Stem Cell Reports Article Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells Chase A. Pagani, 1 Amanda K. Huber, 2 Charles Hwang, 2 Simone Marini, 2 Karthik Padmanabhan, 3 Nicholas Livingston, 1 Johanna Nunez, 1 Yuxiao Sun, 1 Nicole Edwards, 2 Yu-Hao Cheng, 4 Noelle Visser, 2 Pauline Yu, 2 Nicole Patel, 2 Joseph A. Greenstein, 2 Husain Rasheed, 2 Reagan Nelson, 2 Karen Kessel, 2 Kaetlin Vasquez, 2 Amy L. Strong, 2 Geoffrey E. Hespe, 2 Jane Y. Song, 5 Deneen M. Wellik, 5 and Benjamin Levi 1, * 1 Center for Organogenesis and Trauma, Department of Surgery, University of Texas Southwestern, 6000 Harry Hines Boulevard, Dallas, TX 75235, USA 2 Section of Plastic Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA 3 BRCF Epigenomics Core, University of Michigan, Ann Arbor, MI 48109, USA 4 Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA 5 Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53705, USA *Correspondence: benjamin.levi@utsouthwestern.edu https://doi.org/10.1016/j.stemcr.2021.01.011 SUMMARY Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; how- ever, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and induc- ible lineage-tracing mouse (Hoxa11-CreER T2 ;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors spe- cifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and ad- ipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion. INTRODUCTION The correct programming of adult precursor cells is critical to enable normal wound healing after injury. Burn injuries, soft-tissue wounds, and fractures all require a highly orchestrated response of inflammatory, vascular, neural, fibroblast, and mesenchymal stem/progenitor/precursor cells (MSCs) to coordinate proper healing. However, in some cases of severe trauma, proper precursor cell program- ming can be maladaptive, causing pathological healing. This altered programming of precursor cells is manifested in the process of heterotopic ossification (HO). HO is char- acterized by aberrant differentiation of adult tissue-resident MSCs that are ectopic from the adult skeleton. Recent studies have begun to elucidate bone progenitor cells dur- ing normal bone development and repair. The process by which tissue-resident mesenchymal cells undergo aberrant osteogenic and chondrogenic differentiation, however, has yet to be defined (Agarwal et al., 2016a, 2017a; Chan et al., 2018; Comazzetto et al., 2019; Genet et al., 2015; Hsieh et al., 2019; Hwang et al., 2019; Matsushita et al., 2020; Tor- ossian et al., 2017; Wang et al., 2018). In recent years, Cre/LoxP-based lineage-tracing ap- proaches have provided the technical ability to follow the fates of stably marked native cell populations within the injury site and assess these cells’ osteogenic capacity (Agar- wal et al., 2016a; Kan et al., 2018). Although lineage studies have characterized HO as an endochondral process (Foley et al., 2018), studies have yet to elucidate whether several cell populations differentiate into aberrant cartilage and bone or whether HO is derived from one progenitor popula- tion. Mouse lines that mark local MSCs have been used to study HO (Agarwal et al., 2016b, 2017b; Dey et al., 2016; Kan et al., 2013, 2018). However, these Cre models have lim- itations. For example, Prrx1Cre marks the entire lateral plate mesoderm, and each Cre allele is found at many sites outside of the musculoskeletal system (Logan et al., 2002). Given the lack of specificity, use of these Cre systems will not precisely identify the cells likely playing a role in bone formation or in homeostasis (Agarwal et al., 2016a). Cre systems have also been used to conditionally delete genes during develop- ment; however, this can lead to native skeletal abnormalities such as shortened limbs or altered bone mineral density, introducing confounding variables into the measurement of ectopic bone. Therefore, inducible systems are preferred. The available alleles that mark MSCs include PdgfraCreER, Gli1CreER, and ScxCreER lines; however, these alleles also mark MSCs in tissues outside of the extremities (Li et al., 2018; O’Rourke et al., 2016; Qian et al., 2017; Sugimoto et al., 2013b; Zhao et al., 2015). Importantly, these cell line- ages are not specific to one anatomic region, making it diffi- cult to interpret any lineage specific gene-deletion studies. Recent studies have utilized the embryonic patterning gene Hoxa11 to mark the progenitor cells responsible for 626 Stem Cell Reports j Vol. 16 j 626–640 j March 9, 2021 j ª 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).