The small intestine derives from the definitive endoderm, one of the three germ layers formed during gastrulation. At this stage, the mouse embryo is organized in a U-shape rather than a flat sheet of cells. The mouse definitive endoderm is located at the outside of the embryo and is contiguous with the visceral endoderm that gives rise to the yolk sac. At the early somite stage (E8.5), the U-shaped mouse embryo turns, thereby reversing its topography and as a consequence the endoderm relocates into the embryonic body cavity. As a result of these morphogenetic movements the definitive endoderm forms the three-dimensional primitive gut tube by mouse embryonic day 9 (E9.0). Shortly thereafter, the primitive gut tube gives rise to the digestive organs, establishing a functional gastrointestinal tract that is divided into distinct regions: esophagus, stomach, small intestine and large intestine. The small intestine originates from the midgut region by E10.0 as a hairpin loop that grows towards the ventral side of the embryo. Over the next 5 days, the gut extends while rotating to form the convoluted intestinal tract. Due to space constraints, the intestine grows outside the embryonic peritoneal cavity during this period, thereby forming the physiological umbilical hernia. The elongated and tightly wound small intestine withdraws into the peritoneal cavity by E15.5, possibly due to contraction of the musculature of the duodenum and proximal jejunum (Kaufman and Bard, 1999
). Thus, generation of the mature intestinal tract entails dramatic morphogenetic changes. However, despite important advances in the understanding of embryonic development, the cellular and molecular mechanisms that underlie elongation of the small intestine remain obscure.
One pathway that is involved in intestinal organogenesis is Wnt signaling. The multiple Wnt ligands described in vertebrate animals signal through two distinct mechanisms: the Wnt/β-catenin and the non-canonical Wnt pathways. Of the two pathways, the Wnt/β-catenin pathway is well understood, whereas the different arms of the non-canonical Wnt pathway are still not fully elucidated. Originally, different Wnt ligands were assigned to one or the other pathway according to their signaling properties in specific assays. However, more recent evidence suggests that signaling through one or the other pathway is dictated by the composition of the Wnt receptors on receiving cells.
One of the Wnt ligands that traditionally had been assigned to the non-canonical pathway is Wnt5a. In frogs and zebrafish Wnt5a regulates convergence and extension movements during gastrulation in a process likely mediated by Ror2, RhoA GTPase, Jun kinase (JNK), and Ca2+ release. Moreover, Wnt5a has been shown to inhibit the canonical Wnt/β-catenin pathway in different systems, although the mechanisms that mediate this activity remain controversial. In contrast, other results indicate that in the presence of the appropriate receptors, Wnt5a signals through the canonical Wnt/β-catenin pathway. Similar to Wnt5a mutants in frog and zebrafish, mice deficient in Wnt5a show a profound defect in posterior elongation and morphogenesis of outgrowing structures with no alterations in cell fate. In any case, the exact mechanisms of Wnt5a signaling remain unknown and downstream effectors in mice have not been identified.
Here, we have analyzed the requirement for Wnt5a function during formation of the intestinal tract. During mouse development, Wnt5a has been shown to be expressed in the gut mesenchyme. Given its role in other tissues, we hypothesized that Wnt5a might govern intestinal elongation in mouse embryos. Here, we show that Wnt5a mutants display a dramatic shortening of the small intestine accompanied by an aberrant bifurcation of the midgut. This phenotype results from a combination of defective closure of the primitive gut tube at E10.0 and abrogated midgut elongation starting at E10.5. Notably, Wnt5a is not required for the differentiation of the diverse intestinal cell types or for the activation of the canonical Wnt/β-catenin pathway. In contrast, Wnt5a is essential to maintain the architecture of the growing epithelium by regulating re-intercalation of post-mitotic cells into the epithelium after cell division, and by controlling cell proliferation during midgut elongation. Thus, Wnt5a mutant mice reveal critical information about the cellular basis and dynamics of small intestine development.