In this study, we have investigated dynamic gene expression patterns at and around the developing pylorus. Although clear regional patterning of the stomach and intestine occurs prior to E14.5 (e.g., for Sox2, Cdx2, among other genes), this pattern does not play itself out in terms of the global transcriptomes of E14.5 stomach, duodenum, and pylorus tissues, all of which are surprisingly similar. In contrast, at E16.5, a dramatic burst of transcriptional induction occurs in duodenal epithelium and this event generates a distinct compartmentalization of gene expression on the duodenal side of the border. This genetic induction event coordinately activates hundreds of genes involved in absorption and metabolism. As a result, for the first time, epithelial cells of the intestine express genes that unambiguously distinguish their function during digestion from that of the stomach. We call this compartmentalized patterning step intestinalization and note that it occurs strikingly late in fetal development.
Though the vast majority of genes activated in the intestine are epithelial, a few mesenchymal genes are also upregulated at E16.5, several of which are involved in immune response function. This is interesting in light of recent parallel evidence from our laboratory showing that, in adult intestine, decreased Hedgehog signal transduction increases inflammatory signaling (Lees et al., 2008
). Whether there is a direct connection between the downregulation of the Hedgehog pathway that we observe in duodenal mesenchyme at E16.5 (see ) and activation of these mesenchymal inflammatory genes requires further investigation.
An unexpected finding from our array results is that only four genes (Foxa1, Gcg, Mreg and Serpina1c) are specifically downregulated in intestinal epithelium at E16.5 (Supplemental Table 1
). Interestingly, Foxa1 has been shown to be required for Shh expression in the developing lung, another foregut endoderm-derived organ (Wan et al., 2005
). Because of its concomitant downregulation with Shh in intestinal epithelium, it is tempting to postulate that the attenuation of Foxa1 expression is responsible for reduced Shh expression during intestinalization
. If so, it will be important to understand the transcriptional regulation of Foxa1 expression and determine whether downregulation of this gene is required for intestinalization
Given the ample published evidence of a role for Wnt signaling during foregut specification (Okubo and Hogan, 2004
; Kim et al., 2005
; Kim et al., 2007
), we were surprised to find that the Axin2 expression pattern suggests little or no difference in canonical Wnt signaling across the pylorus, either before or after pyloric border formation. In fact, at E14.5, very little canonical Wnt activity is detectable at all in either stomach or intestine, though we observed clear Axin2 activity in the forestomach at this time (data not shown). The lack of canonical Wnt signal in E14.5 duodenum is concordant with the phenotype of Tcf7l2 (Tcf4)-null mice, which exhibit no apparent defect in E14.5 intestine (Korinek et al., 1998
). At E16.5, we see that intestinal Axin2 expression is confined to intervillus regions (see ). This is in accordance with the finding that these proliferative cells are dependent upon canonical Wnt signals, as shown by the loss of this proliferative population in the face of either Tcf4 deficiency (Korinek et al., 1998
) or Dkk1 overexpression (Pinto et al., 2003
; Kuhnert et al., 2004
). Together, these findings and the results of our analysis do not support the idea (Kim et al., 2007
) that a canonical Wnt signaling compartment exists on villus tips at E16.5.
In this regard, the expression pattern of Sfrp5 at E16.5 is interesting. Recent work indicates that Sfrp5 can modulate either canonical or non-canonical Wnt signals in the Xenopus foregut (Li et al., 2008
). In that system, Sfrp5 was able to bind the ortholog of the non-canonical protein Wnt5a. Intriguingly, both overexpression of Sfrp5 (in Xenopus) and deficiency of Wnt5a (in mouse) result in shortened hindgut (Li et al., 2008
; Cervantes et al., 2009). Data presented above show that, at E16.5, Sfrp5 expression is restricted to the intervillus region (see ), the same area that is positive for Axin2 staining (and, hence, canonical pathway activity). Thus, it will be of interest to test whether Sfrp5 functions in the intervillus zone to modulate canonical signals, non-canonical signals, or both.
Several epithelial transcription factors are dramatically upregulated during intestinalization
and may participate directly in large-scale induction of absorptive and metabolic activity in the intestine. The Hnf4γ paralog Hnf4α has been previously implicated in a similarly late developmental maturation event in the liver. In that system, Hnf4α upregulates a large number of structural genes and is thought to be important for the re-epithelialization of hepatic cells following their migration out of the gut tube proper and into the septum transversum (Parviz et al., 2003
). It is tempting to speculate that intestinalization
is a similar event. Even though intestinal epithelial cells never leave the confines of the epithelial sheet, as developing hepatoblasts do, the epithelium itself is drastically reorganized during the process of villus formation. Perhaps Hnf4α and Hnf4γ are critical in the final stabilization of this remodeled state. Certainly, binding sites for these factors are highly enriched in the promoters of intestine-specific and epithelial-specific genes (Li et al., 2007
Two previously unstudied factors were among the most upregulated D16 epithelial transcription factors. Creb3l3 (also known as Creb-H), a member of the bZip family of transcription factors, is involved in the endoplasmic reticulum (ER) stress response (Zhang et al., 2006
), and interestingly, its expression in the developing liver is dependent upon Hnf4α (Luebke-Wheeler et al., 2008
). Since intestinalization
involves transcriptional activation of hundreds of genes, several of which are expressed at tremendously high levels, the ER of intestinal epithelial cells may abruptly require a much higher degree of organization and efficiency to deal with the translational onslaught that follows. Indeed, we show that Creb3l3 is expressed in epithelial cells of the villi, exactly the population in which differentiated gene expression is induced. The idea that ER stress might accompany cell differentiation and might activate mediators of the response pathway in order to coordinate protein biosynthesis remains functionally untested here, but has been well documented for a number of secretory cell types (Wu and Kaufman, 2006
Another transcription factor that is highly induced in E16.5 intestinal epithelium is Tcfec. This bHLH-Zip factor is a member of the MiT family (with Mitf, Tcfeb and Tcfe3), several of which are expressed in a highly tissue- and cell-specific manner. Often these proteins are responsible for the expression of signature proteins that are critical to organ or tissue development and function. For example, MiT family members regulate tartrate-resistant alkaline phosphatase in osteoclasts (Partington et al., 2004
), melanin in pigment cells (Tachibana, 2000
) and proteases in mast cells (Nechushtan and Razin, 2002
). The MiT proteins form both homo- and hetero-dimers, a fact that may explain why mouse knockout models of several family members show no phenotypes, despite the apparent transcriptional importance of these genes (Steingrimsson et al., 2002
). According to our microarray data, the related family member Tcfeb is also expressed in the intestine and is upregulated during intestinalization
, though not as dramatically as Tcfec.
occurs concomitantly with formation of a sharp boundary of epithelial gene expression at the pylorus. For genes like villin, Cdx2 and Sox2, a broad domain of expression with a diffuse boundary is detectable early and reflects the regional divisions of organ territory in the developing gut tube. But at E16.5, the boundary of expression sharpens exquisitely to allow differentiated intestinal cells to lie directly next to future stomach cells. An interesting question for further analysis is whether boundary formation and intestinalization
actually constitute separate events. The process of intestinalization
might reflect maturation of the vertical axis of the villus; differentiating cells exiting the proliferative compartment of the last villus next to the stomach may travel only a set distance from that crypt, coming to rest immediately next to a less differentiated neighbor derived from the stomach progenitor compartment. Alternatively, the pyloric border region itself could propagate a signal that promotes intestinalization
, similar to the function of a classical organizer (Meinhardt, 2008
Our data confirm and extend earlier studies (Smith et al., 2000b
) that reveal a characteristic domain of gene expression at the pylorus. We show that this domain is present both prior to and after intestinalization
. We report here the novel finding that, similar to Nkx2-5, Gata3 is expressed in a narrow band at the pylorus (see ). Given that Nkx and Gata family members are known to interact in other developmental systems, these factors may collaboratively regulate pyloric patterning and organogenesis (Charron and Nemer, 1999
; Peterkin et al., 2003
; Zhang et al., 2007
). In addition, we describe the pyloric expression patterns of gremlin (mesenchyme; see ) and nephrocan (epithelium; see ), two secreted modulators of TGF-β superfamily signaling. To our knowledge, nephrocan is the first secreted signaling protein to be identified in pyloric epithelium. In this regard, pyloric border patterning might be similar to the boundary patterning events observed at the midbrain-hindbrain border in the developing brainstem and the atrioventricular boundary of the heart. These events involve formation of straight, sharp expression boundaries (Joyner et al., 2000
; Canning et al., 2007
; Chi et al., 2008
), and in both cases, the border region itself has signaling activity that influences neighboring tissues (Bai et al., 2002
; Chi et al., 2008
event that we document occurs without a similar maturation process in the stomach, where only limited transcriptional change occurs. The fact that the intestine, a more posterior tissue, matures prior to the stomach is somewhat surprising, given the tendency of embryonic development to progress in an anterior to posterior direction. Indeed, it is possible that this finding has evolutionary roots, as the stomach is believed to be an added character that first appeared in primitive fish (Smith et al., 2000a
). It is possible then, that the stomach epithelium at E16.5 is governed by a program designed to represses the emerging intestinal state in order to preserve the primitive stomach epithelium for the later reception of instructive signals to differentiate as stomach. Though this notion is entirely speculative, it has interesting implications for intestinal metaplasia, a pathological lesion in which patches of epithelium with intestinal character emerge in the stomach. The possibility that active repression of intestinal differentiation in stomach exists during pyloric border formation (and persists throughout adult life) will become amenable to further investigation now that the transcriptomes of stomach and intestine are available during this important developmental event.