5 research outputs found
Shh overexpression results in increased formation of cells expressing Merkel markers.
<p>(A) Schematic diagram showing transgenic mouse and lentiviral constructs for the Shh overexpression experiment. CD1 female mice were mated with male Rosa26-rtTA mice and <i>in utero</i> lentiviral injections were performed in pregnant females E9. Doxycycline treatment was initiated by gavage at E12, and females were continuously fed Doxycycline until embryo collection at E17. In the presence of Doxycycline, lentivirus-injected mouse embryos express both H2B-RFP and Shh proteins. (B-E) IF stainings showing the altered morphology of epidermis infected with Shh+H2B-RFP (C,C’) and the significant increase in the numbers of Krt8(+) (K8) cells in the infected epidermis (C,C’,E) compared to control, un-infected epidermis (E17 control) (B,B’,D). Quantification of Krt8(+) cells per mm of back skin in E17 control and Shh overexpression (right panel) (p<0.0001). (F,F’) IF staining for Phospho-Histone H3 (PH3) showing a significant increase in proliferation in E17 Shh o/exp epidermis (F’) compared to control (F). Quantification of number of PH3(+) per mm<sup>2</sup> of Krt14(+) epidermal cells is represented on the right (p<0.0001). (G,G’) TUNEL staining showing no increase in apoptosis in E17 Shh o/exp epidermis (G’) compared to control (G). Note that cells undergoing cornification are TUNEL(+), as previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006151#pgen.1006151.ref033" target="_blank">33</a>]. (H-I’) IF staining showing an increase in the number of Sox2(+)/Krt8(+) cells in E17 Shh o/exp epidermis (I,I’) compared to control (H,H’). (J-K’) IF stainings showing an increase in the number of Isl1(+)/Krt8(+) cells in E17 Shh o/exp epidermis (K,K’) compared to control (J,J’). Scale bars: (B-C’): 100μm; (D-K’): 25 μm.</p
Concurrent loss of PRC2 and Shh signaling in the epidermis results in dramatically reduced numbers of Merkel Cells.
<p>(A-B) IF stainings for Merkel cell (MC) markers Krt20 (K20) (A), Krt8 (K8) (B), and Sox2 (B) show a dramatic reduction in the number of Merkel cells in P0 EED Smo epidermis-conditional double knockout (EED Smo 2cKO) (K14-Cre; EED<sup>flox/flox</sup>;Smo<sup>flox/flox</sup>) mice compared to control (ctrl) and EED cKO (K14-Cre; EED<sup>flox/flox</sup>; Smo<sup>flox/+</sup>) mice. Quantification of the number of Krt20(+) quantified to the right of A (ctrl vs. Smo cKO, p<0.0001; EED cKO vs. EED Smo 2cKO, p<0.0001). Quantification of number of Krt8(+) quantified to the right of B (ctrl vs. Smo cKO, p<0.0001; EED cKO vs. EED Smo 2cKO, p<0.0001). (C) TUNEL staining shows no increase in apoptosis in the Krt14(+) basal layer of P0 EED Smo 2cKO. Note that cells undergoing cornification in the suprabasal layers stain positive for TUNEL, as previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006151#pgen.1006151.ref033" target="_blank">33</a>]. (D,E) IF stainings for Merkel cell markers Krt8 (D, E), Krt20 (D), and Sox2 (E) show that significantly fewer Merkel cells are present in the glabrous paw skin of P0 EED Smo 2cKO mice compared to EED cKO paws. Quantification of Krt8(+) cells (right panel of D) (ctrl vs. Smo cKO, p = 0.0317; EED cKO vs. EED Smo 2cKO, p<0.0001). (F) Schematic diagram of the signaling events regulating Merkel cell specification in first wave of hair follicle development. Hair follicle dependent intraepidermal signaling promotes Merkel cell specification. These signaling events also occur during second and third wave of hair follicle morphogenesis but these hair follicles do not harbor Merkel cells due to PRC2-mediated repression of Merkel genes. Scale bars: (A-E): 25 μm.</p
Shh signaling activity in the epidermis is required for Merkel cell formation.
<p>(A-C) IF stainings for Merkel cell markers Krt8 (K8) (A,C), Krt20 (K20) (B), Sox2 (B), and Isl1 (C) show a highly significant reduction in the number of Merkel cells in P0 Smoothened epidermis-conditional knockout (Smo cKO) (K14-Cre; Smo<sup>flox/flox</sup>) mice when compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Smo cKO P0 skin (right panel of B) (both p<0.0001). (D) TUNEL staining shows no increase in apoptosis in the skin of P0 Smo cKO mice. Note that cells undergoing cornification are TUNEL(+) as previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006151#pgen.1006151.ref033" target="_blank">33</a>]. (E) At E15, specification of hair follicles is not affected in P0 Smo cKO mice when compared with control. The number of hair follicles is quantified in the right panel (p = 0.3140). IF staining for Krt14 (K14) labels the epidermis (epi) and Sox2 labels the dermal condensate (dc) of the developing hair follicles in E15 WT and Smo cKO mice. (F) IF staining for Merkel cell markers Krt8 and Sox2 shows a complete absence of Merkel cells in E15 Smo cKO compared to control. The number of Krt8(+) Merkel cells is quantified in the right panel (p = 0.0018). (G) IF stainings for Merkel cell markers Krt8 and <i>in situ</i> hybridization for <i>Gli1</i> RNA in E16 WT skin indicates that <i>Gli1</i> is expressed in about 45% of the Krt14(+) (K14) cells (yellow arrowheads) surrounding the Merkel cells and in about 13% of early Merkel cells present in the skin at this time point, quantified in the right panel (p = 0.0022). (H) <i>In situ</i> hybridization for <i>Gli1</i> RNA in E16 WT back skin indicates that <i>Gli1</i> is not expressed in most Krt14(+) cells of the interfollicular epidermis (IFE). (I,J) IF stainings for Merkel cell markers Krt8 (I,J), Krt20 (I), and Sox2 (J) show that significantly fewer Merkel cells are present in the glabrous paw skin of Smo cKO mice compared to control paws. Quantification of Krt8(+) Merkel cells in paws (right panel of J) (p<0.0001). (K,L) IF stainings for Merkel cell markers Krt8 (K,L), Krt20 (K), and Sox2 (L) show that significantly fewer Merkel cells are present in the glabrous paw skin of Shh cKO mice compared to control paws. Quantification of Krt8(+) Merkel cells in paws (right panel of L) (p<0.0001). Unless otherwise indicated, all epidermis represented is dorsal skin. Scale bars: (A,E): 100μm; (B-D,F-L): 25 μm.</p
Loss of PRC2 results in ectopic formation of Merkel cells around all hair follicle types.
<p>(A) Whole-mount immunofluorescence staining showing Merkel cell-specific marker Krt8 (K8) in P0 Ezh1/2 2KO (K14-Cre; Ezh1<sup>del/del</sup>;Ezh2<sup>flox/flox</sup>) and EED cKO (K14-Cre; EED<sup>flox/flox</sup>) epidermis compared to control (ctrl). Clusters of Merkel cells (≥3 Krt8(+) cells) are quantified, and the number of clusters of Merkel cells per mm<sup>2</sup> is presented to the right (Kruskal-Wallis test p<0.0001; ctrl vs. Ezh1/2 2KO, *** p<0.0001; ctrl vs. EED cKO, *** p<0.0001). (B-B’) All hair types can have adjacent Merkel cells in EED cKO (B’), while only first wave hair follicles have adjacent Merkel cells in WT (B) epidermis. IF stainings for Sox2 and integrin α8 (α8) are used to label the dermal papillae (dp) or dermal condensate (dc) of different hair follicle types. The dermal papillae of first (left) and second (middle) wave hair follicles are Sox2(+), and the two types of hair follicles can be discriminated by size. The dermal papillae of the third (right) wave hair follicles are Sox2(-)/α8(+), and these hair follicles are very short at P0. IF staining for Sox2 identifies early-specified Merkel Cells (MC) and Krt20 (K20) identifies mature Merkel cells in the epidermis, which is labeled with E-Cadherin (ECad). (C-D”) Early immature Krt8(+) Merkel cells are found around the WT first wave (C) of developing hair follicles, but not the second wave (D) (control: 0% of placodes, 0.81% of hair germs, 19.65% of hair pegs, and 63.89% of bulbous pegs harbor Merkel cells around them; 148 hair follicles analyzed). (C’,C”,D’,D”) Krt8(+) Merkel cells are found around second wave and first wave developing hair follicles in Ezh1/2 2KO (C’,D’) and EED cKO (C”,D”) E16 embryos (Ezh1/2 2KO: 11.26% of placodes, 23.64% of hair germs, 58.33% of hair pegs, and 100% of bulbous pegs harbor Merkel cells around them; 160 hair follicles analyzed. EED cKO: 12.04% of placodes, 25.76% of hair germs, 70.37% of hair pegs, and 100% of bulbous pegs harbor Merkel cells around them; 213 hair follicles analyzed). Krt14 (K14) labels the developing hair follicles, and integrin α8 labels the dermal condensate in the first stages of hair follicle development; this becomes the dermal papilla in the bulbous peg stage. At E16, the first wave hair follicles are in the hair peg or bulbous peg stages (C-C”), while the second wave hair follicles are in the placode and hair germ stages (D-D”). Scale bars (A): 200 μm; (B-D”) 25 μm.</p
Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes.
<p>(A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED<sup>flox/flox</sup>) compared to control epidermis, while <i>Shh</i> expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006151#pgen.1006151.ref051" target="_blank">51</a>]. (D,E) <i>In situ</i> hybridization for <i>Gli1</i> RNA (D) and <i>Shh</i> RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1<sup>del/del</sup>;Ezh2<sup>flox/flox</sup>) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, <i>Isl1</i>, <i>Sox2</i>, and <i>Atoh1</i>, in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes <i>Isl1</i>, <i>Sox2</i>, and <i>Atoh1</i> in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells (<i>Isl1</i>, p = 0.0004; <i>Sox2</i>, p = 0.0004; <i>Atoh1</i>, p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p<0.0001; MC vs. K14(+), n.s. p>0.05). (J) RT-qPCR in skin epidermal samples showing upregulation of Merkel cell genes <i>Isl1</i>, <i>Atoh1</i> and <i>Sox2</i> in EED cKO animals compared to control. The average of at least three animals is presented compared to Cre(-) siblings (WT) <i>(Isl1</i>, p = 0.0286; <i>Atoh1</i>, p = 0.0286; <i>Sox2</i>, p = 0.0286). (K) ChIP-qPCR analysis in skin epidermal samples from WT and EED cKO animals showing specific H3K27me3 signal at the <i>Isl1</i>, <i>Sox2</i>, and <i>Atoh1</i> promoters. <i>Actin</i> is used as a negative control, and <i>Neuro</i> and <i>Olig3</i> are used as positive controls. Dotted line shows the level of the negative region (all p<0.0001). Scale bars: (B,C,I): 25 μm; (D,E): 100μm.</p