Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation

Abstract

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

Fig. 1

Targeted disruption of the Tmhs gene and replacement with a lacZ reporter gene. A Schematic diagram illustrating the targeting construct, the wild-type Tmhs allele, and the recombinant allele with exons 1 and 2 replaced by a lacZ/Neo cassette. The 15.5-kb targeting construct included the plasmid vector, the 5′ and 3′ homology arms, a lacZ reporter gene and neomycin-resistance (Neo) cassette, and a thymidine kinase (TK) negative selection cassette. B The 5′ and 3′ homology arms of the targeting construct were amplified by highfidelity PCR from genomic DNA of R1 ES cells. C After electroporation of the linearized targeting construct into R1 ES cells and antibiotic selection, positive clones were expanded and screened for recombinant alleles by Southern blot analysis, first with a 3′ probe hybridized to EcoRI-digested DNA followed by verification with a 5′ probe hybridized to PstI-digested DNA. The properly targeted ES cells were then microinjected into B6 blastocyst-stage embryos (3.5 dpc). R1 ES cells are derived from the hybrid strain 129X1/SvJ × 129S1/Sv-p⁺ Tyr⁺ Kitl^Sl-J/J

Fig. 2

Absence of protein expression and hair bundle degeneration in knockout mice (Tmhs -/-). TMHS-specific immunofluorescence was detected in stereocilia (indicated by downward-pointing arrowheads) of inner hair cells (IHC) and outer hair cells (OHC) in +/+ control mice (A), as previously reported (Longo-Guess et al. 2005), but was absent in cochleae of Tmhs -/- mice (B). Organ of Corti surface preparations were stained with phalloidin to visualize hair bundles in control (C) and mutant (D) newborn mice. Normal-appearing stereociliary bundles are clearly present on inner (bottom row) and outer hair cells (three upper rows) of P0 mutant mice. Hair bundle morphology in adult mice was examined by SEM of organ of Corti surface preparations. By P28, stereocilia degeneration is apparent in OHC bundles of Tmhs -/- mice (F) as compared with the normal bundle morphology seen in age-matched +/+ control mice (E). All scale bars, 10 μm

Fig. 3

Tmhs reporter gene expression in the inner ear and sensory ganglia. The lacZ reporter gene for Tmhs expression was visualized by histochemical reaction of its product, β-gal. A,B Two views of whole-mount preparations of inner ears from P0 mice show that Tmhs expression is limited to the neurosensory epithelia: the cristae of the posterior (PC), anterior (AC), and horizontal (HC) semicircular canals, the maculae of the utricle (U) and saccule (S), and the organ of Corti of the cochlea (Co). C-H Surface preparations of the dissected organ of Corti of P2 mice show expression in the three rows of outer hair cells (OHC) and single row of inner hair cells (IHC) along the entire length of the cochlear duct except for the most extreme apical region. I-K Surface preparations of the dissected vestibular sensory regions show expression in the cristae of the posterior (PC), anterior (AC), and horizontal (HC) semicircular canals and in the maculae of the utricle and saccule. Tmhs expression also was detected in cranial and cervical ganglia (L-N), including the geniculate ganglion (GnG, part of the VII cranial nerve), the glossopharyngeal ganglion (IX cranial, see Fig. 4A), the vagal ganglion (X cranial), both proximal and distal parts, the trigeminal ganglion (V cranial), and the superior cervical sympathetic ganglion (SCG), but was not detected in the vestibular ganglion (VG) nor in the spiral ganglion (see Fig. 4A). All scale bars, 100 μm

Fig. 4

Tmhs reporter gene expression in inner-ear cross sections. Midmodiolar cross section through the cochlea of a P0 mouse (A) shows β-gal expression in the hair cells of the basal and middle turns (arrowheads) but not in the apex. Expression also is seen in cells of the glossopharyngeal (IX cranial) ganglion, but not in spiral ganglion (SG) cells. Cochlear expression in inner hair cells (IHC) and outer hair cells (OHC) is strongest at P0 (B), diminishes slightly by P6 (C), and is no longer detectable at P15 (not shown). The Tmhs reporter gene is also strongly expressed in vestibular hair cells of the utricle (not shown), saccule (D), and crista ampularis (E). Expression can be detected as early as E15.5 in vestibular hair cells (E), but not until E17 in cochlear hair cells (not shown). Scale bars: A, 200 μm; B-E, 50 μm

Fig. 5

Molecular characterization of the hscy-2J mutation. A Sequence chromatograms of genomic DNA from a B6 +/+ control and a hscy-2J/hscy-2J mutant mouse. The hscy-2J 6-bp deletion is indicated by dashed lines. The control and mutant 9-bp donor splice site recognition sequences for exon 2 of Tmhs are enclosed in boxes. The hscy-2J deletion inactivates the normal donor splice site. B Genomic structure of Tmhs illustrating the effect of the hscy-2J mutation on transcription. The Tmhs gene comprises four exons, here represented as rectangles with black regions indicating protein-coding sequence and connecting lines representing introns. The hscy-2J mutation causes abnormal splicing from a cryptic, noncanonical donor splice site within exon 2. C DNA sequence analysis of mutant transcripts. The hscy-2J deletion causes the alternative use of a cryptic donor splice site (boxed region) within exon 2. Thus, mutant transcripts do not contain the 153 nt shown in red, which are encoded by the skipped 3′ region of exon 2. Although mutant transcripts are spliced in-frame with exon 3 (splice sites are indicated by arrows), their translated protein product lacks the 51 amino acids shown in blue. D RT-PCR analysis of Tmhs transcripts. Primers corresponding to exon 1 and exon 4 (Table 1) were used to amplify cDNA from brain tissue of two +/+ and two hscy-2J/hscy-2J mice. DNA sequencing verified that the +/+ product (358 bp, lanes 1 and 2) is properly spliced and that the smaller size of the hscy-2J/hscy-2J product (205 bp, lanes 3 and 4) is caused by the abnormal splicing detailed in panels B and C. A negative control without RT is shown in lane 5. E PCR genotyping assay. A simple PCR assay can be used to genotype mice for the 6-bp hscy-2J deletion, with primers that amplify a 104-bp product from wild-type DNA and a 98-bp product from the hscy-2J DNA (Table 1). The PCR products can be separated on a 6% polyacrylamide gel to distinguish +/hscy-2J (lanes 1 and 3), hscy-2J/hscy-2J (lanes 2 and 4), and +/+ (lane 5) genotypes

Fig. 6

Absence of TMHS protein expression in cochleae of hscy-2J mutant mice. TMHS-specific immunofluorescence was detected in stereocilia (indicated by downward-pointing arrowheads) of inner hair cells (IHC) and outer hair cells (OHC) in +/+ control mice at P0 (A), but was absent in age-matched hscy-2J mutant mice (B). Scale bars, 10 μm