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First published online 19 March 2008
doi: 10.1242/dev.014043

Development 135, 1725-1734 (2008)
Published by The Company of Biologists 2008
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Deafness in mice lacking the T-box transcription factor Tbx18 in otic fibrocytes

Mark-Oliver Trowe1, Hannes Maier2, Michaela Schweizer3 and Andreas Kispert1,*

1 Institut für Molekularbiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
2 HNO Klinik, Universitätsklinikum Eppendorf, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany.
3 Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany.


Figure 1
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  		Fig. 1. Diversity of fibrocytes in the cochlea and structure of the stria vascularis. (A) Illustration of the distribution of subtypes of otic fibrocytes on a midmodiolar cochlea section. Subtypes are as indicated in the figure. Based on location, cell morphology and marker gene expression, five major types of fibrocytes can be distinguished in the spiral ligament. (B) Scheme of the cellular structure of the stria vascularis. Cell types are as indicated in the figure. Stria vascularis integrity relies on a three-layered tissue architecture of marginal, intermediate and basal cells that enclose a dense capillary network. The formation of long cell processes by all three cell types and a high degree of interdigitation characterizes strial architecture. OC, Organ of Corti; OtC, otic capsule; SL, spiral ligament; SLB, spiral limbus; StV, stria vascularis.

 

Figure 2
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  		Fig. 2. Tbx18 expression indicates early compartmentalization of otic mesenchyme. (A-R) Comparative analysis of Tbx18, Sox9 and Pou3f4 expression during cochlea development by RNA in situ hybridization on adjacent sagittal sections of wild-type heads. Probes and developmental stages are as indicated in the figure. (A,D,G,J,M,P) Tbx18 expression is confined to the inner zone of periotic mesenchyme representing prospective otic fibrocytes during all developmental stages. (B,E,H,K,N,Q) Mesenchymal Sox9 expression is found in regions of high cell condensation, like the early periotic mesenchyme, the prospective otic capsule, the condensing spiral limbus and the condensing mesenchyme underlying the future stria vascularis. (C,F,I,L,O,R) Developmental expression of Pou3f4 in the inner ear is initially pan-mesenchymal but becomes restricted to prospective otic fibrocytes after E13.5. CD, cochlear duct; OE, otic epithelium; OtC, otic capsule; PM, periotic mesenchyme; RM, Reissner's membrane; SE, sensory epithelium; SG, spiral ganglion; SL, spiral ligament; SLB, spiral limbus; SM, scala media; ST, scala tympani; StV, stria vascularis; SV, scala vestibuli.

 

Figure 3
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  		Fig. 3. Loss of Tbx18 causes deafness. (A-C) Analysis of hearing by ABR (A) and EP (B,C) measurements of Tbx18KO and control mice. (A) ABR thresholds are significantly increased in Tbx18KO mice at three weeks (129.3±5.3 dB pe SPL, n=15) and 12 weeks (130.5±5.8 dB pe SPL, n=10) compared with control animals of the same age (3 weeks: 54.2±19.2 dB pe SPL, n=18; 12 weeks, 54.9±6.4 dB pe SPL, n=8). (B) Registration of the EP in Tbx18KO (dashed line) and control mice (solid line) at P21. After taking the reference DC potential in the fluid meniscus overlying the stria vascularis (M1), the recording electrode was moved forward. Upon penetration of the stria vascularis (P), the EP was measured in the scala media for at least 2 minutes before the animal was sacrificed by the injection of a barbiturate (T61). Within several minutes of the insult, the EP decreased to a negative steady-state-potential (SSP) - reaching a minimum after 8-12 minutes. In contrast to the EP, which is given by the steady state of active and passive ionic current components, the SSP reflects only the passive part of the ionic conductivities, as long as ion concentrations are minimally altered after active transport has come to a halt. As a control for baseline shifts, the reference DC potential was measured after the experiment (M2) to verify its constancy over the entire time. (C) Disruption of the EP and SSP in Tbx18KO mice at three weeks of age (control: EP=102.9±9.1 mV, SSP=-26.4±9.9 mV, n=10; Tbx18KO: EP=1.3±3.1 mV, SSP=-3.8±3.9 mV, n=10). The difference in EP and SSP is significant (P<0.001, Mann-Whitney Rank Sum Test); bars indicate standard deviation.

 

Figure 4
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  		Fig. 4. Histological and ultrastructural defects in the Tbx18KO cochlea. Hematoxylin and Eosin staining (A-J) of midmodiolar sections of the inner ear and ultastructural analysis of the stria vascularis (K,L) of control and Tbx18KO mice at P21. Black rectangles highlight regions of higher magnification. (A,B) Tbx18KO cochleae are normal in shape but reduced in size. (C,D) Higher magnification of the medial region reveals improper encapsulation of the ganglion by bone tissue in Tbx18KO mice (white arrowheads). (E,F) Higher magnification of the basal turn reveals hypoplasia of the lateral wall in Tbx18KO mice. Arrow and arrowhead in F indicate the loss of suprastrial and type IV fibrocytes, respectively. (G,H) Magnified view of the Organ of Corti does not reveal any obvious phenotypic changes in Tbx18KO mice (I,J). Magnification of the lateral wall shows stria vascularis malformations and defects in otic fibrocytes in Tbx18KO mice. White arrow in J indicates extension of the marginal cells into Reissner's membrane (RM). (K,L) Ultrastructural analysis of the stria vascularis in the basal coil uncovers the presence of large gaps between the cellular processes of strial cells. BC, basal cells; IC, intermediate cells; MC, marginal cells; OC, Organ of Corti; SP, spiral prominence; for further abbreviations, see Fig. 2.

 

Figure 5
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  		Fig. 5. Differentiation of otic fibrocytes is disturbed in Tbx18KO mice. (A-J) Localization of otic fibrocyte markers by immunohistochemistry (A,B,E-J) and immunofluorescence (C,D) on midmodiolar sections of cochleae of control and Tbx18KO mice at P21. Figures show the lateral wall in the basal turn. Antibodies against proteins specific for fibrocyte subtypes were used as indicated in the figure. Arrow in B marks the loss of strong Otos expression in the region of type IV fibrocytes. (C,D) Overlay of immunofluorescence and differential interference contrast microscopy. Arrowheads in G mark expression in type III fibrocytes. (K) Schematic summary of expression domains and fibrocyte subtype distribution in wild-type and Tbx18KO cochleae. For abbreviations, see Fig. 2.

 

Figure 6
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  		Fig. 6. Defects in the stria vascularis of Tbx18KO mice. (A-L) Detection of strial proteins by immunofluorescence on midmodiolar sections of cochleae of control and Tbx18KO mice at P21. Figures show the stria vascularis in the basal turn. Antibodies against proteins of marginal, intermediate and basal cells, as well as of basement membrane components, were used as indicated in the figure. (C-F) Immunofluorescence on DAPI-counterstained sections detected by laser scanning microscopy. Insets show higher magnifications of the stria vascularis at regions indicated by rectangles in the figures, and reveal the disturbed formation of cellular processes by marginal cells (C,D) and intermediate cells (E,F). (M) Schematic summary of expression domains and model of the stria vascularis of wild-type and Tbx18KO mice.

 

Figure 7
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  		Fig. 7. Developmental manifestation of the stria vascularis phenotype of Tbx18-/- mice. Analyses of histology by Hematoxylin and Eosin staining (A,B) and of marker gene expression by RNA in situ hybridization (C-L) on sagittal sections of E18.5 cochleae. Figures show the basal coil (C-L) and the stria vascularis region (A,B). Arrowheads mark the lateral extension of the stria vascularis. Tbx18-/- cochleae show normal differentiation of intermediate and marginal cell precursors of the forming stria vascularis, but lack the mesenchymal cell condensation preceding basal cell formation (arrow in B). Probes were used as indicated in the figure. For abbreviations, see Fig. 2.

 

Figure 8
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  		Fig. 8. Disturbed boundary formation between the otic capsule and otic fibrocytes in Tbx18-/- mice. (A-H) Analyses of histology by Hematoxylin/Eosin (A,E) and Picro-Sirius Red collagen staining (B,F), and of marker gene expression by RNA in situ hybridization (C,D,G,H) on sagittal sections of E18.5 cochleae. Figures show the spiral ligament of the basal coil. Genotypes and probes used are as indicated in the figure. (I-N) Analyses of β-galactosidase reporter activity by X-Gal staining on sagittal sections of Tbx18lacZ/Tbx18GFP and Tbx18lacZ/+ (control) mice at E12.5 (I,L), E13.5 (J,M) and E14.5 (K,N). (A,E) HE staining reveals a local expansion of the otic capsule in the basal coil (arrow in E) and altered fibrocyte appearance in the spiral ligament of the mutant (E). (B,F) Collagen staining reveals disturbed boundary formation between spiral ligament fibrocytes and the otic capsule in the Tbx18-/- mice. Arrowhead in B marks the compartment boundary in the control. (C,D,G,H) Spiral ligament fibrocytes show ectopic Postn (G) and a severe reduction of Coch (H) expression in the mutant. White arrow in C,G marks Postn expression lining the outer border of the otic capsule. (I-N) β-Galactosidase activity assay detects (former) Tbx18-expressing cells in the outer compartment of the periotic mesenchyme (asterisk in M) and the otic capsule (asterisk in N). Dashed lines mark the outer boundary of the periotic mesenchyme and the otic capsule. For abbreviations, see Fig. 2.

 

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© The Company of Biologists Ltd 2008