A mutation in Tubb2b, a human polymicrogyria gene, leads to lethality and abnormal cortical development in the mouse.

Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple (brdp) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b (Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.

Focusing forward genetics: a tripartite ENU screen for neurodevelopmental mutations in the mouse.

The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse.

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6(clft1) allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6(clft1) allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family.

Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain.

Organizing centers in the developing brain provide an assortment of instructive patterning cues, including Sonic hedgehog (Shh). Here we characterize the forebrain phenotype caused by loss of Ttc21b, a gene we identified in an ENU mutagenesis screen as a novel ciliary gene required for retrograde intraflagellar transport. The Ttc21b mutant has defects in limb, eye and, most dramatically, brain development. We show that Shh signaling is elevated in the rostral portion of the mutant embryo, including in a domain in or near the zona limitans intrathalamica. We demonstrate here that ciliary defects seen in the Ttc21b mutant extend to the embryonic brain, adding forebrain development to the spectrum of tissues affected by defects in ciliary physiology. We show that development of the Ttc21b brain phenotype is modified by lowering levels of the Shh ligand, supporting our hypothesis that the abnormal patterning is a consequence of elevated Shh signaling. Finally, we evaluate Wnt signaling but do not find evidence that this plays a role in causing the perturbed neurodevelopmental phenotype we describe.

Genome-wide SNP analysis of Tg.AC transgenic mice reveals an oncogenic collaboration between v-Ha-ras and Ink4a, which is absent in p53 deficiency.

Oncogenesis is a progressive process often involving collaboration between various oncogenes and tumor suppressors. To identify those genes that collaborate with oncogenic ras, we took advantage of the Tg.AC transgenic mouse, a line that harbors the v-Ha-ras transgene and spontaneously develops an array of malignant tumors. By crossing Tg.AC mice on an inbred FVB background to other inbred strains, F1 mice were created that could be analysed using genome wide, single nucleotide polymorphism (SNP) screens. Loss of heterozygosity (LOH) in tumors and tumor cell lines marked a somatic event, possibly the inactivation of tumor suppressor gene(s). LOH could also represent DNA damage, a sign of genomic instability in the pretransformed cell. Nonetheless, the screens showed no evidence of such generalized genomic instability. Instead, they revealed a single region of LOH on chromosome 4 that occurred via somatic recombination/gene conversion, generating a region of isoparental disomy. This LOH provided a clue that linked v-Ha-ras to the inactivation of the Ink4a locus in 25 of 32 tumor cell lines. This collaboration is seen regardless of tumor type or genetic background. In contrast, tumors that develop in bitransgenic mice bearing both the v-Ha-ras gene and a heterozygous mutant p53 allele tend to retain the Ink4a locus and instead lose the p53 wild-type allele. This suggests that different strategies can be selected to collaborate with v-Ha-ras in tumorigenesis.

Impaired homeostasis and phenotypic abnormalities in Prdx6-/-mice lens epithelial cells by reactive oxygen species: increased expression and activation of TGFbeta.

PRDX6, a member of the peroxiredoxins (PRDXs) family, is a key player in the removal of reactive oxygen species (ROS). Using targeted inactivation of the Prdx6 gene, we present evidence that the corresponding protein offsets the deleterious effects of ROS on lens epithelial cells (LECs) and regulates gene expression by limiting its levels. PRDX6-depleted LECs displayed phenotypic alterations and elevated alpha-smooth muscle actin and betaig-h3 expression (markers for cataractogenesis), indistinguishable from transforming growth factor beta (TGFbeta)-induced changes. Biochemical assays disclosed enhanced levels of ROS, as well as high expression and activation of TGFbeta1 in Prdx6-/- LECs. A CAT assay revealed transcriptional repression of lens epithelium-derived growth factor (LEDGF), HSP27, and alphaB-crystallin promoter activities in these cells. A gel mobility shift assay demonstrated the attenuation of LEDGF binding to heat shock or stress response elements present in these genes. A supply of PRDX6 toPrdx6-/- LECs reversed these changes. Based on the above data, we propose a rheostat role for PRDX6 in regulating gene expression by controlling the ROS level to maintain cellular homeostasis.

Genomic structure and refined chromosomal localization of the mouse Ptch2 gene.

The vertebrate Patched 2 (Ptch2) gene encodes a putative membrane-embedded protein which may have roles in Hedgehog signaling during development and in tumorigenesis. We determined the genomic structure of the mouse Ptch2 gene and show that Ptch2 is composed of 22 exons spanning approximately 18 kb of genomic DNA. The exon-intron boundaries were found to be conserved within the human and mouse Ptch2 genes. Analysis of the 5' flanking region revealed a CpG island, the putative promoter region and the transcriptional start site while a polyadenylation signal as well as a mRNA destabilizing motif were identified in the 3' flanking region. Single-strand conformation polymorphism analysis was used to map mouse Ptch2 to chromosome 4 between the microsatellite markers D4Mit20 and D4Mit334.

An integrated genetic and physical map of the 650-kb region containing the congenital polycystic kidney (cpk) locus on mouse chromosome 12.

Mice homozygous for the congenital polycystic kidney (cpk) mutation develop a rapidly progressive form of polycystic kidney disease. We report an integrated genetic and physical map of the 650-kb region containing the cpk locus and the exclusion of Rrm2 and Idb2 as candidate cpk genes. Our study establishes the requisite foundation for positional cloning of the cpk gene.

Characterization of the murine Lbx2 promoter, identification of the human homologue, and evaluation as a candidate for Alström syndrome.

The murine Lbx2 gene is a member of the ladybird family of homeobox genes, which is expressed in the developing urogenital system, eye, and brain. Using transgenic mice, we demonstrate that 9 kb of the 5' flanking region of mouse Lbx2 is able to direct expression of a reporter gene in a tissue-specific manner recapitulating the endogenous expression pattern. This regulatory region provides a novel reagent allowing for transgenic expression in the developing urogenital ridge. In addition, we describe the identification of the human homologue, LBX2. Comparison of the human LBX2 and mouse Lbx2 sequences upstream of the coding regions reveals sequence conservation suggesting conserved regulatory regions. Both the human LBX2 and the mouse Lbx2 genes have similar genomic structures and are composed of two exons separated by an intron. We mapped the mouse Lbx2 gene to 35 cM on chromosome 6 and the human LBX2 gene to a homologous region of chromosome 2p13. This is a candidate region for several inherited disorders, including Alström syndrome, a disorder that includes ocular, urogenital, and renal abnormalities. Given the expression pattern of Lbx2, the chromosomal location in humans, and the potential function of mammalian ladybird genes, we have begun to analyze patients with ocular disorders and those with Alström syndrome for mutations in LBX2. Although polymorphisms were identified, our results indicate that mutations in the coding region of LBX2 do not account for Alström syndrome in the six kindreds analyzed.

An Epstein-Barr-related herpesvirus from marmoset lymphomas.

Epstein-Barr virus (EBV) is implicated in the development of human B cell lymphomas and carcinomas. Although related oncogenic herpesviruses were believed to be endemic only in Old World primate species, we now find these viruses to be endemic in New World primates. We have isolated a transforming, EBV-related virus from spontaneous B cell lymphomas of common marmosets (Callithrix jacchus). Sequencing of two-thirds of the genome reveals considerable divergence from the genomes of EBV and Old World primate EBV-related viruses, including differences in genes important for virus-induced cell growth transformation and pathogenesis. DNA related to the C. jacchus herpesvirus is frequently detected in squirrel monkey peripheral blood lymphocytes, indicating that persistent infection with EBV-related viruses is prevalent in both New World primate families. Understanding how these more divergent EBV-related viruses achieve similar biologic outcomes in their natural host is likely to provide important insights into EBV infection, B cell growth transformation, and oncogenesis.

Sequence-based analysis of mutagenized mice.

Treating mice with ethylnitrosourea (ENU) is an efficient means for mutagenizing spermatogonial cells, and this treatment has been proven effective in a variety of screens for both dominant and recessive mutations. However, a significant problem for this technology is that the efficiency of mutagenesis is assessed most often by the empiric determination of a per-locus mutation frequency by using the specific locus test, which is expensive, time-consuming, and logistically difficult. To approach this question more directly and more efficiently, one can utilize methods of PCR-based mutation detection for the characterization of progeny of mutagenized mice. Since this analysis can be done after a single generation of breeding, it is useful as a rapid means for the assessment of the efficiency of mutagen treatment. Furthermore, it is readily imaginable that this strategy can be applied for the general determination of gene function in a systematic manner. Theoretical considerations and empirical analysis suggest that the per-base mutation frequency for a fractionated-dose treatment protocol is on the order of 1 sequence change per 10(5) bp.

Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct.

Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471-6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function.

Germline and somatic loss of function of the mouse cpk gene causes biliary ductal pathology that is genetically modulated.

The mouse cpk mutation is the most extensively characterized murine model of polycystic kidney disease (PKD) and closely resembles human autosomal recessive PKD (ARPKD), with the exception that B6-cpk/cpk homozygotes do not express the biliary ductal plate malformation (DPM) lesion. However, homozygous mutants from outcrosses to other strains, e.g. DBA/2J (D2), CD-1, BALB/c and Mus mus castaneus (CAST), express the DPM. The current study was designed: (i) to characterize the cpk-associated biliary disease in affected F(2) homozygotes from intercrosses with either CAST or D2; and (ii) to evaluate focal biliary cysts identified in heterozygotes from a D2-cpk congenic strain. We found that all F(2) cpk/cpk pups expressed both the typical renal cystic disease and the DPM. The DPM severity, assessed using semi-quantitative histopathological analysis, was markedly variable in these F(2) progeny. We found no correlation between the severity of the DPM and the renal cystic disease in either F(2) cohort. In addition, we identified focal cysts, apparently of biliary origin, in the livers of both aged D2-+/cpk and F(1) heterozygotes. Genetic analysis demonstrated loss of heterozygosity at the cpk interval and supports a loss-of-function model for biliary cysts. We conclude that the cpk allele contains an inactivating mutation which disrupts tubulo-epithelial differentiation in the kidney and biliary tract. Expression of the biliary lesion is modulated by genetic background, and the specific biliary phenotype is determined by whether loss of function of the cpk gene occurs as a germline or a somatic event.

Structure and mapping of the mouse matrilin-3 gene (Matn3), a member of a gene family containing a U12-type AT-AC intron.

The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.

Genetic localization of interacting modifiers affecting severity in a murine model of polycystic kidney disease.

Genetic analysis of mouse disease models provides a means to investigate how modifying loci cause variation in phenotypic expression. We have shown that polycystic kidney disease (PKD) progression in the juvenile cystic kidney (jck) mutation can be influenced by an epistatic interaction between alleles of different strain backgrounds and we localized one of these loci to chromosome 1. Using a chromosome 1 congenic strain, we improved the genetic analysis and mapped the interacting locus to proximal chromosome 4, with a highly significant lod association of 5.5. Re-analysis of the original F(2) cross reveals that in this cohort, while the lod association of the chromosome 4 locus alone is not significant, its effect is apparent when analyzed in combination with the chromosome 1 locus. This result suggests that correlation of paired genotype data with phenotype data will be an effective means to detect epistatic interactions contributing to complex traits, and that these associations can be tested using appropriate congenic lines.

Quantitative trait locus mapping of airway responsiveness to chromosomes 6 and 7 in inbred mice.

Quantitative trait locus (QTL) mapping was used to identify chromosomal regions contributing to airway hyperresponsiveness in mice. Airway responsiveness to methacholine was measured in A/J and C3H/HeJ parental strains as well as in progeny derived from crosses between these strains. QTL mapping of backcross [(A/J x C3H/HeJ) x C3H/HeJ] progeny (n = 137-227 informative mice for markers tested) revealed two significant linkages to loci on chromosomes 6 and 7. The QTL on chromosome 6 confirms the previous report by others of a linkage in this region in the same genetic backgrounds; the second QTL, on chromosome 7, represents a novel locus. In addition, we obtained suggestive evidence for linkage (logarithm of odds ratio = 1.7) on chromosome 17, which lies in the same region previously identified in a cross between A/J and C57BL/6J mice. Airway responsiveness in a cross between A/J and C3H/HeJ mice is under the control of at least two major genetic loci, with evidence for a third locus that has been previously implicated in an A/J and C57BL/6J cross; this indicates that multiple genetic factors control the expression of this phenotype.

Cloning, tissue-specific expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein that catalyzes intermembrane transfer of phosphatidylcholines in vitro. We have cloned a cDNA encoding the human ortholog of PC-TP and have determined its tissue-specific expression as well as genomic organization. Radiation hybrid mapping localized the human gene, PCTP, to chromosome 17q21-22 and PCR-based single strand conformation polymorphism analysis of an interspecific backcross assigned mouse Pctp to the region of syntenic conservation on chromosome 11.

Cloning and expression of a murine histone deacetylase 3 (mHdac3) cDNA and mapping to a region of conserved synteny between murine chromosome 18 and human chromosome 5.

Histone deacetylases (HDACs) are enzymes that play a pivotal role in transcription, differentiation, and cell cycle progression. We previously cloned human HDAC3 cDNA and showed that its transfection into THP-1 cells results in G2/M cell cycle accumulation. Using bioinformatic screening and PCR, we have now cloned the murine Hdac3 cDNA, which encodes a 428-amino-acid protein with near complete identity to its human ortholog. To establish a link to a potential disease locus, we performed PCR-based chromosomal mapping for the mHdac3 gene and chromosomal fluorescence in situ hybridization (FISH) for the human gene. mHdac3 localizes to chromosome 18 and human HDAC3 gene localizes to a syntenic region in chromosome 5 at band q31.3-q32 telomeric to the cytokine gene cluster. Transfection of mHdac3 into HeLa cells led to accumulation in G2/M. Our results suggest a cell cycle function for murine Hdac3, reflecting the complex regulatory roles of this gene family.

Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice.

The mucosal lymphocyte integrin alpha E(CD103)beta 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding alpha E, localized it to chromosome 11, and generated integrin alpha E-deficient mice. In alpha E-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of alpha E beta 7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer's patch, and splenic T lymphocyte numbers were not reduced in alpha E-deficient mice. Thus, alpha E beta 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of alpha E deficiency upon intestinal IEL numbers was greater at 3-4 wk of life than in younger animals, and affected the TCR alpha beta+ CD8+ T cells more than the gamma delta T cells or the TCR alpha beta+ CD4+CD8- population. These findings suggest that alpha E beta 7 is involved in the expansion/recruitment of TCR alpha beta+ CD8+ IEL following microbial colonization. Integrin alpha E-deficient mice will provide an important tool for studying the role of alpha E beta 7 and of alpha E beta 7-expressing mucosal T lymphocytes in vivo.