While genetic evidence has previously suggested a role for Wnt signaling in intestinal development, this report establishes a clear, direct and multifaceted role for Wnt signaling in intestinal specification and patterning. Wnt signaling acts directly on definitive endoderm to induce Cdx2, and application of Wnt to the early foregut endoderm is sufficient to downregulate the anterior endoderm program and to induce a gene expression program that resembles early embryonic intestine. As further proof of its role in intestinal specification, Wnt signaling is sufficient to induce ES cell-derived endoderm to an intestinal fate. Wnt signaling is involved not only in specification of the intestine but in its anterior-posterior patterning, as high-level Wnt and Cdx2 appear to be necessary for large intestine specification.
The effect of Wnt signaling on endoderm is transient. While Wnt possesses the ability to induce Cdx2 between E7.5 and E8.5, electroporation and bead experiments both demonstrate that Wnt signaling after E8.5 is no longer capable of inducing Cdx2. This loss of competence correlates with the time at which BAT-gal mice cease displaying midgut endodermal activity (), and this loss of active posterior endodermal Wnt signaling is seen at a similar embryonic stage in Xenopus
(Schohl and Fagotto, 2002
). E8.5 is also the stage at which Cdx2 and Sox2 expression domains meet at the stomach-intestine border and at which hepatic and pancreatic genes begin to be expressed at this border (Sherwood et al., 2009
). Thus, at E8.5, posterior foregut ceases to receive Wnt signals or inhibitor activity outweighs ligand presence, and the endoderm loses competence to activate intestinal genes in response to such signaling. It has recently been shown that Wnt signaling in the E9.5 endoderm is vital for lung specification (Goss et al., 2009
), and Wnt also plays an important role in the development of the pharyngeal pouches (Balciunaite et al., 2002
), liver and pancreas (Verzi and Shivdasani, 2008
). As the Wnt-Cdx2 axis is dominant over anterior fates, the loss of competence to activate Cdx2 in response to Wnt signaling is a crucial step in endodermal development.
Both the BAT-gal expression in the endoderm () and the anterior creeping of endodermal Cdx2 (Sherwood et al., 2009
) suggest a model in which a posterior source of Wnt diffuses anteriorly, and progressively more anterior cells reach a threshold to activate Cdx2. Our study suggests direct activation of Cdx2 by Wnt signaling, as Cdx2 mRNA is seen within six hours of Wnt treatment in embryonic and ES-derived endoderm, and Tcf/Lef factors have been demonstrated to act directly on the promoters of Cdx1 and Cdx4 (Pilon et al., 2006
; Prinos et al., 2001
), so direct action on Cdx2 is a likely mechanism. The relationship between Wnt and Cdx2 in the nascent intestine may be more complex, however, as evidence from other developmental stages suggests that Cdx2 can induce Wnt signaling (Young et al., 2009
), Cdx2 and the Wnt-responsive transcription factor Tcf4 co-occupy a large number of regulatory sequences (Verzi et al.
), and Cdx2 has been shown to inhibit Wnt responsiveness (Guo et al.
). This model of posterior Wnts patterning the intestinal endoderm provides a convenient explanation for the observation that only higher doses of Wnt in embryos and Cdx2 in ES-derived endoderm are able to induce large intestinal gene expression. However, the inability of high-dose Wnt alone to induce large intestinal gene expression in ES-derived endoderm suggests either that additional signaling mechanisms are required in concert with high-dose Wnt to induce high-level Cdx2 expression and large intestinal fates or that the protocol used for ES cell intestinal endoderm induction fails to provide the temporal competence window required for this induction.
Several experiments suggest that an additional aspect of intestinal organogenesis involves differential adhesion and possibly cell migration. After electroporation of CA β-catenin into foregut endoderm, electroporated cells are found at more posterior positions after longer periods of embryo culture, and after 24 hours, a significant percentage of cells are found near to the endogenous hindgut Cdx2+
domain (, Supplemental Figure 2
). In the endoderm-specific genetic activation of β-catenin, ectopic Cdx2+
cells are found as clusters in the pharyngeal endoderm and as strands in the midgut region anterior to and connected to the endogenous Cdx2+
intestinal domain (). It is clear from the cluster formation in the genetic activation experiments that Wnt-induced Cdx2+
cells have differential adhesion that allows them to cluster and at times disengage from the pharyngeal endodermal epithelium as protrusions, an interesting finding in light of the known role of Cdx2 in controlling cell polarity and cytoskeletal arrangement (Gao and Kaestner
). It is also tempting to speculate that the lack of ectopic clusters in the midgut occurs because Cdx2+
cells close enough to the endogenous intestinal domain migrate to and adhere to the endogenous intestinal domain, which could provide an explanation as well for the electroporation results. These experiments cannot rule out selective survival or proliferation as alternative explanations, so future timelapse imaging experiments will be informative to determine whether migration and differential adhesion play a role in the formation of a strict boundary for the nascent intestinal domain.
Induction of intestinal fate by Wnt seems to rely heavily on Cdx2. Cdx2 is one of the earliest genes activated in the endoderm by activation of Wnt signaling. Wnt signaling has been shown to directly induce Cdx1 in ES cells (Lickert et al., 2000
), and our results strongly suggest a direct activation of Cdx2 in endoderm, as Cdx2 mRNA is induced within 6 hours and Cdx2 protein is induced within 8 hours. As has been recently documented in Cdx2-deficient endoderm (Gao et al., 2009
), Cdx2 orchestrates a network of intestinal transcription, so it is unsurprising that 24 hours after Wnt activation, gene expression has altered significantly toward that of native embryonic intestinal endoderm. Wnt signaling, however, has a more extensive role than simply activating Cdx2, as is demonstrated in experiments comparing genes induced by Wnt or by direct induction of Cdx2 in ES cell-derived endoderm. One potentially crucial difference is that Wnt induces Cdx1 along with Cdx2, and these two genes could have distinct targets during intestinal development. Additionally, Wnt signaling appears to induce Indian Hedgehog independently of Cdx2, and Hedgehog signaling is known to be important in the coordination of intestinal endoderm and mesoderm development (Roberts et al., 1998
In the process of analyzing the role of Wnt in intestinal regional patterning, we have identified several genes differentially expressed in intestinal segments. This process has revealed two surprising results. The first is that, while the small intestine is traditionally divided into three anterior-posterior segments by morphology, gene expression patterns suggest two molecularly distinct small intestinal regions. The duodenum has unique expression of genes such as Ipf1, Onecut1 and 2, Anpep and Tm4sf4, while Osr2, Fzd10 and Cib2 have graded anterior borders at the presumed duodenum-jejunum boundary and are expressed until their graded posterior boundaries slightly past the cecum (, Supplemental Figure 3
). The large intestine-specific genes identified span the entire large intestine posterior to the cecum (, Supplemental Figure 3
), although reports of specific roles for Hox13 members in the anorectal endoderm (Warot et al., 1997
) suggest that some genes may be expressed only in subregions of the large intestine. Second, these regional expression patterns are established at least as early as E10.5 and possibly earlier (, Supplemental Figure 3
) even though morphological distinction among regions starts around E12.5 with the development of the cecum and does not become clear until villus formation after E14.5. The early establishment of regionalization is potentially a consequence of its reliance on differential Wnt levels, as Wnt acts in intestinal specification exclusively between E7.0 and E8.5.
Finally, we demonstrate that activation of Wnt signaling is sufficient to induce intestinal differentiation of ES cell-derived endoderm (). This induction is highly efficient, as greater than 95% of ES cell-derived endoderm cells begin to express Cdx2 after treatment with GSK3iXV. While GSK3 inhibition has been found to affect pathways other than Wnt (Wu and Pan, 2010
), the concordance of phenotypes using GSK3iXV, activated β-catenin, and Wnt3a in multiple assays lends confidence to the conclusion that GSK3 inhibition is exerting its intestinal inductive action primarily through activation of Wnt signaling. Recently, a protocol has been developed demonstrating three-dimensional intestinal organoid differentiation from human embryonic stem cell-derived endoderm by the addition of Fgf4 and Wnt3a (Spence et al., 2011
). In these experiments, Fgf and Wnt signals are required in conjunction to allow for Cdx2 expression and intestinal differentiation; however, equivalent experiments in this work demonstrate that strong Wnt stimulation through GSK3 inhibition is sufficient to induce uniform Cdx2 (), and the discrepancy is likely a result of the increased effectiveness of small molecule GSK3 inhibitors over Wnt3a (). In Spence et al’s work (Spence et al., 2011
), human ES-derived mesoderm is also present and becomes incorporated into the organoids, so Fgf signaling may be acting to pattern this mesenchyme. It will be interesting to replicate these experiments using ES cell-derived populations containing small and large intestinal marker expression to assess the differential morphological and transcriptional properties of these populations.
The gene expression changes induced by Wnt pathway activation are strikingly similar between embryo-derived endoderm and ES cell-derived endoderm. The few differences could result from differences in timing, as the embryonic experiments were performed in E8.25 foregut, which already expresses some specialized genes involved in anterior endoderm organogenesis whereas ES cell-derived endoderm appears by gene expression to resemble early embryonic endoderm, or from indirect effects of non-endodermal tissues in the bead experiments. This finding is significant for the ES cell field, as it lends confidence to the fact that ES cells do respond equivalently to embryonic cells when exposed to extracellular signals.
It will be interesting to investigate ES cell-derived intestine further to determine whether it possesses the ability to differentiate down mature intestinal lineages or to adopt adult intestinal stem cell activity. Derivation of intestinal populations from ES cells will lead to greater understanding of intestinal diseases and will hopefully lead to in vitro models of intestinal diseases and tissue replacement therapy.
- Wnt acts directly on endoderm to specify intestinal fate
- Wnt induces the intestinal master regulator Cdx2
- Wnt signaling induces intestinal gene expression in ES-derived endoderm
- Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses