rabbit anti trpm5  (Alomone Labs)


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    Alomone Labs rabbit anti trpm5
    Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), <t>TRPM5</t> (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.
    Rabbit Anti Trpm5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain"

    Article Title: Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain

    Journal: Chemical Senses

    doi: 10.1093/chemse/bjx032

    Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.
    Figure Legend Snippet: Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.

    Techniques Used: Fluorescence, Mouse Assay, Labeling, Marker, Staining, Injection

    2) Product Images from "Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain"

    Article Title: Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain

    Journal: Chemical Senses

    doi: 10.1093/chemse/bjx032

    Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.
    Figure Legend Snippet: Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.

    Techniques Used: Fluorescence, Mouse Assay, Labeling, Marker, Staining, Injection

    3) Product Images from "Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium"

    Article Title: Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

    Journal: Scientific Reports

    doi: 10.1038/srep38199

    Ascl3-expressing cells are precursors of microvillar cells and Bowman’s glands. Immunohistochemistry was performed on OE isolated from Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice (2 months), using antibodies to tdTomato (RFP) and ( A ) PLC β2, which marks the apical microvilli of microvillar cells, ( B ) IP3R3, ( C ) Trpm5, ( D ) AQP5 and ( E ) OMP. RFP expression colocalized with microvillar cell markers: PLC β2 (arrowheads), IP3R3 and Trpm5 (arrowheads) and Bowman’s glands markers: AQP5 (arrowheads). ( E ) No colocalization was detected between RFP and the mature OSN marker OMP. White asterisks mark Bowman’s gland duct cells. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.
    Figure Legend Snippet: Ascl3-expressing cells are precursors of microvillar cells and Bowman’s glands. Immunohistochemistry was performed on OE isolated from Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice (2 months), using antibodies to tdTomato (RFP) and ( A ) PLC β2, which marks the apical microvilli of microvillar cells, ( B ) IP3R3, ( C ) Trpm5, ( D ) AQP5 and ( E ) OMP. RFP expression colocalized with microvillar cell markers: PLC β2 (arrowheads), IP3R3 and Trpm5 (arrowheads) and Bowman’s glands markers: AQP5 (arrowheads). ( E ) No colocalization was detected between RFP and the mature OSN marker OMP. White asterisks mark Bowman’s gland duct cells. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Techniques Used: Expressing, Immunohistochemistry, Isolation, Mouse Assay, Planar Chromatography, Marker, Staining

    Ablation of Ascl3-expressing cells results in absence of microvillar cells and Bowman’s glands, and decreases GBCs and mature OSNs. ( A ) Ascl3 EGFP-Cre/+ /R26 DTA/+ mice, DTA expression is activated only in the Ascl3-expressing cells. OE was isolated from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at 2 months of age. ( B ) H E staining showed a significant decrease in thickness of the OE in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to Ascl3 +/+ / R26 DTA /+ mice. ( C ) Staining with antibody to PLC β2 (arrowheads) in OE from Ascl3 +/+ / R26 DTA /+ mice and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( D ) Trpm5-positive microvillar cells are present at the apical surface of the OE (arrowheads) in Ascl3 +/+ / R26 DTA /+ mice, but not detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( E ) Antibodies to aquaporin 5 (AQP5) mark the apical surface of the duct cells in the Bowman’s glands extending through the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Ducts cells of the Bowman’s glands were only rarely observed in OE from Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowhead). ( F ) Antibodies to Sox2 revealed no difference in number of sustentacular cells at the apical surface of the OE between mice of the two genotypes. The number of Sox2 + GBCs was decreased (arrowheads). ( G ) p63 + HBC numbers are not changed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( H ) SEC8 antibody labels GBCs near the basal layer of the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Significantly fewer GBCs were detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( I ) Labeling with antibody to OMP showed a significant decrease in the number of labeled mature OSNs in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to controls. ( J ) Antibodies to active caspase-3 showed an increase in number of apoptotic cells in the OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( K ) Quantified results show significant decrease in the thickness of OE and the numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + duct cells of the Bowman gland, but no difference in number of p63 + HBCs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. Quantification also showed a significant decrease in SEC + GBCs and OMP + mature OSNs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. In addition, an increase of caspase-3 + cells was observed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowheads). N ≥ 3 for Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ . *** P
    Figure Legend Snippet: Ablation of Ascl3-expressing cells results in absence of microvillar cells and Bowman’s glands, and decreases GBCs and mature OSNs. ( A ) Ascl3 EGFP-Cre/+ /R26 DTA/+ mice, DTA expression is activated only in the Ascl3-expressing cells. OE was isolated from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at 2 months of age. ( B ) H E staining showed a significant decrease in thickness of the OE in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to Ascl3 +/+ / R26 DTA /+ mice. ( C ) Staining with antibody to PLC β2 (arrowheads) in OE from Ascl3 +/+ / R26 DTA /+ mice and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( D ) Trpm5-positive microvillar cells are present at the apical surface of the OE (arrowheads) in Ascl3 +/+ / R26 DTA /+ mice, but not detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( E ) Antibodies to aquaporin 5 (AQP5) mark the apical surface of the duct cells in the Bowman’s glands extending through the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Ducts cells of the Bowman’s glands were only rarely observed in OE from Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowhead). ( F ) Antibodies to Sox2 revealed no difference in number of sustentacular cells at the apical surface of the OE between mice of the two genotypes. The number of Sox2 + GBCs was decreased (arrowheads). ( G ) p63 + HBC numbers are not changed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( H ) SEC8 antibody labels GBCs near the basal layer of the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Significantly fewer GBCs were detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( I ) Labeling with antibody to OMP showed a significant decrease in the number of labeled mature OSNs in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to controls. ( J ) Antibodies to active caspase-3 showed an increase in number of apoptotic cells in the OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( K ) Quantified results show significant decrease in the thickness of OE and the numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + duct cells of the Bowman gland, but no difference in number of p63 + HBCs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. Quantification also showed a significant decrease in SEC + GBCs and OMP + mature OSNs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. In addition, an increase of caspase-3 + cells was observed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowheads). N ≥ 3 for Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ . *** P

    Techniques Used: Expressing, Mouse Assay, Isolation, Staining, Planar Chromatography, Labeling, Size-exclusion Chromatography

    Ascl3-expressing cells regenerate microvillar cells and Bowman’s gland/ducts after injury. ( A ) Immunohistochemistry with antibodies to EGFP and CK5 on sections of OE isolated from Ascl3 EGFP-Cre /+ after methimazole injection. At day one (1 dpi) post injury, Ascl3-expressing cells marked by EGFP co-localize with HBCs marked by CK5 expression (arrowheads). EGFP + cells gradually migrate away from the basal layer toward the apical OE at 3 and 14 dpi. ( B ) Lineage tracing in Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice after injury. Antibody to RFP detected Ascl3-expressing cells co-localized with HBCs marked by CK5 antibodies at 1 dpi (arrowheads). RFP-labeled cells become apically localized at 3 and 14 dpi. White asterisk marks delaminated OE tissue. ( C) Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice were treated with methimazole and OE was analyzed after 28 days. ( D – H ) Confocal images of OE sections stained with antibodies to RFP and ( D ) PLC β2, ( E ) IP3R3, ( F ) Trpm5, ( G ) AQP5 and ( H ) OMP. RFP expression colocalized with PLC β2, IP3R3, Trpm5 and AQP5 (arrowheads). ( H ) No colocalization was detected between RFP and OMP. Arrowheads indicate Bowman’s gland ducts within the OE. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.
    Figure Legend Snippet: Ascl3-expressing cells regenerate microvillar cells and Bowman’s gland/ducts after injury. ( A ) Immunohistochemistry with antibodies to EGFP and CK5 on sections of OE isolated from Ascl3 EGFP-Cre /+ after methimazole injection. At day one (1 dpi) post injury, Ascl3-expressing cells marked by EGFP co-localize with HBCs marked by CK5 expression (arrowheads). EGFP + cells gradually migrate away from the basal layer toward the apical OE at 3 and 14 dpi. ( B ) Lineage tracing in Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice after injury. Antibody to RFP detected Ascl3-expressing cells co-localized with HBCs marked by CK5 antibodies at 1 dpi (arrowheads). RFP-labeled cells become apically localized at 3 and 14 dpi. White asterisk marks delaminated OE tissue. ( C) Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice were treated with methimazole and OE was analyzed after 28 days. ( D – H ) Confocal images of OE sections stained with antibodies to RFP and ( D ) PLC β2, ( E ) IP3R3, ( F ) Trpm5, ( G ) AQP5 and ( H ) OMP. RFP expression colocalized with PLC β2, IP3R3, Trpm5 and AQP5 (arrowheads). ( H ) No colocalization was detected between RFP and OMP. Arrowheads indicate Bowman’s gland ducts within the OE. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Techniques Used: Expressing, Immunohistochemistry, Isolation, Injection, Mouse Assay, Labeling, Staining, Planar Chromatography

    Time course of OE regeneration in the absence of non-neuronal support cells. Quantification of PLC β2 + , Trpm5 + microvillar cells, duct cells of AQP5 + Bowman’s glands, OE thickness, SEC8 + GBCs and caspase-3 + apoptotic cells from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at days 7, 14, 21, 28 post-injury. ( A – C ) Quantified results showed significantly reduced numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + Bowman gland ducts in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( D ) Decrease in the thickness of OE was detected from day 14 dpi during regeneration. ( E ) Decrease of SEC8 + GBCs was observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( F ) More caspase-3 + apoptosis cells were observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. N ≥ 3 for control and Ascl3-DTA mice. * P
    Figure Legend Snippet: Time course of OE regeneration in the absence of non-neuronal support cells. Quantification of PLC β2 + , Trpm5 + microvillar cells, duct cells of AQP5 + Bowman’s glands, OE thickness, SEC8 + GBCs and caspase-3 + apoptotic cells from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at days 7, 14, 21, 28 post-injury. ( A – C ) Quantified results showed significantly reduced numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + Bowman gland ducts in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( D ) Decrease in the thickness of OE was detected from day 14 dpi during regeneration. ( E ) Decrease of SEC8 + GBCs was observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( F ) More caspase-3 + apoptosis cells were observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. N ≥ 3 for control and Ascl3-DTA mice. * P

    Techniques Used: Planar Chromatography, Mouse Assay

    4) Product Images from "Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium"

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-15-13

    Effect of Skn-1a deficiency on the functional differentiation of Trpm5-positive microvillous cell. (A) In situ hybridization of Trpm5 on coronal sections of the MOE of wild-type and Skn-1a -/- mice. The mRNA signal of Trpm5 was absent in Skn-1a -/- mice. (B and C) Coronal sections of wild-type and Skn-1a -/- MOE of adult mice were immunostained with an anti-Trpm5 antibody (green) and an anti-villin (B) or anti-ChAT (C) antibody (red). Trpm5-positive cells were villin positive in the microvilli in the wild-type MOE (arrowheads), whereas no immunoreactive signal for Trpm5 or villin was observed in the Skn-1a -/- MOE. Trpm5-positive cells were co-immunostained with anti-ChAT antibody in wild-type (arrowheads) but not in Skn-1a -/- mice. Scale bars: 100 μm in A, 10 μm in B and C.
    Figure Legend Snippet: Effect of Skn-1a deficiency on the functional differentiation of Trpm5-positive microvillous cell. (A) In situ hybridization of Trpm5 on coronal sections of the MOE of wild-type and Skn-1a -/- mice. The mRNA signal of Trpm5 was absent in Skn-1a -/- mice. (B and C) Coronal sections of wild-type and Skn-1a -/- MOE of adult mice were immunostained with an anti-Trpm5 antibody (green) and an anti-villin (B) or anti-ChAT (C) antibody (red). Trpm5-positive cells were villin positive in the microvilli in the wild-type MOE (arrowheads), whereas no immunoreactive signal for Trpm5 or villin was observed in the Skn-1a -/- MOE. Trpm5-positive cells were co-immunostained with anti-ChAT antibody in wild-type (arrowheads) but not in Skn-1a -/- mice. Scale bars: 100 μm in A, 10 μm in B and C.

    Techniques Used: Functional Assay, In Situ Hybridization, Mouse Assay

    Characterization of Skn-1a -expressing cells in the main olfactory epithelium. (A) Skn-1a -expressing cells were characterized using two-color in situ hybridization in coronal sections of the MOE at postnatal day 0 with RNA probes for Skn-1a (green) and OSN progenitor/precursor genes Mash1 (neuronal progenitors), Ngn1 (neuronal precursors), and NeuroD (differentiating/postmitotic neurons). Small populations of Skn-1a -potitive cells and Mash1 -positive cells overlapped. The arrowhead indicates a co-labeled cell, and arrows indicate either Skn-1a or Mash1 single-labeled cells . None of Skn-1a -positive cells were co-labeled with Ngn1 and NeuroD (arrows). (B and C) In situ hybridization of Skn-1a (green) with OMP (mature OSNs; B, red) and Trpm5 ( Trpm5 -positive microvillous cells; C, red) in coronal sections of the adult MOE. Neither apical nor basal Skn-1a -expressing cells (arrows) were co-labeled with OMP signals. Trpm5 signals were co-labeled with apical Skn-1a signals (arrowheads) but not with basal Skn-1a signals (arrow). Scale bars, 25 μm. (D and E) Populations of Skn-1a- expressing cells (D) and Mash1- expressing cells (E) were analyzed by two-color in situ hybridization at postnatal day 30. Quantitative analyses revealed that 8.34 ± 2.82% (mean ± SD) of the Skn-1a- expressing cells coexpressed Mash1 (n = 3) , and 77.7 ± 5.95% coexpressed Trpm5 (n = 3). In the OSN-lineage, Mash1- positive olfactory progenitors rarely expressed Skn-1a (1.41 ± 0.564%, n = 3).
    Figure Legend Snippet: Characterization of Skn-1a -expressing cells in the main olfactory epithelium. (A) Skn-1a -expressing cells were characterized using two-color in situ hybridization in coronal sections of the MOE at postnatal day 0 with RNA probes for Skn-1a (green) and OSN progenitor/precursor genes Mash1 (neuronal progenitors), Ngn1 (neuronal precursors), and NeuroD (differentiating/postmitotic neurons). Small populations of Skn-1a -potitive cells and Mash1 -positive cells overlapped. The arrowhead indicates a co-labeled cell, and arrows indicate either Skn-1a or Mash1 single-labeled cells . None of Skn-1a -positive cells were co-labeled with Ngn1 and NeuroD (arrows). (B and C) In situ hybridization of Skn-1a (green) with OMP (mature OSNs; B, red) and Trpm5 ( Trpm5 -positive microvillous cells; C, red) in coronal sections of the adult MOE. Neither apical nor basal Skn-1a -expressing cells (arrows) were co-labeled with OMP signals. Trpm5 signals were co-labeled with apical Skn-1a signals (arrowheads) but not with basal Skn-1a signals (arrow). Scale bars, 25 μm. (D and E) Populations of Skn-1a- expressing cells (D) and Mash1- expressing cells (E) were analyzed by two-color in situ hybridization at postnatal day 30. Quantitative analyses revealed that 8.34 ± 2.82% (mean ± SD) of the Skn-1a- expressing cells coexpressed Mash1 (n = 3) , and 77.7 ± 5.95% coexpressed Trpm5 (n = 3). In the OSN-lineage, Mash1- positive olfactory progenitors rarely expressed Skn-1a (1.41 ± 0.564%, n = 3).

    Techniques Used: Expressing, In Situ Hybridization, Labeling

    Expression of Skn-1a and Trpm5 in the MOE of Mash1 -/- embryos. Expression of Skn-1a and Trpm5 in the Mash1 -/- MOE was examined by in situ hybridization at embryonic day 18.5. The MOE of Mash1 -/- embryos appeared smaller and thinner than that of wild-type littermates, as observed previously. Expression of either Skn-1a or Trpm5 was observed in both the wild-type and Mash1 -/- MOE. Higher-magnification images of the dotted boxes are presented to the right of each image. Scale bars, 100 μm.
    Figure Legend Snippet: Expression of Skn-1a and Trpm5 in the MOE of Mash1 -/- embryos. Expression of Skn-1a and Trpm5 in the Mash1 -/- MOE was examined by in situ hybridization at embryonic day 18.5. The MOE of Mash1 -/- embryos appeared smaller and thinner than that of wild-type littermates, as observed previously. Expression of either Skn-1a or Trpm5 was observed in both the wild-type and Mash1 -/- MOE. Higher-magnification images of the dotted boxes are presented to the right of each image. Scale bars, 100 μm.

    Techniques Used: Expressing, In Situ Hybridization

    Quantification of microvillous cell density in the most superficial layer of the MOE. (A) Image of an MOE dorsal recess from a ChAT-eGFP mouse, showing ChAT / Trpm5 -expressing microvillous cells (GFP + ) in the most superficial layer, a region above the supporting cell nuclei. (B) A higher-magnification view of the DAPI-stained nuclei in the dorsal MOE. Arrowheads point to nuclei of GFP + microvillous cells. (B’) Overlay of GFP signal onto B. (C) Image of an MOE dorsal recess from an Skn-1a -/- mouse. Arrows in B and C point to nuclei that do not belong to GFP + microvillous cells. (D) Plot of the averaged density per surface area of DAPI-stained nuclei and GFP + cells in the most superficial layer of the MOE from ChAT-eGFP mice. Counting was conducted from the dorsal recess and septum of the MOE. Approximately 80% of the cells in the area are GFP + microvillous cells. (E) Comparison of averaged nucleus density, showing approximately 73% reduction in the nucleus density of Skn-1a -/- mice compared with that of ChAT-eGFP mice. Scale bars: 100 μm in A, 20 μm in B-D.
    Figure Legend Snippet: Quantification of microvillous cell density in the most superficial layer of the MOE. (A) Image of an MOE dorsal recess from a ChAT-eGFP mouse, showing ChAT / Trpm5 -expressing microvillous cells (GFP + ) in the most superficial layer, a region above the supporting cell nuclei. (B) A higher-magnification view of the DAPI-stained nuclei in the dorsal MOE. Arrowheads point to nuclei of GFP + microvillous cells. (B’) Overlay of GFP signal onto B. (C) Image of an MOE dorsal recess from an Skn-1a -/- mouse. Arrows in B and C point to nuclei that do not belong to GFP + microvillous cells. (D) Plot of the averaged density per surface area of DAPI-stained nuclei and GFP + cells in the most superficial layer of the MOE from ChAT-eGFP mice. Counting was conducted from the dorsal recess and septum of the MOE. Approximately 80% of the cells in the area are GFP + microvillous cells. (E) Comparison of averaged nucleus density, showing approximately 73% reduction in the nucleus density of Skn-1a -/- mice compared with that of ChAT-eGFP mice. Scale bars: 100 μm in A, 20 μm in B-D.

    Techniques Used: Expressing, Staining, Mouse Assay

    Expression of Skn-1a in the developing main olfactory epithelia. (A) In situ hybridization with RNA probes for Skn-1a in coronal sections of mouse MOE at embryonic days 13.5 and 16.5 and postnatal days 0, 7, 14, and 30. The expression of Skn-1a was first detected at embryonic day 13.5 and was observed during subsequent development. The Skn-1a -expressing cells were located in apical, intermediate, and basal positions in the MOE during embryonic stages and were gradually restricted to apical and basal positions in postnatal development. (B) The expression of Skn-1a in the rostral-caudal axis of the MOE at postnatal day 7. Skn-1a expression was observed throughout the MOE, in terms of the rostral-caudal and the dorsal-ventral axis. (C) In the adult MOE, Skn-1a -expressing cells were distributed in graded fashion: low density in the dorsomedial region to high density in the lateral region. Left and right images are higher-magnification images of the dorsomedial and lateral regions (the areas enclosed by the dashed boxes in the center image), respectively. (D) In situ hybridization of signaling molecules in SCCs on coronal sections of adult MOE. Expression of Tas1r3 , Tas2r105 , Tas2r108 , Gnat3 , and Plcb2 was not observed. Only the signal of Trpm5 mRNA was detected in the superficial layer of the MOE. Scale bars: 50 μm in A and D, 500 μm in B and C.
    Figure Legend Snippet: Expression of Skn-1a in the developing main olfactory epithelia. (A) In situ hybridization with RNA probes for Skn-1a in coronal sections of mouse MOE at embryonic days 13.5 and 16.5 and postnatal days 0, 7, 14, and 30. The expression of Skn-1a was first detected at embryonic day 13.5 and was observed during subsequent development. The Skn-1a -expressing cells were located in apical, intermediate, and basal positions in the MOE during embryonic stages and were gradually restricted to apical and basal positions in postnatal development. (B) The expression of Skn-1a in the rostral-caudal axis of the MOE at postnatal day 7. Skn-1a expression was observed throughout the MOE, in terms of the rostral-caudal and the dorsal-ventral axis. (C) In the adult MOE, Skn-1a -expressing cells were distributed in graded fashion: low density in the dorsomedial region to high density in the lateral region. Left and right images are higher-magnification images of the dorsomedial and lateral regions (the areas enclosed by the dashed boxes in the center image), respectively. (D) In situ hybridization of signaling molecules in SCCs on coronal sections of adult MOE. Expression of Tas1r3 , Tas2r105 , Tas2r108 , Gnat3 , and Plcb2 was not observed. Only the signal of Trpm5 mRNA was detected in the superficial layer of the MOE. Scale bars: 50 μm in A and D, 500 μm in B and C.

    Techniques Used: Expressing, In Situ Hybridization

    5) Product Images from "Skn-1a/Pou2f3 functions as a master regulator to generate Trpm5-expressing chemosensory cells in mice"

    Article Title: Skn-1a/Pou2f3 functions as a master regulator to generate Trpm5-expressing chemosensory cells in mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189340

    Effect of Skn-1a deficiency on the functional differentiation of Trpm5 -expressing brush cells in trachea. A: Skn-1a-expressing cells were characterized using immunohistochemistry with anti-Skn-1a and anti-villin antibodies. Villin-positive brush cells were divided into two types, Skn-1a-positive (arrowhead) and Skn-1a-negative brush cells (arrow). B: Skn-1a -expressing cells were characterized by two-color in situ hybridization with RNA probes for Skn-1a and Trpm5 . Skn-1a- positive brush cells were co-labeled with Trpm5 riboprobe (arrowheads). Scale bars, 20 μm. C: The impact of Skn-1a deficiency on the functional differentiation of Trpm5/Skn1a-positive brush cells in the tracheal epithelium was examined by in situ hybridization using probes for taste signaling molecules of Tas1r3 , Tas2rs ( Tas2r105 , Tas2r108 , Tas2r131 ), Gnat3 , Plcb2 and Trpm5 . The mRNA signals of taste signaling molecules observed in wild-type mice were completely absent in the Skn-1a- /- mice, indicating that Skn-1a is required for the functional differentiation of Trpm5-positive brush cells. Scale bar, 100 μm. D: The expression of taste signaling molecules ( Tas1r3 , Tas2r105 , Tas2r108 , Tas2r131 , Gnat3 , Plcb2 , and Trpm5 ) in wild-type (WT) and Skn-1a -/- (KO) trachea was examined by RT-PCR. The expression of taste signaling molecule genes was not detected in Skn-1a-/- trachea. A housekeeping gene, GAPDH was used as a positive control.
    Figure Legend Snippet: Effect of Skn-1a deficiency on the functional differentiation of Trpm5 -expressing brush cells in trachea. A: Skn-1a-expressing cells were characterized using immunohistochemistry with anti-Skn-1a and anti-villin antibodies. Villin-positive brush cells were divided into two types, Skn-1a-positive (arrowhead) and Skn-1a-negative brush cells (arrow). B: Skn-1a -expressing cells were characterized by two-color in situ hybridization with RNA probes for Skn-1a and Trpm5 . Skn-1a- positive brush cells were co-labeled with Trpm5 riboprobe (arrowheads). Scale bars, 20 μm. C: The impact of Skn-1a deficiency on the functional differentiation of Trpm5/Skn1a-positive brush cells in the tracheal epithelium was examined by in situ hybridization using probes for taste signaling molecules of Tas1r3 , Tas2rs ( Tas2r105 , Tas2r108 , Tas2r131 ), Gnat3 , Plcb2 and Trpm5 . The mRNA signals of taste signaling molecules observed in wild-type mice were completely absent in the Skn-1a- /- mice, indicating that Skn-1a is required for the functional differentiation of Trpm5-positive brush cells. Scale bar, 100 μm. D: The expression of taste signaling molecules ( Tas1r3 , Tas2r105 , Tas2r108 , Tas2r131 , Gnat3 , Plcb2 , and Trpm5 ) in wild-type (WT) and Skn-1a -/- (KO) trachea was examined by RT-PCR. The expression of taste signaling molecule genes was not detected in Skn-1a-/- trachea. A housekeeping gene, GAPDH was used as a positive control.

    Techniques Used: Functional Assay, Expressing, Immunohistochemistry, In Situ Hybridization, Labeling, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

    Loss of the Trpm5-positive chemosensory cells in multiple tissues in Skn-1a -/- mice. A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) in various tissues of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on cryosections of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). The arrows indicate Skn-1a negative and villin-single positive cells. Scale bar, 20 μm. C: Double-label immunohistochemistry of Trpm5 and villin on sections of auditory tube, urethra, thymus, and pancreatic duct of wild-type (top) and Skn-1a -/- mice (bottom) was carried out to examine the impact of Skn-1a deficiency on Trpm5-positive chemosensory cells. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a -/- mice. The immunoreactive signals for villin were detected in Skn-1a -/- urethral epithelium and thymic medulla (arrows), but not in auditory tube and pancreatic duct. Scale bar, 20 μm. D: The expression of taste signaling molecules ( Tas1r3 , Tas2r105 , Tas2r108 , Tas2r131 , Gnat3 , Plcb2 , and Trpm5 ) in auditory tube, urethra, thymus, and pancreatic duct was examined by RT-PCR in wild-type (WT) and Skn-1a -/- (KO) mice. The expression of taste signaling molecules observed in wilt-type mice was not detected in Skn-1a -/- mice. A housekeeping gene, GAPDH was used as a positive control.
    Figure Legend Snippet: Loss of the Trpm5-positive chemosensory cells in multiple tissues in Skn-1a -/- mice. A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) in various tissues of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on cryosections of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). The arrows indicate Skn-1a negative and villin-single positive cells. Scale bar, 20 μm. C: Double-label immunohistochemistry of Trpm5 and villin on sections of auditory tube, urethra, thymus, and pancreatic duct of wild-type (top) and Skn-1a -/- mice (bottom) was carried out to examine the impact of Skn-1a deficiency on Trpm5-positive chemosensory cells. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a -/- mice. The immunoreactive signals for villin were detected in Skn-1a -/- urethral epithelium and thymic medulla (arrows), but not in auditory tube and pancreatic duct. Scale bar, 20 μm. D: The expression of taste signaling molecules ( Tas1r3 , Tas2r105 , Tas2r108 , Tas2r131 , Gnat3 , Plcb2 , and Trpm5 ) in auditory tube, urethra, thymus, and pancreatic duct was examined by RT-PCR in wild-type (WT) and Skn-1a -/- (KO) mice. The expression of taste signaling molecules observed in wilt-type mice was not detected in Skn-1a -/- mice. A housekeeping gene, GAPDH was used as a positive control.

    Techniques Used: Mouse Assay, In Situ Hybridization, Labeling, Immunostaining, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control

    Impact of Skn-1a deficiency on Trpm5-positive tuft cells in digestive tracts. A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) on sections of digestive tracts of stomach, small intestine, and large intestine of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on sections of stomach, small intestine, and large intestine of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). Scale bar, 20 μm. C: The impact of Skn-1a deficiency on Trpm5-positive tuft cells was examined by double-label immunohistochemistry of Trpm5 and villin using sections of stomach, small intestine, and large intestine of wild-type (top) and Skn-1a -/- mice. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a -/- mice. Scale bars, 20 μm. The immunoreactive signals for villin detected in wild-type mice (arrows) were not observed in Skn-1a -/- mice. D: The signals of intestinal tuft cells marker gene, Dclk1 mRNA were observed in wild-type digestive tracts, whereas no signals of Dclk1 mRNA were observed in Skn-1a -/-. Scale bars, 100 μm.
    Figure Legend Snippet: Impact of Skn-1a deficiency on Trpm5-positive tuft cells in digestive tracts. A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) on sections of digestive tracts of stomach, small intestine, and large intestine of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on sections of stomach, small intestine, and large intestine of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). Scale bar, 20 μm. C: The impact of Skn-1a deficiency on Trpm5-positive tuft cells was examined by double-label immunohistochemistry of Trpm5 and villin using sections of stomach, small intestine, and large intestine of wild-type (top) and Skn-1a -/- mice. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a -/- mice. Scale bars, 20 μm. The immunoreactive signals for villin detected in wild-type mice (arrows) were not observed in Skn-1a -/- mice. D: The signals of intestinal tuft cells marker gene, Dclk1 mRNA were observed in wild-type digestive tracts, whereas no signals of Dclk1 mRNA were observed in Skn-1a -/-. Scale bars, 100 μm.

    Techniques Used: In Situ Hybridization, Mouse Assay, Labeling, Immunostaining, Immunohistochemistry, Expressing, Marker

    Skn-1a is required for the functional differentiation of Trpm5-positive tracheal brush cells. A: Immunostaining of Trpm5 and ChAT on coronal sections of the trachea of wild-type and Skn-1a -/- mice. Trpm5-positive brush cells were ChAT positive in the wild-type trachea (arrows), whereas no immunoreactive signals for Trpm5 and ChAT was observed in the Skn-1a -/- trachea. B: Immunostaining of Trpm5 and villin on coronal sections of the trachea of wild-type and Skn-1a -/- mice. In wild-type mice, both Trpm5 and villin-double positive (arrowhead) and villin-single positive (arrow) brush cells were observed. In Skn-1a -/- mice, Trpm5-positive brush cells were absent and only villin-single positive brush cells (arrows) were observed. Scale bars, 20 μm. C: Quantification of the number of immunosignals for Trpm5 and villin in the wild-type and Skn-1a -/- tracheal epithelium. The signals of Trpm5 were completely absent in the Skn-1a -/- tracheal epithelium, and the number of villin-single positive cells was significantly decreased in Skn-1a -/- mice. Each symbol represents an individual mouse. The error bars represent the mean ± SEM (n = 3, *P
    Figure Legend Snippet: Skn-1a is required for the functional differentiation of Trpm5-positive tracheal brush cells. A: Immunostaining of Trpm5 and ChAT on coronal sections of the trachea of wild-type and Skn-1a -/- mice. Trpm5-positive brush cells were ChAT positive in the wild-type trachea (arrows), whereas no immunoreactive signals for Trpm5 and ChAT was observed in the Skn-1a -/- trachea. B: Immunostaining of Trpm5 and villin on coronal sections of the trachea of wild-type and Skn-1a -/- mice. In wild-type mice, both Trpm5 and villin-double positive (arrowhead) and villin-single positive (arrow) brush cells were observed. In Skn-1a -/- mice, Trpm5-positive brush cells were absent and only villin-single positive brush cells (arrows) were observed. Scale bars, 20 μm. C: Quantification of the number of immunosignals for Trpm5 and villin in the wild-type and Skn-1a -/- tracheal epithelium. The signals of Trpm5 were completely absent in the Skn-1a -/- tracheal epithelium, and the number of villin-single positive cells was significantly decreased in Skn-1a -/- mice. Each symbol represents an individual mouse. The error bars represent the mean ± SEM (n = 3, *P

    Techniques Used: Functional Assay, Immunostaining, Mouse Assay

    6) Product Images from "TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2"

    Article Title: TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20082012

    The Ca 2+ -binding site of transient receptor potential melastatin member 4 (TRPM4) proposed by a cryo-electron microscopy (cryo-EM) structure analysis. ( A ) A cryo-EM structure of Ca 2+ -bound human TRPM4 (hTRPM4) after [ 11 ] TRPM4 forms a tetramer. Each monomer is shown in different colors. ( B ) A membrane topology of TRPM4. ( C ) An enlarged bottom view of the Ca 2+ -binding site. Black letters and red letters indicate the amino acid residues of hTRPM4 and rat TRPM4 (rTRPM4), respectively. The gray ball is Ca 2+ . ( D ) An alignment of the amino acid sequences of hTRPM4 (GenBank #AAI32728.1), rTPRM4 (NP_001129701.1) and rat TRPM5 (rTRPM5) (NP_001178825.1) around the Ca 2+ -binding site. Magenta letters indicate the amino acids which were mutated in this study.
    Figure Legend Snippet: The Ca 2+ -binding site of transient receptor potential melastatin member 4 (TRPM4) proposed by a cryo-electron microscopy (cryo-EM) structure analysis. ( A ) A cryo-EM structure of Ca 2+ -bound human TRPM4 (hTRPM4) after [ 11 ] TRPM4 forms a tetramer. Each monomer is shown in different colors. ( B ) A membrane topology of TRPM4. ( C ) An enlarged bottom view of the Ca 2+ -binding site. Black letters and red letters indicate the amino acid residues of hTRPM4 and rat TRPM4 (rTRPM4), respectively. The gray ball is Ca 2+ . ( D ) An alignment of the amino acid sequences of hTRPM4 (GenBank #AAI32728.1), rTPRM4 (NP_001129701.1) and rat TRPM5 (rTRPM5) (NP_001178825.1) around the Ca 2+ -binding site. Magenta letters indicate the amino acids which were mutated in this study.

    Techniques Used: Binding Assay, Electron Microscopy, Cryo-EM Sample Prep

    PI(4,5)P 2 restored TRPM4 currents but not TRPM5 currents after their desensitization. ( A ) Typical time courses of the inside-out patch currents of WT rTRPM4 (left) or WT rTRPM5 (right) at +100 mV (filled circles) or −100 mV (open inverted triangles). The time of patch excisions were indicated by arrows. Twenty seconds after the patch excisions, 30 μM diC8-PI(4,5)P 2 (water-soluble PI(4,5)P 2 ) was applied (lower) or not applied (control, upper). The Ca 2+ concentration of bath solutions (i.e., intracellular side, shaded bar) was 0.3 mM. At the end of measurements, the bath solution was changed to the Ca 2+ -free EGTA-containing solution (EGTA, hatched bar). ( B ) Ratios of the current amplitudes at 60 s after patch excision to the initial peak current amplitudes (I 60 sec /I peak ) at +100 mV in the absence (Control) or the presence (PI(4,5)P 2 ) of 30 μM diC8-PI(4,5)P 2 ( n = 6–10). * p
    Figure Legend Snippet: PI(4,5)P 2 restored TRPM4 currents but not TRPM5 currents after their desensitization. ( A ) Typical time courses of the inside-out patch currents of WT rTRPM4 (left) or WT rTRPM5 (right) at +100 mV (filled circles) or −100 mV (open inverted triangles). The time of patch excisions were indicated by arrows. Twenty seconds after the patch excisions, 30 μM diC8-PI(4,5)P 2 (water-soluble PI(4,5)P 2 ) was applied (lower) or not applied (control, upper). The Ca 2+ concentration of bath solutions (i.e., intracellular side, shaded bar) was 0.3 mM. At the end of measurements, the bath solution was changed to the Ca 2+ -free EGTA-containing solution (EGTA, hatched bar). ( B ) Ratios of the current amplitudes at 60 s after patch excision to the initial peak current amplitudes (I 60 sec /I peak ) at +100 mV in the absence (Control) or the presence (PI(4,5)P 2 ) of 30 μM diC8-PI(4,5)P 2 ( n = 6–10). * p

    Techniques Used: Concentration Assay

    The amino acid residues of TRPM5 corresponding to those forming the Ca 2+ -binding site of TRPM4 were also necessary for the normal Ca 2+ -sensitivity of rTRPM5. ( A ) Numbers of corresponding amino acids of rTRPM5 are labeled in the illustration of the Ca 2+ -binding site of hTRPM4. ( B ) CRCs for the effect of Ca 2+ on the current densities mediated by WT rTRPM5 (black circles), D802A (red squares), N805D (dark red triangles), Q782A (green diamonds), D808E (dark yellow circles), E779D (dark yellow horizontal bars), E779Q (purple open diamonds), N805A (light green asterisks), D808N (brown crosses) and the empty vector (gray circles) ( n = 3 or 4 each). ( C ) A result of surface biotinylation assay. The proteins of rTRPM5 expressed in the plasma membrane were biotinylated and precipitated with streptavidin beads (Surface). Non-precipitated fractions contain intracellular proteins (Intracellular). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The mutants showed similar surface expression levels to WT rTRPM5 ( n = 3).
    Figure Legend Snippet: The amino acid residues of TRPM5 corresponding to those forming the Ca 2+ -binding site of TRPM4 were also necessary for the normal Ca 2+ -sensitivity of rTRPM5. ( A ) Numbers of corresponding amino acids of rTRPM5 are labeled in the illustration of the Ca 2+ -binding site of hTRPM4. ( B ) CRCs for the effect of Ca 2+ on the current densities mediated by WT rTRPM5 (black circles), D802A (red squares), N805D (dark red triangles), Q782A (green diamonds), D808E (dark yellow circles), E779D (dark yellow horizontal bars), E779Q (purple open diamonds), N805A (light green asterisks), D808N (brown crosses) and the empty vector (gray circles) ( n = 3 or 4 each). ( C ) A result of surface biotinylation assay. The proteins of rTRPM5 expressed in the plasma membrane were biotinylated and precipitated with streptavidin beads (Surface). Non-precipitated fractions contain intracellular proteins (Intracellular). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The mutants showed similar surface expression levels to WT rTRPM5 ( n = 3).

    Techniques Used: Binding Assay, Labeling, Plasmid Preparation, Surface Biotinylation Assay, Expressing

    PIP 3 also did not restore TRPM5 currents. ( A ) Typical time courses of the inside-out patch currents of WT rTRPM4 (left) or WT rTRPM5 (right) at +100 mV (filled circles) or −100 mV (open inverted triangles). The time of patch excisions were indicated by arrows. Twenty seconds after the patch excisions, 30 μM diC8-PI(3,4,5)P 3 (water-soluble PIP 3 ) was applied. The Ca 2+ concentration of bath solutions (i.e., intracellular side, shaded bar) was 0.3 mM. ( B ) A summary of ratios of the current amplitudes of rTRPM4 and rTRPM5 in the presence of PIP 3 at 60 s after patch excisions to their initial peak current amplitudes (I 60 sec /I peak ) at +100 mV. n = 4 and 6. * p
    Figure Legend Snippet: PIP 3 also did not restore TRPM5 currents. ( A ) Typical time courses of the inside-out patch currents of WT rTRPM4 (left) or WT rTRPM5 (right) at +100 mV (filled circles) or −100 mV (open inverted triangles). The time of patch excisions were indicated by arrows. Twenty seconds after the patch excisions, 30 μM diC8-PI(3,4,5)P 3 (water-soluble PIP 3 ) was applied. The Ca 2+ concentration of bath solutions (i.e., intracellular side, shaded bar) was 0.3 mM. ( B ) A summary of ratios of the current amplitudes of rTRPM4 and rTRPM5 in the presence of PIP 3 at 60 s after patch excisions to their initial peak current amplitudes (I 60 sec /I peak ) at +100 mV. n = 4 and 6. * p

    Techniques Used: Concentration Assay

    7) Product Images from "CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes"

    Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes

    Journal: Nature

    doi: 10.1038/nature11906

    CALHM1 is selectively expressed in type II taste bud cells ( a ) RT-PCR of mRNA of Calhm1 , Actb (β-actin) and taste cell marker genes from laser micro-dissected circumvallate papillae (CVP) taste buds (TB) and lingual epithelium (LE) in wild-type (+/+) and Calhm1 knock-out (−/−) mouse tongues. RT, reverse transcriptase. ( b–d ) in situ hybridization of Calhm1 in CVP TB of WT ( b ), Calhm1 −/− ( c ) and Skn-1a −/− ( d ) mice. Scale bar, 50 μm. Calhm1 is expressed in subsets of CVP ( e ), fungiform ( f ), and palate ( g ) TB cells. ( h ) Double-label in situ hybridization directly illustrates cellular co-expression of Calhm1 and Trpm5 in CVP TB. Most cells expressing Trpm5 also express Calhm1 , with Calhm1 expression absent in Trpm5 negative cells. ( i ) CVP TB illustrating that Tas1r3 is expressed in a subset of Calhm1 positive cells. Scale bars for ( e–i ), 20 μm.
    Figure Legend Snippet: CALHM1 is selectively expressed in type II taste bud cells ( a ) RT-PCR of mRNA of Calhm1 , Actb (β-actin) and taste cell marker genes from laser micro-dissected circumvallate papillae (CVP) taste buds (TB) and lingual epithelium (LE) in wild-type (+/+) and Calhm1 knock-out (−/−) mouse tongues. RT, reverse transcriptase. ( b–d ) in situ hybridization of Calhm1 in CVP TB of WT ( b ), Calhm1 −/− ( c ) and Skn-1a −/− ( d ) mice. Scale bar, 50 μm. Calhm1 is expressed in subsets of CVP ( e ), fungiform ( f ), and palate ( g ) TB cells. ( h ) Double-label in situ hybridization directly illustrates cellular co-expression of Calhm1 and Trpm5 in CVP TB. Most cells expressing Trpm5 also express Calhm1 , with Calhm1 expression absent in Trpm5 negative cells. ( i ) CVP TB illustrating that Tas1r3 is expressed in a subset of Calhm1 positive cells. Scale bars for ( e–i ), 20 μm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Knock-Out, In Situ Hybridization, Mouse Assay, Expressing

    CALHM1 is required for taste-evoked ATP release from taste cells ( a – c ) Electrophysiological phenotypes of types I, II and III cells identified in WT (red) and Calhm1 −/− (blue) taste cells. Cells held at −70 mV and pulsed from −80 to +80 mV in 20 mV increments with 1 sec duration. I Na (●), I slow at end of pulses (■), and I tail (▲) measured in ( d – f ) for type II (n = 9 WT, 10 Calhm1 −/− ), and ( g ) type III (n = 9 WT, 6 Calhm1 −/− ) cells. Type I current recorded from 16 WT, 9 Calhm1 −/− cells. ( h ) Sensitivities of I slow in GFP-positive cells from TRPM5-GFP mice to Gd 3+ (100 μM), probenecid (1 mM), 1-heptanol (1 mM) (n = 4). ( i ) [Ca 2+ ] i in type II cells from WT (left, 9 cells) and Calhm1 −/− (right, 12 cells) mice. Type II cells identified by robust [Ca 2+ ] i response to a mix of sweet and bitter substances (gray bar). Basal ( j ) and taste-evoked responses ( k ) are comparable in WT and Calhm1 −/− cells. ( l ) Taste-evoked ATP release from gustatory CVP tissue and non-gustatory LE. Bitter mix elicits considerable ATP release from CVP vs. LE in WT mice that is abolished in Calhm1 −/− mice and by 1 μM tetrodotoxin (TTX). Error bars, s.e.; * P
    Figure Legend Snippet: CALHM1 is required for taste-evoked ATP release from taste cells ( a – c ) Electrophysiological phenotypes of types I, II and III cells identified in WT (red) and Calhm1 −/− (blue) taste cells. Cells held at −70 mV and pulsed from −80 to +80 mV in 20 mV increments with 1 sec duration. I Na (●), I slow at end of pulses (■), and I tail (▲) measured in ( d – f ) for type II (n = 9 WT, 10 Calhm1 −/− ), and ( g ) type III (n = 9 WT, 6 Calhm1 −/− ) cells. Type I current recorded from 16 WT, 9 Calhm1 −/− cells. ( h ) Sensitivities of I slow in GFP-positive cells from TRPM5-GFP mice to Gd 3+ (100 μM), probenecid (1 mM), 1-heptanol (1 mM) (n = 4). ( i ) [Ca 2+ ] i in type II cells from WT (left, 9 cells) and Calhm1 −/− (right, 12 cells) mice. Type II cells identified by robust [Ca 2+ ] i response to a mix of sweet and bitter substances (gray bar). Basal ( j ) and taste-evoked responses ( k ) are comparable in WT and Calhm1 −/− cells. ( l ) Taste-evoked ATP release from gustatory CVP tissue and non-gustatory LE. Bitter mix elicits considerable ATP release from CVP vs. LE in WT mice that is abolished in Calhm1 −/− mice and by 1 μM tetrodotoxin (TTX). Error bars, s.e.; * P

    Techniques Used: Size-exclusion Chromatography, Mouse Assay

    8) Product Images from "Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice"

    Article Title: Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27113-x

    Simulated motoneurons supplemented with Trpm5 channels display self-sustained spiking activity and predict a role of Trpm5 in amplifying motor outputs. a – c Superpositions of voltages generated by simulated motoneuron with diminished conductance of Na + and K + channels (TTX/TEA condition) in response to depolarizing 2-s pulses (bottom insets) applied at the soma initially held at −60 mV. The sADP (arrow in a ) followed the spiking evoked by suprathreshold stimuli (red) in case of warm temperature (33 °C) and intact Trpm5 channels (Trpm5+) but disappeared (black) after reducing stimulation to subthreshold values ( a ), cooling to 22 °C ( b ), or blockade of Trpm5 channels ( c ). d – f Self-sustained spiking activity (red) of simulated motoneuron with intact Na + and K + channels triggered by a 2-s depolarizing stimuli in case of pre-holding at −60 mV, warm temperature of 33 °C, and intact Trpm5 channels (Trpm5+). Self-sustained spiking did not occur if Trpm5 channels were not sufficiently activated due to low holding (background) potential of −73 mV ( d ), temperature decrease to 22 °C ( e ), or the Trpm5 channels were totally blocked ( f ) although the cell remained capable of firing in response to a depolarizing pulse (black traces). g – i Integrated firing activity in a population of 50 uncoupled motoneurons with randomized Trpm5 expression (normal distribution of maximum conductivity G Trpm5 , mean ± s.d. = 55 ± 11 mS/cm 2 ) generated in response to 1-Hz sinusoid synaptic excitation of the soma ( g ) or dendrites ( h ). i same as in ( h ), but for an ~4-fold reduced Trpm5 expression ( G Trpm5 mean ± s.d. = 12.75 ± 2.0 mS/cm 2 ). Panels top to bottom: raster plots of spiking; mean firing rate in spikes per 1 s per neuron; synaptic conductivity G syn associated with 0-mV reversal potential; normalized cycle-to-cycle firing rate in percentage of response to first effective cycle. Arrows in the top panel indicate scatter plots of firing of individual neurons, exemplified below in j – l and marked by asterisks of the corresponding color.
    Figure Legend Snippet: Simulated motoneurons supplemented with Trpm5 channels display self-sustained spiking activity and predict a role of Trpm5 in amplifying motor outputs. a – c Superpositions of voltages generated by simulated motoneuron with diminished conductance of Na + and K + channels (TTX/TEA condition) in response to depolarizing 2-s pulses (bottom insets) applied at the soma initially held at −60 mV. The sADP (arrow in a ) followed the spiking evoked by suprathreshold stimuli (red) in case of warm temperature (33 °C) and intact Trpm5 channels (Trpm5+) but disappeared (black) after reducing stimulation to subthreshold values ( a ), cooling to 22 °C ( b ), or blockade of Trpm5 channels ( c ). d – f Self-sustained spiking activity (red) of simulated motoneuron with intact Na + and K + channels triggered by a 2-s depolarizing stimuli in case of pre-holding at −60 mV, warm temperature of 33 °C, and intact Trpm5 channels (Trpm5+). Self-sustained spiking did not occur if Trpm5 channels were not sufficiently activated due to low holding (background) potential of −73 mV ( d ), temperature decrease to 22 °C ( e ), or the Trpm5 channels were totally blocked ( f ) although the cell remained capable of firing in response to a depolarizing pulse (black traces). g – i Integrated firing activity in a population of 50 uncoupled motoneurons with randomized Trpm5 expression (normal distribution of maximum conductivity G Trpm5 , mean ± s.d. = 55 ± 11 mS/cm 2 ) generated in response to 1-Hz sinusoid synaptic excitation of the soma ( g ) or dendrites ( h ). i same as in ( h ), but for an ~4-fold reduced Trpm5 expression ( G Trpm5 mean ± s.d. = 12.75 ± 2.0 mS/cm 2 ). Panels top to bottom: raster plots of spiking; mean firing rate in spikes per 1 s per neuron; synaptic conductivity G syn associated with 0-mV reversal potential; normalized cycle-to-cycle firing rate in percentage of response to first effective cycle. Arrows in the top panel indicate scatter plots of firing of individual neurons, exemplified below in j – l and marked by asterisks of the corresponding color.

    Techniques Used: Activity Assay, Generated, Expressing

    Overview of ionic cascades leading to bistability in spinal motoneurons. Schematic relationships between currents underlying the different phases of the self-sustained firing mode. Trpm5 transient receptor potential cation channel subfamily M member 5, Nav voltage-gated sodium channels, Cav voltage-gated calcium channels, depol depolarization, RyR ryanodine receptors, Serca sarco/endoplasmic reticulum Ca 2+ -ATPase.
    Figure Legend Snippet: Overview of ionic cascades leading to bistability in spinal motoneurons. Schematic relationships between currents underlying the different phases of the self-sustained firing mode. Trpm5 transient receptor potential cation channel subfamily M member 5, Nav voltage-gated sodium channels, Cav voltage-gated calcium channels, depol depolarization, RyR ryanodine receptors, Serca sarco/endoplasmic reticulum Ca 2+ -ATPase.

    Techniques Used:

    Trpm5 channels amplify motor outputs. a Schematic representation of the ventral spinal cord side up with the stimulating (DR L5L) and recording (VR L5L) glass electrodes. b , c Ventral root (L5) responses to 1-Hz ipsilateral dorsal root stimuli, recorded from wild-type ( n = 11 mice) and Trpm5 −/− ( n = 7 mice) spinal cords ( b ) or from wild-type spinal cords before and after bath-applying triphenylphosphine oxide (TPPO, 30 µM, n = 8 mice) ( c ). d , e Quantification of the response as a function of the pulse number. Values are relative to the area of the initial post-stimulation response measured during the first inter-pulse interval. f Schematic representation of the whole-mount spinal cord with the recording glass electrodes from the ipsilateral (L2R, L5R) and contralateral (L5L, L5R) sides. The yellow solid line represents the Vaseline barrier separating the rostral (L2) from the caudal (L5) segments. g Ventral root recordings of NMA/5-HT-induced rhythmic activity generated before and after adding triphenylphosphine oxide (TPPO, 30 µM, n = 6 mice) to caudal lumbar segments. h Quantification of locomotor burst parameters. n.s., no significance; * P
    Figure Legend Snippet: Trpm5 channels amplify motor outputs. a Schematic representation of the ventral spinal cord side up with the stimulating (DR L5L) and recording (VR L5L) glass electrodes. b , c Ventral root (L5) responses to 1-Hz ipsilateral dorsal root stimuli, recorded from wild-type ( n = 11 mice) and Trpm5 −/− ( n = 7 mice) spinal cords ( b ) or from wild-type spinal cords before and after bath-applying triphenylphosphine oxide (TPPO, 30 µM, n = 8 mice) ( c ). d , e Quantification of the response as a function of the pulse number. Values are relative to the area of the initial post-stimulation response measured during the first inter-pulse interval. f Schematic representation of the whole-mount spinal cord with the recording glass electrodes from the ipsilateral (L2R, L5R) and contralateral (L5L, L5R) sides. The yellow solid line represents the Vaseline barrier separating the rostral (L2) from the caudal (L5) segments. g Ventral root recordings of NMA/5-HT-induced rhythmic activity generated before and after adding triphenylphosphine oxide (TPPO, 30 µM, n = 6 mice) to caudal lumbar segments. h Quantification of locomotor burst parameters. n.s., no significance; * P

    Techniques Used: Mouse Assay, Activity Assay, Generated

    Trpm5 channels ensure motor control of hindlimbs. a Surface righting response as a function of postnatal day in wild-type (black) and Trpm5 −/− (red) mice. Values represent the time spent for rotating from a supine position to a prone position on their four paws. Picture illustrates a Trpm5 −/− mouse that fails to right itself within 2 min. b Quantification of the base of support between hindlimb paws during walking as a function of age in wild-type (black) and Trpm5 −/− (red) mice. c Latency to fall from a rod rotating at accelerated speed (4–40 rpm) in young adult mice (4 weeks and > 5 weeks old), either wild-type (black) or Trpm5 −/− (red). d Latency to fall from a rod rotating at constant speed in young adult mice (4 weeks and > 5 weeks old), either wild-type (black) or Trpm5 −/− (red). e , f Mean swimming traveled distance ( e ) and velocity ( f ) of neonates (P5–P12) and young adult (3–4 weeks old) wild-type (black) and Trpm5 −/− (red) mice during three consecutive trials. g Heatmap representation of the swimming of neonates (P12) and young adult (3 weeks old) in wild-type (top) and Trpm5 −/− mice (bottom). Scale bar, 10 cm. h Top and side views of 12-day-old wild-type mice transduced either with the scramble shRNA (left) or with the Trpm5-shRNA (right). i Surface righting response during postnatal development in wild-type mice transduced either with the scramble shRNA (black) or with a Trpm5-shRNA (green). j Swimming activity of 12-day-old wild-type mice transduced either with the scramble shRNA (black) or with the Trpm5-shRNA (green) Left: Swimming distance and velocity were averaged from three consecutive swimming trials. Right: Heatmaps illustrate swimming activity. Scale bar, 10 cm. The numbers in the brackets indicate the numbers of mice. n.s., no significance; * P
    Figure Legend Snippet: Trpm5 channels ensure motor control of hindlimbs. a Surface righting response as a function of postnatal day in wild-type (black) and Trpm5 −/− (red) mice. Values represent the time spent for rotating from a supine position to a prone position on their four paws. Picture illustrates a Trpm5 −/− mouse that fails to right itself within 2 min. b Quantification of the base of support between hindlimb paws during walking as a function of age in wild-type (black) and Trpm5 −/− (red) mice. c Latency to fall from a rod rotating at accelerated speed (4–40 rpm) in young adult mice (4 weeks and > 5 weeks old), either wild-type (black) or Trpm5 −/− (red). d Latency to fall from a rod rotating at constant speed in young adult mice (4 weeks and > 5 weeks old), either wild-type (black) or Trpm5 −/− (red). e , f Mean swimming traveled distance ( e ) and velocity ( f ) of neonates (P5–P12) and young adult (3–4 weeks old) wild-type (black) and Trpm5 −/− (red) mice during three consecutive trials. g Heatmap representation of the swimming of neonates (P12) and young adult (3 weeks old) in wild-type (top) and Trpm5 −/− mice (bottom). Scale bar, 10 cm. h Top and side views of 12-day-old wild-type mice transduced either with the scramble shRNA (left) or with the Trpm5-shRNA (right). i Surface righting response during postnatal development in wild-type mice transduced either with the scramble shRNA (black) or with a Trpm5-shRNA (green). j Swimming activity of 12-day-old wild-type mice transduced either with the scramble shRNA (black) or with the Trpm5-shRNA (green) Left: Swimming distance and velocity were averaged from three consecutive swimming trials. Right: Heatmaps illustrate swimming activity. Scale bar, 10 cm. The numbers in the brackets indicate the numbers of mice. n.s., no significance; * P

    Techniques Used: Mouse Assay, shRNA, Activity Assay

    The thermosensitive I CaN -mediated sADP is driven by Trpm5 channels. a – c Left: superimposition of voltage traces in motoneurons recorded under TTX/TEA from wild-type mice in response to a depolarizing pulse before and after bath-applying linoleic acid ( a , L.A., 50 µM, n = 3 mice) or triphenylphosphine oxide ( b , TPPO, 50 µM, n = 4 mice), or recorded in motoneurons from Trpm5 −/− mice ( n = 5 mice) ( c ), right: mean amplitude of the peak sADP. The numbers in brackets indicate the numbers of recorded motoneurons. Each circle represents an individual motoneuron. d Relationship between the peak amplitude of the sADP and the number of spikes emerging during a 2-s depolarizing current pulse in control (black, n = 9 mice) vs Trpm5 −/− mice (red, n = 5 mice). e qRT-PCR analysis assessing the efficiency of the shRNA to knockdown Trpm5 mRNA in HEK-293 cell cultures ( n = 2) and spinal cords ( n = 7) from ~P12 mice. The expression level of the Trpm5 mRNA in cell cultures or spinal cords was normalized to scramble shRNA values with GAPDH or ACTB as internal references, respectively. Each circle represents the mean value from one cell culture or one spinal cord. f Up: Trpm5 immunoblots of lumbar segments from P12 mice intrathecally injected at birth with an adeno-associated virus (AAV9) encoding either a scramble shRNA ( n = 4 mice) or a Trpm5-targeting shRNA ( n = 4 mice). One mice per lane. Bottom: group mean quantification of the ~130 kDa band normalized to scramble-injected controls. g Left: schematic representation of the experimental design, right: acquisition of a transverse spinal slice (L4) from a P10 mouse intrathecally injected at birth with an AAV9 encoding Trpm5-targeting shRNA and eGFP. Scale bar = 100 μm. The experiment was repeated four independent times with similar results. h High magnification of the ventral horn showing native fluorescence of motoneurons (single arrow) transduced by AAV9 (upper left) and immunostained for choline acetyltransferase (upper right, ChAT antibody; bottom left, merged images). Some astrocytes (double arrow) were also GFP+. Histograms (bottom right): group mean quantification of the proportion of 299 motoneurons (green) and 640 astrocytes (orange) from 4 mice transfected by AAV9-shRNA-Trpm5-eGFP. Each circle represents one mouse. Scale bar = 50 μm. i , j Left: superimposition of voltage ( i ) or current ( j ) traces from GFP + motoneurons recorded under TTX/TEA and transduced either with scramble shRNA (black, n = 6 mice) or with a Trpm5-targeting shRNA (green, n = 5 mice), right: mean amplitude of the peak sADP ( i ) and the peak amplitude of the I CAN current ( j ). k Left: superimposition of voltage traces from GFP + astrocytes recorded in normal aCSF and transduced either with scramble shRNA (black, n = 3 mice) or with a Trpm5-targeting shRNA (green, n = 3 mice), right: mean amplitude of the astrocytic resting membrane potential (left) and the input resistance (right). The numbers in brackets indicate the numbers of recorded cells. Each circle represents an individual motoneuron or astrocyte. * P
    Figure Legend Snippet: The thermosensitive I CaN -mediated sADP is driven by Trpm5 channels. a – c Left: superimposition of voltage traces in motoneurons recorded under TTX/TEA from wild-type mice in response to a depolarizing pulse before and after bath-applying linoleic acid ( a , L.A., 50 µM, n = 3 mice) or triphenylphosphine oxide ( b , TPPO, 50 µM, n = 4 mice), or recorded in motoneurons from Trpm5 −/− mice ( n = 5 mice) ( c ), right: mean amplitude of the peak sADP. The numbers in brackets indicate the numbers of recorded motoneurons. Each circle represents an individual motoneuron. d Relationship between the peak amplitude of the sADP and the number of spikes emerging during a 2-s depolarizing current pulse in control (black, n = 9 mice) vs Trpm5 −/− mice (red, n = 5 mice). e qRT-PCR analysis assessing the efficiency of the shRNA to knockdown Trpm5 mRNA in HEK-293 cell cultures ( n = 2) and spinal cords ( n = 7) from ~P12 mice. The expression level of the Trpm5 mRNA in cell cultures or spinal cords was normalized to scramble shRNA values with GAPDH or ACTB as internal references, respectively. Each circle represents the mean value from one cell culture or one spinal cord. f Up: Trpm5 immunoblots of lumbar segments from P12 mice intrathecally injected at birth with an adeno-associated virus (AAV9) encoding either a scramble shRNA ( n = 4 mice) or a Trpm5-targeting shRNA ( n = 4 mice). One mice per lane. Bottom: group mean quantification of the ~130 kDa band normalized to scramble-injected controls. g Left: schematic representation of the experimental design, right: acquisition of a transverse spinal slice (L4) from a P10 mouse intrathecally injected at birth with an AAV9 encoding Trpm5-targeting shRNA and eGFP. Scale bar = 100 μm. The experiment was repeated four independent times with similar results. h High magnification of the ventral horn showing native fluorescence of motoneurons (single arrow) transduced by AAV9 (upper left) and immunostained for choline acetyltransferase (upper right, ChAT antibody; bottom left, merged images). Some astrocytes (double arrow) were also GFP+. Histograms (bottom right): group mean quantification of the proportion of 299 motoneurons (green) and 640 astrocytes (orange) from 4 mice transfected by AAV9-shRNA-Trpm5-eGFP. Each circle represents one mouse. Scale bar = 50 μm. i , j Left: superimposition of voltage ( i ) or current ( j ) traces from GFP + motoneurons recorded under TTX/TEA and transduced either with scramble shRNA (black, n = 6 mice) or with a Trpm5-targeting shRNA (green, n = 5 mice), right: mean amplitude of the peak sADP ( i ) and the peak amplitude of the I CAN current ( j ). k Left: superimposition of voltage traces from GFP + astrocytes recorded in normal aCSF and transduced either with scramble shRNA (black, n = 3 mice) or with a Trpm5-targeting shRNA (green, n = 3 mice), right: mean amplitude of the astrocytic resting membrane potential (left) and the input resistance (right). The numbers in brackets indicate the numbers of recorded cells. Each circle represents an individual motoneuron or astrocyte. * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, shRNA, Expressing, Cell Culture, Western Blot, Injection, Fluorescence, Transfection

    Ryanodine-operated Ca 2+ release activates Trpm5 to promote bistability in motoneurons. a – h Left: superimposition of voltage traces from motoneurons in response to a 2-s depolarizing current pulse recorded with ( a – c , e , g , h ) or without ( d , f ) TTX/TEA before and after bath-applying U73122 ( a , 10 µM, n = 2 mice), xestospongin C ( b , 1–2.5 µM, n = 2 mice), dantrolene ( c , d , 30 µM, n = 5 mice), caffeine ( e , f , 30 µM–5 mM, n = 5 mice), chelerythrin ( g , 10 µM, n = 3 mice), or thapsigargin ( h , 1 µM, n = 3 mice), right: quantification of the area and/or amplitude of the sADP ( a – c , e , g , h ) or of the Δ V ( d , f ) defined as the difference between the most depolarized pre-stimulus holding potential and the most hyperpolarized holding potential for which self-sustained firing can be triggered (see Supplementary Fig. 1 ). Numbers in brackets indicate the numbers of recorded motoneurons. n.s., no significance; * P
    Figure Legend Snippet: Ryanodine-operated Ca 2+ release activates Trpm5 to promote bistability in motoneurons. a – h Left: superimposition of voltage traces from motoneurons in response to a 2-s depolarizing current pulse recorded with ( a – c , e , g , h ) or without ( d , f ) TTX/TEA before and after bath-applying U73122 ( a , 10 µM, n = 2 mice), xestospongin C ( b , 1–2.5 µM, n = 2 mice), dantrolene ( c , d , 30 µM, n = 5 mice), caffeine ( e , f , 30 µM–5 mM, n = 5 mice), chelerythrin ( g , 10 µM, n = 3 mice), or thapsigargin ( h , 1 µM, n = 3 mice), right: quantification of the area and/or amplitude of the sADP ( a – c , e , g , h ) or of the Δ V ( d , f ) defined as the difference between the most depolarized pre-stimulus holding potential and the most hyperpolarized holding potential for which self-sustained firing can be triggered (see Supplementary Fig. 1 ). Numbers in brackets indicate the numbers of recorded motoneurons. n.s., no significance; * P

    Techniques Used: Mouse Assay

    Bistability of motoneurons relies on Trpm5 channels. a – h Left: superimposition of voltage traces recorded in motoneurons from wild-type mice in response to a single ( a – d ) or repetitive (1 Hz, e – h ) depolarizing current pulses before (black) and after (red) bath-applying linoleic acid (L.A., 30 µM) ( a , e , n = 5 mice), or triphenylphosphine oxide (TPPO, 30 µM, n = 7 mice) ( b , f ), or recorded in motoneurons from Trpm5 −/− mice ( c , h , n = 7 mice) or from eGFP+ motoneurons transduced either with the scramble (black, n = 5 mice) or with a Trpm5-targeting shRNA (green, n = 7 mice) ( d , g ), right: group mean quantification of the proportion of bistable motoneurons and/or Δ V ( a – d ) and of the sADP windup ( e – h ). Numbers in brackets indicate the numbers of recorded motoneurons. Each circle represents an individual motoneuron. * P
    Figure Legend Snippet: Bistability of motoneurons relies on Trpm5 channels. a – h Left: superimposition of voltage traces recorded in motoneurons from wild-type mice in response to a single ( a – d ) or repetitive (1 Hz, e – h ) depolarizing current pulses before (black) and after (red) bath-applying linoleic acid (L.A., 30 µM) ( a , e , n = 5 mice), or triphenylphosphine oxide (TPPO, 30 µM, n = 7 mice) ( b , f ), or recorded in motoneurons from Trpm5 −/− mice ( c , h , n = 7 mice) or from eGFP+ motoneurons transduced either with the scramble (black, n = 5 mice) or with a Trpm5-targeting shRNA (green, n = 7 mice) ( d , g ), right: group mean quantification of the proportion of bistable motoneurons and/or Δ V ( a – d ) and of the sADP windup ( e – h ). Numbers in brackets indicate the numbers of recorded motoneurons. Each circle represents an individual motoneuron. * P

    Techniques Used: Mouse Assay, shRNA

    9) Product Images from "Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals"

    Article Title: Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0610201104

    Analysis of spot size for STED images of TRPM5 immunoreactivity in the cilia layer. ( A and B ) Confocal ( A ) and STED ( B ) images of TRPM5 immunofluorescence in the cilia layer of the olfactory epithelium. ( A Inset ) Confocal image at a lower magnification taken with a confocal microscope. ( B Inset ) Smallest spot gained from the faintest antibody cluster observed in the sample is indicative of the maximum size of the effective point-spread function in the confocal (189-nm) and STED (35-nm) imaging modes. ( C and D ) Higher-magnification images of the areas enclosed by the dashed boxes in A and B , respectively. The image in D is the STED image after a linear deconvolution (LD). ( E ) Histogram showing the distribution of full width at half maxima (FWHM) for the clusters in three separate images (130 individual clusters from three separate images). To estimate the FWHM, background was subtracted from the STED images, and each cluster was fit with Lorentz-shaped profiles.
    Figure Legend Snippet: Analysis of spot size for STED images of TRPM5 immunoreactivity in the cilia layer. ( A and B ) Confocal ( A ) and STED ( B ) images of TRPM5 immunofluorescence in the cilia layer of the olfactory epithelium. ( A Inset ) Confocal image at a lower magnification taken with a confocal microscope. ( B Inset ) Smallest spot gained from the faintest antibody cluster observed in the sample is indicative of the maximum size of the effective point-spread function in the confocal (189-nm) and STED (35-nm) imaging modes. ( C and D ) Higher-magnification images of the areas enclosed by the dashed boxes in A and B , respectively. The image in D is the STED image after a linear deconvolution (LD). ( E ) Histogram showing the distribution of full width at half maxima (FWHM) for the clusters in three separate images (130 individual clusters from three separate images). To estimate the FWHM, background was subtracted from the STED images, and each cluster was fit with Lorentz-shaped profiles.

    Techniques Used: Immunofluorescence, Microscopy, Imaging

    EOG recordings from wild-type (TRPM5 +/+) and TRPM5 knockout (TRPM5 −/−) mice. ( A ) Representative EOG traces. ( B ) Average peak EOG responses to different odors ( n = 4–12). There was no significant difference between knockout and control ( P
    Figure Legend Snippet: EOG recordings from wild-type (TRPM5 +/+) and TRPM5 knockout (TRPM5 −/−) mice. ( A ) Representative EOG traces. ( B ) Average peak EOG responses to different odors ( n = 4–12). There was no significant difference between knockout and control ( P

    Techniques Used: Knock-Out, Mouse Assay

    TRPM5 promoter-driven GFP and TRPM5-like antigenicity in the MOE. ( A ) TRPM5 promoter drove GFP expression in two populations of cells with distinct morphology: sparsely distributed short cells
    Figure Legend Snippet: TRPM5 promoter-driven GFP and TRPM5-like antigenicity in the MOE. ( A ) TRPM5 promoter drove GFP expression in two populations of cells with distinct morphology: sparsely distributed short cells

    Techniques Used: Expressing

    Glomeruli targeted by TRPM5-expressing OSNs were detected as GFP-positive glomeruli in TRPM5-GFP mice. ( A and B ) Whole-mount images showing GFP-positive axons and targeted glomeruli in medial ( A ) and lateral ( B ) surfaces of the olfactory bulb. ( C ) A transverse section showing GFP-positive glomeruli. A line drawn through the subventricular zone is the axis used to map glomeruli in D and E . m, medial; l, lateral. ( D ) A representative 2D map showing location of GFP-positive glomeruli along the rostrocaudal distance and angle around the transverse section. ( E ) A pseudocolor rendering of the average number of glomeruli in bins of 10° and 72 μm (average from six bulbs), showing that the highest density of GFP-positive glomeruli was located in the ventral region of the bulb. ( F–H ) Representative glomeruli that were activated by the pheromone DMP ( F ), the sex-specific odorant MTMT ( G ), and soiled bedding from a mating pair ( H ). Activated glomeruli were identified by numerous Fos-expressing periglomerular neurons (red). GFP antibody immunohistochemistry was used in C and F–H . (Scale bars: A and B , 1 mm; C , 100 μm; F–H , 20 μm.)
    Figure Legend Snippet: Glomeruli targeted by TRPM5-expressing OSNs were detected as GFP-positive glomeruli in TRPM5-GFP mice. ( A and B ) Whole-mount images showing GFP-positive axons and targeted glomeruli in medial ( A ) and lateral ( B ) surfaces of the olfactory bulb. ( C ) A transverse section showing GFP-positive glomeruli. A line drawn through the subventricular zone is the axis used to map glomeruli in D and E . m, medial; l, lateral. ( D ) A representative 2D map showing location of GFP-positive glomeruli along the rostrocaudal distance and angle around the transverse section. ( E ) A pseudocolor rendering of the average number of glomeruli in bins of 10° and 72 μm (average from six bulbs), showing that the highest density of GFP-positive glomeruli was located in the ventral region of the bulb. ( F–H ) Representative glomeruli that were activated by the pheromone DMP ( F ), the sex-specific odorant MTMT ( G ), and soiled bedding from a mating pair ( H ). Activated glomeruli were identified by numerous Fos-expressing periglomerular neurons (red). GFP antibody immunohistochemistry was used in C and F–H . (Scale bars: A and B , 1 mm; C , 100 μm; F–H , 20 μm.)

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

    Colocalization of TRPM5 or GFP immunostaining with OMP or CNGA2 immunoreactivity. ( A ) OMP immunoreactivity (green) was seen in mature OSNs. A subset also labeled with TRPM5 antibody (red). ( B ) A confocal image from a section cut perpendicular to the dendrites at the level of the dendritic knobs and cilia displays immunolabel for OMP and TRPM5. ( C ) Sagittal image of the ventral portion of the olfactory bulb of a TRPM5-GFP mouse. Axons from TRPM5-expressing OSNs (green) project to a subset of glomeruli that were immunopositive for OMP (red, overlap appears as yellow). ( D ) An antibody against CNGA2 (red) labeled the apical layer and soma of the majority of OSNs in a TRPM5-GFP mouse. GFP (green) was present in many such OSNs, an indication of coexpression of these ion channels. ( E ) Immunoreactivity for CNGA2 (red) and GFP (green) in the ventral olfactory bulb (saggital section) in a TRPM5-GFP mouse. Most of the GFP-positive glomeruli also were immunoreactive for CNGA2. A relatively small number of glomeruli displayed GFP expression but stained weakly for CNGA2. (Scale bars: A and D , 20 μm; B , 5 μm; C and E , 50 μm.)
    Figure Legend Snippet: Colocalization of TRPM5 or GFP immunostaining with OMP or CNGA2 immunoreactivity. ( A ) OMP immunoreactivity (green) was seen in mature OSNs. A subset also labeled with TRPM5 antibody (red). ( B ) A confocal image from a section cut perpendicular to the dendrites at the level of the dendritic knobs and cilia displays immunolabel for OMP and TRPM5. ( C ) Sagittal image of the ventral portion of the olfactory bulb of a TRPM5-GFP mouse. Axons from TRPM5-expressing OSNs (green) project to a subset of glomeruli that were immunopositive for OMP (red, overlap appears as yellow). ( D ) An antibody against CNGA2 (red) labeled the apical layer and soma of the majority of OSNs in a TRPM5-GFP mouse. GFP (green) was present in many such OSNs, an indication of coexpression of these ion channels. ( E ) Immunoreactivity for CNGA2 (red) and GFP (green) in the ventral olfactory bulb (saggital section) in a TRPM5-GFP mouse. Most of the GFP-positive glomeruli also were immunoreactive for CNGA2. A relatively small number of glomeruli displayed GFP expression but stained weakly for CNGA2. (Scale bars: A and D , 20 μm; B , 5 μm; C and E , 50 μm.)

    Techniques Used: Immunostaining, Labeling, Immunolabeling, Expressing, Staining

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    Alomone Labs rabbit anti trpm5
    Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), <t>TRPM5</t> (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.
    Rabbit Anti Trpm5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.

    Journal: Chemical Senses

    Article Title: Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain

    doi: 10.1093/chemse/bjx032

    Figure Lengend Snippet: Long-term lineage tracing of Sox2 + cells in the gustatory areas of oral epithelium. ( A ) Fluorescence of tdTomato in the oral epithelium of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom ) and nuclei (stained with DAPI, blue, bottom ) in the soft palate ( left ), fungiform papillae (FuP, middle ), and circumvallate papillae (CvP, right ) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. ( B ) Fluorescent labeling of SOX2 (green, left ), TRPM5 (green, middle ), and DDC (green, right ) and tdTomato (red) in the CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. ( C ) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2 CreERT2/+ ; Rosa26 lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.

    Article Snippet: As primary antibodies, rabbit anti-KCNQ1 (1:1000, Millipore), goat anti-KCNQ1 (1:300, Santa Cruz Biotechnology), goat anti-SOX2 (1:300, Santa Cruz Biotechnology), rabbit anti-TRPM5 (1:3000, Alomone Labs), rabbit anti-DDC (1:2000, GeneTex), mouse anti-PCNA (1:100 Millipore), and mouse anti-GFP (1:1000, Clontech Laboratories) antibodies were used.

    Techniques: Fluorescence, Mouse Assay, Labeling, Marker, Staining, Injection

    Ascl3-expressing cells are precursors of microvillar cells and Bowman’s glands. Immunohistochemistry was performed on OE isolated from Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice (2 months), using antibodies to tdTomato (RFP) and ( A ) PLC β2, which marks the apical microvilli of microvillar cells, ( B ) IP3R3, ( C ) Trpm5, ( D ) AQP5 and ( E ) OMP. RFP expression colocalized with microvillar cell markers: PLC β2 (arrowheads), IP3R3 and Trpm5 (arrowheads) and Bowman’s glands markers: AQP5 (arrowheads). ( E ) No colocalization was detected between RFP and the mature OSN marker OMP. White asterisks mark Bowman’s gland duct cells. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Journal: Scientific Reports

    Article Title: Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

    doi: 10.1038/srep38199

    Figure Lengend Snippet: Ascl3-expressing cells are precursors of microvillar cells and Bowman’s glands. Immunohistochemistry was performed on OE isolated from Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice (2 months), using antibodies to tdTomato (RFP) and ( A ) PLC β2, which marks the apical microvilli of microvillar cells, ( B ) IP3R3, ( C ) Trpm5, ( D ) AQP5 and ( E ) OMP. RFP expression colocalized with microvillar cell markers: PLC β2 (arrowheads), IP3R3 and Trpm5 (arrowheads) and Bowman’s glands markers: AQP5 (arrowheads). ( E ) No colocalization was detected between RFP and the mature OSN marker OMP. White asterisks mark Bowman’s gland duct cells. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Article Snippet: Antibodies used: rabbit anti-βIII tubulin (TuJ1) (Abcam), chicken anti-GFP (Abcam), rabbit anti-Keratin-5 (BioLegend), rabbit anti-NPY (Bachem), rabbit anti-PLC β2 (Santa Cruz), rabbit anti-RFP (RocklandTM antibodies & assay), goat anti-Aquaporin 5 (Santa Cruz), rabbit anti-caspase-3 (Abcam), mouse anti-IP3R3 (BD Bioscience), goat anti-OMP (Wako Chemicals), mouse anti-p63 (Santa Cruz), mouse anti-SEC8 (BD Bioscience), rabbit anti-Sox2 (EMD Millipore) and rabbit anti-Trpm5 (Alomone Labs).

    Techniques: Expressing, Immunohistochemistry, Isolation, Mouse Assay, Planar Chromatography, Marker, Staining

    Ablation of Ascl3-expressing cells results in absence of microvillar cells and Bowman’s glands, and decreases GBCs and mature OSNs. ( A ) Ascl3 EGFP-Cre/+ /R26 DTA/+ mice, DTA expression is activated only in the Ascl3-expressing cells. OE was isolated from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at 2 months of age. ( B ) H E staining showed a significant decrease in thickness of the OE in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to Ascl3 +/+ / R26 DTA /+ mice. ( C ) Staining with antibody to PLC β2 (arrowheads) in OE from Ascl3 +/+ / R26 DTA /+ mice and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( D ) Trpm5-positive microvillar cells are present at the apical surface of the OE (arrowheads) in Ascl3 +/+ / R26 DTA /+ mice, but not detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( E ) Antibodies to aquaporin 5 (AQP5) mark the apical surface of the duct cells in the Bowman’s glands extending through the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Ducts cells of the Bowman’s glands were only rarely observed in OE from Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowhead). ( F ) Antibodies to Sox2 revealed no difference in number of sustentacular cells at the apical surface of the OE between mice of the two genotypes. The number of Sox2 + GBCs was decreased (arrowheads). ( G ) p63 + HBC numbers are not changed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( H ) SEC8 antibody labels GBCs near the basal layer of the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Significantly fewer GBCs were detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( I ) Labeling with antibody to OMP showed a significant decrease in the number of labeled mature OSNs in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to controls. ( J ) Antibodies to active caspase-3 showed an increase in number of apoptotic cells in the OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( K ) Quantified results show significant decrease in the thickness of OE and the numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + duct cells of the Bowman gland, but no difference in number of p63 + HBCs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. Quantification also showed a significant decrease in SEC + GBCs and OMP + mature OSNs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. In addition, an increase of caspase-3 + cells was observed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowheads). N ≥ 3 for Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ . *** P

    Journal: Scientific Reports

    Article Title: Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

    doi: 10.1038/srep38199

    Figure Lengend Snippet: Ablation of Ascl3-expressing cells results in absence of microvillar cells and Bowman’s glands, and decreases GBCs and mature OSNs. ( A ) Ascl3 EGFP-Cre/+ /R26 DTA/+ mice, DTA expression is activated only in the Ascl3-expressing cells. OE was isolated from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at 2 months of age. ( B ) H E staining showed a significant decrease in thickness of the OE in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to Ascl3 +/+ / R26 DTA /+ mice. ( C ) Staining with antibody to PLC β2 (arrowheads) in OE from Ascl3 +/+ / R26 DTA /+ mice and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( D ) Trpm5-positive microvillar cells are present at the apical surface of the OE (arrowheads) in Ascl3 +/+ / R26 DTA /+ mice, but not detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( E ) Antibodies to aquaporin 5 (AQP5) mark the apical surface of the duct cells in the Bowman’s glands extending through the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Ducts cells of the Bowman’s glands were only rarely observed in OE from Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowhead). ( F ) Antibodies to Sox2 revealed no difference in number of sustentacular cells at the apical surface of the OE between mice of the two genotypes. The number of Sox2 + GBCs was decreased (arrowheads). ( G ) p63 + HBC numbers are not changed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( H ) SEC8 antibody labels GBCs near the basal layer of the OE in Ascl3 +/+ / R26 DTA /+ mice (arrowheads). Significantly fewer GBCs were detected in OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( I ) Labeling with antibody to OMP showed a significant decrease in the number of labeled mature OSNs in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice compared to controls. ( J ) Antibodies to active caspase-3 showed an increase in number of apoptotic cells in the OE of Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. ( K ) Quantified results show significant decrease in the thickness of OE and the numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + duct cells of the Bowman gland, but no difference in number of p63 + HBCs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. Quantification also showed a significant decrease in SEC + GBCs and OMP + mature OSNs in the Ascl3 EGFP-Cre /+ / R26 DTA /+ mice. In addition, an increase of caspase-3 + cells was observed in Ascl3 EGFP-Cre /+ / R26 DTA /+ mice (arrowheads). N ≥ 3 for Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ . *** P

    Article Snippet: Antibodies used: rabbit anti-βIII tubulin (TuJ1) (Abcam), chicken anti-GFP (Abcam), rabbit anti-Keratin-5 (BioLegend), rabbit anti-NPY (Bachem), rabbit anti-PLC β2 (Santa Cruz), rabbit anti-RFP (RocklandTM antibodies & assay), goat anti-Aquaporin 5 (Santa Cruz), rabbit anti-caspase-3 (Abcam), mouse anti-IP3R3 (BD Bioscience), goat anti-OMP (Wako Chemicals), mouse anti-p63 (Santa Cruz), mouse anti-SEC8 (BD Bioscience), rabbit anti-Sox2 (EMD Millipore) and rabbit anti-Trpm5 (Alomone Labs).

    Techniques: Expressing, Mouse Assay, Isolation, Staining, Planar Chromatography, Labeling, Size-exclusion Chromatography

    Ascl3-expressing cells regenerate microvillar cells and Bowman’s gland/ducts after injury. ( A ) Immunohistochemistry with antibodies to EGFP and CK5 on sections of OE isolated from Ascl3 EGFP-Cre /+ after methimazole injection. At day one (1 dpi) post injury, Ascl3-expressing cells marked by EGFP co-localize with HBCs marked by CK5 expression (arrowheads). EGFP + cells gradually migrate away from the basal layer toward the apical OE at 3 and 14 dpi. ( B ) Lineage tracing in Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice after injury. Antibody to RFP detected Ascl3-expressing cells co-localized with HBCs marked by CK5 antibodies at 1 dpi (arrowheads). RFP-labeled cells become apically localized at 3 and 14 dpi. White asterisk marks delaminated OE tissue. ( C) Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice were treated with methimazole and OE was analyzed after 28 days. ( D – H ) Confocal images of OE sections stained with antibodies to RFP and ( D ) PLC β2, ( E ) IP3R3, ( F ) Trpm5, ( G ) AQP5 and ( H ) OMP. RFP expression colocalized with PLC β2, IP3R3, Trpm5 and AQP5 (arrowheads). ( H ) No colocalization was detected between RFP and OMP. Arrowheads indicate Bowman’s gland ducts within the OE. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Journal: Scientific Reports

    Article Title: Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

    doi: 10.1038/srep38199

    Figure Lengend Snippet: Ascl3-expressing cells regenerate microvillar cells and Bowman’s gland/ducts after injury. ( A ) Immunohistochemistry with antibodies to EGFP and CK5 on sections of OE isolated from Ascl3 EGFP-Cre /+ after methimazole injection. At day one (1 dpi) post injury, Ascl3-expressing cells marked by EGFP co-localize with HBCs marked by CK5 expression (arrowheads). EGFP + cells gradually migrate away from the basal layer toward the apical OE at 3 and 14 dpi. ( B ) Lineage tracing in Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice after injury. Antibody to RFP detected Ascl3-expressing cells co-localized with HBCs marked by CK5 antibodies at 1 dpi (arrowheads). RFP-labeled cells become apically localized at 3 and 14 dpi. White asterisk marks delaminated OE tissue. ( C) Ascl3 EGFP-Cre /+ / R26 tdTomato /+ mice were treated with methimazole and OE was analyzed after 28 days. ( D – H ) Confocal images of OE sections stained with antibodies to RFP and ( D ) PLC β2, ( E ) IP3R3, ( F ) Trpm5, ( G ) AQP5 and ( H ) OMP. RFP expression colocalized with PLC β2, IP3R3, Trpm5 and AQP5 (arrowheads). ( H ) No colocalization was detected between RFP and OMP. Arrowheads indicate Bowman’s gland ducts within the OE. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm.

    Article Snippet: Antibodies used: rabbit anti-βIII tubulin (TuJ1) (Abcam), chicken anti-GFP (Abcam), rabbit anti-Keratin-5 (BioLegend), rabbit anti-NPY (Bachem), rabbit anti-PLC β2 (Santa Cruz), rabbit anti-RFP (RocklandTM antibodies & assay), goat anti-Aquaporin 5 (Santa Cruz), rabbit anti-caspase-3 (Abcam), mouse anti-IP3R3 (BD Bioscience), goat anti-OMP (Wako Chemicals), mouse anti-p63 (Santa Cruz), mouse anti-SEC8 (BD Bioscience), rabbit anti-Sox2 (EMD Millipore) and rabbit anti-Trpm5 (Alomone Labs).

    Techniques: Expressing, Immunohistochemistry, Isolation, Injection, Mouse Assay, Labeling, Staining, Planar Chromatography

    Time course of OE regeneration in the absence of non-neuronal support cells. Quantification of PLC β2 + , Trpm5 + microvillar cells, duct cells of AQP5 + Bowman’s glands, OE thickness, SEC8 + GBCs and caspase-3 + apoptotic cells from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at days 7, 14, 21, 28 post-injury. ( A – C ) Quantified results showed significantly reduced numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + Bowman gland ducts in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( D ) Decrease in the thickness of OE was detected from day 14 dpi during regeneration. ( E ) Decrease of SEC8 + GBCs was observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( F ) More caspase-3 + apoptosis cells were observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. N ≥ 3 for control and Ascl3-DTA mice. * P

    Journal: Scientific Reports

    Article Title: Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

    doi: 10.1038/srep38199

    Figure Lengend Snippet: Time course of OE regeneration in the absence of non-neuronal support cells. Quantification of PLC β2 + , Trpm5 + microvillar cells, duct cells of AQP5 + Bowman’s glands, OE thickness, SEC8 + GBCs and caspase-3 + apoptotic cells from Ascl3 +/+ / R26 DTA /+ and Ascl3 EGFP-Cre /+ / R26 DTA /+ mice at days 7, 14, 21, 28 post-injury. ( A – C ) Quantified results showed significantly reduced numbers of PLC β2 + and Trpm5 + microvillar cells, AQP5 + Bowman gland ducts in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( D ) Decrease in the thickness of OE was detected from day 14 dpi during regeneration. ( E ) Decrease of SEC8 + GBCs was observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. ( F ) More caspase-3 + apoptosis cells were observed in the Ascl3-DTA mice at days 7, 14, 21, 28 post-injury. N ≥ 3 for control and Ascl3-DTA mice. * P

    Article Snippet: Antibodies used: rabbit anti-βIII tubulin (TuJ1) (Abcam), chicken anti-GFP (Abcam), rabbit anti-Keratin-5 (BioLegend), rabbit anti-NPY (Bachem), rabbit anti-PLC β2 (Santa Cruz), rabbit anti-RFP (RocklandTM antibodies & assay), goat anti-Aquaporin 5 (Santa Cruz), rabbit anti-caspase-3 (Abcam), mouse anti-IP3R3 (BD Bioscience), goat anti-OMP (Wako Chemicals), mouse anti-p63 (Santa Cruz), mouse anti-SEC8 (BD Bioscience), rabbit anti-Sox2 (EMD Millipore) and rabbit anti-Trpm5 (Alomone Labs).

    Techniques: Planar Chromatography, Mouse Assay

    Effect of Skn-1a deficiency on the functional differentiation of Trpm5-positive microvillous cell. (A) In situ hybridization of Trpm5 on coronal sections of the MOE of wild-type and Skn-1a -/- mice. The mRNA signal of Trpm5 was absent in Skn-1a -/- mice. (B and C) Coronal sections of wild-type and Skn-1a -/- MOE of adult mice were immunostained with an anti-Trpm5 antibody (green) and an anti-villin (B) or anti-ChAT (C) antibody (red). Trpm5-positive cells were villin positive in the microvilli in the wild-type MOE (arrowheads), whereas no immunoreactive signal for Trpm5 or villin was observed in the Skn-1a -/- MOE. Trpm5-positive cells were co-immunostained with anti-ChAT antibody in wild-type (arrowheads) but not in Skn-1a -/- mice. Scale bars: 100 μm in A, 10 μm in B and C.

    Journal: BMC Neuroscience

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    doi: 10.1186/1471-2202-15-13

    Figure Lengend Snippet: Effect of Skn-1a deficiency on the functional differentiation of Trpm5-positive microvillous cell. (A) In situ hybridization of Trpm5 on coronal sections of the MOE of wild-type and Skn-1a -/- mice. The mRNA signal of Trpm5 was absent in Skn-1a -/- mice. (B and C) Coronal sections of wild-type and Skn-1a -/- MOE of adult mice were immunostained with an anti-Trpm5 antibody (green) and an anti-villin (B) or anti-ChAT (C) antibody (red). Trpm5-positive cells were villin positive in the microvilli in the wild-type MOE (arrowheads), whereas no immunoreactive signal for Trpm5 or villin was observed in the Skn-1a -/- MOE. Trpm5-positive cells were co-immunostained with anti-ChAT antibody in wild-type (arrowheads) but not in Skn-1a -/- mice. Scale bars: 100 μm in A, 10 μm in B and C.

    Article Snippet: The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M.

    Techniques: Functional Assay, In Situ Hybridization, Mouse Assay

    Characterization of Skn-1a -expressing cells in the main olfactory epithelium. (A) Skn-1a -expressing cells were characterized using two-color in situ hybridization in coronal sections of the MOE at postnatal day 0 with RNA probes for Skn-1a (green) and OSN progenitor/precursor genes Mash1 (neuronal progenitors), Ngn1 (neuronal precursors), and NeuroD (differentiating/postmitotic neurons). Small populations of Skn-1a -potitive cells and Mash1 -positive cells overlapped. The arrowhead indicates a co-labeled cell, and arrows indicate either Skn-1a or Mash1 single-labeled cells . None of Skn-1a -positive cells were co-labeled with Ngn1 and NeuroD (arrows). (B and C) In situ hybridization of Skn-1a (green) with OMP (mature OSNs; B, red) and Trpm5 ( Trpm5 -positive microvillous cells; C, red) in coronal sections of the adult MOE. Neither apical nor basal Skn-1a -expressing cells (arrows) were co-labeled with OMP signals. Trpm5 signals were co-labeled with apical Skn-1a signals (arrowheads) but not with basal Skn-1a signals (arrow). Scale bars, 25 μm. (D and E) Populations of Skn-1a- expressing cells (D) and Mash1- expressing cells (E) were analyzed by two-color in situ hybridization at postnatal day 30. Quantitative analyses revealed that 8.34 ± 2.82% (mean ± SD) of the Skn-1a- expressing cells coexpressed Mash1 (n = 3) , and 77.7 ± 5.95% coexpressed Trpm5 (n = 3). In the OSN-lineage, Mash1- positive olfactory progenitors rarely expressed Skn-1a (1.41 ± 0.564%, n = 3).

    Journal: BMC Neuroscience

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    doi: 10.1186/1471-2202-15-13

    Figure Lengend Snippet: Characterization of Skn-1a -expressing cells in the main olfactory epithelium. (A) Skn-1a -expressing cells were characterized using two-color in situ hybridization in coronal sections of the MOE at postnatal day 0 with RNA probes for Skn-1a (green) and OSN progenitor/precursor genes Mash1 (neuronal progenitors), Ngn1 (neuronal precursors), and NeuroD (differentiating/postmitotic neurons). Small populations of Skn-1a -potitive cells and Mash1 -positive cells overlapped. The arrowhead indicates a co-labeled cell, and arrows indicate either Skn-1a or Mash1 single-labeled cells . None of Skn-1a -positive cells were co-labeled with Ngn1 and NeuroD (arrows). (B and C) In situ hybridization of Skn-1a (green) with OMP (mature OSNs; B, red) and Trpm5 ( Trpm5 -positive microvillous cells; C, red) in coronal sections of the adult MOE. Neither apical nor basal Skn-1a -expressing cells (arrows) were co-labeled with OMP signals. Trpm5 signals were co-labeled with apical Skn-1a signals (arrowheads) but not with basal Skn-1a signals (arrow). Scale bars, 25 μm. (D and E) Populations of Skn-1a- expressing cells (D) and Mash1- expressing cells (E) were analyzed by two-color in situ hybridization at postnatal day 30. Quantitative analyses revealed that 8.34 ± 2.82% (mean ± SD) of the Skn-1a- expressing cells coexpressed Mash1 (n = 3) , and 77.7 ± 5.95% coexpressed Trpm5 (n = 3). In the OSN-lineage, Mash1- positive olfactory progenitors rarely expressed Skn-1a (1.41 ± 0.564%, n = 3).

    Article Snippet: The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M.

    Techniques: Expressing, In Situ Hybridization, Labeling

    Expression of Skn-1a and Trpm5 in the MOE of Mash1 -/- embryos. Expression of Skn-1a and Trpm5 in the Mash1 -/- MOE was examined by in situ hybridization at embryonic day 18.5. The MOE of Mash1 -/- embryos appeared smaller and thinner than that of wild-type littermates, as observed previously. Expression of either Skn-1a or Trpm5 was observed in both the wild-type and Mash1 -/- MOE. Higher-magnification images of the dotted boxes are presented to the right of each image. Scale bars, 100 μm.

    Journal: BMC Neuroscience

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    doi: 10.1186/1471-2202-15-13

    Figure Lengend Snippet: Expression of Skn-1a and Trpm5 in the MOE of Mash1 -/- embryos. Expression of Skn-1a and Trpm5 in the Mash1 -/- MOE was examined by in situ hybridization at embryonic day 18.5. The MOE of Mash1 -/- embryos appeared smaller and thinner than that of wild-type littermates, as observed previously. Expression of either Skn-1a or Trpm5 was observed in both the wild-type and Mash1 -/- MOE. Higher-magnification images of the dotted boxes are presented to the right of each image. Scale bars, 100 μm.

    Article Snippet: The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M.

    Techniques: Expressing, In Situ Hybridization

    Quantification of microvillous cell density in the most superficial layer of the MOE. (A) Image of an MOE dorsal recess from a ChAT-eGFP mouse, showing ChAT / Trpm5 -expressing microvillous cells (GFP + ) in the most superficial layer, a region above the supporting cell nuclei. (B) A higher-magnification view of the DAPI-stained nuclei in the dorsal MOE. Arrowheads point to nuclei of GFP + microvillous cells. (B’) Overlay of GFP signal onto B. (C) Image of an MOE dorsal recess from an Skn-1a -/- mouse. Arrows in B and C point to nuclei that do not belong to GFP + microvillous cells. (D) Plot of the averaged density per surface area of DAPI-stained nuclei and GFP + cells in the most superficial layer of the MOE from ChAT-eGFP mice. Counting was conducted from the dorsal recess and septum of the MOE. Approximately 80% of the cells in the area are GFP + microvillous cells. (E) Comparison of averaged nucleus density, showing approximately 73% reduction in the nucleus density of Skn-1a -/- mice compared with that of ChAT-eGFP mice. Scale bars: 100 μm in A, 20 μm in B-D.

    Journal: BMC Neuroscience

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    doi: 10.1186/1471-2202-15-13

    Figure Lengend Snippet: Quantification of microvillous cell density in the most superficial layer of the MOE. (A) Image of an MOE dorsal recess from a ChAT-eGFP mouse, showing ChAT / Trpm5 -expressing microvillous cells (GFP + ) in the most superficial layer, a region above the supporting cell nuclei. (B) A higher-magnification view of the DAPI-stained nuclei in the dorsal MOE. Arrowheads point to nuclei of GFP + microvillous cells. (B’) Overlay of GFP signal onto B. (C) Image of an MOE dorsal recess from an Skn-1a -/- mouse. Arrows in B and C point to nuclei that do not belong to GFP + microvillous cells. (D) Plot of the averaged density per surface area of DAPI-stained nuclei and GFP + cells in the most superficial layer of the MOE from ChAT-eGFP mice. Counting was conducted from the dorsal recess and septum of the MOE. Approximately 80% of the cells in the area are GFP + microvillous cells. (E) Comparison of averaged nucleus density, showing approximately 73% reduction in the nucleus density of Skn-1a -/- mice compared with that of ChAT-eGFP mice. Scale bars: 100 μm in A, 20 μm in B-D.

    Article Snippet: The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M.

    Techniques: Expressing, Staining, Mouse Assay

    Expression of Skn-1a in the developing main olfactory epithelia. (A) In situ hybridization with RNA probes for Skn-1a in coronal sections of mouse MOE at embryonic days 13.5 and 16.5 and postnatal days 0, 7, 14, and 30. The expression of Skn-1a was first detected at embryonic day 13.5 and was observed during subsequent development. The Skn-1a -expressing cells were located in apical, intermediate, and basal positions in the MOE during embryonic stages and were gradually restricted to apical and basal positions in postnatal development. (B) The expression of Skn-1a in the rostral-caudal axis of the MOE at postnatal day 7. Skn-1a expression was observed throughout the MOE, in terms of the rostral-caudal and the dorsal-ventral axis. (C) In the adult MOE, Skn-1a -expressing cells were distributed in graded fashion: low density in the dorsomedial region to high density in the lateral region. Left and right images are higher-magnification images of the dorsomedial and lateral regions (the areas enclosed by the dashed boxes in the center image), respectively. (D) In situ hybridization of signaling molecules in SCCs on coronal sections of adult MOE. Expression of Tas1r3 , Tas2r105 , Tas2r108 , Gnat3 , and Plcb2 was not observed. Only the signal of Trpm5 mRNA was detected in the superficial layer of the MOE. Scale bars: 50 μm in A and D, 500 μm in B and C.

    Journal: BMC Neuroscience

    Article Title: Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

    doi: 10.1186/1471-2202-15-13

    Figure Lengend Snippet: Expression of Skn-1a in the developing main olfactory epithelia. (A) In situ hybridization with RNA probes for Skn-1a in coronal sections of mouse MOE at embryonic days 13.5 and 16.5 and postnatal days 0, 7, 14, and 30. The expression of Skn-1a was first detected at embryonic day 13.5 and was observed during subsequent development. The Skn-1a -expressing cells were located in apical, intermediate, and basal positions in the MOE during embryonic stages and were gradually restricted to apical and basal positions in postnatal development. (B) The expression of Skn-1a in the rostral-caudal axis of the MOE at postnatal day 7. Skn-1a expression was observed throughout the MOE, in terms of the rostral-caudal and the dorsal-ventral axis. (C) In the adult MOE, Skn-1a -expressing cells were distributed in graded fashion: low density in the dorsomedial region to high density in the lateral region. Left and right images are higher-magnification images of the dorsomedial and lateral regions (the areas enclosed by the dashed boxes in the center image), respectively. (D) In situ hybridization of signaling molecules in SCCs on coronal sections of adult MOE. Expression of Tas1r3 , Tas2r105 , Tas2r108 , Gnat3 , and Plcb2 was not observed. Only the signal of Trpm5 mRNA was detected in the superficial layer of the MOE. Scale bars: 50 μm in A and D, 500 μm in B and C.

    Article Snippet: The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M.

    Techniques: Expressing, In Situ Hybridization