Characterization of a novel murine Sost ER^T2 Cre model targeting osteocytes

Abstract

Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5’ and 3’ regions of the Sost gene (Sost ER^T2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky’ with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ER^T2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.

Animal model: Transgenic mice offer platform to study mature osteocytes

A new transgenic mouse allows DNA modifications to be targeted to mature bone cells known as osteocytes, enabling researchers to better study the function of this cell population, although the model has some weaknesses that may restrict its use. Lynda Bonewald from Indiana University, Indianapolis, USA, and colleagues engineered mice to express introduced genetic material only in cells in which a gene called Sost is normally active — namely, osteocytes — and only when experimenters introduce a drug called tamoxifen. In general, transgene activity was contained to the osteocytes, but a full characterization of the mice showed some issues. For one, spontaneously transgene expression occurred in older mice in the absence of tamoxifen, although this was limited to the mature bone cells. With tamoxifen, the researchers observed some transgene expression outside of the bone. Most problematically, there were some broad muscle defects.

References

[1] Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell… and more. Endocr. Rev., 2013, 34:658-690

[2] Javaheri B et al. Deletion of a single beta-catenin allele in osteocytes abolishes the bone anabolic response to loading. J. Bone Miner. Res., 2014, 29:705-715

[3] Tatsumi S et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell. Metab., 2007, 5:464-475

[4] Das S, Sakthiswary R. Bone metabolism and histomorphometric changes in murine models treated with sclerostin antibody: a systematic review. Curr. Drug. Targets., 2013, 14:1667-1674

[5] Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J. Bone Miner. Res., 2011, 26:19-26

[6] Delmas PD. Clinical potential of RANKL inhibition for the management of postmenopausal osteoporosis and other metabolic bone diseases. J. Clin. Densitom., 2008, 11:325-338

[7] Shimada T, Fukumoto S. FGF23 as a novel therapeutic target. Adv. Exp. Med. Biol., 2012, 728:158-170

[8] Elefteriou F, Yang X. Genetic mouse models for bone studies--strengths and limitations. Bone, 2011, 49:1242-1254

[9] Kalajzic I et al. In vitro and in vivo approaches to study osteocyte biology. Bone, 2013, 54:296-306

[10] Dallas SL, Xie Y, Shiflett LA, Ueki Y. Mouse Cre Models for the Study of Bone Diseases. Curr. Osteoporos. Rep., 2018, 16:466-477

[11] Abou-Khalil R, Colnot C. Cellular and molecular bases of skeletal regeneration: what can we learn from genetic mouse models? Bone, 2014, 64:211-221

[12] Yang W et al. Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo. J. Biol. Chem., 2005, 280:20680-20690

[13] Lu Y et al. DMP1-targeted Cre expression in odontoblasts and osteocytes. J. Dent. Res., 2007, 86:320-325

[14] Gorski JP et al. Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age. J. Biol. Chem., 2016, 291:4308-4322

[15] Lim J, Burclaff J, He G, Mills JC, Long F. Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice. Bone Res., 2017, 5:16049

[16] Bivi N et al. Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation. J. Bone Miner. Res., 2012, 27:374-389

[17] Powell WF Jr et al. Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses. J. Endocrinol., 2011, 209:21-32

[18] Brunkow ME et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am. J. Hum. Genet., 2001, 68:577-589

[19] Balemans W et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum. Mol. Genet., 2001, 10:537-543

[20] Balemans W, Van Hul W. Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev. Biol., 2002, 250:231-250

[21] van Bezooijen RL et al. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J. Exp. Med., 2004, 199:805-814

[22] van Bezooijen RL et al. Sclerostin in mineralized matrices and van Buchem disease. J. Dent. Res., 2009, 88:569-574

[23] Winkler DG et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J., 2003, 22:6267-6276

[24] Poole KE et al. Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J., 2005, 19:1842-1844

[25] Xiong J et al. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone. PLoS One, 2015, 10:e0138189

[26] Amrein K et al. Sclerostin and its association with physical activity, age, gender, body composition, and bone mineral content in healthy adults. J. Clin. Endocrinol. Metab., 2012, 97:148-154

[27] Mödder UI et al. Relation of age, gender, and bone mass to circulating sclerostin levels in women and men. J. Bone Miner. Res., 2011, 26:373-379

[28] Moester MJ, Papapoulos SE, Löwik CW, van Bezooijen RL. Sclerostin: current knowledge and future perspectives. Calcif. Tissue Int., 2010, 87:99-107

[29] Zimmermann C et al. Histological characterization and biochemical analysis of paraspinal muscles in neuromuscularly healthy subjects. Muscle Nerve, 2015, 52:45-54

[30] Lynch GS, Hinkle RT, Chamberlain JS, Brooks SV, Faulkner JA. Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old. J. Physiol., 2001, 535 Pt 2 591-600

[31] Thornton AM et al. Store-operated Ca(2+) entry (SOCE) contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle. Aging, 2011, 3:621-634

[32] Brotto M, Abreu EL. Sarcopenia: pharmacology of today and tomorrow. J. Pharmacol. Exp. Ther., 2012, 343:540-546

[33] Mann CJ et al. Aberrant repair and fibrosis development in skeletal muscle. Skelet Muscle, 2011, 1

[34] Indra AK et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res., 1999, 27:4324-4327

[35] Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res., 2005, 33:e36

[36] Fry CS et al. Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy. FASEB J., 2014, 28:1654-1665

[37] Bouxsein ML et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res., 2010, 25:1468-1486

[38] Zhao X et al. Compromised store-operated Ca2 + entry in aged skeletal muscle. Aging Cell, 2008, 7:561-568

[39] Dobin A et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013 15-21 p

[40] Breese MR, Liu Y. NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets. Bioinformatics, 2013, 29:494-496 p

[41] Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2014, 30:923-930 p

[42] Ewels P, Magnusson M, Lundin S, Kaller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics, 2016, 32:3047-3048 p

[43] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 2010, 26:139-140 p

[44] McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res., 2012, 40:4288-4297 p

[45] Mi H et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucl. Acids Res, 2017, 45 D1 D183-D189

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(1PO1AG039355)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)(RC2 AR058962)