Kidney-specific cadherin (cdh16) is expressed in embryonic kidney, lung, and sex ducts.

Cdh16 was initially described as a truncated cadherin expressed in the adult rabbit kidney. We have analyzed the expression pattern of cdh-16 during mouse embryogenesis, and show that cdh-16 transcripts are present in ureter-derived epithelia of the metanephric kidney. In addition, we demonstrate that cdh-16 is also transiently expressed in the epithelia of embryonic sex ducts and the lung of the embryo.

Large-scale screen for genes involved in gonad development.

The vertebrate gonad develops from the intermediate mesoderm as an initially bipotential organ anlage, the genital ridge. In mammals, Sry acts as a genetic switch towards testis development. Sox9 has been shown to act downstream of Sry in testis development, while Dax1 appears to counteract Sry. Few more genes have been implicated in early gonad development. However, the genetic networks controlling early differentiation events in testis and ovary are still far from being understood. In order to provide a broader basis for the molecular analysis of gonad development, high-throughput gene expression analysis was utilized to identify genes specifically expressed in the gonad. In total, among 138 genes isolated which showed tissue specific expression in the embryo, 79 were detected in the developing gonad or sex ducts. Twenty-seven have not been functionally described before, while 40 represent known genes and 12 are putative mouse orthologues. Forty-five of the latter two groups (86%) have not been described previously in the fetal gonad. In addition, 21 of the gonad specific genes showed sex-dimorphic expression suggesting a role in sex determination and/or gonad differentiation. Eighteen of the latter (86%) have not been described previously in the fetal gonad. In total we provide new data on 72 genes which may play a role in gonad or sex duct development and/or sex determination. Thus we have generated a large gene resource for the investigation of these processes, and demonstrate the suitability of high-throughput gene expression screening for the genetic analysis of organogenesis.

Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

More than one component of the Newcastle disease virus particle is capable of interferon induction.

The interferon (IFN)-inducing capacities of intact NDV virions, beta-propiolactone-inactivated particles and several structural components were compared, using human PBML as the IFN producing cells. Intact and inactivated virions as well as the nucleocapsid fraction did not differ significantly in their IFN-inducing capacity. In contrast, genomic RNA as well as M protein fraction and envelopes induced IFN titres to a level of about 10% of those achieved with virions. NDV-induced IFN production could be blocked specifically by incubation with polyclonal anti-NDV-monoclonal antibodies (mAbs) and with two of three anti-HN-mAbs, but not with anti-NDV-mAbs directed against the F, M or NP protein. In addition, IFN induction by fixed MDBK cells, expressing NDV surface proteins after infection with NDV Ulster, was inhibited by one of two anti-F-mAbs. The results suggest that the induction of IFN synthesis in human PBML is a complex process involving not only the HN protein but also the uncleaved F protein precursor, a component of the M protein fraction and--once having entered the cell--the genomic RNA.

Interferon induction in peripheral blood mononuclear leukocytes of man and farm animals by poxvirus vector candidates and some poxvirus constructs.

Prototypes of three poxvirus genera--orthopoxvirus (OPV), parapoxvirus (PPV), avipoxvirus (APV)--and Newcastle disease virus (NDV) as a control, as well as three recombinant OPV strains and one recombinant APV strain, were incubated in vitro with peripheral blood mononuclear leukocytes (PBML) of man, sheep and swine. Antiviral activity was determined in PBML culture supernatants at different time intervals after virus cell interaction using a cytopathic effect inhibition bioassay. Additionally, supernatants derived from human PBML were screened for interferons (IFN) alpha and gamma as well as for tumor necrosis factor by enzyme-linked immunosorbent assay. IFN titers reached a maximum 24 h after PBML stimulation at a multiplicity of infection (MOI) greater than 1. IFN alpha/beta was found to be responsible for the antiviral effect. Using a MOI > or = 1 the highly attenuated strain MVA was the only representant of vaccinia virus (VV) that induced significant amounts of IFN also as a lacZ recombinant. Replicable virus from five well-known VV strains as well as the Chinese VV strain Tien Tan (VVTT) as a recombinant vaccine failed to induce leukocyte IFN. Inactivated VV strain Elstree and the recombinant TT strain induced high titers of leukocyte IFN. Supernatants derived from human, porcine and ovine PBML stimulated with replicable PPV, native VV MVA and MVA lacZ recombinant or native APV and APV lacZ recombinant virus regularly contained IFN alpha. In contrast to NDV, neither specific antisera nor monoclonal antibodies were able to block the INF induction by VV and PPV.

Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene.

During a gene trap screen, an insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene, has been discovered. Because the ROSA beta geo vector was used, the new allele is called Dmd(mdx-beta geo). The insertion occurred 3' of exon 63 of the dystrophin gene, resulting in a mutation that affects all presently known dystrophin isoforms. In contrast to spontaneous or ENU-induced alleles, Dmd(mdx-beta geo) can be used to follow dystrophin expression by staining for beta-galactosidase activity. The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with other methods. Dystrophin promoters are active predominantly in the dermamyotome, limb buds, telencephalon, floor plate, eye, liver, pancreas anlagen, and cardiovascular system. Adult Dmd(mdx-beta geo) mice show reporter gene expression in brain, eye, liver, pancreas, and lung. In skeletal and heart muscle, beta-galactosidase activity is not detectable, confirming Western blot data that indicate the absence of the mutant full-length protein in these tissues. Hemizygous Dmd(mdx-beta geo) mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located nuclei. Some mutant animals develop a dilated esophagus, probably due to constriction by the hypertrophic crura of the diaphragm.

[Interferon in sheep]. / Interferon beim Schaf.

There is only limited information on sheep interferon available. Recent publications have reported on: 1. an interferon (IFN) alpha subtype, which is secreted by the fetal trophectoderm into the lumen of the uterus between the 10th and 21st day of gestation. It was therefore named ovine trophoblast protein (oTP-1), and is responsible for signalling pregnancy to the ewe via high affinity receptors in the endometrium. It is thought that oTP-1 acts by directly influencing prostaglandin metabolism. 2. the role of lentivirus-induced interferon (LV-IFN) in the pathogenesis of Maedi/Visna. The results indicate that LV-IFN limits viral replication and therefore contributes to virus persistence and is also responsible for a chronic inflammatory process. 3. the mitogen- or antigen-dependent induction of ovine interferon gamma (IFN gamma) and its characterization.

M-twist is an inhibitor of muscle differentiation.

The twist gene encodes a transcription factor containing a conserved basic helix-loop-helix domain. During development, transcription factors of this type are normally associated with the induction of differentiation. Yet the expression pattern of the murine M-twist suggests an inhibitory role during muscle differentiation. Following stable transfection of myogenic mouse cells with an M-twist expression vector, 75% of M-twist-expressing clones were impaired in their ability to differentiate. In contrast, only 15% of control clones were unable to differentiate. Treatment with antisense oligonucleotides restored differentiation capacity in a concentration-dependent manner. Control oligonucleotides had no effect. These experiments show that the mouse twist gene can act as an inhibitor of muscle differentiation and that this inhibition is reversible.

A protein kinase encoded by the t complex responder gene causes non-mendelian inheritance.

Males heterozygous for the t-haplotype form of mouse chromosome 17 preferentially transmit the t-chromosome to their progeny. Several distorter/sterility loci carried on the t-haplotype together impair flagellar function in all spermatozoa whereas the responder, Tcr, rescues t-sperm but not wild-type sperm. Thus, t-sperm have an advantage over wild-type sperm in fertilizing egg cells. We have isolated Tcr by positional cloning and show that it is a member of a novel protein kinase gene family, designated Smok, which is expressed late during spermiogenesis. Smok kinases are components of a signal cascade which may control sperm motility. Tcr has a reduced kinase activity, which may allow it to counterbalance a signalling impairment caused by the distorter/sterility loci. Tcr transgene constructs cause non-mendelian transmission of chromosomes on which they are carried, which leads to sex-ratio distortion when Tcr cosegregates with the Y chromosome.

The mouse Aire gene: comparative genomic sequencing, gene organization, and expression.

Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.

3849 + 10 kb C --> T splicing mutation in Hispanic CF patients.

Seven patients compound heterozygous for the 3849 + 10kb C --> T mutation in the CFTR gene were found among the 152 patients attending the CHLA CF Clinic. The frequency of this mutation accounts for 2.3 and 3.9% of thetotal and Hispanic CF alleles of CHLA patients. These are significantly higher than the 0.6% of the general CF population. The average age of diagnosis of this group of Hispanics is 3.1 years, which is much younger than that reported for CF patients of other ethnicities with the same mutation. Both pancreatic sufficient and pancreatic insufficient patients were observed. It is concluded that the 3849 + 10kb C --> T mutation is associated with a variable but potentially mild type of CF.