Viable Dominant Spotting Research Articles

Dystroglycan expression in the mouse cochlea

Viable dominant spotting (W v/W v) mice have a c-kit gene mutation, which impedes the migration of neural crest cells to the developing cochlea where they normally differentiate into intermediate cells (ICs). A prominent pathological feature shared by these mutants and the aging human and gerbil cochlea is thickening of the basement membrane (BM) of strial capillaries. Atrophy of strial capillaries in the aging gerbil has been associated with changes in the expression of dystroglycan (DG), a cell-surface receptor that regulates BM assembly. Here we evaluated the expression of DG in W v/W v mutant and C57BL/6J wild-type mice to investigate the possible role of ICs in regulating strial capillary BM homeostasis. The DG gene product was identified in lateral wall dissections from both W v/W v mutant and wild-type mice by reverse transcription-polymerase chain reaction. Subunit-specific antibodies were employed to localize the α and β subunits of the DG heterodimer. Some sites in both wild-type and mutant mice, such as the subepithelial BM lining the scala media and regions of contact between selected epithelial cells, expressed α-DG alone. Other sites such as the perineural BM and the perivascular BM subtending strial capillaries and capillaries in the central portion of the auditory nerve coexpressed α- and β-DG. The strong diffuse staining for α-DG along the basolateral membrane of strial marginal cells disappeared with advancing strial degeneration in abnormal turns of W v/W v mutants. Variations in staining intensity for both α- and β-DG also occurred in the subendothelial BM of strial capillaries in turns lacking ICs and appeared to correspond with the degree of capillary atrophy. The results support the possibility that ICs play a role in the homeostasis of the strial capillary BM.

An ATP-Dependent Inwardly Rectifying Potassium Channel, KAB-2 (Kir4.1), in Cochlear Stria Vascularis of Inner Ear: Its Specific Subcellular Localization and Correlation with the Formation of Endocochlear Potential

Cochlear endolymph has a highly positive potential of approximately +80 mV. This so-called endocochlear potential (EP) is essential for hearing. Although pivotal roles of K+ channels in the formation of EP have been suggested, the types and distribution of K+ channels in cochlea have not been characterized. Because EP was depressed by vascular perfusion of Ba2+, an inhibitor of inwardly rectifying K+ (Kir) channels, but not by either 4-aminopyridine or tetraethylammonium, we examined the expression of Kir channel subunits in cochlear stria vascularis, the tissue that is supposed to play the central role in the generation of positive EP. Of 11 members of the Kir channel family examined with reverse transcription-PCR, we could detect only expression of KAB-2 (Kir4.1) mRNA in stria vascularis. KAB-2 immunoreactivity was specifically localized at the basolateral membrane of marginal cells but not in either basal or intermediate cells. Developmental expression of KAB-2 in marginal cells paralleled formation of EP. Furthermore, deaf mutant mice (viable dominant spotting; WV/WV) expressed no KAB-2 in their marginal cells. These results suggest that KAB-2 in marginal cells may be critically involved in the generation of positive EP.

Expression of α and β subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the W v or Sl d loci

Mice homozygous for mutations at the viable dominant spotting (W v) and Steel-dickie (Sl d) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of W v W v and Sl d Sl d mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the α1 and β1 subunits. Vestibular dark cells showed no reactivity for β1 but expressed abundant β2, whereas, strial marginal cells stained strongly for both β isoforms. The only qualitative difference between mutant and wild-type mice was the absence of β1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the β1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K + leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K + normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K + from perilymph to the intrastrial compartment.

Characteristics of stria vascularis melanocytes of viable dominantspotting ( [formula omitted]) mouse mutants

The W v mutation lies in the kinse domain of the proto-oncogene c-kit which is expressed in a variety of cells including neural crest derived melanoblasts The mutation results in the abnormal migration, proliferation, survival and/or differentiation of melanoblasts. Viable Dominant Spotting ( W v W v ) mouse mutants have a white coat due to the absence of melanocytes. The majority of these animals have no melanocytes within the stria vasculans and no endocochlear potential (EP). A proportion of homozygous mutants partially escape the effects of the mutation 47 2% of pinnae and 21% of vestibular regions were pigmented and 10 8% of ears had an EP. All ears with an EP that were available for histology had some pigmentation of the stria. There was no obvious correlation between external and internal spotting in W v W v mice, and asymmetrical pigmentation of the ears was common. Both light and dark intermediate cells (which are derived from melanocytes) were present in the middle and/or basal turns of these cochlear ducts and they appeared to function normally in enabling the stria to produce an EP (although the EP was usually lower than normal). This suggests that the c-kit gene product is needed only during development of the stria, and not for mature melanocyte function because the melanocytes present in the mutant strias were carrying the mutant version of the c-kit gene. Melanocytes were similar in appearance in controls and mutants, except that fewer melanin granules were observed in the strias of W v W v mice. The observations that strial melanocytes with very few melanin granules in W v W v mutants are able to support EP production, together with previous observations that albino animals with strial melanocytes but no melanin have a normal EP, suggest that melanocytes but not melanin are essential for normal strial function.

Cytogenetic characterization of radiosensitive mouse mutants

In order to develop mouse models for human mutagen-sensitive syndromes, we carried out cytogenetic characterization of several mouse mutants and MS/Ae mice showing enhanced radiosensitivities. The applied cytogenetic techniques include chromosomal analysis of in vitro cell cultures and lymphocyte cultures as well as in vivo UDS in hepatocytes, induction of micronuclei in polychromatic erythrocytes and translocation induction in spermatogonial stem cells. Among the mutations studied, namely the contrasted allele of steel ( Sl con ), viable dominant spotting ( W υ ), wasted ( wst), varitint-waddler ( Va) and dystonia musculorum ( dt) as well as MS/Ae mice, various iso-, hyper- or hypo-sensitive conditions were recorded. Only Va and dt appear to be associated with some deficiency in DNA repair.

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: Structural analysis in the viable dominant spotting mouse mutant

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear

The stria vascularis of the mammalian cochlea is composed primarily of three types of cells. Marginal cells line the lumen of the cochlear duct and are of epithelial origin. Basal cells also form a continuous layer and they may be mesodermal or derived from the neural crest. Intermediate cells are melanocyte-like cells, presumably derived from the neural crest, and are scattered between the marginal and basal cell layers. The marginal cells form extensive interdigitations with the basal and intermediate cells in the normal adult stria. The stria also contains a rich supply of blood vessels. We investigated the role of melanocytes in the stria vascularis by studying its development in a mouse mutant, viable dominant spotting, which is known to have a primary neural crest defect leading to an absence of recognisable melanocytes in the skin. Melanocytes were not found in the stria of most of the mutants examined, and from about 6 days of age onwards a reduced amount of interdigitation amongst the cells of the stria was observed. These ultrastructural anomalies were associated with strial dysfunction. In the normal adult mammal, the stria produces an endocochlear potential (EP), a resting dc potential in the endolymph in the cochlear duct, which in mice is normally about +100 mV. In our control mice, EP rose to adult levels between 6 and 16 days after birth. In most of the mutants we studied, EP was close to zero at all ages from 6 to 20 days. Melanocyte-like cells appear to be vital for normal stria vascularis development and function. They may be necessary to facilitate the normal process of interdigitation between marginal and basal cell processes at a particular stage during development, and the lack of adequate interdigitation in the mutants may be the cause of their strial dysfunction. Alternatively, melanocytes may have some direct, essential role in the production of an EP by the stria. Melanocytes may be important both for normal strial development and for the production of the EP. We believe this is the clearest demonstration yet of a role for migratory melanocytes other than their role in pigmentation.

A search for radiosensitive mouse mutants by use of the micronucleus technique

In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel ( Sl con), scabby ( scb), viable dominant spotting ( W v), quaking ( qk). fidget ( fi) and postaxial hemimelia ( px), heterozygous lurcher ( Lc), hemizygous gyro ( Gy), the compounds Sl con/grizzle-belly ( Sl gbH) and W v/rumpwhite ( Rw) and MS/Ae mice, were regarded as radiosensitive, with Sl con/ Sl con the highest in rank order. Homozygous wabbler-lethal ( wl) and wasted ( wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.

Strial dysfunction in mice with cochleo-saccular abnormalities

Most viable dominant spotting (Wv/Wv) mutant mice, which show cochleo-saccular degeneration, were found to have an endocochlear potential (EP) around zero together with a structurally abnormal stria vascularis. Inner hair cells were well preserved, but outer hair cells in the basal half of the cochlea were degenerating, possibly as a result of primary strial dysfunction. Thresholds for the detection of a compound action potential were raised to around 100 dB SPL in the mutants with no EP, and there was little if any cochlear microphonic at the round window. Of the 20 Wv/Wv mice studied, five partially escaped the effects of the mutation and had measurable positive potentials (15-86 mV) in scala media in the basal turn; responses in these animals were intermediate between control responses and those of mutants with no EP. These findings confirm that the pathological processes in this mutant, with cochleo-saccular abnormalities, are fundamentally different from the pathological processes in animals with neuroepithelial abnormalities reported previously [see Steel and Bock (1983) Arch. Otolaryngol. 109, 22-29, for references].

The endolymphatic sac in a mouse mutant with cochleo-saccular degeneration. Electrophysiological and ultrastructural correlations.

The response of the endolymphatic sac to a disturbance in endolymph homeostasis was investigated by examining the sac in a mouse mutant, viable dominant spotting, which is known to exhibit primary strial dysfunction and cochleo-saccular degeneration. The function of the vascular stria was assessed by measuring the endocochlear potential and the sacs were then studied by light and transmission electron microscopy. The endolymphatic sac was found to be morphologically abnormal in these mutants, the main abnormality being the presence of granular epithelial cells, which showed clear histological signs of secretory activity. A stainable precipitate, believed to be secreted by the granular cells, was observed in the lumen of the endolymphatic sac in the mutants. The findings strengthen the view that the sac is involved in the regulation of endolymph volume and pressure.

Central auditory function in a hearing-impaired white mouse

The dysfuction of the stria vascularis in the viable dominant spotting mutant mouse results in the reduction or the absence of the endocochlear potential. However, these mutants respond to an intense acoustic stimulus with a Preyer reflex (pinna twitch). This study used 14C autoradiography and electromyography to investigate central auditory responses in this mutant. There were three main findings: (1) autoradiography demonstrated an increase in the metabolic rate within each of the central auditory nuclei during noise exposure compared with silence; (2) electromyographic recordings indicated that there was no tensor tympani muscle reflex; (3) the mutants were found to be susceptible to audiogenic seizures. It was concluded that the central auditory pathway of the viable dominant spotting mutant could be activated despite the abnormal striai function. Absence of the tensor tympani muscle reflex, together with auditory deprivation, might contribute to the susceptibility to audiogenic seizures.

C57BL-6J mice with inherited retinal degeneration.

New stocks of mice carrying the autosomal recessive gene, retinal degeneration ( rd ), have been developed and are characterized in this report. The aim was to generate mutant and control mice that are (1) littermates, (2) distinguishable by linked marker gene(s) prior to recognizable phenotypic expression of the mutation, and (3) nearly identical at all genetic loci outside the chromosomal segment bearing rd and the marker mutations. C57BL/6J congenic strains carrying the linked genes rd , light ear ( le ), and viable dominant spotting ( W ) meet these criteria. The recombination percentages between these loci and their probable order is Wv -12- rd -1- le . The rd and le phenotypes on the C57BL/6J background appear not to interact with respect to expression of the photoreceptor cell degeneration. A comparison is given of the advantages and disadvantages of several breeding schemes for propagating the rd and related genes.

The role of the tissue environment in the expression of spotting genes in the mouse

ABSTRACT Genes causing white spotting in the mouse act in two major ways: some affect the melano-blasts, while others affect the tissue environment of the melanoblasts. The question is whether the normal tissue environment plays any role in the origin of spots in those mutants in which the melanoblasts are believed to be the site of gene activity. An earlier study, using the genotypes + /mi, Miwhl+, s/s, Wv/ + and Wv/Wv(mi = microphthalmia; Miwh= white; s = piebald; Wv= viable dominant spotting), indicated that it probably does, the evidence largely consisting in the occurrence of extremely precise pigmentation patterns on a minute scale. It seemed that more direct evidence could be obtained by comparing the pigmentation of the iris with that of the choroid and the retina in the same eye in these and other genotypes. The outer and inner layers of the iris derive their pigment cells from the choroid and the retina respectively; therefore any clear and consistent differences between the behaviour of these cells in their original and their secondary place of activity would constitute evidence for the role of the tissue environment. Such differences were found. It was also found that in another genotype, Miwh/mi the retinal pigment cells, although unpigmented, are clearly distinguishable. This casts serious doubt on the widespread assumption that melanoblasts which do not differentiate always die.