Progressive loss of PAX9 expression correlates with increasing malignancy of dysplastic and cancerous epithelium of the human oesophagus.

in Journal of Pathology (2002), 197(3), 293-7

Pax genes encode a family of transcription factors that play key roles in embryonic development. Whereas the functions of Pax genes in the adult organism are largely unknown, upregulated Pax gene ... [more ▼]

Pax genes encode a family of transcription factors that play key roles in embryonic development. Whereas the functions of Pax genes in the adult organism are largely unknown, upregulated Pax gene expression has been implicated in tumourigenesis. In this study, PAX9-specific monoclonal antibodies have been generated and it has been shown that PAX9 protein is expressed in the normal epithelium of the adult human oesophagus. PAX9 expression was either lost or significantly reduced in the majority of invasive carcinomas and epithelial dysplasias, the latter representing precancerous lesions. Notably, the percentage of PAX9-positive cells within the epithelium decreased with increasing malignancy of the epithelial lesion. These results identify PAX9 as a sensitive marker for deregulated differentiation of oesophageal keratinocytes and indicate a role for PAX9 in the normal differentiation process of internal stratified squamous epithelia. These data suggest that upregulated PAX9 expression is not required for the formation of the majority of squamous cell carcinomas of the human oesophagus. [less ▲]

Thymopoiesis requires Pax9 function in thymic epithelial cells.

in European Journal of Immunology (2002), 32(4), 1175-81

The epithelial thymic anlage develops from the third pharyngeal pouch. Pax9 is expressed in the entire pharyngeal endoderm, and its function is required for normal development of organs derived from ... [more ▼]

The epithelial thymic anlage develops from the third pharyngeal pouch. Pax9 is expressed in the entire pharyngeal endoderm, and its function is required for normal development of organs derived from pharyngeal pouches. Here, we show that in Pax9 null mice, the thymic anlage develops as an ectopic polyp-like structure in the larynx. It expresses Whn/Foxn1, a marker of thymic epithelium, but fails to perform the normal caudo-ventral movement to the upper mediastinum. The thymic rudiment contains mesenchymal cells, blood vessels and is colonized by T cell progenitors. However, from embryonic day 14.5 onwards, the size of the Pax9 mutant thymus is severely reduced. Whereas expression of TCRbeta chain genes is readily detectable in the mutant thymus, no expression of the TCRgamma chain was detectable. Our results identify a new genetically defined control point of thymopoiesis. [less ▲]

Respiratory mechanics in mice: strain and sex specific differences.

in Acta Physiologica Scandinavica (2002), 174(4), 367-75

To assess the contribution of genetic background to respiratory mechanics, we developed a ventilator unit to measure lung function parameters in the mouse. We studied two commonly used inbred mice strains ... [more ▼]

To assess the contribution of genetic background to respiratory mechanics, we developed a ventilator unit to measure lung function parameters in the mouse. We studied two commonly used inbred mice strains originating from Mus musculus domesticus (C57BL/6 and C3HeB/FeJ) and a third strain derived from Mus musculus molossinus [Japanese fancy mouse 1 (JF1)]. The ventilator allows for accurate performance of the different breathing manoeuvres required for measuring in- and expiratory reserve capacity, quasi-static and dynamic compliance, and airway resistance. In combination with a mass spectrometer for monitoring gas concentrations, single-breath manoeuvres were performed and He-expirograms obtained, from which dead space volume and slope of phase III were determined. From each strain and each sex, 10, 2-month old animals were studied immediately after being killed by an intraperitoneal overdose of xylazine and ketamine. C3HeB/FeJ and C57BL/6 exhibited comparable lung volumes. In male C3HeB/FeJ mice, e.g. vital capacity (VC) was 1072 +/- 79 microL, inspiratory reserve capacity 782 +/- 88 microL, and dead space volume at total lung inflation 216 +/- 18 microL. Lung volumes of JF1 were significantly lower (e.g. VC 611 +/- 53 microL, P < 0.01) even when normalized to body weight. In all three strains, specific lung volumes were significantly higher in females than in males, possibly explained by a higher oxygen demand during pregnancy and lactation, both of which fill most of their life times. Static compliance in C3HeB/FeJ was 64.3 +/- 5.4 microL cmH2O-1. It was smaller in C57BL/6 and JF1 mice, even when related to the lung volume. Analysis of the degree of genetic vs. non-genetic components of the phenotypic variation revealed that at least 80% of the total variation of lung volumes and static compliance in the mixed population is attributable to genetic differences between individuals. These differences will be verified in further studies by segregation and genetic linkage analysis. [less ▲]

Systematic approaches to mouse mutagenesis.

in Current Opinion in Genetics and Development (2001), 11(3), 268-73

A major challenge in post-genomics is the systematic determination of mammalian gene function. A variety of mouse mutagenesis technologies, both gene- and phenotype-driven, are being used to underpin ... [more ▼]

A major challenge in post-genomics is the systematic determination of mammalian gene function. A variety of mouse mutagenesis technologies, both gene- and phenotype-driven, are being used to underpin systematic and comprehensive approaches to mammalian gene function studies. Recently, a number of centres have completed large-scale ENU mutagenesis programmes that employ a phenotype-driven approach to the generation of mouse mutants. The use of ENU mutagenesis represents a powerful and efficient approach to mammalian gene-function studies, but many parallel developments are needed in downstream technologies to properly harness the new enlarged mouse-mutant resources that are being created. [less ▲]

Comparative analysis of the genomic organization of Pax9 and its conserved physical association with Nkx2-9 in the human, mouse, and pufferfish genomes.

in Mammalian Genome (2001), 12(3), 232-7

As a first step towards the identification of cis-regulatory elements of Pax9 by means of comparative genomics, we have analyzed genome regions encompassing the Pax9 gene in three vertebrate species ... [more ▼]

As a first step towards the identification of cis-regulatory elements of Pax9 by means of comparative genomics, we have analyzed genome regions encompassing the Pax9 gene in three vertebrate species, humans, mice (Mus musculus), and the Japanese pufferfish (Fugu rubripes). We show the genomic organization of Pax9 and its physical association with Nkx2-9 conserved in the three species. We discuss about possible implications of the conserved synteny between Pax9 and Nkx2-9 in a context of vertebrate evolution. This report also includes the first description of the primary structures of Fugu Pax9 and Nkx2-9. Furthermore, we report the identification of a novel upstream exon and putative transcription start sites in mouse Pax9. Our results suggest that transcription of Pax9 may be initiated at two alternative start sites and driven by TATA-less promoters. [less ▲]

Characterization of a new, dominant V124E mutation in the mouse alphaA-crystallin-encoding gene.

in Investigative Ophthalmology and Visual Science (2001), 42(12), 2909-15

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screening, mice were tested for the occurrence of dominant cataracts. The purpose of the study was morphologic description, mapping of the mutant gene ... [more ▼]

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screening, mice were tested for the occurrence of dominant cataracts. The purpose of the study was morphologic description, mapping of the mutant gene, and characterization of the underlying molecular lesion in a particular mutant, Aey7. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed with a set of microsatellite markers covering all autosomal chromosomes. cDNA was amplified after reverse transcription of lens mRNA. For PCR, cDNA or genomic DNA was used as a template. RESULTS: Nuclear opacity and posterior suture anomaly were visible at eye opening and progressed to a nuclear and zonular cataract at 2 months of age. The opacity as well as the microphthalmia was more pronounced in the homozygotes than in the heterozygotes. The mutation was mapped to chromosome 17 between the markers D17Mit133 and D17Mit180. This position made the alphaA-crystallin-encoding gene (Cryaa) an excellent candidate gene. Sequence analysis revealed a mutation of a T to an A at position 371 in the Cryaa cDNA. The mutation was confirmed by an additional MnlI restriction site in the genomic DNA of homozygous mutants leading to replacement of Val with Glu at codon 124 affecting the C-terminal region of the alphaA-crystallin. CONCLUSIONS: The Aey7 mutant represents the first dominant mouse cataract mutation affecting the Cryaa gene. The mutation leads to progressive opacification of the lens. Compared with the beta- and gamma-crystallin-encoding genes, mutations in the alpha-crystallin-encoding genes are rare. [less ▲]

Ethylnitrosourea-induced mutation in mice leads to the expression of a novel protein in the eye and to dominant cataracts.

in Genetics (2001), 157(3), 1313-20

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the ... [more ▼]

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the chromosomal position, the gamma-crystallin encoding gene cluster (Cryg) and the betaA2-crystallin encoding gene Cryba2 were tested as candidate genes. An A --> T mutation destroys the start codon of the Cryge gene in the mutants; this mutation was confirmed by the absence of a restriction site for NcoI in the corresponding genomic fragment of homozygous mutants. The next in-frame start codon is 129 bp downstream; this predicted truncated gammaE-crystallin consists of 131 amino acids, resulting in a molecular mass of 14 kD. However, another open reading frame was observed just 19 bp downstream of the regular Cryge start codon, resulting in a protein of 119 amino acids and a calculated molecular weight of 13 kD. Western blot analysis using polyclonal antibodies against gamma-crystallins or the novel Aey1-specific protein demonstrated the specific expression of the Aey1 protein in the cataractous lenses only; the truncated form of the gammaE-crystallin could not be detected. Therefore, it is concluded that the novel protein destroys the sensitive cellular structure of the eye lens. [less ▲]

Sequence interpretation. Functional annotation of mouse genome sequences.

in Science (2001), 291(5507), 1251-5

Characterization of a mutation in the lens-specific MP70 encoding gene of the mouse leading to a dominant cataract.

in Experimental Eye Research (2001), 73(6), 867-76

During an ethylnitrosourea mutagenesis screen, Aey5, a new mouse mutation exhibiting an autosomal dominant congenital cataract was isolated. The cataractous phenotype is visible at the eye opening and ... [more ▼]

During an ethylnitrosourea mutagenesis screen, Aey5, a new mouse mutation exhibiting an autosomal dominant congenital cataract was isolated. The cataractous phenotype is visible at the eye opening and progresses to a nuclear and zonular cataract at 2 months of age with no difference in onset or severity between heterozygous and homozygous mutants. Histological analysis revealed that fiber cell differentiation continues at the lens bow region, but the cell nuclei do not degrade normally and remain in the deeper cortex. Further, the lens nucleus has clefts of various sizes while the remainder of the eye was morphologically normal. The mutation was mapped to chromosome 3 between the markers D3Mit101 and D3Mit77 near the connexin encoding genes Gja5 and Gja8. Sequence analysis revealed no differences in the Gja5 gene, but identified a T-->C mutation at position 191 in the Gja8 gene, which was confirmed by an additional Mva 12691 restriction site in the genomic DNA of homozygous mutants. This mutation results in Val-->Ala substitution at codon 64 of connexin50 (Cx50) also known as lens membrane protein 70 (MP70). Aey5 represents the second dominant mouse cataract mutant affecting Cx50, a membrane protein preferentially expressed in the lens. Since both mutations affect similar regions in the first extracellular domain this region appears to be critically important for its function in lens transparency. [less ▲]

The biochemical metabolite screen in the Munich ENU Mouse Mutagenesis Project: determination of amino acids and acylcarnitines by tandem mass spectrometry.

in Mammalian Genome (2000), 11(7), 547-51

BACKGROUND: Gene mutations often result in altered protein expression and, in turn, lead to changes in metabolite levels in one or more distinct biochemical pathways. Traditional analytical methods for ... [more ▼]

BACKGROUND: Gene mutations often result in altered protein expression and, in turn, lead to changes in metabolite levels in one or more distinct biochemical pathways. Traditional analytical methods for metabolite determination are usually time consuming, expensive, and, thus, not suitable for high throughput analysis. However, recent developments in electrospray-tandem-mass-spectrometry allow comprehensive metabolite scanning from very small amounts of blood with high speed, cost effectiveness, and accuracy. METHODS: A blood spot from a filter paper equivalent to 3 microl of blood was punched out and transferred to a 96-well microtiter plate. After addition of a set of 14 stable isotope-labeled internal standards, amino acids and acylcarnitines were extracted with methanol. The dried residue was derivatized with butanolic hydrochloric acid and subjected to MSMS analysis. RESULTS: Acyl-carnitines were all determined by a precursor ion scan of 85 Da. Neutral loss scanning of 102 Da was suitable for the quantitation of threonine, serine, proline, histidine, alanine, aspartic acid, glutamic acid, methionine, tyrosine, phenylalanine, isoleucine/leucine and valine. Glycine was detected by a loss of a 56-Da fragment, whereas a 119-Da loss was suitable for the measurement of citrulline, ornithine, arginine, and lysine. Specific problems encountered: owing to their identical molecular weight, isoleucine and leucine could not be quantitated separately, and, owing to their instability, glutamine and asparagine were found to be decarboxylated to their respective acids. Determination was linear over the concentration range tested (20 to 1000 micromol/L), and intraassay and interassay coefficients of variation were in the range of 10-15%. CONCLUSION: ESI-MSMS proved to be a highly sensitive, linear, and sufficiently precise method for the quantitative determination of amino acids and acylcarnitines in mouse blood, allowing large-scale screening applications when speed and cost effectiveness are mandatory. [less ▲]