Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: Sox2 expressing tooth epithelial stem cells (green) and their conventional differentiation pathway, marked by production of early progeny which express Sfrp5. The Sfrp5+ cells become transit-amplifying cells (TACs, blue), which express high levels of P-cadherin, and at the ultimate stage also express Sonic Hedgehog. TACs give rise to ameloblasts and stratum intermedium cells, which is marked by loss of P-cadherin expression. A second cell population, marked by P-cadherin expression (purple), arises from Sox2+ stem cells and extends toward the outer enamel epithelium.

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: List of antibodies used

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: List of PCR probes used

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: Proximal ends of the incisors were isolated and cultured in the presence of 10 M cyclopamine or 100ng/ml of Shh. (A-C) EdU staining was used to detect cell proliferation, which was analyzed 24h after the initiation of treatments, which include DMSO treated control (A), cyclopamine (B) and Shh (C). (D-F) Caspase3 staining was performed to analyze cell death 12h after the initiation of treatments, which include DMSO treated control (D), cyclopamine (E) and Shh (F). (G-H) Immunostaining for Sox2 to determine the number of Sox2+ cells in DMSO control (G), cyclopamine (H) and Shh (I) treated cultures, 48h after the treatment was initiated. (J-L) Sox-GFP expression analyzed in live cultures 24h after the initiation of DMSO (J), cyclopamine (K), and Shh (L) treatments. Asterisk marks the Sox2+ SCs which are significantly reduced in cyclopamine treated samples, while white arrow points to the Sox2+ domain which appears in cyclopamine treated samples, but in controls only few Sox2+ cells are detected. (M) Flow cytometry analysis of Sox2-GFP + cells in control and cultures treated with Shh for 48h. (N) RT-qPCR analysis of Sox2 expression in control and cultures treated with Shh for 48h. (O-Q) Immunostaining for Lgr5 in DMSO control (G), cyclopamine (H) and Shh (I) treated cultures, 48h after the treatment was initiated. *p value < 0.05, n=7. Scale bar 100 m. Scale bar 100um.

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: Isolation, Cell Culture, Staining, Immunostaining, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: (A-C) Immunolabeling for P-cadherin (white) and label retaining cells (LRC, green) in explants treated with cyclopamine or Shh for 2 days. White arrow in A-C indicates P-cadherin expression in the outer enamel epithelium (OEE). Yellow arrowheads in B points to the region which contains Sox2+ SCs, while red arrowheads in B points to the region where TACs reside. (D, E) RT-qPCR analysis of P-cadherin and E-cadherin expression. N=3. ( F, H) Immunostaining for Shh and (I-K) YAP proteins. White arrowheads in (G) indicate lack of Shh signal in the presumptive ameloblast region. White arrowheads in (J) indicate lack of polarized preameloblast and ameloblast cells, and green asterisk indicates location which correlates with the location of LRCs (B) in the cyclopamine treated sample. The yellow arrow in (J) points to a region which corresponds to the region of upregulated P-cadherin (yellow arrow in (B). Scale bar 100um.

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: Immunolabeling, Expressing, Quantitative RT-PCR, Immunostaining

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: (A-D) Radioactive in situ hybridization for Shh, Gli1, Ptch1 and Ptch2. (E-H) Immunostaining for Ptch1 and Gli1 on frozen sections of non-decalcified mandibles. Yellow asterisk marks the presumptive Sox2+ SC domain. Arrows in F point to cytoplasmic expression of Gli1 in TAC region. (I, J, K) Immunolabeling of the peripheral nerve in the close proximity to the labial cervical loop using myelin binding protein (MBP) (green, I) and Dhh antibody (red, J). Arrows point to the Dhh expression in the mandibular nerve, while the yellow arrowheads indicate Dhh protein detected at the entry of the neurovascular bundle (NVB) into tooth mesenchyme. (L) Western blot analysis of the neurovascular bundle and proximal portion of the incisor for Dhh and Shh expression. Scale bar 100um.

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: In Situ Hybridization, Immunostaining, Expressing, Immunolabeling, Binding Assay, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: (A-D): RT-qPCR analysis of Gli1,Sox2, P-cadherin, and E-cadherin in mouse embryonic fibroblast cultures. n ≥ 4; *, p < .01; **, p < .005; ****, p < .0001 were determined by Mann–Whitney’s nonparametric test. (E–G): Immunolabeling for Sox2 in organ cultures treated with 100 ng/ml of Desert Hedgehog (Dhh; F) or Sonic Hedgehog (Shh; G) for 24 hours. (H, I): RT-qPCR analysis of Sox2 (n ≥ 3) and P-cadherin (n ≥ 4) expression in organ cultures treated with Dhh and Shh for 24 hours. *, p = .015; **, p = .009 were determined by Mann–Whitney’s nonparametric test. Scale bar: 100 μm.

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: Quantitative RT-PCR, Immunolabeling, Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Functionally distinctive Ptch receptors establish multimodal Hedgehog signaling in the tooth epithelial stem cell niche

doi: 10.1002/stem.3042

Figure Lengend Snippet: List of antibodies used

Article Snippet: Samples were visualized using a Zeiss LSM 700 confocal microscope and images analyzed with Zen 2012 (Carl Zeiss, Germany). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Vendor and catalogue number Dilution Sox2 Santa Cruz, sc-17320 1:250 Sox2 Millipore, ab5603 1:250 YAP Cell Signaling, 14074S 1:200 P-cadherin R&D Systems, AF761 1:100 Shh Santa Cruz, sc9024 1:250 Shh R&D Systems AF464 1:250 Gli1 Santa Cruz, sc-20687 1:100 Ptch1 Santa Cruz, sc-6149 1:100 Dhh R&D Systems, MAB733-SP 1:500 Lgr5 Santa Cruz, SC-68580 1:250 pSMAD5/8 Santa Cruz, SC-12353-R 1:250 Casp3 Cell Signaling, 96615 1:500 Open in a separate window Abbreviations: Dhh, Desert Hedgehog; Shh, Sonic Hedgehog.

Techniques: