In situ hybridization at E14.5 shows the expression of Atoh1 in a wild-type (Atoh1^+/+) mouse (a) which is less profound in the heterozygous KI (Atoh1^+/KINeurog1) littermate (b). There is no in situ signal for Atoh1 mRNA in the homozygous KI (Atoh1^{KINeurog1/KINeurog1}) (c) and in the Atoh1 conditional knockout mice (CKO, Pax2cre; Atoh1^f/f) (d). Neurog1 in situ hybridization in the wild-type control mice represents expression only in the delaminating neurons emanating from the utricle and saccule but not in the sensory epithelia (e, e’,e”). In heterozygous KI mice, Neurog1 is expressed in the vestibular sensory epithelia, but not yet in the cochlea and is diminished in the delaminating neuroblasts near the utricle and saccule compared to the wild-type littermate (compare red arrows in e, e’,e” and f, f’, f”). Both homozygous KI and Atoh1 CKO mice show expression of Neurog1 in the delaminating neuroblasts and in the epithelia of the utricle and saccule (g, g’, g”, h, h’,h”). In addition, Neurog1 is expressed in the canal cristae and the mid-base of the cochlea of homozygous KI mice (white arrow in g) which is not observed in the Atoh1 CKO cochlea (h). The expression of Neurog1 in the sensory epithelia recapitulates Atoh1 expression (compare a, g) whereas the more obvious expression of Neurog1 in delaminating neurons is observed in Atoh1 CKO mice (h). Co-expression of Atoh1 with Neurog1 in the developing hair cells restricts Neurog1 expression in delaminating neurons in the heterozygous KI mice below the level encountered in wild-type, homozygous KI and Atoh1 CKO mice (Red arrows indicate the delaminating neuroblasts; asterisks indicate the sensory epithelia; yellow dotted lines demarcate the boundaries of the utricle and saccule in e’,e”, f’, f”, g’, g”, h’,h”). AC, anterior crista; Co, Cochlea; HC, horizontal crista; PC, posterior crista; S, saccule; U, utricle. Bar indicates 100 µm.

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.

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.

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’.

These images of the comparable segments of E18.5 cochlea display expression differences of several genes in and around the organ of Corti for four different mouse genotypes. In situ hybridization of Atoh1 shows a reduced expression in the heterozygous KI mice (a’) compared to wild-type littermates (a) and complete loss of Atoh1 in the homozygous KI mice and Atoh1 CKO mice (a”-a””). Fgf8 is expressed only in inner hair cells in wild-type mice (b), shows reduction and loss in some inner hair cells (arrow in b’) in heterozygous KI mice, is absent in homozygous KI mice (b”,b”’) but is faintly expressed in some organ of Corti cells in the absence of Atoh1 (arrow in b””). Neurog1 is absent in the organ of Corti in wild-type mice (c) and Atoh1 CKO mice (c””), but is present in the hair cells in heterozygous KI mice (c’) and shows a patchy distribution in the organ of Corti cells in the homozygous KI mice (c”, c”’). Neurod1 and Nhlh1 are downstream genes of Neurog1 and Atoh1 and are absent in Atoh1 CKO mice (d””, e””) but present in wild-type and heterozygous KI mice (d,d’, e, e’). In the homozygous KI mice both genes are present in clusters of cells with progressive reduction towards the base (d”,d”’, e”, e”’). Jag1, a Notch ligand, is widely expressed in wild-type (f) and in heterozygous KI mice (f’), is expressed in patches of cells in the homozygous KI mice (f”, f”’) and is completely absent in Atoh1 CKO mice (f””). Notch effector gene, Hes5, shows expression in the supporting cells and in GER both in wild-type and in heterozygous KI mice (g, g’). Hes5 is massively diminished in the homozygous KI cochlea with some patchy retention only in the apex of the cochlea (g”, g”’) and is completely absent in the Atoh1 CKO (g””). Gata3 is expressed in a wide area in the cochlear epithelium in wild-type and heterozygous KI mice (h, h’), shows retention in the homozygous KI mice with a wavy appearance near the presumed organ of Corti (h”,h”’) and is much reduced in Atoh1 CKO mice (h””) compared to the base of the homozygous KI cochlea (h”’). Fgf10 and Bmp4 are markers of the medial and lateral boundary of the organ of Corti (i, j) and are expressed almost identical in wild-type and heterozygous knockin mice (i, i’, j, j’). Expression of these markers are reduced in homozygous KI mice and show a wavy and discontinuous appearance (arrows in i”, j”, j”’), probably flanking the remaining patches of organ of Corti cells still expressing organ of Corti marker genes. Both Bmp4 and Fgf10 expressions are further reduced in the base of Atoh1 CKO mice, reflecting the near complete loss of the organ of Corti in these mice (i””, j””). GER, greater epithelial ridge, He/Cl, Hensens’ and Claudius cells; IHC, inner hair cells, ‘OC’, putative organ of Corti in Atoh1 CKO mice. ‘{‘ indicates the differentiated organ of Corti and ‘[‘ marks the presumed organ of Corti in the homozygous KI mice. Arrows indicate gaps in expression. Bar indicates 100 µm.

Immunochemistry of activated Caspase3 in homozygous KI mice reveal the labeling of patches of cells in the putative organ of Corti, mostly in the basal half of the cochlea at E16.5 (a, a’,b). These dying cells are typically 1-2 cells in one patch situated more medially (neural side) of the organ of Corti (white arrow in b, c, d), typically on the medial edge of the Sox2 positive presumed supporting cells (c), most likely the precursors of inner hair cells or the first row of outer hair cells (a-c). Neurog1 in situ positive patches of cells in the homozygous KI mice are progressively reduced from E16.5 to E18.5 particularly in the base (d and e) consistent with the appearance of activated Caspase3 labeled cells in the base. Neurog1 positive cells shown in thin plastic sections performed after whole mount Neurog1 in situ hybridization (red arrows in f and f’) form compact clusters of 2-3 cells. The histological localization of the Neurog1 positive cells in homozygous KI mice is comparable to LacZ positive cells in the Atoh1 null mice (f’, g). ACasp3, activated Caspase3; GER, greater epithelial ridge; HS, Hoechst stain; Stv. Stain, Stevenel’s blue staining; ‘SC’, putative supporting cells expressing Sox2; Tm, tectorial membrane. ‘[‘ indicates position of the putative organ of Corti. Bar indicates 10 µm except 100 µm in a.

In E18.5 wild-type mice, Sox2 is present in the GER and in all supporting cells (a) and many radial fibers project to inner hair cells. Anti-Tubulin antibody labeling shows Type II fibers projecting in a regular pattern to outer hair cells (a’) between the Sox2 immunopositive supporting cells (a”). Heterozygous KI mice reveal some disorientation in Sox2 positive supporting cells with disorganized projection of type II fibers between the disorganized supporting cells (arrows in b, b’, b”). In homozygous KI littermates Sox2 positive cells are organized as multiple patches along the organ of Corti with a gradient of decreased density from apex to base (c-e”). Tubulin immunolabeling reveals fibers reaching to these patches and extending between Sox2 positive cells (arrows in e’ and e”). Atoh1 CKO mice have few occasional patches of Sox2 positive cells (f) and tubulin positive fibers mostly form loops with some reaching with rare projections to Sox2 positive cells of the organ of Corti (compare e’ and f’). Neurovue dye tracing of efferent innervation from the brainstem shows formation of intraganglionic spiral bundles (IGSB) in homozygous KI mice comparable to wild-type littermate (compare g and h). Multiple efferents in the homozygous KI mice enter into the undifferentiated organ of Corti (h-h”) where fibers ramify apparently between the remaining patches of organ of Corti cells (arrows in h’,h”). GER, greater epithelial ridge; OC, organ of Corti; ‘OC’, presumed organ of Corti in Atoh1 CKO; RF, radial fibers; SC, supporting cells; ‘SC’, putative supporting cells expressing Sox2; ‘[‘ indicates position of the putative organ of Corti. Bar indicates 50 µm in all except 100 µm in d-d”, g, h and 10 µm in h’ and h” .

Ntf3 expression is uniform throughout the organ of Corti but appears more prominent in heterozygous KI mice compared to the wild-type littermate (a, a’, b, b’). In contrast, homozygous KI mice exhibit patchy Ntf3 expression in the middle and base, but continuous in the apex and in basal tip of the cochlea (c,c’), consistent with Sox2 distribution and the pattern of residual innervation. In contrast, Bdnf which is predominantly expressed in the hair cells is somewhat elevated in heterozygotic KI mice compared to wild-type littermates (d,e) but shows expression only in the apical turn in homozygotic KI mice (f; insert in f). ‘{‘ indicates the area of differentiated organ of Corti ‘[‘ indicates the putative organ of Corti in the homozygous KI mice. Bar indicates 100 µm.

This diagram shows the interactions of Atoh1 protein with the its enhancer to increase levels of Atoh1 mRNA expression (a). Atoh1 in part regulates Neurod1 and presumably also Fgf8 (a’, [74]) whereas Neurod1 in turn suppresses Atoh1 and Fgf8 as reported previously in Neurod1 null mice [24] and shown here. In wild-type mice inner hair cells (IHC) express all three genes (color coded as, red, Atoh1; blue, Neurod1 and lilac, Fgf8; a’) and outer hair cells (OHC) express Atoh1 and Neurod1 but not Fgf8 (red and blue; a’, a”). In heterozygous KI mice (Atoh1^+/KINeurog1) (b-b”’) enhancer activation of Atoh1 as well as activation of the Neurod1 and Fgf8 will be retained with expression of Atoh1. However, Neurog1 protein mediated enhanced expression of Neurod1 suppresses Atoh1 and Fgf8 more strongly. Consistent with the proposed regulation changes are our observed changes in mRNA expression pattern and the effects on hair cell development in heterozygous KI mice such as extra rows of outer hair cells, misalignment and progressive loss of inner hair cells (b”,b’”). In heterozygous KI mice, all four genes including Neurog1 are expressed in IHC (green) but have reduced expression of Atoh1 and Fgf8 (b”, b’”) and the three genes are expressed in OHC (b”, b’”). In homozygous KI mice (Atoh1^{KINeurog1/KINeurog1}) there is no Atoh1 expression and thus no activation of the Atoh1 enhancer (c). Therefore, enhanced expression of Atoh1 driven by the Atoh1 protein binding to the enhancer will be disrupted, leaving only non-Atoh1 protein mediated activation to drive Neurog1 expression. Neurog1 protein mediated Neurod1 expression apparently suppresses all Fgf8 expression (c’). Homozygous KI mice show undifferentiated organ of Corti cells (which express only Neurog1 and Neurod1) that may survive but form only microvilli (c”, c’”) and are surrounded by a reduced expression of Bmp4 (c”). In the Atoh1 CKO mice (d) the recombined Atoh1 locus (At) does not produce a protein able to activate the enhancer or expression of Neurod1. However, a limited expression of Fgf8 is possible indicating that other factors are involved in regulating that gene. The reduced expression domains of Fgf10 and Bmp4 approximate each other (d”) and no organ of Corti cell remains in the ‘flat epithelium’ (d”, d’”).