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First published online May 30, 2007
doi: 10.1242/10.1242/dev.001206

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Wnt signaling is a key mediator of Cdx1 expression in vivo

Nicolas Pilon^1,*, Karen Oh¹, Jean-René Sylvestre¹, Joanne G. A. Savory² and David Lohnes^2,

¹ Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
² Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H 8M5, Canada.

View larger version (55K): [in this window] [in a new window]   Fig. 1. Targeting of the LRE and RARE in the mouse Cdx1 promoter. (A) Schematic of the wild-type (WT) Cdx1 locus, targeting vector, targeted allele and Cre-recombined allele. Probe A (5' external probe) was used to screen for targeted ES cells, probe B (5' internal probe) was used to confirm the predicted targeting event and probe C (3' internal probe) was used to confirm excision of the floxed neomycin selection cassette. E, EcoRI; H, HindIII; K, KpnI; S, SacI; E1, Exon 1; *, mutated RARE; ++, mutated LRE. (B) Southern blot analysis of DNA from wild-type (left), heterozygous targeted (middle) and heterozygous Cre-recombined (right) offspring using probe B and the indicated restriction endonucleases (see A). Concomitant integration of mutated LRE and LRE+RARE within the targeted allele was determined by the introduction of novel restriction sites: for the RARE mutation, a SacI restriction site was observed by Southern blot analysis (last lane of middle and right panels); for the LRE mutation, novel SfuI restriction sites were assessed by restriction of a PCR product spanning the LRE (lower left panel; see also Materials and methods).

View larger version (53K): [in this window] [in a new window]   Fig. 2. Skeletal analysis of vertebral patterning defects. Cervical region of whole-mount mouse skeletal preparations from (A) wild-type, (B) LRE+/-, (C) LRE+RARE+/-, (D) Cdx1+/-, (E) RARE-/-, (F,I) LRE-/-, (G,J) LRE+RARE-/- and (H) Cdx1-/- offspring. Note the C2 to C1 transformations evidenced by a broader C2 neural arch and an ectopic anterior arch of the atlas (*AAA in C-E). Anterior transformation of C1 was assessed by loss of the anterior arch of the atlas and anterior malposition (C1 arrow in F,G,H) and fusion with the basioccipital bone (small bracket in F,G,I). Anterior transformation of C2, C3 and C7 are denoted "C1", "C2" and "C6", respectively, in F-H as determined by morphological criteria as described in Material and methods. A partial rib associated with presumptive T1, which did not reach the sternum, is indicated by the short arrow (F,H). Fusions between adjacent vertebrae in LRE and LRE+RARE-null mutants are indicated by the long bracket (I,J). AAA, anterior arch of the atlas; *AAA, ectopic anterior arch of the atlas; TA, tuberculum anterior; C, cervical vertebrae; T, thoracic vertebrae. Quotation marks indicate presumptive anterior transformations.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Cdx1 expression in LRE and LRE+RARE mutants. Whole-mount in situ hybridization analysis of Cdx1 expression in wild-type mouse embryos (A-C), and in LRE (D-F) and LRE+RARE (G-I) homozygous mutant embryos. All embryos were processed in parallel, although mutant specimens were stained four times longer than the wild type to reveal residual expression. Approximate chronological stage is shown above. (A,D,G). The onset of Cdx1 expression at late primitive streak in wild-type controls (A) was delayed to early head-fold stage in the mutants (D,G), and subsequently transcript levels were reduced (compare wild-type controls in B with mutants in E,H). Cdx1 transcripts became undetectable after the 4- to 5-somite stage in both mutant lines (E,F,H,I), whereas expression was readily detected in wild-type controls (B,C). Arrows in C indicate Cdx1 expression in mesoderm in the tailbud region at E9.5.

View larger version (101K): [in this window] [in a new window]   Fig. 4. Hox gene expression in LRE and LRE+RARE mutants. Expression of (A-C) Hoxa3, (D-F) Hoxd3, (G-I) Hoxb4 and (J-L) Hoxd4 in E9.5 wild-type (A,D,G,L), LRE mutant (B,E,H,K) and LRE+RARE mutant (C,F,I,L) mouse embryos. The rostral somitic expression boundary (arrows) was posteriorized by one somite for each Hox gene examined in both mutant backgrounds, as compared with wild-type controls. S, somite.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Regulation of Cdx1 by Wnt3a. Expression of Cdx1 in E8.5 wild-type (A,B), LRE mutant (C,D) and LRE+RARE mutant (E,F) mouse embryos following ex vivo culture with control (A,C,E) or Wnt3a-conditioned (B,D,F) media. Embryos of a given genotype were cultured and stained in parallel. Mutants were stained four times longer than controls to reveal residual expression of Cdx1.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Regulation of Cdx1 by RA. Expression of Cdx1 in E7.5 (A,B,E,F) or E8.5 (C,D,G,H) wild-type (A-D) and LRE mutant (E-H) mouse embryos following in utero treatment with DMSO vehicle (A,C,E,G) or RA (100 mg/kg; B,D,F,H). Embryos were processed and stained in parallel, with mutants stained four times longer than controls to reveal residual expression.

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