior development is significantly diminished. (H) Transverse section of G shows a rudimentary gut tube, with chaotic development of neural and mesodermal structures along the body axis. da, dorsal aorta; nt, neural tube; fg, foregut; mg, midgut; hg, hindgut.

View larger version (104K): [in a new window]   Fig. 4. Loss of anterior primitive streak derivatives in Smad2+/–;Smad3–/– mutant embryos. Whole-mount in situ hybridization of (A,C,E,G,I,K,K',M,O,S, U,W,Y) wild-type (WT) and (B,D,F,H,J,L,L',N,P,T,V,X,Z) Smad2+/–;Smad3–/– mutant (mut) embryos at (A,B,Q) E7.25, (C-J,R) E7.5, (M-P) E7.75 and (K-L,S-Z) E8.5. (A-F) Foxa2 transcripts first identify the AVE (red line), then AME cells emerging from the anterior streak and later demarcate the node (n). (A,B) Foxa2 and (Q) Cer1 expression in the AVE confirms the normal establishment of the primary anteroposterior axis. However, mutant embryos show few (B) to no (D,F) Foxa2-expressing AME cells extending anteriorly. Similar results were obtained with Shh (G-J) that is also diagnostic for the AME and node. Note that Foxa2 expression is retained in the patent node (D,F). In both control and mutant embryos at the early somite stage (K-L), Shh expression persists in the node and identifies its derivative the notochord (nc). At the early headfold stage, (M,N) Nog and (O,P) Chrd mark the node, midline and posterior PCP (bracket), and are downregulated and truncated anteriorly in mutant embryos (N,P). Note the expanded anterior neuroectoderm in N,P, and lack of neural groove in P. (Q,R) Lower levels of the nascent definitive endoderm markers Cer1 and Hhex are observed during early gastrulation. At E8.5, Hhex marks involuting foregut endoderm and lateral angioblasts (ab) (S). Foregut Hhex expression is absent, but retained in angioblasts in mutant embryos (T). Similarly, Shh expression in the hindgut (hg) endoderm of mutants is largely lost (K',L'). (U-X) The loss of anterior structures is revealed by diminished Six3 and Otx2 expression. (Y,Z) Fgf8 is expressed in the anterior neural ridge (arrowhead) and mid/hindbrain boundary (asterisk) in the wild type. In mutant embryos, anteriormost Fgf8 transcripts are absent, whereas expression in the mid/hindbrain region is normal (Z). (K,L) Hindbrain formation is unperturbed as assessed by Krox20 expression in rhombomeres 3 and 5. (W-Z) Posterior T expression indicates ongoing gastrulation.

View larger version (88K): [in a new window]   Fig. 5. Conditional removal of Smad2 in the context of Smad3 deficiency leads to gastrulation and streak patterning defects. Whole-mount views or sections as indicated of (A) Sox2Cre;Smad2Robm1/CA (S2 ca/-), (B-D) Sox2Cre;Smad2Robm1/CA;Smad3null/+ (S2 ca/-;S3+/–), (E-H',K-L'',O,P) Sox2Cre; Smad2Robm1/CA;Smad3null/null (S2 ca/-;S3–/–) and (I-J',M-N) wild-type (WT) embryos at (I-L'') E7.5 and (A-H',M-P) E8.5. (A) Sox2Cre;Smad2Robm1/CA mutants display anterior truncations similar to Smad2+/–;Smad3–/– embryos and normal bilateral somites (s) (A'). (B) Combined reduction of Smad3 gene dose to one wild-type allele and loss of Smad2 in the epiblast leads to consistent anterior truncations and elimination of notochord (nc), node and definitive endoderm, which results in somite fusions across the midline (B'). (C,D) Failure to detect Shh transcripts by whole-mount in situ hybridization confirms absence of midline structures in Sox2Cre;Smad2Robm1/CA;Smad3null/+ mutants. (E-H') Combined loss of Smad2 and Smad3 in the epiblast significantly impacts mesoderm formation and patterning. Mutant embryos are mainly composed of neuroectoderm (ne). (I-L'') T expression in the presumptive posterior marks nascent mesoderm forming in the primitive streak (ps). However, production of embryonic mesoderm is greatly diminished, as evidenced by restricted T expression and absence of the paraxial mesoderm marker Meox1 (M-P). Small pockets of presumptive heart mesoderm are occasionally observed. By contrast, extra-embryonic mesoderm (xm) is formed, lining the visceral yolk sac and forming a compact structure resembling an allantois (F-H').

View larger version (69K): [in a new window]   Fig. 6. Synergistic requirement for Smad2 and Smad3 in the patterning and organization of the early post-implantation embryo. Whole-mount views or transverse sections of embryos within the deciduum of E7.5 (A,E,I,M,Q) wild-type (WT; anterior towards the left), (B,F,J,N,R) Smad2–/–, (C,G,K,O,S) Smad2–/–;Smad3+/– and (D,H,L,P,T) Smad2–/–;Smad3–/– embryos hybridized with (I-L) Oct4, (M-P) Bmp4 or (Q-T) Sox2 riboprobes. (A,E) Wild-type embryos have distinct embryonic and extra-embryonic regions (separated by broken line), with embryonic (m) (E) and Bmp4-expressing extra-embryonic (xm) mesoderm formation (E,M), and Oct4-expressing epiblast (ep) (I). (M) Bmp4 and (Q) Sox2 transcripts identify the extra-embryonic ectoderm (xc) (E) and its derivative the chorion (ch). Smad2–/– embryos express little Oct4 (J) and form exclusively Bmp4-positive extra-embryonic mesoderm (F,N), which displaces the extra-embryonic ectoderm normally to form the chorion (ch) (F,J,N,R). Smad2–/–;Smad3+/– mutant embryos undergo cavitation, but show no clear embryonic/extra-embryonic boundary, and are enveloped by a prominently folded and thickened layer of visceral endoderm (C,G,K,O,S) that surrounds disorganized extra-embryonic ectoderm (G,O,S), with little to no formation of presumptive extra-embryonic mesoderm (xm*) (G). (D,L,P,T) Smad2–/–;Smad3–/– embryos develop as a small disorganized tissue mass within the parietal yolk sac endoderm (pe). Sections reveal rudimentary epithelial development with no mesoderm formation (H). Double mutants lack Oct4 (L), but consistently show expression of Bmp4 (P) and Sox2 (T), suggesting loss of embryonic ectoderm and unique formation of extra-embryonic ectoderm (xc*) (H). ae, anterior ectoderm.

View larger version (23K): [in a new window]   Fig. 7. Dose-dependent Smad2 and Smad3 signals pattern the primitive streak. Columns are grouped according to genotypes derived from intercrossing Smad2;Smad3 double heterozygous mice (left) and specific elimination of Smad2 in the epiblast in the context of progressive loss of Smad3 (right). The identical phenotypes of Smad2+/–;Smad3–/– and Sox2Cre;Smad2CA/Robm1 mutant embryos (columns with black perimeter) suggest that Smad2 is the predominant intracellular effector of Nodal signaling during patterning of the primitive streak. This observation establishes the hypothetical dose relationship whereby one Smad3 wild-type allele (red) is 50% less active than a Smad2 wild-type allele (blue). Accordingly, the combined Smad2/3 dosage is then calculated for each genotype analyzed in our studies. Three phenotypic thresholds emerge below which patterning of the primitive streak is sequentially compromised. The first eliminates the anterior AME followed by the node, remaining axial mesoderm (notochord) and definitive endoderm, and finally the lateral and paraxial mesoderm.

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