To test whether transgenic Cre mouse lines are

To test whether transgenic Cre mouse lines are capable of specific targeting APCs in vivo, mice carrying the Cre recombinase-responsive Enhanced Yellow Fluorescent Protein (EYFP) allele in the Rosa locus (Rosa-EYFP) (Srinivas et al., 2001) were crossed to one of three Cre lines. These lines were chosen for their common usage in studies of adipose tissue (Fabp4-Cre) (Mullican et al., 2013; Berry et al., 2014) or because of previous reports of activity of these Cre lines in adipose or mesenchymal progenitors (PdgfRα-Cre, Prx1-Cre) (Calo et al., 2010; Lee et al., 2012; Berry and Rodeheffer, 2013). The flow cytometry analysis scheme (Figure 1A) was applied to the SVF of fat pads from Cre;Rosa-EYFP double transgenic mice.
Discussion Recent studies have elucidated surface markers that can be used to distinguish and isolate APCs in adipose tissue from other stromal cell populations (Rodeheffer et al., 2008). However, continued progress in the field relies on the identification of tools for targeted manipulation of APCs in vivo. In this study, we assessed the ability of different Cre mouse lines to target APCs relative to other adipose stromal populations. While prior studies have examined recombination in selected stromal phorbol directed by either Fabp4-Cre (Shan et al., 2013) or PdgfRα-Cre (Berry and Rodeheffer, 2013), here we provide a quantitative report of in vivo Cre recombinase activity in all stromal populations of WAT and BAT depots. Comparative analysis of the expression patterns directed by the various Cre lines reveals important distinctions. In particular, the most widely used line for directing adipose-specific recombination, Fabp4-Cre, is poorly suited to target APCs. A previous study of Fabp4-Cre;Rosa-TdTomato mice reported substantially higher numbers of recombined precursors (∼60% in both WAT and BAT) than reported here, using the same cell surface marker profile (Shan et al., 2013). However, Shan and colleagues evaluated cells that were cultured after sorting, whereas in our study, the EYFP reporter expression is quantified in vivo. Fabp4-Cre is neither active in the majority of APCs of any depot, nor is it specific for APCs: other stromal cells types, particularly ECs, display similar or even higher levels of recombination (Figures 2A and 2B). Since Fabp4 expression increases with adipogenic differentiation, it is plausible that the Fabp4-Cre recombination of APCs marks a more committed subset of cells (Liu et al., 2013), although additional studies are required to substantiate this hypothesis. Despite low levels of recombination in APCs, substantial activity of Fabp4-Cre is apparent in mature adipose tissues, consistent with the initial characterization (He et al., 2003), and this should contribute to the phenotypes observed in studies that utilize this promoter. However, the lack of fidelity to the adipose lineage is clear, as recombination has been detected in muscle and liver (Figures 2E and S2D, respectively; Morán-Salvador et al., 2013; Mullican et al., 2013), embryonic limb bud and dorsal root ganglion (Urs et al., 2006), adrenal medulla and brain (Martens et al., 2010), and other tissues (Mullican et al., 2013). These other sites of expression could be a feature of this particular transgenic line, but analysis of another independently generated Fabp4-Cre line (Abel et al., 2001) also identified many nonadipose sites of recombination (Lee et al., 2013). Together, these findings suggest that Fabp4-Cre is not a suitable tool for effective and specific targeting of APCs. In contrast to Fabp4-Cre, PdgfRα-Cre is highly active in both WAT and BAT Lin−SCA1+ cells, demonstrating the highest recombination efficiency among all examined Cre lines. However, we observed PdgfRα-Cre activity in other stromal populations (Figures 3A–3C). While further evaluation will be required, the expression of Cre in other lineages could confound the results of in vivo experiments utilizing this line to study adipose tissue biology. The PdgfRα-Cre line characterized here was generated using a large genomic fragment, from a bacterial artificial chromosome, in an effort to capture the regulatory elements required for the complex endogenous PdgfRα expression pattern (Zhang et al., 1998). However, it would be highly useful to isolate regulatory elements for particular progenitor populations. Such findings should allow more precise targeting of each cell group and may inform the lineage hierarchy among progenitors.