Tbx18 Targets Dermal Condensates for Labeling, Isolation, and Gene Ablation during Embryonic Hair Follicle Formation Laura Grisanti, Carlos Clavel, Xiaoqiang.

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Tbx18 Targets Dermal Condensates for Labeling, Isolation, and Gene Ablation during Embryonic Hair Follicle Formation Laura Grisanti, Carlos Clavel, Xiaoqiang Cai, Amelie Rezza, Su-Yi Tsai, Rachel Sennett, Melanie Mumau, Chen-Leng Cai, Michael Rendl Journal of Investigative Dermatology Volume 133, Issue 2, Pages 344-353 (February 2013) DOI: 10.1038/jid.2012.329 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Tbx18LacZ expression in embryonic skin. (a) Schematic of embryonic hair follicle development. Dermal condensates are visible at embryonic day (E)14.5. (b) Top: schematic of Tbx18LacZ reporter. Bottom: whole-mount X-Gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) staining in embryos. (c-f) Top: LacZ expression in skin sections at different ages. Bottom: pseudo-colored X-Gal (red) overlaid with 4 ,6-diamidino-2-phenylindole (DAPI) to highlight nuclei. The dotted line marks the basement membrane. (c) LacZ in dermal condensates at E14.5 (arrows). (d) LacZ expression in dermal papilla (DP) cells of downgrowing guard hair follicles at E16.5 (arrow) and in second-wave DPs (open arrowheads). (e) At E18.5, all DPs express LacZ in downgrowing follicles (arrow, open arrowheads) and third-wave dermal condensates (open arrows). (d, e) Note weak LacZ expression in the dermis (filled arrowheads) and arrector pili muscles (asterisks). (f) Weak LacZ expression in dermal cells in lower back skin at E14.5 (arrowheads). (g) Schematic summarizing Tbx18 expression in embryonic skin at E14.5. Blue dots illustrate Tbx18 expression in DP precursor cells, and the blue line illustrates widespread expression in the dermis (lower bracket). Bars=50μm. Journal of Investigative Dermatology 2013 133, 344-353DOI: (10.1038/jid.2012.329) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Labeling and isolation of dermal condensates with Tbx18H2BGFP. (a) Schematic of Tbx18H2BGFP reporter. Below embryonic day (E)14.5 embryo (left) showing green fluorescent protein (GFP) expression in hair follicles. (b-e) Sox2 colocalization with GFP in dermal papilla (DP) precursors and more mature DPs. (b) Whole-mount skin and (c) sagittal section at E14.5. Arrows point to double-labeled dermal condensates. (d) GFP expression in first- and second-wave DP follicles (arrows) and dermis (arrowheads) at E16.5. (e) GFP and Sox2 coexpression in mature DP. Integrin-beta 4 (Itgb4) marks the basement membrane. 4',6-Diamidino-2-phenylindole (DAPI) highlights all nuclei (blue). (f) Quantification of Sox2- and GFP-positive DPs in all developmental stages (n=4). (g) FACS isolation of DP precursors at E14.5. Three cell populations were isolated: GFPhi, dermal condensates (green); GFPlow, dermal cells from lower backskin (blue); and GFP−, negative cells (black). (h) Real-time PCR analysis of isolated cells. Only GFPhi cells were enriched in dermal condensate genes. Data shown are mean±SD (n=2). Bar=50μm. Journal of Investigative Dermatology 2013 133, 344-353DOI: (10.1038/jid.2012.329) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Tbx18Cre activity in dermal papilla (DP) precursor cells during hair follicle formation. (a) Schematic of Tbx18Cre, R26RLacZ, and R26ACTB-mT/mG reporter lines. (b) Whole-mount X-Gal (5-bromo-4-chloro-indolyl-b-D-galactopyranoside) staining of Tbx18Cre/R26RLacZ embryos at embryonic day (E)14.5 showed Cre activity in a hair follicle pattern. Few labeled follicles were visible at E14.0 (arrowheads). (c) Cre activity in DP precursor cells at E14.5 (arrows). (d, e) At E16.5 and E18.5, Cre activity is present in all DP cells (arrows, open arrowheads) and dermal condensates (open arrows), and is more widespread in the dermis and arrector pili muscle (filled arrowheads, asterisks). (f) Immunofluorescence for Sdc1 (Syndecan-1) confirmed the identity of membrane green fluorescent protein (mG)-positive dermal condensates with the R26ACTB-mT/mG reporter. (g) Quantification of mG and Sdc1 double-labeled dermal condensates (n=2). Data shown are mean±SD. Bars=50μm. Journal of Investigative Dermatology 2013 133, 344-353DOI: (10.1038/jid.2012.329) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Inducible Cre activity in Tbx18MerCreMer mice targets guard hair dermal papilla (DP) cells and their progeny. (a) Schematic of tamoxifen (TAM)-inducible Tbx18MerCreMer (MCM) mice crossed with R26RLacZ or R26ACTB-mT/mG reporters. (b) Timeline of Cre induction and reporter analysis. (c) Whole-mount X-Gal staining of Tbx18MCM/R26RLacZ embryo showed reporter activation in hair follicle pattern at embryonic day (E)14.5. (d) Inducible Cre activity in dermal condensates. (e) Quantification of labeled follicles in Tbx18Cre and Tbx18MCM embryos. (f) Coexpression of Sox2 and membrane green fluorescent protein (mG) in DPs of Tbx18MCM/R26ACTB-mT/mG whole-mount skin at E14.5 (arrows). Few dermal cells were labeled (arrowheads); asterisks mark autofluorescence; right: high magnification. (g) Colocalization in sagittal section. (h) mG expression in DP (arrows) and dermal sheath cells (open arrows) of guard hair follicles at E18.5. Few dermal cells were mG positive (arrowheads). (i) Quantification of follicles with mG-positive DP cells (n=3). (j) Quantification of mG-positive DP cells per DP in embryo sections. In ∼25% of sectioned guard hairs, 100% DP cells were labeled (n=3). Data shown are mean±SD. Bar=50μm. Journal of Investigative Dermatology 2013 133, 344-353DOI: (10.1038/jid.2012.329) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Tbx18 in dermal papilla (DP) precursor cells are not required for hair follicle formation. (a) Schematic of generating Tbx18 knockouts (KO). (b) Newborn Tbx18 KO and heterozygous (HET) pups. (c) Verification of Tbx18 ablation by real-time PCR in FACS-isolated DP precursor cells at embryonic day (E)14.5 (n=3). (d) Hematoxylin and eosin staining of newborn back skins of HET and KO. (e) Unchanged total hair follicle numbers (n=3). (f) All hair follicle subtypes are formed in KO (n=3). (g) Normal hair shaft formation in skin grafts of newborn HET (Tbx18LacZ) and KO (Tbx18LacZ/H2BGFP). (h) Hematoxylin and eosin staining showed normal follicle morphologies in KO skin grafts. (i) LacZ and green fluorescent protein (GFP) expression in DPs confirming donor origin of grafted skins. (j, k) Real-time PCRs of FACS-isolated DPs at E14.5 for DP signature genes (j) and T-box family members (k) (n=3). Data shown are mean±SD. **, P<0.01. Bar=50μm. Journal of Investigative Dermatology 2013 133, 344-353DOI: (10.1038/jid.2012.329) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions