Journal: Scientific Reports

Article Title: Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice

doi: 10.1038/s41598-023-48702-4

Figure Lengend Snippet: Experimental design, body weights, and FABP7 expression in the cochlea. Schematic of the experiments using wild-type (WT) and Fabp7 knockout (KO) mice ( A ). The body weight (BW) of Fabp7 KO mice was not significantly different from that of WT mice ( B ). Immunohistochemistry of FABP7 in the cochlea of WT mice revealed that FABP7 was expressed throughout the spiral ganglion (SG), organ of Corti (OC), spiral limbus (SLim), and spiral ligament (SLig) ( C ). Scale bar, 200 μm ( C – D ). No expression of FABP7 was observed in the cochlea of Fabp7 KO mice ( D ). Maximum projection images of the OC showing high expression of FABP7 in Schwann cells and SOX2-positive supporting cells around the MYO7a-positive outer hair cells (OHCs) in the cochlea of WT mice ( E ). In the SG, FABP7 was expressed in satellite cells surrounding TUJ-1-positive neurons ( F ). Scale bars, 50 μm ( E – F ). Auditory brainstem response; ABR, weeks; W, post-noise exposure day; PNED, inner hair cell; IHC. Statistical significance was determined using two-way analysis of variance, followed by Šídák multiple comparison test. Error bars represent standard deviation.

Article Snippet: After rinsing with PBS, tissue sections were blocked with 3% bovine serum albumin/0.3% Triton X-100/ PBS for 30 min at room temperature, the unconjugated AffiniPure Fab fragment of anti-mouse IgG (1:10, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature, and incubated overnight with the following primary antibodies: (1) rabbit IgG anti-FABP7 (1:1000, Merck, Darmstadt, Germany #PRS4259), (2) mouse IgG anti-TUJ-1 (1:500, Abcam, Cambridge, UK #ab78078), (3) mouse IgG2a anti-Myosin 7a (MYO7a) (1:200, Santa Cruz Biotechnology, Dallas, TX, USA #SC-74516), and (4) goat IgG anti-SOX2 (1:500, R&D Systems, Minneapolis, MN, USA #AF2018).

Techniques: Expressing, Knock-Out, Immunohistochemistry, Comparison, Standard Deviation

Journal: Scientific Reports

Article Title: Gata3 is required in late proneurosensory development for proper sensory cell formation and organization

doi: 10.1038/s41598-023-39707-0

Figure Lengend Snippet: Deletion of Gata3 results in loss of HCs in a basal to apical gradient ( A – F′ ) Representative images from the basal, middle, and apical regions of the cochlea for HCs indicated by MYOSIN7A + staining. Two different controls were used, Gata3 f/f and Sox2-cre ERT2 , in order to account for the haploinsufficent phenotype of the Cre line used. Both the heterozygous and homozygous mutant show IHC duplets (white circles) and missing rows of OHCs (white brackets), while the homozygous mutant also shows ectopic HCs in the GER. ( G ) Total hair cell quantification was performed in the base, middle, and apex in 100 µm sections using a One-way ANOVA with post hoc Dunnett’s test (P** ≤ 0.01; P*** ≤ 0.001) Scale bar: 50 µm.

Article Snippet: The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOIN7A Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 (NF200) HC Chicken (Aves; 1:200), and SOX2 Rabbit (Sigma; 1:500).

Techniques: Staining, Mutagenesis

Journal: The Journal of Physiology

Article Title: Gata3 is required for the functional maturation of inner hair cells and their innervation in the mouse cochlea

doi: 10.1113/JP277997

Figure Lengend Snippet: A–D , maximum intensity projections of confocal z ‐stacks taken from the apical cochlear region of control ( A , gata3 fl/fl ; C , gata3 +/+ ) and either gata3 fl/fl otof‐cre +/− ( B ) or gata3 +/− ( D ) mice using antibodies against CtBP2 (ribbon synaptic marker: red) and PSD95 (postsynaptic density marker: green). Myosin 7a (Myo7a, blue) was used as a hair cell marker. Scale bars 10 μm. E and F , number of CtBP2 puncta in the gata3 fl/fl otof‐cre +/− ( E ) and gata3 +/− ( F ) mice at P48. Note that in addition to the mean values (large circles), the individual IHC counts are also shown (smaller symbols). In E , the ribbon number in gata3 fl/fl otof‐cre +/− cells (14.4 ± 2.7, n = 28 IHCs, 3 mice, P = 0.468) was no different to that in control gata3 fl/fl IHCs (13.9 ± 3.1, n = 30 IHCs, 3 mice). In IHCs from gata3 +/− mice ( F ) the number of CtBP2 puncta (11.3 ± 2.2, n = 44 IHCs, 4 mice) was significantly reduced compared to control cells (13.5 ± 2.5, n = 44 IHCs, 4 mice, P < 0.0006). Averages are means ± SEM.

Article Snippet: Primary antibodies were: mouse anti‐myosin7a (1:100, DSHB, Iowa City, IA, USA; no. 138‐1S), rabbit anti‐myosin7a (1:500, Proteus Biosciences, Nottingham, UK; no. 25‐6790), mouse anti‐CtBP2 (1:200, BD Biosciences, Berkshire, UK; no. 612044), rabbit anti‐SK2 (1:500, Sigma‐Aldrich, no. P0483), goat anti‐choline acetyltransferase (ChAT, 1:500, Millipore, Hertfordshire, UK; no. AB144P) and mouse anti‐PSD95 (1:1000, Millipore, no. MABN68).

Techniques: Marker

Journal: PLoS Biology

Article Title: Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

doi: 10.1371/journal.pbio.3001445

Figure Lengend Snippet: (A) Experimental paradigm: DT was injected into P1 wild-type or Lgr5 DTR/+ mice. EdU was injected daily from P3 to P5, and cochleae were examined at P4 or P7. (B-D, I-K) Representative images of the apical, middle, and basal turns of DT-treated wild-type cochleae showing no EdU + or Ki67 + Sox2 + SCs at P4 or P7. IPhC region is outlined by dashed lines. (E-G) In each turn of the P4 DT-treated Lgr5 DTR/+ cochlea, EdU + and/or Ki67 + Sox2 + cells were detected in the GER. Some EdU + and/or Ki67 + Sox2 + cells were also found in the IPhC and PC/DC regions. E’-G”“represent single channel images. Insets in E and E’ are high magnification images of a cell in metaphase. G”‘ and G”“are high magnification images from G’ and G”. (H) Significantly more EdU + Sox2 + cells were found in the P4 GER regions throughout the DT-treated Lgr5 DTR/+ cochlea relative to controls. Numbers decrease in an apical–basal gradient. (L-N) In all 3 turns of the P7 DT-treated Lgr5 DTR/+ cochlea, EdU + Sox2 + cells were primarily found in IPhC region, and only few were found in the GER or PC/DC region. Ki67 + Sox2 + cells were rarely detected at this age. L’-N”“represent single channel images. N”‘ and N”“are high magnification images from N’ and N”. (O) Significantly more EdU + Sox2 + cells were detected throughout the P7 IPhC regions of DT-treated Lgr5 DTR/+ cochlea relative to controls, decreasing in an apical–basal gradient. Data represent mean ± SD. ** p < 0.01, *** p < 0.001. Two-way ANOVA with Tukey’s multiple comparisons test. n = 4–7. See for H, O. DC, Deiters’ cell; DT, diphtheria toxin; GER, greater epithelial ridge; IPhC, inner phalangeal cell; PC, pillar cell; SC, supporting cell.

Article Snippet: The following primary antibodies were used: rat anti-CD44 (1:200; BD Biosciences), goat anti-CtBP2 (1:500; Santa Cruz Biotechnology), chicken anti-GFP (1:1,000; Aves Labs), rabbit anti-GLAST (1:200; Abcam), mouse anti-GluR2 (1:1,000; Millipore), rabbit anti-Ki67 (1:1,000; Abcam), rat anti-Ki67 (1:400; ThermoFisher Scientific), rabbit anti-Myosin7a (1:1,000; Proteus Bioscience), mouse anti-Myosin7a (1:1,000; Developmental Studies Hybridoma Bank), mouse anti-Na + /K + ATPase α-1, α6F (1:500; Developmental Studies Hybridoma Bank), goat anti-Sox2 (1:200; Santa Cruz Biotechnology or R&D Systems), goat anti-Sparcl1 (1:500; R&D Systems), mouse anti-Tuj1 (1:1,000; Neuromics), rabbit anti-Fabp7 (1:200; Abcam), and rabbit anti-Vglut3 (1:1,000; Synaptic Systems).

Techniques: Injection