Characterization of a new, inducible transgenic mouse model with GFP expression in melanocytes and their precursors

Abstract

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties.

ComplementaryIn VivoApproaches Reveal Quiescent Melanocytes in Anagen Outside the Hair Follicle Bulge

“…To characterize comprehensively quiescence of McSCs throughout telogen, Dct-H2BGFP mice (Joshi et al 2018) were intraperitoneally injected with BrdU every 12 hours throughout early 5 (P49-P56), mid-(P56-P63), and late telogen (P63-P70) ( Figure 1a Moreover, 34% of HFs analyzed at P70 had entered anagen, consistent with the enhanced uptake of BrdU in the SHG noted previously (Greco et al 2009). The incorporation of BrdU administered from 7 days prior in three distinct regions of the anagen follicle, the bulge/upper outer root sheath (ORS); the lower ORS, here defined as the region between the bulge and the bulb; and the bulb were quantified.…”

“…Dct-H2BGFP mice (Joshi et al 2018) were used to identify melanocytic cells. To label proliferating cells, Dct-H2BGFP mice were intraperitoneally injected with 10mM BrdU (50µg/g body weight, Molecular Probes) every 12h from P21-P30 or for one-week periods during telogen.…”

“…In this study, we dissect proliferative differences between McSCs located in the HF bulge and other HF regions during both telogen and anagen. We utilize BrdU incorporation studies in conjunction with a bitransgenic mouse strain, Dct-H2BGFP (Joshi et al 2019;Joshi et al 2018), for identification of McSCs. We reveal the unexpected presence of a quiescent population of melanocytes residing outside the bulge region during HF anagen.…”

Increased PHGDH expression uncouples hair follicle cycle progression and promotes inappropriate melanin accumulation

“…The resulting mice were then exposed to a dox diet for 30 days, plucked, and skin was collected in anagen II. Adapting previously described protocols (Joshi et al, 2017;Zaidi, Davis, et al, 2011), this skin was then dissociated into single-cell suspension and sorted by flow cytometry into GFP-positive and GFP-negative populations in order to quantitate the effect of PHGDH expression on the relative abundance of GFP-positive melanocytes (Supplemental Figure 5A). In order to validate that the GFPpositive cells were indeed melanocytes, we performed qPCR for tyrosinase, a melanocyte marker, which was present in GFP-positive cells, and nearly undetectable in GFP-negative cells ( Figure 4E).…”

CD34 defines melanocyte stem cell subpopulations with distinct regenerative properties

“…Recently, by using tetracycline-regulated expression of a stable H2BGFP fusion protein from the Dct promoter in bitransgenic Dct- tTA;TRE-H2BGFP ( Dct- H2BGFP) mice, we localized subbulge McSCs to the SHG, a transient structure at the base of the telogen HF [15]. Given this localization of McSCs to anatomically separate telogen HF compartments whose epithelial stem cells possess distinct characteristics, we wondered whether McSCs occupying these distinct sections were functionally different or interchangeable.…”