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Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles Resource Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles Graphical Abstract Highlights d RNA-seq identifies transcriptomes of 14 skin populations during hair growth d SC precursors, progenitors, and hair-type-specific DP niche signatures are defined d Comparison with embryonic and adult signatures shows dynamic gene expression d Signaling interaction network reveals a complex web of intercellular exchanges Rezza et al., 2016, Cell Reports 14, 3001–3018 March 29, 2016 ª2016 The Authors http://dx.doi.org/10.1016/j.celrep.2016.02.078 Authors Amélie Rezza, Zichen Wang, Rachel Sennett, ..., Peter Zandstra, Avi Ma’ayan, Michael Rendl Correspondence
[email protected] In Brief Rezza et al. examine signal exchange among bulge stem cell precursors, proliferating progeny, and their niche during morphogenetic hair growth. The authors isolate and characterize 14 specialized skin cell populations, defining signature genes and revealing numerous cell-cell interactions in the hair follicle bulb. Accession Numbers GSE77197 mailto:
[email protected] http://dx.doi.org/10.1016/j.celrep.2016.02.078 http://crossmark.crossref.org/dialog/?doi=10.1016/j.celrep.2016.02.078&domain=pdf Cell Reports Resource Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles Amélie Rezza,1,2 Zichen Wang,1,4,5 Rachel Sennett,1,2,4 Wenlian Qiao,7 Dongmei Wang,6 Nicholas Heitman,1,2,4 Ka Wai Mok,1,2 Carlos Clavel,1,2,8 Rui Yi,6 Peter Zandstra,7 Avi Ma’ayan,1,4,5 and Michael Rendl1,2,3,4,* 1Black Family Stem Cell Institute 2Department of Developmental and Regenerative Biology 3Department of Dermatology 4Graduate School of Biomedical Sciences 5Department of Pharmacology and Systems Therapeutics, BD2K-LINCS Data Coordination and Integration Center, Knowledge Management Center for Illuminating the Druggable Genome (KMC-IDG) Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 6Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA 7Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada 8Present address: A*Star Institute of Medical Biology, Singapore 138648, Singapore *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.celrep.2016.02.078 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). SUMMARY The hair follicle (HF) is a complex miniorgan that serves as an ideal model system to study stem cell (SC) interactions with the niche during growth and regeneration. Dermal papilla (DP) cells are required for SC activation during the adult hair cycle, but signal exchange between niche and SC precursors/ transit-amplifying cell (TAC) progenitors that regu- lates HF morphogenetic growth is largely unknown. Here we use six transgenic reporters to isolate 14 major skin and HF cell populations. With next-gener- ation RNA sequencing, we characterize their tran- scriptomes and define unique molecular signatures. SC precursors, TACs, and the DP niche express a plethora of ligands and receptors. Signaling interaction network analysis reveals a bird’s-eye view of pathways implicated in epithelial-mesen- chymal interactions. Using a systematic tissue-wide approach, this work provides a comprehensive plat- form, linked to an interactive online database, to identify and further explore the SC/TAC/niche cross- talk regulating HF growth. INTRODUCTION Embryonic hair follicle (HF) formation, hair growth after birth, and regulation of the adult hair cycle involve complex signaling inter- actions among epithelial stem cells (SCs), progenitors, and a dermal specialized niche compartment, the dermal papilla (DP) (Lee and Tumbar, 2012; Rezza et al., 2014; Rompolas and Greco, 2014; Sennett and Rendl, 2012). At the end of the resting phase of the hair cycle, DP cells signal to bulge/germ SCs to acti- Cell vate new HF growth, and recent ligand supplementation exper- iments and receptor ablation studies in the SCs indicate an important role for DP-derived TGFB2, FGF7, and inhibitory BMP signals (Greco et al., 2009; Kobielak et al., 2003; Oshimori and Fuchs, 2012). Laser-mediated ablation established the ab- solute requirement of these cells for SC activation during the hair cycle (Rompolas et al., 2012). Similarly, during hair growth, DP cells are thought to act as a core signaling center for surrounding epithelial progenitors (transit-amplifying cells or TACs) within the wider matrix (Mx) compartment that proliferate, migrate upward, and differentiate into the multiple layers of the hair shaft and the inner root sheath channel (Hsu et al., 2014a). The outer root sheath (ORS) lines the epithelial HF compartment, is contiguous with the Mx and epidermis, and contains the SC precursors of the adult HF bulge during early hair growth (Schlake, 2007). FGF signals from theDP have been implicated in controlling hair growth (Petiot et al., 2003), while BMP and WNT signaling play an important role in hair shaft progenitor differentiation; but, the precise source of BMP and WNT ligands is unclear (DasGupta and Fuchs, 1999; Kobielak et al., 2003). SHH is produced by a subpopulation of TAC progenitors that reside right next to the DP compartment (Gambardella et al., 2000; Hsu et al., 2014b). It is still unclear if a broader requirement for TAC-derived signals interacting with the DP niche exists, as pure TACs of growing HFs have not been isolated and characterized. Finally, the third major cellular component in the HF bulb is melanocytes (Mc) that provide pigment to the epithelial cells and are thought to receive regula- tory signals from the DP niche (Enshell-Seijffers et al., 2008, 2010). Whether Mc signal with TAC progenitors and Mx cells is currently unclear. Previous studies have tried to identify signals involved in driving HF growth using a global transcriptomic approach (Dris- kell et al., 2009; Rendl et al., 2005). Isolation of HF cell popula- tions during the morphogenetic growth phase and subsequent Reports 14, 3001–3018, March 29, 2016 ª2016 The Authors 3001 mailto:
[email protected] http://dx.doi.org/10.1016/j.celrep.2016.02.078 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1016/j.celrep.2016.02.078&domain=pdf gene expression analysis with early microarrays identified en- riched genes for the DP, as well as for Mc, Mx, and ORS cells (Rendl et al., 2005). Although this study provided important in- sights into the molecular composition of major HF cell types, it was not yet possible to distinguish TAC progenitors from Mx, HFSC precursors from the remaining ORS, or hair type-specific DP subpopulations. Indeed, during early postnatal hair growth, four hair types can be recognized that form in three embryonic developmental waves and have different HF lengths within the skin (Figure 1A), as well as different hair shaft sizes, kinks, and bends externally (Schlake, 2007). Guard (G) HFs, which develop first, are the largest and extend deepest in the dermis. Awl and Auchene (AA) follicles develop subsequently, are thinner, and reach the lower part of the reticular dermis. Zigzag (ZZ) HFs appear last and are ultimately the shortest. While these morphological and developmental idiosyncrasies have been noted for years, it is unclear if hair type-specific DP subpopula- tions are molecularly distinct and control regulation of hair type sizes and shapes. In previous work, ZZ-enriched DP cells were isolated and ZZ-enriched DP genes were described with a possible role in controlling ZZ hair type (Driskell et al., 2009). Conversely, another recent study manipulated the total number of DP cells per HF resulting in hair type switching, suggesting that the cumulative signaling output from the niche determines hair type rather than its intrinsic molecular features (Chi et al., 2013). Here, we comprehensively define the molecular traits of all DP subpopulations, SHH-expressing TAC progenitors, and HFSC precursors from developing HFs, in conjunction with other major skin/HF cell types, and identify signaling interac- tions potentially involved in HF growth. For this we utilized six different fluorescent transgenic mouse reporter lines combined with immunofluorescence to isolate a total of 14 distinct skin/ HF populations from postnatal day (P)5 back skin, and we per- formed genome-wide transcriptome analysis by multiplexed RNA deep sequencing (RNA-seq). We defined molecular signa- tures of uniquely enriched genes for each population, establish- ing a comprehensive set of markers and identifying interacting ligand/receptor combinations for key HF cell types during hair growth. Molecular characterization of hair type-specific DP subpopulations showed only few specific signature genes, revealing a remarkable molecular relatedness at the mRNA level. We further defined a core DP molecular signature of genes uniquely enriched and expressed by all DP subpopula- tions. HFSC precursors from growing HFs showed common features with adult HFSCs but mostly expressed unique signa- ture genes as they matured during development. TAC progen- itors expressed numerous uniquely enriched genes, including many signaling factors, as was the case for DP, suggesting a rich crosstalk between these populations. Finally, our global unbiased analysis of intercellular signaling interaction revealed a network of multiple ligand/receptor interaction pairs involving all cell types during HF growth, with a particular density in the HF bulb. With this study we establish a comprehensive bird’s- eye view of the complex signaling interactions in growing HFs of developing skin, and we share it with the community on the Hair-GEL online database for further validation and investi- gation (http://hair-gel.net). 3002 Cell Reports 14, 3001–3018, March 29, 2016 ª2016 The Author RESULTS Isolation of Key Cell Populations from Growing Skin and HFs To purify and molecularly characterize all major cellular con- stituents of developing HFs during the first hair growth phase, we devised an integrated approach that utilized pairwise combinations of six different transgenic reporter mouse lines together with three specific immunofluorescence stainings. In this manner we were able to isolate by fluorescence-activated cell sorting (FACS) of P5 back skins a total of 14 distinct skin/ HF cell populations and subpopulations, including SC precur- sors and TACs as well as hair type-specific DP niche cells (Figure 1A). First, to purify seven core skin and hair cell types, we revisited, improved, and expanded cell isolations from K14-H2BGFP; Lef1-RFP transgenic mice previously utilized to obtain HF Mx, ORS, DP cells andMc (Rendl et al., 2005). In these reporters, nu- clear GFP is expressed in all epithelial cells of the epidermis and HFs under the keratin-14 promoter, while red fluorescent protein (RFP) is present in DP, Mc, and upper dermal fibroblasts (DFs, Figure 1B) driven by a Lef1 promoter fragment. P5 back skins were harvested, and the epidermis and dermis were enzymati- cally separated and processed to obtain epidermal and HF-en- riched dermal preparations of single cells. From the epidermal sample, we selected basal epidermal cells (Epi) as the K14- H2BGFP+ population (81% of live cells) (Figure S1A). The dermal sample was subjected to further immunofluorescence marker stainings (Figure 1B). Based on GFP expression alone, we selected Mx (35%) and ORS (21%) cells as K14-H2BGFPLow and K14-H2BGFPHigh populations, respectively, as previously described (Rendl et al., 2005). The RFP+ population was sub- divided to obtain CD117+ Mc (2.24%), and DP cells (1.1%) that