Single-cell transcriptomic analysis of adult mouse pituitary reveals sexual dimorphism and physiologic demand-induced cellular plasticity

Abstract

The anterior pituitary gland drives highly conserved physiologic processes in mammalian species. These hormonally controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current cellular models of mammalian anteiror pituitary, largely built on candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex relationship between hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with complementary imaging-based single-cell analyses of mRNAs and proteins, to systematically map both cell-type diversity and functional state heterogeneity in adult male and female mouse pituitaries at single-cell resolution and in the context of major physiologic demands. These quantitative single-cell analyses reveal sex-specific cell-type composition under normal pituitary homeostasis, identify an array of cells associated with complex complements of hormone-enrichment, and undercover non-hormone producing interstitial and supporting cell-types. Interestingly, we also identified a Pou1f1-expressing cell population that is characterized by a unique multi-hormone gene expression profile. In response to two well-defined physiologic stresses, dynamic shifts in cellular diversity and transcriptome profiles were observed for major hormone producing and the putative multi-hormone cells. These studies reveal unanticipated cellular complexity and plasticity in adult pituitary, and provide a rich resource for further validating and expanding our molecular understanding of pituitary gene expression programs and hormone production.

Keywords

mouse pituitary / cellular plasticity / sexual dimorphism / single-cell RNA sequencing

References

[1] Andersen B, Pearse RV 2nd, Jenne K, Sornson M, Lin SC, Bartke A, Rosenfeld MG (1995) The Ames dwarf gene is required for Pit-1 gene activation. Dev Biol 172:495–503

[2] Andoniadou CL, Matsushima D, Mousavy Gharavy SN, Signore M, Mackintosh AI, Schaeffer M, Gaston-Massuet C, Mollard P, Jacques TS, Le Tissier P (2013) Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell 13:433–445

[3] Barbosa JA, Gill BM, Takiyyuddin MA, O’Connor DT (1991) Chromogranin A: posttranslational modifications in secretory granules. Endocrinology 128:174–190

[4] Beliveau BJ, Boettiger AN, Avendano MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J (2015) Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 6:7147

[5] Budry L, Balsalobre A, Gauthier Y, Khetchoumian K, L’Honore A, Vallette S, Brue T, Figarella-Branger D, Meij B, Drouin J (2012) The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling. Genes Dev 26:2299–2310

[6] Campbell JN, Macosko EZ, Fenselau H, Pers TH, Lyubetskaya A, Tenen D, Goldman M, Verstegen AM, Resch JM, McCarroll SA (2017) A molecular census of arcuate hypothalamus and median eminence cell types. Nat Neurosci 20:484–496

[7] Camper SA, Saunders TL, Katz RW, Reeves RH (1990) The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation. Genomics 8:586–590

[8] Cao D, Ma X, Cai J, Luan J, Liu AJ, Yang R, Cao Y, Zhu X, Zhang H, Chen YX et al (2016) ZBTB20 is required for anterior pituitary development and lactotrope specification. Nat Commun 7:11121

[9] Castrique E, Fernandez-Fuente M, Le Tissier P, Herman A, Levy A (2010) Use of a prolactin-Cre/ROSA-YFP transgenic mouse provides no evidence for lactotroph transdifferentiation after weaning, or increase in lactotroph/somatotroph proportion in lactation. J Endocrinol 205:49–60

[10] Cawley NX, Li Z, Loh YP (2016) 60 YEARS OF POMC: biosynthesis, trafficking, and secretion of pro-opiomelanocortin-derived peptides. J Mol Endocrinol 56:T77–97

[11] Chen R, Wu X, Jiang L, Zhang Y (2017) Single-cell RNA-seq reveals hypothalamic cell diversity. Cell Rep 18:3227–3241

[12] Cheung LYM, George AS, McGee SR, Daly AZ, Brinkmeier ML, Ellsworth BS, Camper SA (2018) Single-cell RNA sequencing reveals novel markers of male pituitary stem cells and hormoneproducing cell types. Endocrinology 159:3910–3924

[13] Childs GV (2000) Green fluorescent proteins light the way to a better understanding of the function and regulation of specific anterior pituitary cells. Endocrinology 141:4331–4333

[14] Davis SW, Ellsworth BS, Perez Millan MI, Gergics P, Schade V, Foyouzi N, Brinkmeier ML, Mortensen AH, Camper SA (2013) Pituitary gland development and disease: from stem cell to hormone production. Curr Top Dev Biol 106:1–47

[15] Davis SW, Keisler JL, Perez-Millan MI, Schade V, Camper SA (2016) All hormone-producing cell types of the pituitary intermediate and anterior lobes derive from Prop1-expressing progenitors. Endocrinology 157:1385–1396

[16] Devnath S, Inoue K (2008) An insight to pituitary folliculo-stellate cells. J Neuroendocrinol 20:687–691

[17] Durand D, Pampillo M, Caruso C, Lasaga M (2008) Role of metabotropic glutamate receptors in the control of neuroendocrine function. Neuropharmacology 55:577–583

[18] Fletcher PA, Smiljanic K, Maso Prévide R, Iben J, Li T, Rokic MB, Sherman A, Coon SL, Stojilkovic SS (2019) Cell type-and sexdependent transcriptome profiles of rat anterior pituitary cells. Front Endocrinol 10:623

[19] Frawley LS, Boockfor FR (1991) Mammosomatotropes: presence and functions in normal and neoplastic pituitary tissue. Endocr Rev 12:337–355

[20] Gage PJ, Brinkmeier ML, Scarlett LM, Knapp LT, Camper SA, Mahon KA (1996) The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation. Mol Endocrinol 10:1570–1581

[21] Gaylinn BD (2002) Growth hormone releasing hormone receptor. Recept Channels 8:155–162

[22] Gill BM, Barbosa JA, Dinh TQ, Garrod S, O’Connor DT (1991) Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing. Regul Pept 33:223–235

[23] Hammer RE, Brinster RL, Rosenfeld MG, Evans RM, Mayo KE (1985) Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315:413–416

[24] Han X, Wang R, Zhou Y, Fei L, Sun H, Lai S, Saadatpour A, Zhou Z, Chen H, Ye F (2018) Mapping the mouse cell atlas by microwell-seq. Cell 173:1307

[25] Ho Y, Elefant F, Cooke N, Liebhaber S (2002) A defined locus control region determinant links chromatin domain acetylation with longrange gene activation. Mol Cell 9:291–302

[26] Ho Y, Tadevosyan A, Liebhaber SA, Cooke NE (2008) The juxtaposition of a promoter with a locus control region transcriptional domain activates gene expression. EMBO Rep 9:891–898

[27] Ho Y, Liebhaber SA, Cooke NE (2011) The role of the hGH locus control region in somatotrope restriction of hGH-N gene expression. Mol Endocrinol 25(5):877–884

[28] Ho Y, Shewchuk BM, Liebhaber SA, Cooke NE (2013) Distinct chromatin configurations regulate the initiation and the maintenance of hGH gene expression. Mol Cell Biol 33:1723–1734

[29] Hu P, Fabyanic E, Kwon DY, Tang S, Zhou Z, Wu H (2017) Dissecting cell-type composition and activity-dependent transcriptional state in mammalian brains by massively parallel single-nucleus RNA-seq. Mol Cell 68:1006–1015

[30] Hu P, Liu J, Zhao J, Wilkins BJ, Lupino K, Wu H, Pei L (2018) Singlenucleus transcriptomic survey of cell diversity and functional maturation in postnatal mammalian hearts. Genes Dev 32:1344–1357

[31] Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL (1994) The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312

[32] Janknecht R (2010) Multi-talented DEAD-box proteins and potential tumor promoters: p68 RNA helicase (DDX5) and its paralog, p72 RNA helicase (DDX17). Am J Transl Res 2:223–234

[33] Kelberman D, Rizzoti K, Lovell-Badge R, Robinson IC, Dattani MT (2009) Genetic regulation of pituitary gland development in human and mouse. Endocr Rev 30:790–829

[34] Lamberts SW, Macleod RM (1990) Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279–318

[35] Lamolet B, Pulichino AM, Lamonerie T, Gauthier Y, Brue T, Enjalbert A, Drouin J (2001) A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104:849–859

[36] Le Tissier PR, Hodson DJ, Martin AO, Romano N, Mollard P (2015) Plasticity of the prolactin (PRL) axis: mechanisms underlying regulation of output in female mice. Adv Exp Med Biol 846:139–162

[37] Li S, Crenshaw EB 3rd, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG (1990) Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528–533

[38] Lin SC, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE, Rosenfeld MG (1993) Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature 364:208–213

[39] Liu NA, Liu Q, Wawrowsky K, Yang Z, Lin S, Melmed S (2006) Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs. Mol Endocrinol 20:871–880

[40] Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161:1202–1214

[41] Mayo KE, Hammer RE, Swanson LW, Brinster RL, Rosenfeld MG, Evans RM (1988) Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene. Mol Endocrinol 2:606–612

[42] Mayo KE, Miller T, DeAlmeida V, Godfrey P, Zheng J, Cunha SR (2000) Regulation of the pituitary somatotroph cell by GHRH and its receptor. Recent Prog Horm Res 55:237–266 discussion 266–237

[43] Mayran A, Khetchoumian K, Hariri F, Pastinen T, Gauthier Y, Balsalobre A, Drouin J (2018) Pioneer factor Pax7 deploys a stable enhancer repertoire for specification of cell fate. Nat Genet 50:259–269

[44] Michael SD, Kaplan SB, Macmillan BT (1980) Peripheral plasma concentrations of LH, FSH, prolactin and GH from birth to puberty in male and female mice. J Reprod Fertil 59:217–222

[45] Nakajima T, Yamaguchi H, Takahashi K (1980) S100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res 191:523–531

[46] Nakane PK (1970) Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J Histochem Cytochem 18:9–20

[47] Nishida Y, Yoshioka M, St-Amand J (2005) Sexually dimorphic gene expression in the hypothalamus, pituitary gland, and cortex. Genomics 85:679–687

[48] Ohta K, Nobukuni Y, Mitsubuchi H, Fujimoto S, Matsuo N, Inagaki H, Endo F, Matsuda I (1992) Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency. Biochem Biophys Res Commun 189:851–855

[49] Peel MT, Ho Y, Liebhaber SA (2018) Transcriptome analyses of female somatotropes and lactotropes reveal novel regulators of cell identity in the pituitary. Endocrinology 159:3965–3980

[50] Perez Millan MI, Brinkmeier ML, Mortensen AH, Camper SA (2016) PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells. Elife 5:e14470

[51] Philips A, Maira M, Mullick A, Chamberland M, Lesage S, Hugo P, Drouin J (1997) Antagonism between Nur77 and glucocorticoid receptor for control of transcription. Mol Cell Biol 17:5952–5959

[52] Radovick S, Nations M, Du Y, Berg LA, Weintraub BD, Wondisford FE (1992) A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 257:1115–1118

[53] Ragoczy T, Bender MA, Telling A, Byron R, Groudine M (2006) The locus control region is required for association of the murine betaglobin locus with engaged transcription factories during erythroid maturation. Genes Dev 20:1447–1457

[54] Rizzoti K, Akiyama H, Lovell-Badge R (2013) Mobilized adult pituitary stem cells contribute to endocrine regeneration in response to physiological demand. Cell Stem Cell 13:419–432

[55] Seuntjens E, Hauspie A, Roudbaraki M, Vankelecom H, Denef C (2002a) Combined expression of different hormone genes in single cells of normal rat and mouse pituitary. Arch Physiol Biochem 110:12–15

[56] Seuntjens E, Hauspie A, Vankelecom H, Denef C (2002b) Ontogeny of plurihormonal cells in the anterior pituitary of the mouse, as studied by means of hormone mRNA detection in single cells. J Neuroendocrinol 14:611–619

[57] Shekhar K, Lapan SW, Whitney IE, Tran NM, Macosko EZ, Kowalczyk M, Adiconis X, Levin JZ, Nemesh J, Goldman M (2016) Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics. Cell 166(1308–1323): e1330

[58] Smith MS, Fox SR (1984) Regulation of gonadotropin secretion during lactation. Arch Biol Med Exp 17:231–238

[59] Stuart T, Satija R (2019) Integrative single-cell analysis. Nat Rev Genet 20:257–272

[60] Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, Hao Y, Stoeckius M, Smibert P, Satija R (2019) Comprehensive integration of single-cell data. Cell 177:1888–1902.e1821

[61] Tanay A, Regev A (2017) Scaling single-cell genomics from phenomenology to mechanism. Nature 541:331–338

[62] Theogaraj E, John CD, Christian HC, Morris JF, Smith SF, Buckingham JC (2005) Perinatal glucocorticoid treatment produces molecular, functional, and morphological changes in the anterior pituitary gland of the adult male rat. Endocrinology 146:4804–4813

[63] Vazquez-Borrego MC, Gahete MD, Martinez-Fuentes AJ, Fuentes-Fayos AC, Castano JP, Kineman RD, Luque RM (2018) Multiple signaling pathways convey central and peripheral signals to regulate pituitary function: lessons from human and non-human primate models. Mol Cell Endocrinol 463:4–22

[64] Villalobos C, Nunez L, Garcia-Sancho J (2004a) Anterior pituitary thyrotropes are multifunctional cells. Am J Physiol Endocrinol Metab287:E1166–1170

[65] Villalobos C, Nunez L, Garcia-Sancho J (2004b) Phenotypic characterization of multi-functional somatotropes, mammotropes and gonadotropes of the mouse anterior pituitary. Pflugers Arch 449:257–264

[66] Young MD, Behjati S (2018) SoupX removes ambient RNA contamination from droplet based single cell RNA sequencing data. BioRxiv. https://doi.org/10.1101/303727

[67] Zahuczky G, Kristof E, Majai G, Fesus L (2011) Differentiation and glucocorticoid regulated apopto-phagocytic gene expression patterns in human macrophages. Role of Mertk in enhanced phagocytosis. PLoS ONE 6:e21349

[68] Zheng GX, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, Ziraldo SB, Wheeler TD, McDermott GP, Zhu J (2017) Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049

[69] Zhu X, Gleiberman AS, Rosenfeld MG (2007) Molecular physiology of pituitary development: signaling and transcriptional networks. Physiol Rev 87:933–963

[70] Zhu X, Tollkuhn J, Taylor H, Rosenfeld MG (2015) Notch-dependent pituitary SOX2(+) stem cells exhibit a timed functional extinction in regulation of the postnatal gland. Stem Cell Rep 5:1196–1209

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