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First published online 17 October 2007
doi: 10.1242/dev.010991

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Hox gene function in vertebrate gut morphogenesis: the case of the caecum

¹ National Research Centre `Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland.
² School of Life Sciences, Ecole Polytechnique Fédérale, Lausanne, Switzerland.

View larger version (60K): [in this window] [in a new window]   Fig. 1. Co-expression of seven Hoxd genes in posterior midgut. (A) Whole-mount RNA in situ hybridization detection of Hoxd10 transcripts in E13 mouse embryo, showing some sites of expression, including the intestinal hernia. (B) Anatomical subdivisions of the mid-gestation murine gastrointestinal system at E12. (C-K) Detection of Hoxd13 (C), Hoxd12 (D), Hoxd11 (E), Hoxd10 (F), Hoxd9 (G), Hoxd8 (H), Hoxd4 (I), Hoxd3 (J) and Hoxd1 (K) transcripts in dissected gut of E12.5 mouse embryos. The contiguous loci Hoxd1 to Hoxd10 are all co-expressed in the posterior midgut, in the region that involves the incipient caecum bud (F-K). Hoxd11 is excluded from the anterior (ileal) part, but is expressed in the posterior (colonic) part of the caecum bud (E). Hoxd12 (D) and Hoxd13 (C) expression is not detected in this region. ce, caecum; co, colon; oes, oesophagus; sb, small bowel; st, stomach.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Defect in the gut of Del(1-10) mutants. Whole-mount RNA in situ hybridization analysis of gene expression in dissected posterior midgut of E12 mouse embryos. Hoxd11 (A-C), Hoxd12 (D-F), Hoxd13 (G-I) Hoxa10 (J-L), Hoxa6 (M-O) and Fgf10 (P-R). Caecum budding is well underway in wild-type controls (A,D,G,J,M,P), perceptibly delayed in heterozygotes (B,E,H,K,N,Q) and is absent from homozygous specimens (C,F,I,L,O,R). In heterozygous specimens, ectopic Hoxd11 and Hoxd12 expression is detected in the anterior part and in the entire caecum bud, respectively (B,E). In homozygotes, Hoxd11 and Hoxd12 expression is detectable in the presumptive area for caecum budding (asterisks in C,F). In all three genotypes, including homozygous embryos, the demarcation between Hoxa10-negative and Hoxa10-positive regions seems to be rather faithfully maintained (J-L). Retarded bud growth in heterozygous specimens is correlated with reduced hybridisation signals for Hoxa6 (N) and Fgf10 (Q). Absence of bud growth correlates with the massive reduction of the expression domain of both these genes, leaving only of a tiny expression domain for both Hoxa6 (O) and Fgf10 (R).

View larger version (47K): [in this window] [in a new window]   Fig. 3. Role of Hoxd12 in caecum agenesis. (A) Derivation of the Del(4-11) allele by TAMERE. (B-J) Whole-mount RNA in situ hybridization of Hoxd12 (B-D), Fgf10 (E-G) and Pitx1 (H-J) transcripts in wild-type control (B,E,H), Del(4-11)-deficient HoxD cluster mutant heterozygous (C,F,I) and homozygous (D,G,J) posterior midguts at E12.5. In wild-type controls, the Hoxd12 transcript is always absent from the caecum bud (B), whereas localized expression of both Fgf10 (E) and Pitx1 (H) are always detectable. Caecum bud growth is conspicuously retarded in Del(4-11) heterozygous specimens and is correlated with robust Hoxd12 transcript accumulation (C), reduced hybridization signals for Fgf10 (F) and undetectable signal for Pitx1 (I) in bud mesenchyme. Absence of bud growth in homozygous specimens is correlated with extremely reduced of Fgf10 (asterisk in G) and complete absence of localized Pitx1 (J).

View larger version (92K): [in this window] [in a new window]   Fig. 4. `Anterior' Hoxd genes promote adult caecum size in mice. (A-I) Dissected ileo-caecal transition zones of three genotypes, illustrating key examples of the phenotypic series presented in Table 1. A,A',D,G depict X-Gal-stained newborn specimens, to confirm the identity of the complementing alleles. Arrowheads in A,A',D point to the anterior limit of Hoxd11/lac marker gene expression in mesenchyme, associated with the respective alleles, depicted in the line diagrams on the right. In gut mesenchyme, del(8i-13) shows a more anterior Hoxd11/lac expression involving the posterior ileum (A,A'), while the reporter gene associated with the del(11-13) allele is limited at the ileo-caecal junction, reminiscent of endogenous Hoxd11. Blue staining in G is due to endogenous activity in enterocytes, which does not involve the gut mesenchyme. The lac fusion protein does not have Hoxd11 function, consistent with lack of caecum growth promotion by the del(8i-13) allele (A,A',B) in the presence of ectopic Hoxd12. By contrast, in the case of del(11-13), the presence of the caecum (D,E) indicates growth promotion by Hoxd8, Hoxd9 and Hoxd10 present in addition to the Hoxd1, Hoxd3 and Hoxd4 loci, which alone were not sufficient to neutralize ectopic Hoxd12. Control guts are shown in G and H for comparison.