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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00889

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Retinoic acid regulates a subset of Cdx1 function in vivo

¹ Department of Molecular Biology, Université de Montréal, 110 ave des Pins, ouest, Montréal, Québec H2W 1R7, Canada
² The Institut de Recherches Cliniques de Montréal, 110 ave des Pins, ouest, Montréal, Québec H2W 1R7, Canada
³ Division of Experimental Medicine, McGill University, 110 ave des Pins, ouest, Montréal, Québec H2W 1R7, Canada

View larger version (26K): [in a new window]   Fig. 1. Targeting of the Cdx1 RARE. (A) Schematic representation of the 5' genomic region of the Cdx1 locus, targeted allele (targeting vector sequences shaded) and anticipated Cre-recombination product. The sequences external to the targeting vector used for screening initial recombinants are indicated below the targeted allele, as is the internal probe used to screen for Cre-catalyzed recombination. (B) A multi-enzyme Southern blot demonstration of the predicted targeting event using the internal probe denoted in A with the restriction endonucleases used indicated above each lane. (C) Characterization of Cre-mediated recombination. Primers (arrows in A) were used to amplify over the RARE region by PCR. Products were resolved by agarose gel electrophoresis and characterized by Southern blot analysis using oligonucleotide probes specific for wild-type or recombination products. As anticipated, the wild-type (WT, lower panel) band is present in all samples with the exception of the negative control (lane 7), while the Cre-generated product is observed only in mice from the appropriate mating (lane 2) or from a cell line transfected with a Cre expression vector (lane 6). S, SacI; H, HindIII; R, EcoRI; K, KpnI; X, XhoI.

View larger version (8K): [in a new window]   Fig. 2. The targeted mutation of the RARE abrogates RA-response in tissue culture. F9 embryocarcinoma cells were transfected with 1.5 µg of wild-type (Wt) Cdx1 reporter, a reporter with a mutated RARE (Mut) or an RARE equivalent to the targeted mutation after Cre-mediated recombination (Lox mut). Twenty-four hours after transfection, cells were treated with vehicle or 10-6 M RA and luciferase activity assessed 24 hours post-treatment. Results, from independent triplicate experiments, were expressed as fold RA-induction relative to vehicle treated cells. Each transfection was repeated at least twice with similar results.

View larger version (56K): [in a new window]   Fig. 3. Loss of RARE function affects Cdx1 expression in vivo. Whole-mount in situ hybridization analysis of Cdx1 expression in wild type (A,C,E) and RARE mutants (B,D,F). All stages to be compared were processed and stained in parallel. (A,B) Analysis of early E7.0 to late head folds stages demonstrates onset of Cdx1 expression at late primitive streak stages (E7.5) in both wild-type (A) and RARE mutant embryos (B). Dark field micrographs are shown for ease of stage comparison. (C,D) Wild-type (C) and RARE mutant (D) embryos ranging from 1 (left) to 7 (right) somites. Note the diminution of expression in mutant embryos relative to stage matched controls. (E,F) Wild-type (E) and RARE mutant (F) embryos ranging from 7 (left) to 13 or 14 (right) somites. Note the near loss of expression in the mutants relative to stage matched controls.

View larger version (105K): [in a new window]   Fig. 4. Cdx1 is induced by RA in RARE-null mutants. In situ hybridization analysis of Cdx1 expression of wild-type (A,B,E,F,I,J,M,N) or Rare-/- (C,D,G,H,K,L,O,P) embryos following treatment with vehicle (A,C,E,G,I,K,M,O) or RA (B,D,F,H,J,L,N,P) at E7 (A-D), E7.5 (E-H), E8.0 (I-L) or E8.5 (M-P). All embryos were recovered 4 hours after in utero treatment with RA and equivalent stage matched samples processed in parallel for analysis. Note that Cdx1 was induced by RA at gastrulation stages, with induction seen in both wild-type (A versus B) and RARE mutants (C versus D). Induction also occurred at all later stages irrespective of genotype.

View larger version (124K): [in a new window]   Fig. 5. RA induction of Cdx1 in RARE-null mutants is independent of de novo protein synthesis. RARE-null mutant E8.5 embryos were cultured for 4 hours with vehicle (A), 10-6 M RA (B), cycloheximide (C) or cycloheximide plus RA (D; all cycloheximide cultures were initiated 30 minutes prior to addition of RA). Note that cycloheximide did not prevent RA induction of Cdx1 in the RARE-null mutants (compare D with B). The embryos are arranged to represent stage-matched samples between the four panels, with those on top possessing 8-12 somites and those on the bottom having between one and eight somites.

View larger version (48K): [in a new window]   Fig. 6. Skeletal defects in RARE and RARE-Cdx1 compound mutants. Representative skeletal preparations from neonatal offspring; genotypes are indicated in each panel. Asterisk indicates ectopic anterior arch of the atlas (AAA) indicative of C2 to C1 transformation (C-F). Brackets (E,F) indicate narrower then normal neural arches (NA) on C1, indicative of a transformation to an exoccipital-like structure, while the arrowhead and broad arrow (E,F) indicate a broader C2 neural arch, suggestive of C2 to C1 and C3 to C2 transformations, respectively. TA (E,F) indicates the anterior tubercles, normally found ventral to C6, that have been shifted posteriorly to the seventh cervical element. Thin arrow (E,F) indicates posterior extension of the basioccipital.

View larger version (60K): [in a new window]   Fig. 7. Skeletal defects in RAR and RAR-RARE compound mutants. Representative RAR and RAR-RARE vertebral phenotypes with genotypes indicated in each panel. (B,D-F) Malformed neural arches on C1 are indicated by a bracket. (B-F) Widened neural arches on C2 are indicated by arrowheads, whereas neural arch fusions between C1 and C2 or C2 to C3 are indicated by a bracket in F. (E,F) Ectopic anterior arch of the atlas associated with C2 is indicated by an asterisk.

View larger version (65K): [in a new window]   Fig. 8 . Hox paralog group three gene expression is altered in Cdx1, but not RARE, null mutants. (A,D,G) Wild-type, (B,E,H) Rare-/- and (C,F,I) Cdx1-/- specimens were assessed for expression of Hoxa3 (A-C), Hoxb3 (D-F) or Hoxd3 (G-I) by whole-mount in situ hybridization analysis. Somite number is indicated by numbering in each panel, starting with the anterior-most limit of expression of each Hox gene. All three Hox genes were posteriorized by one somite in the Cdx1-/- mutants (C,F,I) but were unaffected in RARE-null samples (B,E,H).

View larger version (96K): [in a new window]   Fig. 9. Hox paralog group four gene expression is altered in Cdx1-and RARE-null mutants. In situ hydridization analysis of (A,D,G) wild type, (B,E,H) Rare-/- and (C,F,I) Cdx1-/- specimens for Hoxa4 (A-C), Hoxb4 (D-F) or Hoxd4 (G-I). Somite number is indicated in each panel commencing with the anterior-most limit of detection of each Hox gene. Hoxb4 and Hoxd4 exhibited similar posteriorization by one somite in both RARE (E,H) and Cdx1 (F,I) mutants, while Hoxa4 expression was unaffected (B,C). Note the low residual expression of Hoxb4 and Hoxd4 in somite 6 in both classes of mutants (E,F and H,I).

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