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Volume 52 (4): 509-516, 2004
Copyright ©The Histochemical Society, Inc.
Expression of Notch Receptors and Ligands in the Adult Gut
Child Health Research Institute, Womens and Childrens Hospital, Adelaide, South Australia (GRS,BCP), and Department of Paediatrics, University of Adelaide, Adelaide, South Australia (BCP)
Correspondence to: G.R. Sander, Child Health Research Institute, 72 King William Road, North Adelaide, South Australia 5006, Australia. E-mail: guy.sander{at}adelaide.edu.au
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The Notch signaling pathway has become recognized as a vitally important pathway in regulating proliferative/differentiative decisions and cell fate. To explore the involvement of the Notch pathway in adult gut, we investigated the expression of Notch receptors and their ligands by Northern blotting and in situ hybridization. Notch receptors and ligands were expressed in both proliferative and post-mitotic cells throughout adult rat gut, variously in epithelial, immune, and endothelial cells. Expression of Notch1, Jagged1, and Jagged2 frequently overlapped, whereas Notch2 expression was restricted to specific crypt cells, the lamina propria of the large intestine, and Peyer's patch lymphocytes. We propose that the expression of multiple Notch receptors and ligands in a range of different intestinal cell types indicates that this signaling pathway underpins many of the processes involved in the maintenance and function of the adult gut.
(J Histochem Cytochem 52:509–516, 2004)
Key Words: Notch • Jagged • Delta1 • receptor • ligand • mucosal immune system
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Introduction |
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THE GASTROINTESTINAL (GI) tract is a complex organ system. To perform its primary functions of digestion, absorption, and excretion, diverse types of cells are assembled into the elaborate form of the adult gut. Epithelial cells provide protective, secretory, and absorptive functions and mucosal immune cells are vital in the development of oral tolerance and responses to infection. The molecular pathways that underlie the development and maintenance of the cell types involved in these functions are poorly understood.
The Notch signaling pathway is involved in determining cell fates in a variety of tissues (Weinmaster et al. 1992
; Mitsiadis et al. 1995; Lindsell et al. 1996; Lewis et al. 1998; Powell et al. 1998; Artavanis–Tsakonas et al. 1999; Hoyne et al. 2001). The Notch genes encode transmembrane receptors that interact with ligands, also transmembrane proteins, on adjacent cells. In vertebrates there are four receptors and five ligands. Receptor–ligand interaction results in cleavage of the intracellular domain of the Notch receptor, its transduction to the nucleus, and transcriptional activation. The cell contact-dependent nature of Notch signaling enables the coordination of proliferative/differentiative decisions and cell fates in a group of otherwise uncommitted cells. Depending on the context, signaling can either inhibit or induce differentiation (Artavanis–Tsakonas et al. 1999).
In the immune system, Notch signaling has key roles in the maintenance and differentiation of precursor cells. Notch appears to regulate lymphocyte development in the bone marrow and thymus, promoting development of T-cells from bipotential T/B precursors, the development of the T-cell 
ß lineage, and the maturation of the CD4 and CD8 lineages (Pui et al. 1999; Radtke et al. 1999; Hoyne et al. 2001). The study by Wilson et al. (2000) in conditional Notch1-deficient mice was the first to indicate that Notch1 is essential for maturation of extrathymically-derived T-cells in the gut.
Information on Notch expression in the mammalian GI tract is limited to description of selected receptors or ligands primarily in embryonic development (Weinmaster et al. 1992; Zagouras et al. 1995; Valsecchi et al. 1997; Imatani and Callahan 2000; Jensen et al. 2000; Fusse and Hoch 2002; Schroder and Gossler 2002) and more superficially in the adult gut (Zagouras et al. 1995; Imatani and Callahan 2000). These reports provide only snapshots of Notch expression and do not indicate whether Notch signaling plays an ongoing role throughout the GI tract.
Given the morphological complexity of the GI tract and the key roles of Notch signaling in other tissues, there is a clear lack of knowledge of the involvement of this important signaling pathway in gut function. Here we investigated the expression of the Notch1, Notch2, and Notch3 receptors and the Delta1, Jagged1, and Jagged2 ligands in the adult GI tract. We report that the Notch receptors and ligands are differentially expressed throughout the GI tract, in the intestinal epithelium, the endothelium, and the peripheral immune system. To our knowledge, this is the first report detailing the localization of Notch ligands and receptors in a diverse range of cell types of the adult rat esophagus, small intestine, and large intestine.
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Materials and Methods |
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Tissue Preparation
Four-month-old female Sprague–Dawley rats in the weight range of 200–400 g were humanely sacrificed and tissue segments from the stomach (fundic and body regions), esophagus (proximal and distal), and duodenum, jejunum, ileum, cecum, and colon (proximal, middle, and distal for each) were removed, rinsed in 1 x PBS and snap-frozen in liquid nitrogen. Segments were also fixed in 4% paraformaldehyde and processed for in situ hybridization (ISH) (Powell et al. 1998
). All experiments were approved by the Animal Ethics Committee of the Womens and Childrens Hospital, Adelaide, South Australia.
Probes for RNA Expression Analysis
The Jagged1 and Jagged2 probes used for ISH and Northern blotting analysis have been previously described (Powell et al. 1998). The Delta1 probe was a 2795-bp EcoRI/XhoI DNA fragment from clone pRat Delta1 (Lindsell et al. 1996). The Notch1 probe was a 444-bp EcoR1/HindIII fragment from clone SN6-7 that includes the intracellular domain from the transmembrane region to part of the ankyrin repeats (Weinmaster et al. 1992). The Notch2 probe for ISH was a 2462-bp fragment from clone H10-6 including some EGF repeats, the transmembrane region, and some of the ankyrin repeats (Weinmaster et al. 1992). For Northern blotting analysis, a 641-bp fragment that spans the end of the LNR region to the beginning of the ankyrin repeats was isolated from clone H10-6 using SalI/HindIII. The Notch3 probe was a 746-bp fragment from clone 5'PCRN3 that spans some of the EGF repeats (Lindsell et al. 1996). All Notch receptor and ligand probes were gifts of G. Weinmaster, (University of California). The rat ß-actin RNA probe was synthesized from pTri-Bactin-125 (Ambion; Austin, TX). The histone H3 probe was a gift of M. Chou, Taiwan (Chou et al. 1990).
Northern Blotting Analysis
Total RNA was prepared from 1 g of female Sprague–Dawley rat gut segments. PolyA+ RNA was isolated using the Poly-A Tract IV system (Promega; Madison, WI) and 1–2 µg was fractionated in a 1% agarose gel containing formaldehyde. RNA was transferred to a nylon Zeta-GT probe membrane (BioRad; Hercules, CA), then UV cross-linked. Blots were prehybridized in 10 ml of Ultrahyb hybridization solution (Ambion) at 42C for 2 hr and probed consecutively with [32P]-dCTP radiolabeled rat Notch1, Notch2, Notch3, Jagged1, Jagged2, and Delta1 probes synthesized with the Megaprime labeling kit (Amersham Biosciences; Piscataway, NJ). Blots were incubated overnight at 42C and washed to a final stringency of 0.1 x SSC/0.1% SDS at 65C. Radioactive signals were detected with a Typhoon 4100 phosphorimager (Amersham Biosciences) after 16-hr exposure. Between consecutive probings, membranes were stripped of radioactive probe by boiling in 0.1 x SSC/0.1% SDS for 30 min and lack of residual signal checked by overnight exposure.
In Situ Hybridization
Riboprobes were labeled to high specific activity by incorporation of [33P]-rUTP using a SP6/T3/T7 riboprobe kit (Promega). Antisense and sense radiolabeled riboprobes were hybridized to tissue sections (Powell et al. 1998). Hybridized sections were washed at a final stringency of 0.1 x SSPE at 65C for 30 min and exposed for 10 days at 4C. Tissues were stained with hematoxylin and images captured under brightfield and darkfield illumination with an Olympus BH2 microscope fitted with an Olympus darkfield condenser (model # U-DCW) and a Sony digital video camera (model #SSC-DC50P) and were analyzed with Image Pro Plus analysis software (Media Cybernetics; Carlsbad, CA).
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Northern Blotting Analyses
To begin an investigation of Notch expression in the GI tract, we undertook Northern blotting analysis using gene-specific riboprobes from the Notch1, Notch2, and Notch3 receptors and the ligands Jagged1, Jagged2, and Delta1 (Figure 1). GI tract tissue was divided into recognized anatomical zones and then subdivided into smaller segments to enable coverage of the entire gut. Transcripts of the expected sizes for the Notch receptors and ligands were detected in the stomach, duodenum, jejunum, ileum, cecum, and colon. Rat ß-actin was used to normalize for loading differences between lanes. Because the sensitivity of each riboprobe is unknown, they cannot be used to infer the relative expression levels of different Notch receptors and ligands. However, the relative expression of any single gene in different gut compartments can be compared. The expression level for the receptors Notch1–3 were relatively constant in the duodenum, jejunum, ileum, cecum, and colon, with the exception that Notch1 and Notch3 were elevated in the colon. The ligands Jagged1, 2, and Delta1 showed some variation in these regions, with a marked increase of Delta1 in the jejunum and colon. In the fundic region of the stomach there was a higher level of Notch3 and Jagged2 and a lower level of Delta1 compared with the intestine. Notch2, Notch3, Jagged1, and Jagged2 were also markedly more abundant in the body region of the stomach. There was no change in expression within an anatomic region (data not shown).
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In Situ Hybridization
We used ISH to locate the expression of Notch1, Notch2, Jagged1, and Jagged2. It was particularly interesting to look at the Notch1 and Notch2 receptors in the large and complex immune system of the gut because of their key roles in T- and B-cell function elsewhere in the body. These analyses revealed discrete or overlapping expression, depending on the region and cell type (summarized in Table 1).
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Esophagus and Forestomach
Notch1, Notch2, Jagged1, and Jagged2 were all expressed in the stratified squamous epithelium of the esophagus and forestomach (Figure 2). In general, expression was highest in the proliferative basal layer, particularly noticeable for Notch1 and Jagged2. With all probes some expression was evident in suprabasal cells, although it was decreased compared with the activity in basal cells. Rat forestomach has a keratinized architecture similar to that of the esophagus and displayed similar expression patterns (data not shown).
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Intestine
Notch1, Notch2, Jagged1, and Jagged2 showed a mixture of overlapping and spatially different expression patterns in the small intestine (Figure 3). Results were identical in the duodenum, jejunum, and ileum. Notch1 (Figures 3A and 3B), Jagged1 (Figures 3N and 3O), and Jagged2 (Figures 3R–3U) were expressed in the lower third of all crypts, a region that contains the proliferative cells, as shown in Figure 3E by the proliferation marker histone H3. In striking contrast, Notch2 was expressed in only a few crypt cells (Figures 3J and 3K). Notch1 was also abundantly expressed in the lamina propria (Figure 3B, arrows). In the colon, Notch1 (Figures 4A and 4B) and Jagged1 (Figures 4E and 4F) were abundantly expressed in the proliferative region located in the mid-third of the crypts (compare with proliferative marker in Figure 4K), whereas Notch2 was absent (Figures 4C and 4D). Jagged2 appeared to be more uniformly expressed in the crypt. Notch1, Notch2, and Jagged2 were detected in colon lamina propria cells, whereas Jagged1 appeared to be absent. Although Notch1 and Notch2 were expressed in most lamina propria cells, Jagged2 was detected mainly in cells located beneath surface colonocytes (Figures 4G and 4H, arrows).
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Peyer's Patches
Of the receptors and ligands studied, only Notch1 and Notch2 were detected in lymphocytes of the Peyer's patches; Jagged1 and Jagged2 were absent (Figure 5). Notch1 was expressed at a low level in Peyer's patches compared with its expression in adjacent intestinal crypts, and appeared to be localized to coronal cells in the upper region of the patch (Figures 5A–5D). In contrast, Notch2 expression was clearly evident throughout Peyer's patches, in the germinal center, and in the corona (Figures 5G and 5H). Jagged1 was not detected in Peyer's patch lymphocytes but was expressed in cells associated with high endothelial venules (Figures 5I–5L). Whether these are isolated endothelial cells or trafficking lymphocytes is not clear.
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Blood Vessels
Notch1, Jagged1, and Jagged2, but not Notch2, were expressed in small muscular arteries and arterioles throughout the GI tract but not in veins (Figure 6). Notch1 (Figures 6A and 6B) and Jagged2 (Figures 6E–6J) were restricted to endothelial cells and were not detected in the muscular wall surrounding arteries, unlike Jagged1 (Figures 6C and 6D). Notch1, Jagged1 (data not shown), and Jagged2 expression was also detected in capillary-like structures in the middle of mucosal folds in the colon (Figures 6E and 6F).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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We have shown that the receptors Notch1, Notch2, and Notch3 and the ligands Delta1, Jagged1, and Jagged2 express mRNA transcripts throughout the adult GI tract in diverse cell types. Our ISH analyses revealed novel expression data for Notch1, Notch2, Jagged1, and Jagged2 in the mucosal immune system and in epithelial cells throughout the adult rat gut. Absence of the Jagged ligands from Peyer's patches of the immune system indicate that delta family ligands may act there. These data provide a basis for specific studies on the downstream targets and modifiers of the Notch pathway in the gut.
Recently, Schroder and Gossler (2002) described the expression of Notch pathway genes in mouse small intestine during embryonic development and in the young adult. Our data extend their studies to the esophagus, stomach, and colon and provide enhanced definition of the expression zones. Our data in the small intestine are in general agreement with those of Schroder and Gossler (2002), although we found Notch1 and Jagged1 expression to be more expansive. We observed Jagged1 to be expressed in most if not all crypt cells rather than just a few cells, and we found Notch1 to be very abundant in the crypt and present in many villous mesenchymal cells rather than a few scattered cells. We attribute these differences to the superior sensitivity of radioactive ISH but note that species (rat vs mouse) and age (mature vs young adult) differences may impact, too. In the current study, expression of Notch1, Jagged1, and Jagged2 (and Notch2 in the esophagus) in various proliferative compartments of the GI tract suggests an important role in sustaining populations of gut cells in an undifferentiated state, a classic function of Notch signaling.
In the intestinal crypts, four cell types arise from the stem cells, i.e., enterocytes, goblet, enteroendocrine, and Paneth cells. The recent report by Yang et al. (2001) provides evidence for a role for the Notch pathway in early determination of their fate. In knocking out Math1, the secretory cell types such as goblet, enteroendocrine, and Paneth cells were abolished but enterocyte development was unaffected. Because Math1 can be negatively regulated by the Notch downstream effector Hes (Kageyama et al. 1997), one would predict that Notch and Math1 expression might be inversely correlated in the crypts. This is supported by our data from colon crypts that show high Notch1 expression in the proliferative zone and low expression in the non-proliferative bottom third of the crypts where goblet cells predominate, the opposite of Math1 expression (Yang et al. 2001). In striking contrast to ubiquitous Notch1 expression in intestinal crypts, Notch2 was absent from colon crypts and was restricted to a few as yet uncharacterized small intestinal crypt cells. This tends to implicate Notch1 involvement, and to exclude Notch2, in blocking the specification of secretory cell types in favor of enterocyte formation. A more specialized role for Notch2 is predicted, perhaps in the specification of Paneth cells, which are the only cells of the three secretory types expressed in the small intestine but not the large intestine.
Whereas a role for Notch1 in T-cell development and function in the bone marrow and thymus is becoming clear (Pui et al. 1999; Radtke et al. 1999; Wilson et al. 2000), the role of Notch signaling in the peripheral immune system is largely unexplored (Hoyne et al. 2001). We have shown that Notch1, Notch2, Jagged1, and Jagged2 are differentially expressed in the inductive (Peyer's patch) and effector (lamina propria) compartments of the gut mucosal immune system, indicating that they probably have a wider role in immune function than previously recognized. That Notch1 appears to be more restricted to the coronal cells of the Peyer's patch, which are typically of T-cell origin, is consistent with the capability of Notch1 to promote development of T-cells from bipotential T/B precursors (Pui et al. 1999; Radtke et al. 1999). In contrast, Notch2 is found throughout the predominantly B-cell region of Peyer's patches, suggesting a fundamental role in B-cell function. To gain further insight into Notch function in the peripheral immune system, it will be important to identify the immune cell types resident in the gut that are actively engaged in Notch signaling, their repertoire of Notch receptors and ligands, and what the functional consequences of that signaling are.
It is clear from this account of the distribution of Notch receptors and ligands in adult gut tissue that the Notch signaling pathway is likely to have diverse roles in the maintenance of normal gut function.
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Acknowledgments |
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Supported in part by a Cooperative Research Centre grant from the Australian Government.
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Footnotes |
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Received for publication September 17, 2003; accepted December 17, 2003
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