Journal: Nature

Article Title: Tbx2 is a master regulator of inner versus outer hair cell differentiation

doi: 10.1038/s41586-022-04668-3

Figure Lengend Snippet: Fgf8CreER; Tbx2F/F; R26LSL-tdTomato/+ newborn (P0) pups were treated with tamoxifen at P0 and collected for examination at each subsequent day from P1 to P8. (a-c) Immunohistochemistry at P5 reveals that hair cells in the position of the IHCs express the IHC functional marker VGLUT3 (and not the OHC developmental market BCL11b) (a; confocal optical section), but also the OHC functional markers Prestin (b; confocal optical section) and PMCA2 (c; confocal projection). In (c), former IHCs are identified by the expression of TdTomato. Hence, at this time transdifferentiating hair cells display features of both IHCs and OHCs. (d) Immunohistochemistry at P1, displayed as a confocal radial optical section, reveals that cells transdifferentiating from IHCs to OHCs (at this point ambiguously termed ic-HCs; TdTomato+ in the lower panel) do not express SOX2, a nuclear marker of both supporting cells and nascent hair cells, and are beginning to express PMCA2 in their stereocilia (bottom panel). For a better visualization of SOX2, the top panel displays its immunoreactivity only with the nuclear counterstain DAPI. (e,f) In situ hybridization combined with immunohistochemistry (for Myosin VIIa, to label IHCs and OHCs) on cryostat sections from P1 cochleae reveal that ic-HCs transdifferentiating from IHCs to ic-OHCs do not express Bcl11b (e) or Insm1 (f) mRNAs, which are expressed by OHCs during their early (embryonic to postnatal) differentiation. Images were taken at apical, middle and basal cochlear positions at P1 and subsequent postnatal days. Images shown are examples of results obtained in at least three separate tissue samples (n=3 biologically independent samples). All scale bars are 20 μm. (g) Schematic summary of all the stainings performed on mice at P1 to P8 in which Tbx2 ablation had been induced by tamoxifen administration at P0. Blue shading denotes the period (P1 to P7) during which transdifferentiating ic-HCs co-express VGLUT3 with OHC markers PMCA and/or Prestin. At no point from P1 to P8 do ic-HCs express the nascent HC marker SOX2 or the OHC differentiation markers Bcl11b (mRNA or protein) or Insm1. Expression levels are subjectively categorized as strong (+++) to undetectable above background (−). Expression in ic-HCs (red font) is provided by comparison to expression in the following control cells (blue font) in the same tissues or stages: Wild type (WT) IHCs for VGLUT3; adjacent OHCs for Prestin, PMCA2, Bcl11b and Insm1; and Supporting Cells (SC) for SOX2. Supporting and other cells labeled in (d) as expressing SOX2 are epithelial cells of Kölliker’s Organ (KO), Inner Border (IBC), Inner Phalangeal (IPhC), Inner Pillar (IPC), Outer Pillar (OPC), Deiters’ (DC) and Hensen’s (HeC) cells.

Article Snippet: Nuclear volumes were measured on the basis of DAPI fluorescence using Imaris, as detailed in ref. 2 .

Techniques: Immunohistochemistry, Functional Assay, Marker, Expressing, In Situ Hybridization, Comparison, Control, Labeling

Journal: Nature

Article Title: Tbx2 is a master regulator of inner versus outer hair cell differentiation

doi: 10.1038/s41586-022-04668-3

Figure Lengend Snippet: a–e′, In situ hybridizations (red) with immunohistochemistry for hair cells using myosin VIIa (green) on E17.5 cochleae after conditional Tbx2 deletion. In mutants, embryonic hair cells in the position of IHCs (square brackets) express markers of developing OHCs (a–b′) but not those of IHCs (c–e′). Expression of Brip1 and Msx1 in supporting cells near IHCs is unaltered in conditional knockouts (cKOs) (n ≥ 3 biologically independent samples). f, ABRs are absent (up-facing arrows) from mature or weaned (P26–P39) mice with embryonic ablation of Tbx2 (red; n = 11: 5 Atoh1cre/+; Tbx2F/F plus 6 Gfi1cre/+; Tbx2F/F). Littermate controls (black; n = 15: Tbx2F/F, Atoh1cre/+; Tbx2F/+, Gfi1cre/+; Tbx2F/+ and Gfi1cre/+) had normal ABRs. Error bars, s.d. g–p′, Immunohistochemistry of mature cochleae indicating that, after embryonic Tbx2 ablation, all hair cells in the position of IHCs (square brackets) displayed all examined features of OHCs but none for IHCs. These features include expression of prestin (g, g′, k, k′), KCNQ4 (i, i′) and PMCA2 (p, p′); lack of VGLUT3 (h, h′), BK, parvalbumin (j, j′), CALB2 (l, l′) and nuclear CtBP2 (m–n′); few synaptic ribbons (Ribeye+ puncta; k–n′); small nuclei (DAPI+; m, m′) located at the base and not the middle of the hair cell (h, i′, j); and shorter stereocilia tightly bundled as in OHCs rather than the long and fanning pattern for IHCs (o–p′). Dotted lines (p) delineate IHCs, which lack PMCA2 (n ≥ 3 biologically independent samples). q–t, Whole-cell currents from ic-OHCs (of Fgf8creER; Tbx2F/F; R26LSL-tdTomato/+ mice treated with tamoxifen at birth; tdTomato+) and from control IHCs and OHCs. q, r, Current responses to voltage steps (−140 mV to +80 mV) in ic-OHCs (n = 7) were characteristic of OHCs (n = 7) but not IHCs (n = 12). s, t, Electromotility assessment from nonlinear capacitance (s) and video recordings (t). Two-state Boltzmann fits. Similar to control OHCs (n = 9), ic-OHCs (n = 10) were electromotile. Hair cells originated from the apical one-fourth of the cochlea at P25–P29. The cells were from ≥3 animals. Scale bars, 20 μm.

Article Snippet: Nuclear volumes were measured on the basis of DAPI fluorescence using Imaris, as detailed in ref. 2 .

Techniques: In Situ, Immunohistochemistry, Expressing, Control

Journal: Nature

Article Title: Tbx2 is a master regulator of inner versus outer hair cell differentiation

doi: 10.1038/s41586-022-04668-3

Figure Lengend Snippet: Immunohistochemistry in cochleae of mice (P20 in d, d′; P29 in f–g′; P26 in all other panels) in which both Tbx2 and Insm1 were conditionally ablated (cDKOs: Atoh1cre/+; Insm1F/F; Tbx2F/F; a′–g′) or in control littermates in which both genes were functional (Insm1F/F; Tbx2F/F; a–g). Staining with antibodies to prestin and parvalbumin (PV; a, a′), oncomodulin and calmodulin (b, b′), PMCA2 and CtBP2 (Ribeye and nuclear isoforms; c, c′), VGLUT3 (d, d′), CALB2 (e, e′), BK (f, f′) and KCNQ4 (g, g′) is shown. Nuclei are labelled with DAPI (d–f′) or are outlined with a dotted white line (IHCs in g). In cDKOs, the phenotype is exactly as in Tbx2-knockout mice (both IHCs and OHCs express OHC markers). By contrast, cDKOs do not display the Insm1-knockout phenotype, in which nearly half of the hair cells in the position of OHCs expressing IHC markers, as previously shown2,15. Expression of prestin, oncomodulin and PMCA2, few CtPB2+ synaptic ribbons and smaller nuclei at the basal end of the hair cell are all features of normal OHCs and are also displayed by all of the examined cells in the position of IHCs in cDKO mice. Expression of calmodulin, CALB2 and nuclear CtBP2, numerous CtPB2+ synaptic ribbons and larger nuclei located in the middle of the hair cell are features of normal IHCs that are missing in the cDKO cells in the position of IHCs. All images are from middle portions of the cochlea (spanning approximately 31–82% of the cochlear length, from base to apex) except those in f–g′, which are more basal (approximately 10–31%). The images shown are examples of results obtained with three separate tissue samples (n = 3 biologically independent samples). Scale bars, 20 μm.

Article Snippet: Nuclear volumes were measured on the basis of DAPI fluorescence using Imaris, as detailed in ref. 2 .

Techniques: Immunohistochemistry, Control, Functional Assay, Staining, Knock-Out, Expressing

Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: Cell reports

Article Title: Glial progenitor heterogeneity and key regulators revealed by single-cell RNA sequencing provide insight to regeneration in spinal cord injury

doi: 10.1016/j.celrep.2023.112486

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Next, tdT+/DAPI-cells for scRNA library preparation were collected by FACS using a BD FACS Aria II flow cytometer (BD Biosciences) with a 100 μm nozzle at 4°C.

Techniques: Purification, Recombinant, Multiplex Assay, Negative Control, Sequencing, Software, Flow Cytometry, Microscopy

Journal: Cells

Article Title: Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment

doi: 10.3390/cells8080937

Figure Lengend Snippet: Scatter plots of leukocyte (Panel A ), platelet (Panel B ), and red blood cell frequencies (Panel C ) in 1 μL of whole blood of 10 healthy individuals as estimated by fluorescence imaging and ACCEPT enumeration ( x -axis) and by the hematology analyzer ( y -axis). Correlation between the measurements of the two techniques was found as indicated by the R 2 . The mean counts of each population of 4–6 technical replicates were used in the case of the fluorescence imaging approach.

Article Snippet: Image data sets of 55–65 frames/channel were acquired to cover the whole surface of each well using a semi-automated inverted fluorescence microscope (Eclipse Ti-E, Nikon Instruments, Amsterdam, The Netherlands) equipped with a 10×/0.45 numerical aperture (NA) objective, a camera (Orca flash 4.0 LT, C11440, Hamamatsu, Almere, The Netherlands) and fluorescence filter cubes (DAPI, FITC, PerCP, APC filter sets for the detection of Hoechst, CD61-Alexa 488, CD45-PerCP and CD235a-Alexa 647, respectively).

Techniques: Fluorescence, Imaging