Philomena Mburu

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31

The whirler mouse mutant (wi) does not respond to sound stimuli, and detailed ultrastructural analysis of sensory hair cells in the organ of Corti of the inner ear indicates that the whirler gene encodes a protein involved in the elongation and maintenance of stereocilia in both inner hair cells (IHCs) and outer hair cells (OHCs). BAC-mediated transgene correction of the mouse phenotype and mutation analysis identified the causative gene as encoding a novel PDZ protein called whirlin. The gene encoding whirlin also underlies the human autosomal recessive deafness locus DFNB31. In the mouse cochlea, whirlin is expressed in the sensory IHC and OHC stereocilia. Our findings suggest that this novel PDZ domain–containing molecule acts as an organizer of submembranous molecular complexes that control the coordinated actin polymerization and membrane growth of stereocilia.

Quiet as a mouse: dissecting the molecular and genetic basis of hearing

Mouse genetics has made crucial contributions to the understanding of the molecular mechanisms of hearing. With the help of a plethora of mouse mutants, many of the key genes that are involved in the development and functioning of the auditory system have been elucidated. Mouse mutants continue to shed light on the genetic and physiological bases of human hearing impairment, including both early- and late-onset deafness. A combination of genetic and physiological studies of mouse mutant lines, allied to investigations into the protein networks of the stereocilia bundle in the inner ear, are identifying key complexes that are crucial for auditory function and for providing profound insights into the underlying causes of hearing loss.

Gelsolin plays a role in the actin polymerization complex of hair cell stereocilia.

A complex of proteins scaffolded by the PDZ protein, whirlin, reside at the stereocilia tip and are critical for stereocilia development and elongation. We have shown that in outer hair cells (OHCs) whirlin is part of a larger complex involving the MAGUK protein, p55, and protein 4.1R. Whirlin interacts with p55 which is expressed exclusively in outer hair cells (OHC) in both the long stereocilia that make up the stereocilia bundle proper as well as surrounding shorter microvilli that will eventually regress. In erythrocytes, p55 forms a tripartite complex with protein 4.1R and glycophorin C promoting the assembly of actin filaments and the interaction of whirlin with p55 indicates that it plays a similar role in OHC stereocilia. However, the components directly involved in actin filament regulation in stereocilia are unknown. We have investigated additional components of the whirlin interactome by identifying interacting partners to p55. We show that the actin capping and severing protein, gelsolin, is a part of the whirlin complex. Gelsolin is detected in OHC where it localizes to the tips of the shorter rows but not to the longest row of stereocilia and the pattern of localisation at the apical hair cell surface is strikingly similar to p55. Like p55, gelsolin is ablated in the whirler and shaker2 mutants. Moreover, in a gelsolin mutant, stereocilia in the apex of the cochlea become long and straggly indicating defects in the regulation of stereocilia elongation. The identification of gelsolin provides for the first time a link between the whirlin scaffolding protein complex involved in stereocilia elongation and a known actin regulatory molecule.

Otitis media in the Tgif knockout mouse implicates TGFβ signalling in chronic middle ear inflammatory disease

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Genetic mapping of the whirler mutation.

Abstract. The whirler (wi) mutation on mouse Chromosome (Chr) 4 results in an autosomal recessive neuroepithelial deafness and vestibular dysfunction exhibited as a characteristic shaker-waltzer behavior (deafness, circling, and head-bobbing). We have constructed a genetic linkage map across the wi region in both an interspecific [(wi/wi× CAST/Ei)F1×wi/wi] backcross (n = 817) and an intraspecific [(wi/wi× CBA/Ca)F1×wi/wi)] backcross (n = 335). In the interspecific backcross, wi was found to be non-recombinant with Orm1, 0.12 cM distal of D4Mit87 and Ambp, and 0.12 cM proximal of CD301. In the intraspecific backcross, wi was found to be non-recombinant with Orm1 and D4Mit244, 0.3 cM distal of Mup1, and 0.6 cM proximal of Tnc. We also report a family from the interspecific backcross that shows evidence of multiple recombinations across the region of mouse Chr 4 around the wi locus. These rearrangements appear specific to both the region and the family.

Towards a mutant map of the mouse – new models of neurological, behavioural, deafness, bone, renal and blood disorders

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al., 2000). As of 2003, 35,000 mice have been produced to date in a genome-wide screen for dominant mutations and screened using a variety of screening protocols. Nearly 200 mutants have been confirmed as heritable and added to the mouse mutant catalogue and, overall, we can extrapolate that we have recovered over 700 mutants from the screening programme. For further information on the project and details of the data, see http://www.mgu.har.mrc.ac.uk/mutabase.

ENU mutagenesis and the search for deafness genes

The availability of mouse mutant models for known human deafness loci is limited. Moreover, it is unlikely that the current mouse archives hold mutants for the full panoply of genes involved in auditory system development and transduction. A large-scale ENU mutagenesis is currently underway to increase significantly the number of mouse deafness mutants available, employing specific screens for both deafness and balance defects. In the MRC Harwell screen, 13 mice have been identified so far with deafness, a balance defect or both. Mutagenized mice from the programme are also being used to search for modifiers of a known deafness gene, myosin VIIA (mutated in the Shaker 1 mutant mouse). The progress and encouraging results of the programme indicate that the combination of ENU mutagenesis and effective phenotype screens will lead to a significant contribution to the understanding of the genes and mechanisms involved in hereditary deafness.

Protein 4.1 expression in the developing hair cells of the mouse inner ear.

Protein 4.1 (band 4.1 or 4.1R) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. Subsequently, several new family members, 4.1N, 4.1G and 4.1B, have been identified, which are expressed in many cell types, in particular at cell-cell junctions. We previously reported that 4.1R and 4.1N are expressed in the inner ear hair cells with specific localization patterns, and that 4.1R forms a complex with the membrane-associated guanylate kinase (MAGUK) protein p55 and two deafness gene products, myosin XV and whirlin. To determine the functions of the other family members, 4.1G and 4.1B, we observed their expression patterns in developing stereocilia in mice inner ear hair cells. 4.1G is expressed in the basal tapers of the stereocilia bundle in early postnatal stages. 4.1B was specifically and constantly expressed in the stereocilia tips during postnatal development. Additionally, we found that 4.1B is ablated in the hair cells of both myosin XV and whirlin mutant mice at all stages in hair cell development. These results suggest that 4.1 family members play important roles in the development and maintenance of the inner ear hair cells, and that 4.1B may be a member of the myosin XV-whirlin complex that is important for stereocilia maturation.

The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.