利用谱系追踪方法探究Lgr5在胰腺组织及其类器官中的表达

doi:10.16288/j.yczz.22-022

遗传 ›› 2022, Vol. 44 ›› Issue (5): 432-441.doi: 10.16288/j.yczz.22-022

利用谱系追踪方法探究Lgr5在胰腺组织及其类器官中的表达

闫炳儒¹(), 艾显辉¹, 刘淼¹, 田丽红², 梁洋¹, 滕春波¹()

1. 东北林业大学生命科学学院,哈尔滨 150040
2. 东北林业大学野生动物保护与自然保护地学院,哈尔滨 150040

收稿日期:2022-01-26 修回日期:2022-03-26 出版日期:2022-05-20 发布日期:2022-04-18
通讯作者: 滕春波 E-mail:heylr5553@163.com;chunboteng@nefu.edu.cn
作者简介:闫炳儒,在读硕士研究生,专业方向：生物学。E-mail: heylr5553@163.com
基金资助:
国家自然科学基金面上项目资助(32072801)

Study on Lgr5 expression in pancreas tissues and organoids by lineage tracing

Bingru Yan¹(), Xianhui Ai¹, Miao Liu¹, Lihong Tian², Yang Liang¹, Chun-Bo Teng¹()

1. College of Life Science, Northeast Forestry University, Harbin 150040, China
2. College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China

Received:2022-01-26 Revised:2022-03-26 Online:2022-05-20 Published:2022-04-18
Contact: Teng Chun-Bo E-mail:heylr5553@163.com;chunboteng@nefu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(32072801)

摘要/Abstract

摘要：

富含亮氨酸重复序列G蛋白偶联受体5 (leucine-rich repeat containing G protein-coupled receptor 5, Lgr5)在体内分布广泛,可以作为多种上皮组织(包括小肠、结肠、胃和毛囊)中干细胞的标记物。为了探究小鼠(Mus musculus)胰腺发育过程中导管上皮细胞及体外培养的胰腺导管类器官中Lgr5的表达情况,本研究利用Lgr5-Cre^ERT2+/-和Rosa26-mTmG杂交后的转基因小鼠,经Tamoxifen (他莫昔芬)诱导后,观察不同发育阶段胰腺组织切片的荧光表达情况,并通过三维培养建立成体小鼠胰腺导管类器官,观察诱导后类器官细胞中的荧光变化。结果显示：Tamoxifen诱导的正常成体转基因小鼠胰腺导管内未检测到表达Lgr5的细胞;通过对孕鼠及哺乳母鼠注射Tamoxifen,在胚胎发育15.5 d和新生小鼠胰腺中也未发现Lgr5阳性细胞;但是将4-hydroxy- Tamoxifen (4-羟基-他莫昔芬)添加到培养基中,在Lgr5-Cre^ERT2+/-;Rosa26-mTmG转基因小鼠胰腺导管来源的类器官中检测到部分细胞表达Lgr5。本研究结果证实,在成体及胚胎胰腺组织中没有检测到Lgr5表达,但在体外培养的胰腺导管类器官细胞中检测到Lgr5表达。本研究为探索胰腺发育过程中干/祖细胞特异性表达基因奠定了基础。

关键词: 胰腺, 导管, Lgr5, 类器官, 谱系追踪

Abstract:

Leucine rich repeat containing G protein-coupled receptor 5(Lgr5) is widely expressed in multiple tissues and can be used as a stem cell marker in a variety of epithelial organs (including the small intestine, colon, stomach and hair follicles). In this study, we used Lgr5-Cre^ERT2+/- and Rosa26-mTmG hybridized transgenic mice to investigate the expression of Lgr5 in both ductal epithelial cells during pancreas development and in vitro cultured pancreatic duct organoids. After induction with Tamoxifen, the Lgr5 expression was analyzed by detecting the enhanced green fluorescence protein in the pancreatic tissue sections in adult animals and embryos at different developmental stages. The results showed that Lgr5 expression was detected neither in adult pancreatic duct epithelia nor in the embryonic pancreatic tissues at day 15.5 or in newborn mice. However, when 4-hydroxy-Tamoxifen was supplemented to the culture medium, EGFP could be detected in the primary pancreatic duct organoids from Lgr5-Cre ^ERT2+/-; Rosa26-mTmG mice. These results suggested that Lgr5 was not expressed in adult and embryonic pancreatic tissues; but could be expressed in the cultured pancreas ductal organoids. The research lays the foundation for exploring specific gene expression patterns in stem/progenitor cells during pancreatic development.

Key words: pancreas, duct, Lgr5, organoid, lineage tracing

闫炳儒, 艾显辉, 刘淼, 田丽红, 梁洋, 滕春波. 利用谱系追踪方法探究Lgr5在胰腺组织及其类器官中的表达[J]. 遗传, 2022, 44(5): 432-441.

Bingru Yan, Xianhui Ai, Miao Liu, Lihong Tian, Yang Liang, Chun-Bo Teng. Study on Lgr5 expression in pancreas tissues and organoids by lineage tracing[J]. Hereditas(Beijing), 2022, 44(5): 432-441.

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参考文献 28

[1]	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. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature, 2007, 449(7165):1003-1007. doi: 10.1038/nature06196
[2]	Koo BK, Clevers H. Stem cells marked by the R-spondin receptor LGR5. Gastroenterology, 2014, 147(2):289-302. doi: 10.1053/j.gastro.2014.05.007
[3]	Nagano K. R-spondin signaling as a pivotal regulator of tissue development and homeostasis. Jpn Dent Sci Rev, 2019, 55(1):80-87. doi: 10.1016/j.jdsr.2019.03.001
[4]	Cardinale V, Wang YF, Carpino G, Mendel G, Alpini G, Gaudio E, Reid LM, Alvaro D. The biliary tree--a reservoir of multipotent stem cells. Nat Rev Gastroenterol Hepatol, 2012, 9(4):231-240. doi: 10.1038/nrgastro.2012.23 pmid: 22371217
[5]	Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas. Science, 2008, 322(5907):1490-1494. doi: 10.1126/science.1161431
[6]	Slack JM. Developmental biology of the pancreas. Development, 1995, 121(6):1569-1580. pmid: 7600975
[7]	Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development, 2002, 129(10):2447-2457. doi: 10.1242/dev.129.10.2447
[8]	Wang YF, Lanzoni G, Carpino G, Cui CB, Dominguez- Bendala J, Wauthier E, Cardinale V, Oikawa T, Pileggi A, Gerber D, Furth ME, Alvaro D, Gaudio E, Inverardi L, Reid LM. Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis. Stem Cells, 2013, 31(9):1966-1979. doi: 10.1002/stem.1460
[9]	Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MMA, Ellis E, van Wenum M, Fuchs SA, de Ligt J, van de Wetering M, Sasaki N, Boers SJ, Kemperman H, de Jonge J, Ijzermans JN, Nieuwenhuis EE, Hoekstra R, Strom S, Vries RR, van der Laan LJ, Cuppen E, Clevers H. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell, 2015, 160(1-2):299-312. doi: 10.1016/j.cell.2014.11.050
[10]	Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng HK. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci, 2010, 13(1):133-140. doi: 10.1038/nn.2467 pmid: 20023653
[11]	Sohal DS, Nghiem M, Crackower MA, Witt SA, Kimball TR, Tymitz KM, Penninger JM, Molkentin JD. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ Res, 2001, 89(1):20-25. pmid: 11440973
[12]	Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T, Stange DE, Begthel H, van den Born M, Danenberg E, van den Brink S, Korving J, Abo A, Peters PJ, Wright N, Poulsom R, Clevers H. Lgr5^+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell, 2010, 6(1):25-36. doi: 10.1016/j.stem.2009.11.013 pmid: 20085740
[13]	Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgård R. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet, 2008, 40(11):1291-1299. doi: 10.1038/ng.239
[14]	Aguayo-Mazzucato C, Andle J, Lee TB Jr, Midha A, Talemal L, Chipashvili V, Hollister-Lock J, van Deursen J, Weir G, Bonner-Weir S. Acceleration of β cell aging determines diabetes and senolysis improves disease outcomes. Cell Metab, 2019, 30(1): 129-142.e4. doi: S1550-4131(19)30246-3 pmid: 31155496
[15]	Rezanejad H, Lock JH, Sullivan BA, Bonner-Weir S. Generation of pancreatic ductal organoids and whole-mount immunostaining of intact organoids. Curr Protoc Cell Biol, 2019, 83(1):e82.
[16]	Ho BX, Pek NMQ, Soh BS. Disease modeling using 3D organoids derived from human induced pluripotent stem cells. Int J Mol Sci, 2018, 19(4):936. doi: 10.3390/ijms19040936
[17]	Pan FC, Evans T, Chen SB. Modeling endodermal organ development and diseases using human pluripotent stem cell-derived organoids. J Mol Cell Biol, 2020, 12(8):580-592. doi: 10.1093/jmcb/mjaa031
[18]	Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo LQ. A global double-fluorescent Cre reporter mouse. Genesis, 2007, 45(9):593-605. doi: 10.1002/dvg.20335 pmid: 17868096
[19]	Kretzschmar K, Watt FM. Lineage tracing. Cell, 2012, 148(1-2):33-45 doi: 10.1016/j.cell.2012.01.002 pmid: 22265400
[20]	Hoffman RM. Recent advances on in vivo imaging with fluorescent proteins. Methods Cell Biol, 2008, 85:485-495.
[21]	Vintersten K, Monetti C, Gertsenstein M, Zhang PZ, Laszlo L, Biechele S, Nagy A. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis, 2004, 40(4):241-246. doi: 10.1002/gene.20095 pmid: 15593332
[22]	Megason SG, Fraser SE. Digitizing life at the level of the cell: high-performance laser-scanning microscopy and image analysis for in toto imaging of development. Mech Dev, 2003, 120(11):1407-1420. doi: 10.1016/j.mod.2003.07.005
[23]	Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett, 1997, 407(3):313-319. doi: 10.1016/s0014-5793(97)00313-x pmid: 9175875
[24]	Snippert HJ, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, Clevers H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell, 2010, 143(1):134-144. doi: 10.1016/j.cell.2010.09.016 pmid: 20887898
[25]	Laugwitz KL, Moretti A, Lam J, Gruber P, Chen YH, Woodard S, Lin LZ, Cai CL, Lu MM, Reth M, Platoshyn O, Yuan JX, Evans S, Chien KR. Postnatal isl1 + cardioblasts enter fully differentiated cardiomyocyte lineages. Nature, 2005, 433(7026):647-653. doi: 10.1038/nature03215
[26]	Huch M, Bonfanti P, Boj SF, Sato T, Loomans CJM, van de Wetering M, Sojoodi M, Li VSW, Schuijers J, Gracanin A, Ringnalda F, Begthel H, Hamer K, Mulder J, van Es JH, de Koning E, Vries RG, Heimberg H, Clevers H. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J, 2013, 32(20):2708-2721. doi: 10.1038/emboj.2013.204
[27]	Huch M, Dorrell C, Boj SF, van Es JH, Li VSW, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature, 2013, 494(7436):247-250. doi: 10.1038/nature11826
[28]	Rodriguez UA, Socorro M, Criscimanna A, Martins CP, Mohamed N, Hu J, Prasadan K, Gittes GK, Esni F. Conversion of α-cells to β-cells in the postpartum mouse pancreas involves Lgr5 progeny. Diabetes, 2021, 70(7):1508-1518. doi: 10.2337/db20-1059 pmid: 33906911

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基因名称	引物序列(5′→3′)
GAPDH	F：CACGGCAAATTCAACGGCACAGTCAAGG
GAPDH	R：GTTCACACCCATCACAAACATGG
Cre	F：GCCTGCATTACCGGTCGATGC
Cre	R：TCCCACCGTCAGTACGTGAGATA
mTmG	F：CTCTGCTGCCTCCTGGCTTCT
	R1：CGAGGCGGATCACAAGCAATA
	R2：TCAATGGGCGGGGGTCGTT
Lgr5	F：CTGCTCTCTGCTCCCAGTCT
	R1：ATACCCCATCCCTTTTGAGC
	R2：GAACTTCAGGGTCAGCTTGC

利用谱系追踪方法探究Lgr5在胰腺组织及其类器官中的表达

Study on Lgr5 expression in pancreas tissues and organoids by lineage tracing

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