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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Summary |
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(J Histochem Cytochem 52:509516, 2004)
Key Words: Notch Jagged Delta1 receptor ligand mucosal immune system
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Introduction |
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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
; ArtavanisTsakonas 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. Receptorligand 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 (ArtavanisTsakonas 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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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 SpragueDawley rat gut segments. PolyA+ RNA was isolated using the Poly-A Tract IV system (Promega; Madison, WI) and 12 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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Results |
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Discussion |
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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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Footnotes |
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Literature Cited |
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