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  • It was previously reported that Prx

    2018-11-02

    It was previously reported that Prx1-Cre expresses in the precursors of all brown and white adipocytes in 4- to 6-week-old mice (Calo et al., 2010). This report did not indicate which depots were examined, so we could not do a perfect comparison. Nevertheless, neither our study nor Krueger et al. (2014) detect broad labeling of precursor or mature brown adipocytes. The differences between our studies could reflect the use of different reporters or possibly strain background differences (the mice in Calo et al., 2010 are on a mixed background compared to the C57Bl/6 background used here and in Krueger et al., 2014). Labeling mature adipocytes with cytoplasmic reporters such as LacZ can also be less reliable than using membrane-targeted reporters because adipocytes have little cytoplasm, making it difficult to detect which hopefully express β-galactosidase (Berry et al., 2014; Berry and Rodeheffer, 2013; Sanchez-Gurmaches and Guertin, 2014; Sanchez-Gurmaches et al., 2012). There are noteworthy comparisons between Prx1-Cre and the Myf5-Cre and Pax3-Cre-labeling patterns in adipose tissue (Sanchez-Gurmaches and Guertin, 2014). First, in the asWAT, Prx1-Cre marks mature adipocytes in a ventral to dorsal pattern. This labeling pattern is opposite to the Myf5-Cre;R26R-mTmG and Pax3-Cre;R26R-mTmG patterns in asWAT, in which cells label in a dorsal to ventral gradient. Thus, one possibility is that Myf5-Cre/Pax3-Cre and Prx1-Cre may mark two different adipocyte progenitor pools that converge in asWAT. Second, Prx1-Cre labels a significant number of periaortic brown adipocytes whereas Myf5-Cre and Pax3-Cre do not (Sanchez-Gurmaches and Guertin, 2014). paBAT may have an important role in diabetes and cardiovascular disease (Fitzgibbons and Czech, 2014; Fitzgibbons et al., 2011; Guilherme et al., 2008). These results are consistent with a model in which brown adipocytes may arise from multiple origins. Finally, the observation that Myf5-Cre, Pax3-Cre, and Prx1-Cre label populations of both brown and white adipocytes suggests some brown and white adipocytes may share a common precursor, although further experiments are required to verify this. Interestingly, Prx1-Cre does not distinguish between white (unilocular) and brite/beige (multilocular) adipocytes induced by CL316,243 in the psWAT. Currently, it is unclear as to whether brite/beige adipocytes arise de novo from precursors of a different lineage or form from preexisting unilocular white adipocytes (Barbatelli et al., 2010; Lee et al., 2015; Rosenwald et al., 2013; Wang et al., 2013). Our results suggest that, at least after CL316,243 treatment, any brite/beige adipocytes that might form de novo originate from either the same Prx1-Cre precursor pool as the resident subcutaneous white adipocytes or they express Prx1-Cre during differentiation. Recent studies suggest that it is likely the case that the newly observed brown-like adipocytes appearing in psWAT are being derived largely from the preexisting Prx1-Cre-marked unilocular adipocytes (Lee et al., 2015). One interesting possibility is that Prx1-Cre marks a progenitor pool in development that gives rise to a subcutaneous white adipocyte lineage. However, there are important caveats that need to be considered when interpreting data generated from Cre-mediated cell marking or lineage tracing. For example, whereas Cre-Lox-based recombination is currently one of the best methods for lineage tracing in mice (Kretzschmar and Watt, 2012), this approach is strictly a measure of promoter activity and does not provide any information as to whether the endogenous gene products actually express in the early developmental precursors of adipocytes and/or functionally contribute to adipose tissue development. In addition, in reporter-based cell-marking/lineage-tracing studies, in which multiple tissue types label positive with a particular Cre driver, it cannot be determined whether the recombination occurred in a single lineage or alternatively in multiple independent lineages that give rise to the different tissues. Nor can it be determined when a constitutively active Cre driver first expresses. This last caveat may be addressable with the use of inducible Cre drivers, such as tamoxifen-inducible CreER drivers. However, CreER drivers are limited in many cases by their efficiency and because tamoxifen reportedly has negative effects on fat (Liu et al., 2015). The use of doxycycline-driven Cre drivers might offer a better alternative (Hudak et al., 2014; Jiang et al., 2014; Wang et al., 2013). For example, a recent study inducing β-galactosidase in adipocytes with doxycycline reports that brite/beige adipocytes in psWAT are generated by de novo adipogenesis from an undefined APC pool (Wang et al., 2013); however, other studies using inducible drivers but different reporters find that psWAT brite/beige adipocytes arise from existing white adipocytes, suggesting more work is needed to optimize methods of cell marking and lineage tracing in adipose tissue (Lee et al., 2015; Rosenwald et al., 2013). Lastly, whereas Myf5-Cre and Pax3-Cre are knockin alleles (i.e., the Cre is driven from the endogenous Myf5 and Pax3 promoter) and therefore more ideal for cell-labeling experiments, the Prx1-Cre used here is a transgene, and thus positional elements might also influence promoter activity for this driver.