Abstract
The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.
[1] Huizinga, J.D., Thuneberg, L., Kluppel, M., Malysz, J., Mikkelsen, H.B. and Bernstein, A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373 (1995) 347–349. DOI: 10.1038/373347a0. http://dx.doi.org/10.1038/373347a010.1038/373347a0Search in Google Scholar
[2] Koh, S.D., Sanders, K.M. and Ward, S.M. Spontaneous electrical rhythmicity in cultured interstitial cells of cajal from the murine small intestine. J. Physiol. 513(Pt 1) (1998) 203–213. http://dx.doi.org/10.1111/j.1469-7793.1998.203by.x10.1111/j.1469-7793.1998.203by.xSearch in Google Scholar
[3] Thomsen, L., Robinson, T.L., Lee, J.C., Farraway, L.A., Hughes, M.J., Andrews, D.W. and Huizinga, J.D. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat. Med. 4 (1998) 848–851. http://dx.doi.org/10.1038/nm0798-84810.1038/nm0798-848Search in Google Scholar
[4] Koh, S.D., Ward, S.M., Ordog, T., Sanders, K.M. and Horowitz, B. Conductances responsible for slow wave generation and propagation in interstitial cells of Cajal. Curr. Opin. Pharmacol. 3 (2003) 579–582. http://dx.doi.org/10.1016/j.coph.2003.09.00210.1016/j.coph.2003.09.002Search in Google Scholar
[5] Sanders, K.M., Koh, S.D. and Ward, S.M. Interstitial cells of cajal as pacemakers in the gastrointestinal tract. Annu. Rev. Physiol. 68 (2006) 307–343. DOI: 10.1146/annurev.physiol.68.040504.094718. http://dx.doi.org/10.1146/annurev.physiol.68.040504.09471810.1146/annurev.physiol.68.040504.094718Search in Google Scholar
[6] Huizinga, J.D., Berezin, I., Chorneyko, K., Thuneberg, L., Sircar, K., Hewlett, B.R. and Riddell, R.H. Interstitial cells of Cajal: pacemaker cells? Am. J. Pathol. 153 (1998) 2008–2011. http://dx.doi.org/10.1016/S0002-9440(10)65715-X10.1016/S0002-9440(10)65715-XSearch in Google Scholar
[7] Huizinga, J.D. Gastrointestinal peristalsis: joint action of enteric nerves, smooth muscle, and interstitial cells of Cajal. Microsc. Res. Tech. 47 (1999) 239–247. DOI: 10.1002/(SICI)1097-0029(19991115)47:4 〈 239::AID-JEMT3 〉 3.0.CO;2-0. http://dx.doi.org/10.1002/(SICI)1097-0029(19991115)47:4<239::AID-JEMT3>3.0.CO;2-010.1002/(SICI)1097-0029(19991115)47:4<239::AID-JEMT3>3.0.CO;2-0Search in Google Scholar
[8] Huizinga, J.D., Robinson, T.L. and Thomsen, L. The search for the origin of rhythmicity in intestinal contraction; from tissue to single cells. Neurogastroenterol. Motil. 12 (2000) 3–9. http://dx.doi.org/10.1046/j.1365-2982.2000.00177.x10.1046/j.1365-2982.2000.00177.xSearch in Google Scholar
[9] Chen, H., Ordog, T., Chen, J., Young, D.L., Bardsley, M.R., Redelman, D., Ward, S.M. and Sanders, K.M. Differential gene expression in functional classes of interstitial cells of Cajal in murine small intestine. Physiol. Genomics 31 (2007) 492–509. DOI: 10.1152/physiolgenomics.00113.2007. http://dx.doi.org/10.1152/physiolgenomics.00113.200710.1152/physiolgenomics.00113.2007Search in Google Scholar
[10] Zhu, M.H., Kim, T.W., Ro, S., Yan, W., Ward, S.M., Koh, S.D. and Sanders, K.M. A Ca(2+)-activated Cl(-) conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity. J. Physiol. 587 (2009) 4905–4918. DOI: 10.1113/jphysiol.2009.176206. http://dx.doi.org/10.1113/jphysiol.2009.17620610.1113/jphysiol.2009.176206Search in Google Scholar
[11] Sanders, K.M., Zhu, M.H., Britton, F., Koh, S.D. and Ward, S.M. Anoctamins and gastrointestinal smooth muscle excitability. Exp. Physiol. 97 (2012) 200–206. DOI: 10.1113/expphysiol.2011.058248. http://dx.doi.org/10.1113/expphysiol.2011.05824810.1113/expphysiol.2011.058248Search in Google Scholar PubMed PubMed Central
[12] Zhu, M.H., Sung, I.K., Zheng, H., Sung, T.S., Britton, F.C., O’Driscoll, K., Koh, S.D. and Sanders, K.M. Muscarinic activation of Ca2+-activated Cl-current in interstitial cells of Cajal. J. Physiol. 589 (2011) 4565–4582. DOI: 10.1113/jphysiol.2011.211094. http://dx.doi.org/10.1113/jphysiol.2011.21109410.1113/jphysiol.2011.211094Search in Google Scholar PubMed PubMed Central
[13] Eberwine, J. Single-cell molecular biology. Nat. Neurosci. 4Suppl (2001) 1155–1156. DOI: 10.1038/nn1101-1155. http://dx.doi.org/10.1038/nn1101-115510.1038/nn1101-1155Search in Google Scholar PubMed
[14] Takeda, Y., Koh, S.D., Sanders, K.M. and Ward, S.M. Differential expression of ionic conductances in interstitial cells of Cajal in the murine gastric antrum. J. Physiol. 586 (2008) 859–873. DOI: 10.1113/jphysiol.2007.140293. http://dx.doi.org/10.1113/jphysiol.2007.14029310.1113/jphysiol.2007.140293Search in Google Scholar PubMed PubMed Central
[15] Wouters, M.M., Gibbons, S.J., Roeder, J.L., Distad, M., Ou, Y., Strege, P.R., Szurszewski, J.H. and Farrugia, G. Exogenous serotonin regulates proliferation of interstitial cells of Cajal in mouse jejunum through 5-HT2B receptors. Gastroenterology 133 (2007) 897–906. DOI: 10.1053/j.gastro.2007.06.017. http://dx.doi.org/10.1053/j.gastro.2007.06.01710.1053/j.gastro.2007.06.017Search in Google Scholar PubMed
[16] Li, H.H., Gyllensten, U.B., Cui, X.F., Saiki, R.K., Erlich, H.A. and Arnheim, N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335 (1988) 414–417. DOI: 10.1038/335414a0. http://dx.doi.org/10.1038/335414a010.1038/335414a0Search in Google Scholar PubMed
[17] Wang, B., Kunze, W.A., Zhu, Y. and Huizinga, J.D. In situ recording from gut pacemaker cells. Pflugers Arch. 457 (2008) 243–251. DOI: 10.1007/s00424-008-0513-6. http://dx.doi.org/10.1007/s00424-008-0513-610.1007/s00424-008-0513-6Search in Google Scholar PubMed
[18] Parsons, S.P., Kunze, W.A. and Huizinga, J.D. Maxi-channels recorded in situ from ICC and pericytes associated with the mouse myenteric plexus. Am. J. Physiol. Cell Physiol. 302 (2012) C1055–1069. DOI: 10.1152/ajpcell.00334.2011. http://dx.doi.org/10.1152/ajpcell.00334.201110.1152/ajpcell.00334.2011Search in Google Scholar PubMed
[19] Dubois, C.M., Ruscetti, F.W., Stankova, J. and Keller, J.R. Transforming growth factor-beta regulates c-kit message stability and cell-surface protein expression in hematopoietic progenitors. Blood 83 (1994) 3138–3145. Search in Google Scholar
[20] Gomez-Pinilla, P.J., Gibbons, S.J., Bardsley, M.R., Lorincz, A., Pozo, M.J., Pasricha, P.J., Van de Rijn, M., West, R.B., Sarr, M.G., Kendrick, M.L., Cima, R.R., Dozois, E.J., Larson, D.W., Ordog, T. and Farrugia, G. Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract. Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G1370–1381. DOI: 10.1152/ajpgi.00074.2009. http://dx.doi.org/10.1152/ajpgi.00074.200910.1152/ajpgi.00074.2009Search in Google Scholar PubMed PubMed Central
[21] Kashyap, P., Gomez-Pinilla, P.J., Pozo, M.J., Cima, R.R., Dozois, E.J., Larson, D.W., Ordog, T., Gibbons, S.J. and Farrugia, G. Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation. Neurogastroenterol. Motil. 23 (2011) 760–765. DOI: 10.1111/j.1365-2982.2011.01729.x. http://dx.doi.org/10.1111/j.1365-2982.2011.01729.x10.1111/j.1365-2982.2011.01729.xSearch in Google Scholar PubMed PubMed Central
[22] Parsons, S.P. and Huizinga, J.D. Transient outward potassium current in ICC. Am. J. Physiol. Gastrointest. Liver Physiol. 298 (2010) G456–466. DOI: 10.1152/ajpgi.00340.2009. http://dx.doi.org/10.1152/ajpgi.00340.200910.1152/ajpgi.00340.2009Search in Google Scholar
[23] Wright, G.W., Parsons, S.P. and Huizinga, J.D. Ca2+ sensitivity of the maxi chloride channel in interstitial cells of Cajal. Neurogastroenterol. Motil. 24 (2012) e221–234. DOI: 10.1111/j.1365-2982.2012.01881.x. http://dx.doi.org/10.1111/j.1365-2982.2012.01881.x10.1111/j.1365-2982.2012.01881.xSearch in Google Scholar
[24] Phillips, J.K. and Lipski, J. Single-cell RT-PCR as a tool to study gene expression in central and peripheral autonomic neurones. Auton. Neurosci. 86 (2000) 1–12. DOI: 10.1016/S1566-0702(00)00245-9. http://dx.doi.org/10.1016/S1566-0702(00)00245-910.1016/S1566-0702(00)00245-9Search in Google Scholar
[25] Hwang, S.J., Blair, P.J., Britton, F.C., O’Driscoll, K.E., Hennig, G., Bayguinov, Y.R., Rock, J.R., Harfe, B.D., Sanders, K.M. and Ward, S.M. Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles. J. Physiol. 587 (2009) 4887–4904. DOI: 10.1113/jphysiol.2009.176198. http://dx.doi.org/10.1113/jphysiol.2009.17619810.1113/jphysiol.2009.176198Search in Google Scholar
[26] Espinosa-Luna, R., Collins, S.M., Montano, L.M. and Barajas-Lopez, C. Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine. Can. J. Physiol. Pharmacol. 77 (1999) 598–605. http://dx.doi.org/10.1139/y99-05710.1139/y99-057Search in Google Scholar
[27] Okada, Y., Shimizu, T., Maeno, E., Tanabe, S., Wang, X. and Takahashi, N. Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J. Membr. Biol. 209 (2006) 21–29. DOI: 10.1007/s00232-005-0836-6. http://dx.doi.org/10.1007/s00232-005-0836-610.1007/s00232-005-0836-6Search in Google Scholar
[28] Shen, M.R., Yang, T.P. and Tang, M.J. A novel function of BCL-2 overexpression in regulatory volume decrease. Enhancing swelling-activated Ca(2+) entry and Cl(-) channel activity. J. Biol. Chem. 277 (2002) 15592–15599. DOI: 10.1074/jbc.M111043200. http://dx.doi.org/10.1074/jbc.M11104320010.1074/jbc.M111043200Search in Google Scholar
[29] Ponce, A., Jimenez-Pena, L. and Tejeda-Guzman, C. The role of swellingactivated chloride currents (I(CL,swell)) in the regulatory volume decrease response of freshly dissociated rat articular chondrocytes. Cell Physiol. Biochem. 30 (2012) 1254–1270. DOI: 10.1159/000343316. http://dx.doi.org/10.1159/00034331610.1159/000343316Search in Google Scholar
[30] West, R.B., Corless, C.L., Chen, X., Rubin, B.P., Subramanian, S., Montgomery, K., Zhu, S., Ball, C.A., Nielsen, T.O., Patel, R., Goldblum, J.R., Brown, P.O., Heinrich, M.C. and van de Rijn, M. The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am. J. Pathol. 165 (2004) 107–113. DOI: 10.1016/S0002-9440(10)63279-8. http://dx.doi.org/10.1016/S0002-9440(10)63279-810.1016/S0002-9440(10)63279-8Search in Google Scholar
[31] Robinson T.L., Sircar, K., Hewlett B.R., Chorneyko K., Riddell R.H. and Huizinga J.D. Gastrointestinal stromal tumors may originate from a subset of CD34-positive interstitial cells of Cajal. Am. J. Pathol. 156 (2000) 1157–1163. http://dx.doi.org/10.1016/S0002-9440(10)64984-X10.1016/S0002-9440(10)64984-XSearch in Google Scholar
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