Biomedical subjects

Jana Löster

Publications and source records attributed to Jana Löster.

13 recordsLinked to original sources

PubMed2005

Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.

PURPOSE: To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS: Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS: The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS: Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.

Alleles↗

PubMed2004

Electroretinography as a screening method for mutations causing retinal dysfunction in mice.

PURPOSE: To detect mice with hereditary retinal impairment, a high-throughput electroretinography (ERG) screening system was established. METHOD: Mice from eight different strains without known retinal disorders (102, 129/SvJ, AKR, C57BL/6J, C57BL/6JIco, CBA/CaJ, and DBA/2NCrlBR) and one control strain with retinal degeneration (C3HeB/FeJ) were fixed on a specially constructed sled, ERG electrodes were placed on the cornea, and mice were moved into a Ganzfeld stimulator. From a luminance range of 0.0125 to 500 cd-s/m(2) in a pretest series two levels (5 and 125 cd-s/m(2)) were chosen to shorten examination times. The root mean square (RMS) of the ERG-recording was analyzed to detect animals with abnormal retinal function. ERG responses of the left and right eyes were compared in amplitudes and implicit times of the a- and b-waves. Statistical analysis of the latter parameters was performed in all wild-type animals. Histology was performed on selected mice. RESULTS: ERG recordings of individual animals for the left and right eye revealed good agreement in amplitudes and implicit times of the a- and b-waves (P < 0.05). Comparison of these parameters among the wild-type strains showed several differences. Evaluation of the RMS revealed, in addition to the C3HeB/FeJ mice, a subgroup of mice within the 129/SvJ strain with abnormal retinal function. Molecular analysis of these mice demonstrated the presence of the same retroviral insertion in the Pde6b gene, which is causative of the Pde6b(rd1) allele carried in C3HeB/FeJ mice. Histologic analysis demonstrated good correlation between retinal electrophysiology and morphology. CONCLUSIONS: The present results demonstrate the feasibility of ERG for screening a large number of mice to detect animals with functional retinal impairment.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

Genetic and allelic heterogeneity of Cryg mutations in eight distinct forms of dominant cataract in the mouse.

PURPOSE: The purpose of this study was the characterization of eight new dominant cataract mutations. METHODS: Lenses of mutant mice were described morphologically and histologically. Each mutation was mapped by linkage studies. The candidate genes (the Cryg gene cluster and the closely linked Cryba2 gene) were sequenced. RESULTS: Molecular analysis confirmed all mutations in Cryg genes. Five mutations lead to amino acid exchanges, two are due to premature stop codons, and one is a 10-bp deletion in the Cryge gene. Morphologically, mutant carriers expressed nonsyndromic cataracts, ranging from diffuse lenticular opacities (Crygd(ENU910) and Cryge(ENU449)), to dense nuclear and subcortical opacity (Crygd(K10), Crygc(MNU8), Cryge(Z2), Crygd(ENU4011), and Cryge(ADD15306)), to dense nuclear opacity and ruptured lenses (Cryga(ENU469)). Results of histologic analyses correlate well with the severity of lens opacity, ranging from alterations in the process of secondary fiber nucleus degradation to lens vacuoles, fiber degeneration, and disruption of the lens capsule. CONCLUSIONS: In total, 20 mutations have been described that affect the Cryg gene cluster: Nine mutations affect the Cryge gene, but only one affects the Crygb or Crygf genes. No mutation was observed in the closely linked Cryba2. Two mutations occur at the same site in the Crygd and Cryge genes (Leu45-->Pro). The unequal distribution of mutations suggests hot spots in the Cryg genes. The overall high number of mutations in these genes demonstrates their central role in the maintenance of lens transparency.

Alleles↗

PubMed2003

The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart.

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with "primitive" pacemaker action potentials could be found. However, cardiac cells with "mature" pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.

Action Potentials↗

PubMed2003

Developmental genetics in ophthalmology.

Much of our knowledge about the function of genes in mammalian development has been derived from the molecular analysis of spontaneous or induced mutations in the mouse. Since mutations affecting the mouse eye can be easily identified, a remarkable number of mutant lines provide animal models for congenital anomalies in man. To understand the mechanisms of lens development in detail, the isolation of the corresponding genes and the characterization of the mutations at the molecular level are important. A prerequisite for molecular analysis is the chromosomal localization of the gene. In this review, some mutants from our institute will be discussed according to the embryological time scale of the expression of the affected genes, reflecting also their genetic hierarchy. (1) In the aphakia mouse mutant, two deletions in the promoter of the homeobox transcription factor Pitx3 lead to a loss of its function and to an arrest of eye development at the lens stalk stage. Mutations in the homologous human PITX3 gene have been demonstrated to be causative of cataracts and the dysmorphology of the anterior segment of the eye. (2) Connexin50 is present in the lens vesicle. Later on, it becomes abundant in the anterior part of the fiber cells and in the lens epithelial cells. Mutations in the connexin50-encoding gene Gja8 lead to dominant cataracts. (3) alphaA-crystallin is present in the mouse lens cup, in the posterior half of the lens vesicle, and later in a high concentration in the lens fiber cells. Mutations in the alphaA-crystallin-encoding gene Cryaa lead to recessive and dominant cataracts. (4) Mutations in the gamma-crystallin -encoding genes (Cryg) are the most frequent cause of congenital, dominant nuclear, or total cataracts in the mouse. Indications from our first studies in congenital human cataracts support these data. (5) Some postnatal, progressive cataracts have been characterized by mutations in the beta-crystallin -encoding genes (Cryb). Since at least one of them is also expressed in the retina and the brain, effects on these tissues have to be considered, too.

Animals↗

PubMed2003

Mutation in intron 6 of the hamster Mitf gene leads to skipping of the subsequent exon and creates a novel animal model for the human Waardenburg syndrome type II.

In the course of analysis of ENU-induced mutations in Syrian hamsters, a novel dominant anophthalmic white mutant (Wh(V203)) with hearing loss was recovered. Because of this phenotype and a close linkage to the Tpi gene, the Mitf gene was considered as a candidate gene. In the Mitf cDNA, a deletion of 76 bp covering the entire exon 7 was detected. Further molecular analysis revealed a T --> A exchange 16 bp upstream of the end of intron 6, leading to skipping of exon 7. These 16 bp at the end of intron 6 are identical in hamster, rat, mouse, and humans, indicating high conservation during evolution and a functional importance in splicing. Since the loss of exon 7 changes the open reading frame of the MITF transcript, translation will be stopped after 10 new amino acids. The truncated protein is predicted to contain only a part of the basic region and will miss the two helical domains and the leucine zipper. The Wh(V203) mutation in the Syrian hamster affects the same functional domains of the Mitf transcription factor as the human R124X mutation, causing human Waardenburg syndrome type II. Therefore, the Wh(V203) hamster mutant provides a novel model for this particular syndrome.

Amino Acid Sequence↗

PubMed2003

An in vivo doxycycline-controlled expression system for functional studies of the retina.

PURPOSE: Transgenic mice were developed that express tetracycline-controlled transactivator 1 (tTA1) specifically in photoreceptor cells. In these mice the transcription of the gene of interest can be easily inactivated in the retina in a short time frame. METHODS: A construct was prepared containing tTA1 under control of the murine rhodopsin regulatory region. This construct was used for the generation of transgenic mice. In situ hybridization was performed to study the distribution of the transactivator in the retina. The activity of the transactivator was analyzed by mating the lines with a luciferase reporter transgenic mouse. tTA1 activity and doxycycline's ability to block it were analyzed by luciferase assay. The effects of tTA1 on the retina were assessed by histology and electrophysiology. RESULTS: Two transgenic lines were developed that specifically express tTA1 in photoreceptor cells. The time course of transgene expression replicated transcription of endogenous rhodopsin. tTA1 was not toxic to the retina. Transactivator activity was blocked readily by doxycycline. CONCLUSIONS: An expression system for photoreceptor cells was generated to drive transcription in a cell-specific and time-controllable manner. This system is suitable for the study of factors involved in retinal biology and of mutant forms of genes involved in retinal diseases.

Animals↗

PubMed2002

Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Amyloid↗

PubMed2002

Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.

Pax6 is a key regulator of eye development in vertebrates and invertebrates, and heterozygous loss-of-function mutations of the mouse Pax6 gene result in the Small eye phenotype, in which a small lens is a constant feature. To provide an understanding of the mechanisms underlying this haploinsufficient phenotype, we evaluated in Pax6 heterozygous mice the effects of reduced Pax6 gene dosage on the activity of other transcription factors regulating eye formation. We found that Six3 expression was specifically reduced in lenses of Pax6 heterozygous mouse embryos. Interactions between orthologous genes from the Pax and Six families have been identified in Drosophila and vertebrate species, and we examined the control of Pax6 and Six3 gene expression in the developing mouse lens. Using in vitro and transgenic approaches, we found that either transcription factor binds regulatory sequences from the counterpart gene and that both genes mutually activate their expression. These studies define a functional relationship in the lens in which Six3 expression is dosage-dependent on Pax6 and where, conversely, Six3 activates Pax6. Accordingly, we show a rescue of the Pax6 haploinsufficient lens phenotype after lens-specific expression of Six3 in transgenic mice. This phenotypic rescue was accompanied by cell proliferation and activation of the platelet-derived growth factor alpha-R/cyclin D1 signaling pathway. Our findings thus provide a mechanism implicating gene regulatory interactions between Pax6 and Six3 in the tissue-specific defects found in Pax6 heterozygous mice.

Animals↗

PubMed2002

V76D mutation in a conserved gD-crystallin region leads to dominant cataracts in mice.

During a large-scale ENU mutagenesis screen, a mouse mutant with a dominant cataract was detected and referred to as Aey4. Aim of this study was the morphological description of the mutant, the mapping of the mutation, and the characterization of the underlying molecular lesion. The slit-lamp examination revealed a strong nuclear cataract surrounded by a homogeneous milky opacity in the inner cortex. The histological analysis demonstrated remnants of cell nuclei throughout the entire lens. The mutation was mapped to Chromosome 1 by a genome-wide linkage making the six gamma-crystallin encoding genes and the closely linked betaA2-crystallin encoding gene to relevant candidate genes. Finally, a T-->A exchange in exon 2 of the gammaD-crystallin encoding gene (symbol: Crygd) was demonstrated to be causative for the cataract phenotype; this particular mutation is, therefore, referred to Crygo(Aey4). The alteration in codon 76 leads to an amino acid exchange of Val-->Asp. Val at this position is highly conserved; it is found in all mouse and rat gammaD/E/F-crystallins as well as in the human gammaA- and gammaD-crystallins. It may be replaced solely by Ile, which is present in all bovine gamma-crystallins, in the rat and mouse gammaA/B/C-crystallins, as well as in the human gammaB/C-crystallins. It is predicted that the exchange of a hydrophobic side chain by a polar and acidic one might influence the microenvironment by a dramatic decrease of the isoelectric point by 1.5 pH units in the 10 amino acids surrounding position 76. The Crygd(Aey4) additionally demonstrates the importance of the integrity of the Cryg gene cluster for lens transparency.

Amino Acid Substitution↗

PubMed2002

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

Alkylating Agents↗

PubMed2002

A 6-bp deletion in the Crygc gene leading to a nuclear and radial cataract in the mouse.

PURPOSE: A mouse mutant expressing a bilateral nuclear and radial cataract was found after paternal treatment with chlorambucil. The purpose of this study was to establish the linkage of the mutation to a particular chromosome to allow molecular characterization. Moreover, the mutants were examined morphologically. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. The mutation was localized to chromosome 1 by allelism testing with the Cryge(nz) mutation. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced. RESULTS: A novel mouse cataract was characterized by a nuclear and radial opacification of the lens. The lenses of the mutants are smaller than those of the wild type. The histologic analysis demonstrated degeneration of lens fibers in the lens core. Abnormal remnants of cell nuclei are present throughout the entire lens. Genetic analysis revealed allelism to the Cat2 group of dominant cataracts on mouse chromosome 1; therefore, the cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A 6-bp deletion in exon 3 of the gammaC-crystallin encoding gene (Crygc) is causative for the cataract phenotype; the mutation is therefore designated CrygcChl3. The deletion of the bases 420 to 425 leads to a loss of two amino acids, Gly and Arg, in the fourth Greek-key motif. CONCLUSIONS: The CrygcChl3 is the first mutation in the mouse affecting the Crygc gene. Dominant mutations for five of the six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster for lens transparency.

Alleles↗

PubMed2002

Crygf(Rop): the first mutation in the Crygf gene causing a unique radial lens opacity.

PURPOSE: The Rop (radial opacity) mutation, which was recovered in a mutagenicity screen after paternal treatment with procarbazine, was analyzed to determine phenotype, chromosomal localization, candidate genes, and molecular lesion. METHODS: Native lenses were photographed under a dissecting microscope. Histologic sections of the eye were made according to standard procedures. Fine mapping of the mutation in relation to microsatellite markers for mouse chromosome 1 was performed. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced. RESULTS: The nuclear opacity of the heterozygous mutants showed radial structures, whereas the opacity of the homozygotes was homogenous. The histologic analysis revealed changes in the lens nucleus, which corresponds to the pronounced opacification in lenses of homozygous mutants. The allelism of Rop to the Cat2 group of dominant cataracts on mouse chromosome 1 was confirmed by linkage to microsatellite markers D1Mit156 and D1Mit181. The cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A T-->A exchange in exon 2 of the Crygf gene leads to a Val-->Glu exchange in codon 38 and was considered to be causative for the cataract phenotype; therefore, Crygf(Rop) has been suggested as the designation for the mutation. CONCLUSIONS: Crygf(Rop) is the first mutation affecting the Crygf gene. Dominant cataract mutations for all six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster in lens transparency.

Animals↗