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First published online 28 November 2007
doi: 10.1242/dev.009266

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Wnt3a/β-catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation

¹ Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, NIH. Frederick, MD 21702, USA.
² Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, 606-8501, Japan.

View larger version (95K): [in this window] [in a new window]   Fig. 1. Conditional loss and gain of β-catenin function causes mesoderm and segmentation phenotypes. (A-C) Wnt/β-catenin (BATlacZ) reporter activity in E9 wild-type (A), T-Cre;Ctnnb1flLOF/ (B) and T-Cre;Ctnnb1flGOF/+ (C) embryos. β-galactosidase (β-gal) activity was reduced posteriorly in the truncated T-Cre;Ctnnb1flLOF/ mutants, and highly upregulated in the grossly enlarged PSM of the T-Cre;Ctnnb1flGOF/+ embryo. In addition to the somite defects, kinked neural tubes, and an enlarged alantois were observed in the GOF mutants, whereas enlarged pericardia, and heart-looping defects were found in both LOF and GOF mutants. Bars, segment borders; curved line, the extent of the PSM. (D-Q) BATlacZ expression in 5 ss wild-type (D-F), T-Cre;Ctnnb1flLOF/ (I-K), and T-Cre;Ctnnb1flGOF/+ (N,O) embryos. (E,J) Cross-sections through the PS and PSM (level indicated by dotted lines in F and K) illustrate that the remaining β-gal activity in the T-Cre;Ctnnb1flLOF/ PS (I) was found only in the ectoderm and not in the mesoderm (arrow in J). (G,H,L,M,P,Q) Hematoxylin and Eosin stained sections of E8.5 wild-type (G,H), T-Cre;Ctnnb1flLOF/ (L,M), and T-Cre;Ctnnb1flGOF/+ (P,Q) embryos. High-power magnifications illustrated in H, M and Q are taken from boxed regions in G, L and P. The asterisk in P indicates the kinked neural tube. All embryo images are lateral views, with the exception of F, K, and O, which offer a ventral-posterior perspective. S0, forming somite; SI, first somite; ps, primitive streak; psm, presomitic mesoderm; nt, neural tube. Scale bars: 100 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Examination of mesoderm markers and oscillating clock genes in conditional β-catenin mutants. (A-I) Expression analysis of mesoderm and PS markers. Mox1 (orange), a somite and anterior PSM marker (A), is expressed in the E8.5 T-Cre;Ctnnb1flLOF/ (B) and T-Cre;Ctnnb1flGOF/+ (C) paraxial mesoderm despite the relative lack of segments. T (purple) was expressed in the node and notochord of the T-Cre;Ctnnb1flLOF/ mutant but was absent from the PS and PSM (B). T expression, along with the first segment border B0 (black bar) (Pourquie and Tam, 2001), was anteriorized in the T-Cre;Ctnnb1flGOF/+ streak and PSM (C). Expression of the PSM marker Tbx6 (D), and the PS marker Fgf8 (G), were similarly dependent upon β-catenin, being absent from the T-Cre;Ctnnb1flLOF/ PSM (E,H) but upregulated and anteriorized in the T-Cre;Ctnnb1flGOF/+ mutants (F,I). (J-S) Expression analysis of oscillating genes in the Fgf and Notch pathways. Dusp6/Mkp3, an oscillating component of the Fgf signaling pathway, is not expressed in the PSM in the absence of β-catenin (K), but is expressed in an anteriorized fashion when β-catenin is stabilized (L). Striped domains of cycling Lfng and Hes7 expression in the PSM (arrows in M,Q) were not apparent in T-Cre;Ctnnb1flLOF/ embryos (N,R). By contrast, ectopic Lfng and Hes7 stripes were observed in the elongated T-Cre;Ctnnb1flGOF/+ PSM (O,S). Lfng mRNA stripes were also easily detected in the T-Cre;Ctnnb1flGOF/ mutant PSM (P). All views are lateral, with the exception of the ventral view in (P) which afforded a clear view of the Lfng stripes. Scale bars: 100 µm.

View larger version (141K): [in this window] [in a new window]   Fig. 3. Wnt3a and β-catenin regulate segment polarity and boundary formation by positioning the expression of segment determination genes. (A-F) Uncx4.1 expression in wild-type (A,B), T-Cre;Ctnnb1flLOF/ (C,D), and T-Cre;Ctnnb1flGOF/+ (E,F) 4-6 ss embryos. Note that B and D are ventral-posterior views, whereas F is an anteroventral view. The curved line depicts the extent of the expression domain along the AP axis. (G-J) Two-color WISH depicting Uncx4.1 (orange) and Tbx18 (purple) expression in wild-type (G), Wnt3a-/- (H), T-Cre;Ctnnb1flLOF/ (I), and T-Cre;Ctnnb1flGOF/+ (J) 5-7 ss embryos. (K-R) Two-color WISH depicting Uncx4.1 (orange) and Mesp2 (purple, arrows) expression in wild-type (K,O), Wnt3a-/- (L,P), T-Cre;Ctnnb1flGOF/+ (M,Q), and Wnt3a-/-; T-Cre;Ctnnb1flGOF/+ (N,R) 10 ss embryos. O-R are high-power magnifications of dorsal (O,P) and ventral (Q,R) views. (S-V) Mesp2 expression in S-I (S) is posteriorized, relative to the node (line), in Wnt3a-/- embryos (T), and nearly absent from T-Cre;Ctnnb1flLOF/ 2-5 ss embryos (U). Mesp2 is anteriorized and expressed in multiple stripes (arrows) in T-Cre;Ctnnb1flGOF/+ embryos (V). 3a, Wnt3a-/-; LOF, loss of function; GOF, gain of function. Scale bars: 100 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 4. Transcriptional profiling of 0-2 ss Wnt3a-/- embryos identified the putative segment boundary determination gene Ripply2. (A) Hierarchical clustering of select differentially expressed genes revealed that several previously identified direct Wnt/β-catenin target genes such as Axin2, Tnfrsf9(Troy) and Nkd1 were downregulated (blue) in the Wnt3a-/- mutants, as expected. The Riken EST C030002E08 (Ripply2) was identified in a class of genes that were upregulated (red) in embryos lacking Wnt3a. (B-E) Two-color WISH illustrating striped Ripply2 (purple) expression in the anterior PSM (arrows), overlapping with the Notch pathway components (orange) Dll1 (B) and Hes7 (C). A representative half-embryo culture experiment illustrates that at time 0, Ripply2 (purple) and Mesp2 (orange) are expressed as mutually exclusive domains in adjacent presumptive somites (S-I and S-II, respectively) in one half of the sagitally bisected embryo (D). The solid line represents segment border B0, which is out of the focal plane. After culturing the complementary half-embryo for 1 hour (E), Ripply2 was strongly co-expressed with Mesp2 in S-I, while disappearing in S0. The forming segment border B0 is represented by a dashed line. (F-I) Ripply2 expression in S-I (F) is posteriorized relative to the node (line) in Wnt3a-/- embryos (G), and nearly absent from T-Cre;Ctnnb1flLOF/ embryos (H). Ripply2 is not expressed in T-Cre;Ctnnb1flGOF/+ embryos (I). Scale bars: 100 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Ripply2 expression is regulated by the Notch and Wnt pathways. (A) Comparative Vista plots of the mouse and human Ripply2 loci identify highly conserved putative regulatory regions (red) and coding exons (blue). Sequence identity (%) is indicated on the right. lacZ reporter constructs driven by these Ripply2 regulatory elements are depicted below the Vista plot. Representative transgenic founder embryos expressing the `enhancer/promoter' reporter (1), the `enhancer' reporter (2), or the `promoter' reporter (3) are shown on the right. The number of β-gal-positive transgenic embryos/total number of transgenic founders is shown in brackets. The gradually diminishing reporter expression in anterior somites is probably due to the perdurance of the stable β-gal protein. (B) Luciferase reporter constructs are depicted on the left. The enhancer and promoter elements are the same as in A. Graph displaying activity of Ripply2 luciferase constructs in HEK293 cells is shown on the right. Note that stabilized β-catenin expression had no effect on Mesp2 or Tbx6 protein levels as assessed by western blot (not shown). (C) Highly conserved region (33/34 bp identical) in the mouse (m) and human (h) Ripply2 promoters contains an Ebox and near-consensus Tbx6 binding site (BS). (D) An oligo containing the putative Tbx6 BS bound specifically to FLAG-Tbx6 in EMSA assays. Binding of Flag-Tbx6 protein to the wild-type oligo is specifically competed by adding 125x excess unlabeled oligo to the binding reaction.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Wnt3a-dependent transcriptional networks coordinate mesoderm formation with segment boundary determination. Schematic of selected target genes and pathways activated in the PSM by Wnt3a, see text for details. The red color represents the expression domain of the Wnt3a and β-catenin target genes (Dll1, T, Tbx6) in the PSM. T and Tbx6 are grouped together for the sake of simplicity. Yellow indicates a representative oscillating negative-feedback loop in the Notch (N) pathway in the segmentation clock. Expression of the segment boundary determination genes Ripply2 and Mesp2 (black hatching) in S-I, overlap with the anterior domain of Tbx6 expression. The proposed Wnt3a/β-catenin target repressor (?) is expressed in the posterior PSM where it ensures that boundary determination gene expression is repressed. A, anterior; P, posterior.