Journal: Frontiers in Cellular Neuroscience

Article Title: Age-related alterations in efferent medial olivocochlear-outer hair cell and primary auditory ribbon synapses in CBA/J mice

doi: 10.3389/fncel.2024.1412450

Figure Lengend Snippet: Antibody labeling.

Article Snippet: Anti-MyosinVIIa (1:200) , HIS-tagged synthetic peptide in the human MYO7A sequence (a.a. 927–1,203) , DSHB** – mouse monoclonal , MYO7A 138–1 RRID: AB_2282417 , Immunohistochemistry, immunoblot/Western blot ( ) , Hair cells ( ; ; ) .

Techniques: Labeling, Staining, Recombinant, Expressing, Marker, Sequencing, Immunohistochemistry, Western Blot, Immunoprecipitation, Membrane

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a LacZ reporter is expressed throughout the sensory region in Gpr156 del/+ vestibular organs (ant/lat c., anterior/lateral crista). b LacZ expression is limited to MYO7A + HCs in a saccule cross-section. X-gal signal is trapped in HC vesicles (arrow in magnified inset) but support cells (arrowhead) are negative. c , d P2 wild-type utricle where basal body labeling (PCNT) indicates HC orientation. GPR156 polarization (solid arrowheads) is limited to lateral HCs oriented medially. HCs across the LPR oriented laterally do not show polarized GPR156 (hollow arrowheads). Boxed regions in continuous fields in the left panels are magnified in the central and right panels (saccule: see Supplementary Fig. ). d GPR156 enrichment in the utricle LPR domain at the HC junction opposite (opp. BB) or near (BB) the basal body. HCs oriented medially (left) are analyzed separately from HC oriented laterally (right). GPR156 is expressed as ratio of ZO1 signal (mean ± SD; n , HC numbers in 3 animals; Kruskal-Wallis test with Dunn’s multiple comparisons, **** p < 0.0001; * p = 0.0332). e Summary of HC orientation (arrows), GPR156 protein distribution (magenta) and previously reported Emx2 expression (blue) by vestibular organ in normal and mutant conditions. The scheme in c indicates the position of the domain analyzed in c and d (blue). Scale bars are 100 µm ( a ), 50 µm ( b ), 20 µm ( c ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Expressing, Labeling, Mutagenesis

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a LPR domain in E17.5 utricles. Polarization of GPR156 in lateral HCs (arrowhead) is lost when EMX2 is missing and these HCs fail to reverse. b GPR156 enrichment in the lateral utricle (LES) domain. c Medial domain in E18.5 utricles. Ectopic expression of Emx2 reverses HC orientation and induces polarization of GPR156 (arrowhead) in medial HCs. d GPR156 enrichment in the utricle medial domain. e LPR domain in E18.5 utricles. Polarization of GPR156 in lateral HCs expressing PTXa is intact although these HCs fail to reverse. Utricles are labeled with GPR156, PCNT, and ZO1 ( a , c ) or MYO7A ( e ). In a , c , e boxed areas are magnified in the lower panels, and HC orientation and GPR156 distribution are summarized in a cartoon form. In b , d GPR156 enrichment is measured at the junction opposite (opp. BB) or near (BB) the basal body in the same HC. GPR156 is expressed as ratio of ZO1 signal (mean ± SD; n, HC numbers in 3 or more animals; Kruskal-Wallis test, **** p < 0.0001, ns p > 0.9999). Arrows indicate HC orientation based on PCNT-labeled basal body. Utricle schemes indicate the domain imaged or analyzed (blue). Yellow dashed lines represent the LPR in controls. See Supplementary Fig. for related saccule and crista results. Scale bars are 10 µm ( a , c ), 20 µm ( e ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Expressing, Labeling

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a Schematic of the lateral-line system in a 5 day-post-fertilization zebrafish. Neuromast HCs show binary orientation along the antero-posterior (A–P) or dorso-ventral (D–V) axis as depicted. Emx2 is only expressed in HCs of one orientation in each neuromast (green D to V and A to P HCs). HC orientation is indicated by a black dot representing the off-center basal body. b – i Phalloidin labeling in neuromasts reveals HC orientation by the lack of signal above the off-center basal body. In wild-type siblings ( b , e , f , i ), neuromasts contain an equal proportion of HCs with either orientation. In gpr156 mutants ( c – e , g – i ), there are more P to A ( c – e ) and V to D ( g – i )-oriented HCs compared to wild-type siblings (Tukey’s multiple comparison test, P to A exon2 allele p < 0.0001, sa34566 allele p < 0.0001; V to D exon2 allele p < 0.0001, sa34566 allele p < 0.0001). Green and blue asterisks highlight the two HC orientations in wild-type sibling neuromasts. Magenta and yellow asterisks highlight outlier HCs oriented 180° or 90° compared to the majority of HCs in gpr156 mutants. n = 10 neuromasts and N ≥ 8 animals per genotype, examined at 5 dpf. j , k Emx2 and Myo7a co-labeling in neuromasts. Wild-type siblings and gpr156 mutants neuromasts have a similar number of HCs ( l ) (mean ± SEM; unpaired t-test (two-tailed), A-P p = 0.1686; Mann–Whitney test (two-tailed), D-V p = 0.8547), and a similar proportion of HCs express Emx2 per neuromast ( m ) (mean ± SEM; unpaired t-test (two-tailed), A-P p = 0.5756; Mann–Whitney test (two-tailed), D-V p = 0.4805). In l – m the number of neuromasts (n) examined at 5 dpf in N ≥ 8 animals per genotype is indicated. n Scheme showing the GCaMP6s calcium reporter (blue and green) and the imaging plane in a neuromast. o1 , p1 Baseline gray scale GCaMP6s images of the hair bundle imaging plane in wild-type siblings ( o1 ) and gpr156 mutants ( p1 ; sa34566 allele). o2 , o3 , p2 , p3 Spatial patterns of GCaMP6s calcium signal increases in hair bundles during P to A ( o2 , p2 ) or A to P ( o3 , p3 ) directed fluid-jet stimulation. GCaMP6s signals are colorized according to the ∆F heat maps and superimposed onto prestimulus (prestim) baseline images ( o1 , p1 ). q In wild-type siblings, GCaMP6s signals are detected during both P to A and A to P directed stimulation ( o2 , o3 ). In contrast, compared to wild-type, in gpr156 mutants, significantly more hair bundles respond to P to A directed stimulation ( p2 – p3 ) (Sidak’s multiple comparison test, P to A p = 0.0008; n = 8 neuromasts per genotype and N = 4 wild-type and N = 3 mutant animals, examined at 5 dpf. See Supplementary Fig. for individual HC responses). NM, neuromast; sib, wild-type sibling. Scale bars are 5 µm ( b – d and f – h , j , k , o 1 – 3 , and p 1 – p 3 ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY, Imaging, Mutagenesis

Journal: Hearing research

Article Title: Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA

doi: 10.1016/j.heares.2013.06.011

Figure Lengend Snippet: The number of Jag1 positive SCs decreased in OCs following Hes1 siRNA treatment. Examples of confocal images from untreated OC cultures (A), or OCs treated with Hes 1 siRNALF (B), 4-HNE plus scRNALF (C), or 4-HNE plus Hes 1 siRNALF (D) and labeled with anti-Jagged1 antibody (green, arrowheads), anti-myosin VIIa antibody (red, brackets-OHCs, arrows-IHCs), and DAPI (blue, nuclei) are shown. Note that four rows of OHCs are observed in the OC treated with either Hes1 siRNALF alone or Hes1 siRNALF plus 4-HNE (bracket in B and D) and no OHCs were observed in the cross-sectional view of the OC treated with scRNALF and 4-HNE (C). The number of Jag1 positive SCs was counted in the OCs and statistically analyzed (E). Significantly fewer Jag1 positive SCs were observed in the OC cultures treated with either Hes1 siRNALF alone or Hes1 siRNALF plus 4-HNE compared to the number of Jag1 positive cells observed in OCs treated with scRNALF alone or scRNALF plus 4-HNE treated cultures (decreased by 23.56% and 25.86%, respectively, all p < 0.01). No significant difference in Jag1 positive cell numbers was observed in OCs treated with scRNA compared to untreated cultures (p > 0.05). ** indicate p < 0.01. Scale bar = 10 μm in D for A-D. All images were taken from OCs that had been cultured eight days in vitro.

Article Snippet: The sections were immunolabeled with goat anti-Jagged1 antibody (1:250, Santa Cruz Biotechnology, Inc. Santa Cruz, CA) and rabbit anti-myosin VIIa (1:12.5, Santa Cruz Biotechnology, Inc. Santa Cruz, CA) followed by incubation with Alexa Fluor® 594 donkey anti-rabbit IgG and Alexa Fluor® 488 donkey anti-goat IgG (each at 1:1000, Invitrogen, Carlsbad, CA).

Techniques: Labeling, Cell Culture, In Vitro

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A . Schematic diagram showing the genetic structure of the integrated EIAV vectors used in this study . EIAV-CMV-MYO7A (UshStat) is based on a non-replicating non-human recombinant lentiviral vector based on the non-pathogenic wild type equine infectious anaemia virus (EIAV). The wild-type EIAV virus has 6 distinct genetic units, however, the majority of these EIAV sequences have been removed to produce a minimal vector system that contains less than 10% of the original viral genome and does not contain any viral promoters or enhancers and there are no coding regions for accessory proteins in either the EIAV genome or in the packaging system. SIN LTR: Self inactivating long term repeat. Neo: Neomycin open reading frame (ORF). CMV: Cytomegalovirus promoter (constitutive). RK: Rhodopsin kinase promoter (photoreceptor specific). eGFP: enhanced green fluorescent protein ORF. MYO7A: Myosin VIIa ORF. WPRE: Woodchuk hepatitis virus post-transcriptional regulatory element. B . Expression analysis of myosin VIIa in 4 weeks mouse eye and HeLa cells transfected with the EIAV-CMV-Null (Null) or UshStat constructs. β-actin was used as loading control. RPE: retinal pigment epithelium. NR: neuroretina. IP/Null: immunoprecipitates of HeLa cells transfected with the null vector. IP/UshStat: immunoprecipitates of HeLa cells transfected with the myosin VIIa vector. IB MYO7A: immunoblot with the mouse anti-myosin VIIa. Molecular weight markers are denoted to the left. C–D : Immunocytochemistry studies of HeLa cells transduced with the null ( C ) or the UshStat vector ( D ) and immunostained for myosin VIIa (red). DAPI was used to counter stain the nucleus. Scale bar: 15 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Recombinant, Plasmid Preparation, Virus, Expressing, Transfection, Construct, Control, Western Blot, Molecular Weight, Immunocytochemistry, Transduction, Staining

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Shaker1 mouse retinas were co-transduced by a subretinal injection of UshStat and EIAV-CMV-GFP or EIAV-CMV-Null and EIAV-RK-GFP (the GFP vector was used to identify the transduced region of the retina). After 4 weeks the animals were dark-adapted overnight, then light-adapted for 10 minutes under 200 lux illumination. Retinas were double immunostained with antibodies against GFP (green) and α-transducin (red). Panel I : Low magnification image of a retina transduce with UshStat and GFP, showing that the gradient in α-transducin translocation (left to right) parallels GFP expression (indicative of wild type myosin VIIa). Scale bar: 40 μm. Panel II : A–C : Representative examples of an EIAV transduced region of the retina (determine by the presence of GFP), presenting dual immunostaining for GFP and α-transducin ( C ). In the presence of wild type myosin VIIa, α-transducin is translocated to the inner segment (IS). D–F : An untransduced region of the same retina as in A–C does not show α-transducin translocation to the IS upon illumination. G–I : Retinas co-transduced with EIAV-Null-vector and EIAV-RK-GFP (photoreceptor cell-specific promoter) as a control for the effects of subretinal lentiviral transduction on α-transducin translocation. The region of the retina shown was transduced, evidenced by GFP expression in the photoreceptors ( H ) however there was no translocation of α-transducin to the IS ( G and I ). The qualitative results represented in each of the panels are representative images for at least three replicate experiments. RPE = Retinal Pigment Epithelium; OS = Outer Segments; IS = Inner Segments; ONL = Outer Nuclear Layer; OPL = Outer Plexiform Layer. Scale bar: 25 μm. Arrowheads in D and G indicate translocation of α-transducin in individual photoreceptors. Asterisks in I denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Injection, Plasmid Preparation, Translocation Assay, Expressing, Immunostaining, Transduction, Control

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A–B : Show myosin VIIa immunostaining of shaker1 retinas transduced with the EIAV-CMV-Null vector ( A ) or UshStat ( B ). Myosin VIIa expression can be detected in the RPE, OS and IS regions. C–D : monkey retinas transduced with UshStat ( D ) showed high levels of human myosin VIIa in the RPE and moderate expression in outer and inner segments of the photoreceptor cells. Asterisk in C denotes weak immunostaining of the endogenous myosin VIIa in the RPE layer. Note that the anti-myosin VIIa antibody was titrated so as to only detect the exogenous overexpressed virus-derived wild type myosin VIIa ( B, D ) and not the endogenous protein ( A, C ). Scale bars: 25 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Immunostaining, Transduction, Plasmid Preparation, Expressing, Virus, Derivative Assay

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Retinas were co-transduced with EIAV-CMV-Null vector and EIAV-RK-GFP ( A–C ) or UshStat and EIAV-CMV-GFP ( D–F, J ). After 4 weeks, mice were exposed to 6 days continuous light at 2000 lux illumination. Retinas were harvested and dual immunostained for myosin VIIa (red) and GFP (green) ( A–F, J ). Immunostaining conditions were chosen as to only detect the exogenous overexpressed myosin VIIa. G–H : Eosin and hematoxylin histochemical staining of shaker1 mouse retinas transduced with either the EIAV-CMV-Null vector ( G ) or UshStat ( H ). I : Eosin and hematoxylin staining of wild type (untransduced) retina for comparison of relative light dependent degeneration of the ONL that is typically observed (double headed arrow). J : Low magnification image of an UshStat+GFP co-transduced retina, showing the area used for the studies (bracket) relative to the area of injection (syringe). This area corresponds to the juxtaposed ∼0.2 mm from the site of injection. K : Schematic representation of the area of injection represented by red boxes, where right box area corresponds to the injection in the right eye and left box area injection in the left eye. The inferior retina was always used for the injections and ONL counting. Scale bars A–F : 25 μm, G–I : 50 μm. J : 90 μm. Labels are as in . Asterisks in C , F and J denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Transduction, Plasmid Preparation, Immunostaining, Staining, Comparison, Injection

Journal: EMBO Molecular Medicine

Article Title: Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness

doi: 10.15252/emmm.202013259

Figure Lengend Snippet: Whole‐mount immunofluorescence of a P8 Syne4 −/− organ of Corti showing intact hair cells, as labeled with myosin VIIa. Whole‐mount immunofluorescence of WT and Syne4 −/− organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14. Inner and outer hair cell counts of Syne4 −/− organ of Corti at P8, P10, P12, and P14. FM1‐43 uptake performed on P8+1 DIV (days‐ in‐vitro ) WT and Syne4 −/− organ of Corti. Top shows OHC plane, and bottom shows IHC plane. Data information: Scale bars = 100 μm for (A) and 10 μm for (B and D). Source data are available online for this figure.

Article Snippet: Antibody and stain concentrations were as follows: rabbit polyclonal myosin VIIa (Proteus Biosciences 25‐6790) 1:250, mouse anti‐FLAG (Sigma F3165) 1:1,000, DAPI (Abcam ab228549) 1:1,000, goat anti‐mouse (Abcam ab150119) 1:250, and goat anti‐rabbit Alexa Fluor 488 (Cell Signaling 4412s).

Techniques: Immunofluorescence, Labeling, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness

doi: 10.15252/emmm.202013259

Figure Lengend Snippet: Whole‐mount immunofluorescence of Syne4 −/− organ of Corti from the 8, 16, and 32 kHz regions at P10 and P12, labeled with myosin VIIa. Scale bar = 10 μm. Source data are available online for this figure.

Article Snippet: Antibody and stain concentrations were as follows: rabbit polyclonal myosin VIIa (Proteus Biosciences 25‐6790) 1:250, mouse anti‐FLAG (Sigma F3165) 1:1,000, DAPI (Abcam ab228549) 1:1,000, goat anti‐mouse (Abcam ab150119) 1:250, and goat anti‐rabbit Alexa Fluor 488 (Cell Signaling 4412s).

Techniques: Immunofluorescence, Labeling

Journal: EMBO Molecular Medicine

Article Title: Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness

doi: 10.15252/emmm.202013259

Figure Lengend Snippet: Schematic representation of AAV.Syne4 and AAV.GFP constructs. Whole‐mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, and right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un‐injected mouse. Transduction rates of AAV9‐PHP.B at 8, 16, and 32 kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate. Staining for FLAG at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4. Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHCs were measured. 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell, myosin VIIa and DAPI signals were removed to only show FLAG staining. Data information: Scale bars = 100 μm for (B), 10 μm for (C and F), and 5 μm for (H). Plots show mean ± SD. Source data are available online for this figure.

Article Snippet: Antibody and stain concentrations were as follows: rabbit polyclonal myosin VIIa (Proteus Biosciences 25‐6790) 1:250, mouse anti‐FLAG (Sigma F3165) 1:1,000, DAPI (Abcam ab228549) 1:1,000, goat anti‐mouse (Abcam ab150119) 1:250, and goat anti‐rabbit Alexa Fluor 488 (Cell Signaling 4412s).

Techniques: Construct, Immunofluorescence, Injection, Transduction, Control, Fluorescence, Staining