Reports have suggested that the two Notch ligands, Dll1 and Dll4, are indispensable to maintain the homeostasis of the intestinal epithelium. However, within the intestinal epithelium, the precise distribution of the cells that express those ligands at the protein level remains largely unknown. Here, we show a series of immunohistochemical analysis through which we successfully identified mice intestinal epithelial cells (IECs) that endogenously express Dll1 or Dll4. Results showed that Dll1-positive (Dll1+ve) IECs reside exclusively within the crypt, whereas Dll4-positive (Dll4+ve) IECs can locate both in the crypt and in the villus of the small intestine. Also in the colon, Dll1+ve IECs resided at the lower part of the crypt, whereas Dll4+ve IECs resided at both upper and lower part of the crypt, including the surface epithelium. Both Dll1+ve and Dll4+ve IECs were ATOH1-positive, but Hes1-negative cells, and located adjacent to Hes1-positive cells within the crypts. A sub-population of both Dll1+ve and Dll4+ve IECs appeared to co-express Muc2, but rarely co-expressed other secretory lineage markers. However, as compared to Dll1+ve IECs, Dll4+ve IECs included larger number of Muc2-postive IECs, suggesting that Dll4 is more preferentially expressed by goblet cells. Also, we identified that Dll4 is expressed in the Paneth cells of the small intestine, whereas Dll1 and Dll4 is expressed in the c-kit-positive IECs of the colon, indicating that Dll1+ve and Dll4+ve IECs may contribute to constitute the intestinal stem cell niche. Compared to the normal colon, analysis of DSS-colitis showed that number of Dll1+ve IECs significantly decrease in the elongated crypts of the inflamed colonic mucosa. In sharp contrast, number of Dll4+ve IECs showed a significant increase in those crypts, which was accompanied by the increase in number of Hes1-positive IECs. Those Dll4+ve IECs were mostly found adjacent to the Hes1-positive IECs, suggesting that Dll4 may act as a major Notch ligand in the crypts of the inflamed colonic mucosa. Our results illustrate distinct expression patterns of Dll1 and Dll4 within the intestinal epithelium, and suggest that these two ligands may have different roles in normal and inflamed mucosa.
References
[1]
Artavanis-Tsakonas S, Rand MD, Lake RJ. 1999. Notch signaling: cell fate control and signal integration in development. Science 284(5415):770-776
[2]
Barker N. 2013. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nature Reviews Molecular Cell Biology 15(1):19-33
[3]
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003-1007
[4]
Clevers H. 2013. The intestinal crypt, a prototype stem cell compartment. Cell 154(2):274-284
[5]
Crosnier C, Stamataki D, Lewis J. 2006. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nature Reviews Genetics 7(5):349-359
[6]
Crosnier C, Vargesson N, Gschmeissner S, Ariza-McNaughton L, Morrison A, Lewis J. 2005. Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine. Development 132(5):1093-1104
[7]
Hori K, Sen A, Artavanis-Tsakonas S. 2013. Notch signaling at a glance. Journal of Cell Science 126(10):2135-2140
[8]
Hozumi K, Mailhos C, Negishi N, Hirano KI, Yahata T, Ando K, Zuklys S, Hollander GA, Shima DT, Habu S. 2008. Delta-like 4 is indispensable in thymic environment specific for T cell development. Journal of Experimental Medicine 205(11):2507-2513
[9]
Hozumi K, Negishi N, Suzuki D, Abe N, Sotomaru Y, Tamaoki N, Mailhos C, Ish-Horowicz D, Habu S, Owen MJ. 2004. Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo. Nature Immunology 5(6):638-644
[10]
Kim T-H, Li F, Ferreiro-Neira I, Ho L-L, Luyten A, Nalapareddy K, Long H, Verzi M, Shivdasani RA. 2014. Broadly permissive intestin al chromatin underlies lateral inhibition and cell plasticity. Nature 506(7489):511-515
[11]
Nakamura T, Tsuchiya K, Watanabe M. 2007. Crosstalk between Wnt and Notch signaling in intestinal epithelial cell fate decision. Journal of Gastroenterology 42(9):705-710
[12]
Okamoto R, Tsuchiya K, Nemoto Y, Akiyama J, Nakamura T, Kanai T, Watanabe M. 2009. Requirement of Notch activation during regeneration of the intestinal epithelia. American Journal of Physiology - Gastrointestinal and Liver Physiology 296(1):G23-G35
[13]
Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. 1990. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98(3):694-702
[14]
Pellegrinet L, Rodilla V, Liu Z, Chen S, Koch U, Espinosa L, Kaestner KH, Kopan R, Lewis J, Radtke F. 2011. Dll1- and Dll4-mediated notch signaling are required for homeostasis of intestinal stem cells. Gastroenterology 140(4):1230-1240
[15]
Riccio O, van Gijn ME, Bezdek AC, Pellegrinet L, van Es JH, Zimber-Strobl U, Strobl LJ, Honjo T, Clevers H, Radtke F. 2008. Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2. EMBO Reports 9(4):377-383
[16]
Ridgway J, Zhang G, Wu Y, Stawicki S, Liang W-C, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I, Singh M, Chien M, Tan C, Hongo Jo-Anne S, de Sauvage F, Plowman G, Yan M. 2006. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444(7122):1083-1087
[17]
Rocha SF, Lopes SS, Gossler A, Henrique D. 2009. Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity. Developmental Biology 328(1):54-65
[18]
Rothenberg ME, Nusse Y, Kalisky T, Lee JJ, Dalerba P, Scheeren F, Lobo N, Kulkarni S, Sim S, Qian D, Beachy PA, Pasricha PJ, Quake SR, Clarke MF. 2012. Identification of a cKit+ Colonic Crypt Base Secretory Cell That Supports Lgr5+ Stem Cells in Mice. Gastroenterology 142(5):1195-1205
[19]
Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H. 2012. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469(7330):415-418
[20]
Schrder N, Gossler A. 2002. Gene Expression Patterns 2(3–4):247-250
[21]
Stamataki D, Holder M, Hodgetts C, Jeffery R, Nye E, Spencer-Dene B, Winton DJ, Lewis J. 2011. Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system. PLoS ONE 6(9):e24484
[22]
van Den Brink GR, de Santa Barbara P, Roberts DJ. 2001. Epithelial cell differentiation–a Mather of choice. Science 294(5549):2115-2116
[23]
VanDussen KL, Carulli AJ, Keeley TM, Patel SR, Puthoff BJ, Magness ST, Tran IT, Maillard I, Siebel C, Kolterud , Grosse AS, Gumucio DL, Ernst SA, Tsai Y-H, Dempsey PJ, Samuelson LC. 2012. Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development 139(3):488-497
[24]
van Es JH, Sato T, van de Wetering M, Lyubimova A, Nee ANY, Gregorieff A, Sasaki N, Zeinstra L, van den Born M, Korving J, Martens ACM, Barker N, van Oudenaarden A, Clevers H. 2012. Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nature Cell Biology 14(10):1099-1104
[25]
Vooijs M, Liu Z, Kopan R. 2011. Notch: architect, landscaper, and guardian of the intestine. Gastroenterology 141(2):448-459
[26]
Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M. 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nature Medicine 18(4):618-623
[27]
Zheng X, Tsuchiya K, Okamoto R, Iwasaki M, Kano Y, Sakamoto N, Nakamura T, Watanabe M. 2011. Suppression of hath1 gene expression directly regulated by hes1 Via notch signaling is associated with goblet cell depletion in ulcerative colitis. Inflammatory Bowel Diseases 17(11):2251-2260
