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.

Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing

“…We identified a homozygous, unique SNP in that critical region at 2,815,634 on chromosome 11 that causes a premature stop codon at residue 80 of Lhfpl5a (ENSDARG00000045023). The homolog of lhfpl5a, LHFPL5, causes nonsyndromic deafness in humans and the hurry scurry (hscy) mouse, thus making it a strong candidate (Longo-Guess et al, 2005;Shabbir et al, 2006;Longo-Guess et al, 2007). However, we could not validate this mutation by morpholino phenocopy, possibly because lhfpl5a functions at a late stage of development by which time the morpholinos have been degraded.…”

“…Alagille syndrome pathology also includes inner ear dysplasia (Koch et al, 2006). In contrast to syndromic phenotypes, autosomal recessive mutations in the human orthologs of both cdh23 and lhfpl5a cause nonsyndromic deafness (Bolz et al, 2001;Bork et al, 2001;Longo-Guess et al, 2005;Shabbir et al, 2006;Longo-Guess et al, 2007). Along with Protocadherin 15, Cdh23 has recently been identified as one of the components of the tip links of hair-cell bundles, where mechanical forces are transduced into electrical potentials (Kazmierczak et al, 2007;Sakaguchi et al, 2009).…”

Carbonic anhydrase 2 deficiency leads to increased pyelonephritis susceptibility

“…Car2-deficient (Car2 Ϫ/Ϫ ) mouse embryos and wild-type (Car2 ϩ/ϩ ) C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). The initial coding sequence of the Car2 gene was replaced with a LacZ marker under the direction of the Car2 promoter (25). Heterozygous (Car2 ϩ/Ϫ ) mice were rederived on a C57BL/6 genetic background at the Transgenic Core at the Research Institute of Nationwide Children's Hospital.…”

The tip-link molecular complex of the auditory mechano-electrical transduction machinery

“…These four proteins are Tmc1 (transmembrane channel protein 1), Tmc2 (transmembrane channel protein 2) (Beurg et al, 2014(Beurg et al, , 2015Kawashima et al, 2011;Pan et al, 2013), Tmhs (tetraspan membrane protein of hair cell stereocilia, also know as Lhfpl5) (Beurg et al, 2015;Longo-Guess et al, 2007, 2005Xiong et al, 2012) and Tmie (transmembrane protein of inner ear hair cells) (Karuppasamy et al, 2012;Mitchem et al, 2002;Park et al, 2013; Su CD3-specific sequence (encoded by exon 39) is indicated in yellow, and the sequence encoded by exon 37 is indicated in green. Protein sequences encoded by phases other than the conventional phase are indicated in gray.…”