The role of Cdx genes in the mammalian gut.

O rganisation of multicellular animals involves the action of genes that impart ‘‘positional information’’. All vertebrates are built on a segmental pattern that is most obviously expressed by the appearance of somites during embryonic development. A common feature of genes that impart individual identity (and therefore positional information) to specific segments is the possession of a ‘‘homeobox’’ DNA binding motif coding for a consensus sequence of 60–63 amino acids that acts as a transcriptional regulator of ‘‘downstream’’ genes. The most widely researched homeobox genes are the so-called homeotic selector genes of the Antp-type (the defining gene is named Antennapaedia). In the fruit fly Drosophila, these are situated on chromosome 3 as part of the HOM cluster. HOM-C genes are strongly conserved during evolution and in mammals have been replicated to appear on separate chromosomes in four paralogous complexes called Hox clusters. They are expressed principally in developing ectodermal and mesodermal tissues and in general terms are responsible for segmental specification of the dermatomes, musculoskeletal, and nervous systems. However, Hox genes are not expressed in the greater part of the gut endoderm but in their place, mammalian members of the ParaHox genes—an ‘‘evolutionary sister’’ of the Hox clusters—seem to play an important role in gut patterning. Members of this group are Pdx1 which is required for the correct development of the pancreas and duodenum and three homologues of the Drosophila gene Caudal which in mammals are called Cdx1, Cdx2, and Cdx4. In addition to their own unique domains, the Cdx genes exhibit significant topographical overlap of expression during development, as well as in the adult, and it is reasonable to assume that in such areas a degree of compensation may occur in the event of single gene deficiency. Cdx2 is expressed in the endoderm of the entire postgastric epithelium from the time of its initiation at the stage of hindgut invagination throughout development and adult life. Cdx1 is not expressed in the early definitive gut endoderm but appears at postsomite stages just before transition of the multilayered intestinal endoderm to a single layered epithelium at 14 days post coitum (dpc). Cdx4 is expressed in the earliest hindgut invagination but little is known of its distribution after 10 dpc. It is important to note that in addition to their role in the gut, Cdx genes are active at multiple other sites during early development where, inter alia, they regulate the extent of ecto/mesodermal expression of Hox genes and thus also indirectly influence nonintestinal anteroposterior patterning. However, this review is principally concerned with their role in the gut. Much insight into the function of a gene may be obtained by studying the morphology of mice in which it has been inactivated by homologous recombination. Knockout of the Cdx1 gene produced anterior homeotic shifts (that is, a posterior structure such as a vertebra or rib exhibiting the morphology of a more anterior element) of the axial skeleton but no gut abnormalities. Inactivation of Cdx2 on the other hand produced not only axial homeotic shifts in heterozygotes (one affected allele) but was also found to prevent trophoblast maturation and consequently blastocyst implantation if present in the homozygous (both alleles affected) form. Most importantly in the context of this commentary, Cdx2+/2 embryos exhibited multiple polyps in the caecum and adjacent ileum and proximal colon (that is, in the midgut). 12 Histological examination showed the presence of forestomach epithelium in the substance of the polyps and this was interpreted as evidence of an anterior homeotic shift involving the gut endoderm analogous to the mesodermal shift involving the axial skeleton. In other words, disturbance of ‘‘positional information’’ has resulted in anterior structures (forestomach epithelium) occurring ectopically at more posterior sites. While initially defined as adenomatous polyps, critical histological analysis together with studies on timed development of the polyps established an interesting process of so-called intercalary growth. It was concluded that the function of Cdx2 was to direct the ‘‘default state’’ forestomach endoderm towards a caudal phenotype and that lower levels of expression of Cdx2 in the developing distal intestine of Cdx2+/2 heterozygotes lead to reversion to a more anterior phenotype (that is, to forestomach epithelium). Subsequently, intercalary growth around the gut lesions results in ‘‘filling in’’ of tissue types at the discontinuity between the ectopic gastric and surrounding colonic epithelia and an orderly succession of histologically normal epithelia characteristic of cardia, corpus, antrum, and small intestine, in that order, appeared all around the forestomach epithelium at the junction with colonic epithelium. The molecular basis of the intercalary growth is unclear but probably involves local intercellular communication. Clonal analysis experiments have been performed to establish the origin of the secondarily generated tissue between ectopic forestomach and colonic epithelia. Y chromosome painting in wild-type/Cdx22/2 chimaeric* mice of opposite sex indicated that once differentiated to express Cdx2, host colonic epithelium can only contribute small intestinal epithelium to the secondarily generated tissue while the Cdx2 mutant chimaeric cells give rise to the succession of gastric-type tissue but never to small intestinal morphology. These findings have interesting implications in the context of intestinal regeneration. No information concerning the role of Cdx4 in gut development is currently available as knockout or RNAi knockdown experiments have not been performed and no spontaneous mutants are available. Having shown that Cdx2 is necessary for the establishment of the midgut phenotype, we must now ask whether it is sufficient to convert the ‘‘default’’ stomach phenotype to that of colon. The question has been investigated by making transgenic mice in which Cdx2 is expressed in the stomach (which in wild-type mice is Cdx2 negative). This is achieved by introducing a Cdx2 expressing ‘‘transgene’’ into the genome