Journal: PLoS ONE

Article Title: Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

doi: 10.1371/journal.pone.0030853

Figure Lengend Snippet: Atoh1 in situ hybridization shows a reduced expression in E18.5 heterozygous KI mice compared to the wild-type littermate (a, b) but complete absence in homozygous KI mice (c). Neurog1 is absent in the wild-type cochlea by E18.5 (d) but present in the organ of Corti of the heterozygous KI cochlea (e). Homozygous KI mice show a strong expression of Neurog1 in discontinuous cluster of cells in the organ of Corti, except for a continuous expression in the apex of the cochlea (f). Neurod1 , a downstream gene to Neurog1 and Atoh1 is relatively more advanced toward the apex in the cochlea of heterozygous KI mice than the wild-type littermate (inserts in g, h; compare the distance in g,h). In homozygous KI mice Neurod1 is strongly expressed in discontinuous patches of organ of Corti cells and some spiral neurons (i) and almost reaches the apical tip (compare g, h, i). The distance measured from the apical tip of the cochlea to the Neurod1 expression is marked with yellow dotted lines (g,h,i). Immunochemistry in E18.5 heterozygous KI mice demonstrates both Neurog1 and Myc-tag immunopositivity in the hair cells as well as in the spiral ganglia (j-k’). Neurog1 and Myc-tag immunopositivity in hair cells is shown together with Myo7a, a marker for hair cells (j-k’). DsRed2, a protein marker for Neurog1 expression showed localization only in the hair cells in the heterozygous KI mice which are innervated by the radial fibers labeled with anti-Tubulin antibody (l). OC, organ of Corti; RF, radial fibers; Spg, spiral ganglia. Bar indicates 100 µm except j’ and k’ where it indicates 10 µm.

Article Snippet: Then the primary antibodies for Myo7a (Myosin 7a, Proteus Biosciences), Tubulin (Sigma), Sox2 (Millipore), activated Caspase3 (Cell Signaling Technology), DsRed2 (anti-red fluorescent protein; Genway) and Prox1 (Covance) were used in dilutions of 1∶200, 1∶800, 1∶200, 1∶100, 1∶1000 and 1∶200, respectively. and incubated for 24-48 hours at 4°C.

Techniques: In Situ Hybridization, Expressing, Marker, Labeling

Journal: PLoS ONE

Article Title: Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

doi: 10.1371/journal.pone.0030853

Figure Lengend Snippet: Immunochemistry in E18.5 mice shows no Myo7a expressing cells in the organ of Cori of the homozygous KI mice compared to wild-type mice (a,b). Scanning electron microscopy in the P0 homozygous KI mice (c-g) display patches of organ of Corti cells with formation of microvilli or rudimentary stereocilia (red arrows in c, d, e, e’, f) surrounded by ‘flat epithelium’ composed of Claudius cell-like cells. Some of these cells are adjacent to each other with two central kinocilia (yellow arrows in c’). These clusters of cells are mostly in the position of outer hair cells and are surrounded by organized Claudius cell-like cells (c, d, e). The tectorial membrane is largely confined to the GER but extents selectively to these patches of microvilli bearing cells, many having attachment of protrusions of tectorial membrane on their surfaces (e-g). Lack of Atoh1 in Atoh1 CKO results in a continuous ‘flat epithelium’ without formation of any presumed sensory cells bearing microvilli (h,h’). GER, greater epithelia ridge; He/Cl; Hensen’s cells and Claudius cells; IHC, inner hair cells; OHC, outer hair cells; Tm, tectorial membrane; SL, spiral limbus. Bar indicates 100 µm in a,b; 10 µm in c, d, e, h, h’; and 2 µm in c’, d’, e’, f, g.

Article Snippet: Then the primary antibodies for Myo7a (Myosin 7a, Proteus Biosciences), Tubulin (Sigma), Sox2 (Millipore), activated Caspase3 (Cell Signaling Technology), DsRed2 (anti-red fluorescent protein; Genway) and Prox1 (Covance) were used in dilutions of 1∶200, 1∶800, 1∶200, 1∶100, 1∶1000 and 1∶200, respectively. and incubated for 24-48 hours at 4°C.

Techniques: Expressing, Electron Microscopy, Membrane

Journal: PLoS ONE

Article Title: Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

doi: 10.1371/journal.pone.0030853

Figure Lengend Snippet: Immunochemistry of Myo7a shows normal rows of hair cells with loss of some outer hair cells in P7 heterozygous KI mice (white arrows in a, a’, a””). Co-labeling of Myo7a with the supporting cell-specific markers, Prox1 and Tubulin shows the loss of few outer hair cells in areas of misaligned and/or ectopic pillar cell formation as well as disorganization of Deiters' cells (white arrows in a’-a””; b,b’). Some ectopic hair cells are found in the position of inner hair cells more apparently at P9 and also in between the rows of outer hair cells which are associated with the disorganization of supporting cells (yellow arrows in a- a””, c-c”) followed by loss of some inner hair cells and pillar cells by P26 (arrows in d-d”). Anti-Prox1 specifically labels outer pillar cells and third rows of Deiters’ cells at P7 whereas anti-Tubulin strongly labels outer pillar and all three rows of Deiters' cells. Anti-Tubulin more weakly labels inner pillar cells in heterozygous (a’-a””, c’,c”,d’,d”) and wild-type cochlea (b, b’). Prox1 is shown in cyan in a’ and lilac in a” and b for better contrast. Scanning electron microscopy P1 control ( Pax2-cre; Atoh1 +/f ) mice reveal four parallel rows of hair cells in the organ of Corti with a staircase-pattern of stereocilia (e). SEM in E18.5 to P26 heterozygous KI mice reveals defects in hair cell stereocilia development and patterning (f-l). Hair cells show kinocilia that are not centered in relation to the symmetry of the stereocilia bundle (shown with yellow arrows and red asterisks in f,f’,g). At P1, some abnormal patches of stereocilia form in the inner hair cells (red arrow in g) as well as some ectopic hair cells adjacent to inner hair cells (red arrows in h and h’). At P9, occasionally extra rows of outer hair cells form with unequal length of the ‘W’ end of the stereocilia (i, red asterisk in j). In addition, the stereocilia of inner hair cells are sporadically disrupted or fused abnormally (k, k’) and some inner hair cells at P26 are missing (arrow in l) consistent with gaps seen with the Myo7a immunochemistry. Bar indicates 10 µm except 2 µm in e’, f’, g’, j’.

Article Snippet: Then the primary antibodies for Myo7a (Myosin 7a, Proteus Biosciences), Tubulin (Sigma), Sox2 (Millipore), activated Caspase3 (Cell Signaling Technology), DsRed2 (anti-red fluorescent protein; Genway) and Prox1 (Covance) were used in dilutions of 1∶200, 1∶800, 1∶200, 1∶100, 1∶1000 and 1∶200, respectively. and incubated for 24-48 hours at 4°C.

Techniques: Labeling, Electron Microscopy, Control