ENU-induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.     

Effects of the brachyury (T) mutation on mitotic activity in the neural tube.

Mitotic activity in the neural tube was examined in +/+, $\text{T}\text{+}$ and $\text{T}\text{T}$ mouse embryos. The number of mitotic figures per unit area of ependymal layer (area index) was higher in $\text{T}\text{+}$ embryos than in +/+ embryos. In +/+ embryos, area index was higher in the dorsal part of the neural tube than in the ventral part, whereas this pattern is disordered in $\text{T}\text{+}$ embryos. In $\text{T}\text{T}$ embryos, these values fluctuate. The process of nuclear migration accompanying cell division seems to be normal in the neural tube of $\text{T}\text{+}$ and $\text{T}\text{T}$ embryos.     

Mammary tumor modifiers in BALB/cJ mice heterozygous for p53

BALB/c mice are predisposed to developing spontaneous mammary tumors, which are further increased in a p53 heterozygous state. C57BL/6J mice are resistant to induced mammary tumors and develop less than 1% mammary tumors in both wild-type and p53 ^+/− states. To map modifiers of mammary tumorigenesis, we have established F₁ and F₂ crosses and backcrosses to BALB/cJ (N2-BALB/cJ) and C57BL/6J (N2-C57BL/6J) strains. All cohorts developed mammary carcinomas in p53 ^+/− females, suggesting that multiple loci dominantly and recessively contributed to mammary tumorigenesis. We mapped two modifiers of mammary tumorigenesis in the BALB/cJ strain. Mtsm1 (mammary tumor susceptibility modifier), a dominant-acting modifier, is located on chromosome 7. Mtsm1 is suggestive for linkage to mammary tumorigenesis (p = 0.001). We have analyzed the Mtsm1 region to locate candidate genes by comparing it to a rat modifier region, Mcs3, which shares syntenic conservation with Mtsm1. Expression data and SNPs were also taken into account. Five potential candidate genes within Mtsm1 are Aldh1a3, Chd2, Nipa2, Pcsk6, and Tubgcp5. The second modifier mapped is Mtsm2, a recessive-acting modifier. Mtsm2 is located on chromosome X and is significantly linked to mammary tumorigenesis (p = 1.03 × 10⁻⁷). Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Genetic modifiers of otocephalic phenotypes in Otx2 heterozygous mutant mice

Mice heterozygous for the Otx2 mutation display a craniofacial malformation, known as otocephaly or agnathia-holoprosencephaly complex. The severity of the phenotype is dependent on the genetic background of a C57BL/6 (B6) strain; most of the offspring of Otx2 knock-out chimeras, which are equivalent to the F(1) of CBA and B6 strains, backcrossed with B6 females display reduction or loss of mandible, whereas those backcrossed with CBA females do not show noticeable phenotype at birth. The availability of phenotypically disparate strains renders identification of Otx2 modifier loci possible. In this study, a backcross of chimera with B6 was generated and genome-wide scans were conducted with polymorphic markers for non-mendelian distribution of alleles in Otx2 heterozygous mutant mice displaying abnormalities in the lower jaw. We identified one significant locus, Otmf18, between D18Mit68 and D18Mit120 on chromosomes 18, linked to the mandibular phenotype (LOD score 3.33). A similar replication experiment using a second backcross (N3) mouse demonstrated the presence of another significant locus, Otmf2 between D2Mit164 and D2Mit282 on chromosome 2, linked to the mandibular phenotype (LOD score 3.93). These two modifiers account for the distribution of the craniofacial malformations by the genetic effect between B6 and CBA strains. Moreover, Otmf2 contain a candidate gene for several diseases in mice and humans. These genetic studies involving an otocephalic mouse model appear to provide new insights into mechanistic pathways of craniofacial development. Furthermore, these experiments offer a powerful approach with respect to identification and characterization of candidate genes that may contribute to human agnathia-holoprosencephaly complex diseases.     

tctN-mutation causing taillessness in mice heterozygous for gene T

Screening of wild male mice trapped in Turkmenistan (Middle Asia) revealed a mouse with a mutation causing strong increase in expressivity of T mutation (Brachyury). A novel mutation was designated tctN-t complex tail interaction Novosibirsk. Compound heterozygotes T/tctN have tailless of extremely short tailed phenotype. Homozygotes tctN/tctN were completely viable and fertile. It was shown that the novel mutation was closely linked to T locus. Further genetic analysis showed that the chromosome with tct had no properties of t haplotypes: no lethal factors or the factors influencing fertility and segregation of homologoues; there was no effect on recombination frequency in the proximal part of chromosome 17 in tctN/+mice. The problems of t haplotype evolution are discussed.     

Electron microscopy and histochemistry of tail regression in the brachyury mouse

The dominant Brachyury allele (T), nonlethal in the heterozygous condition, leads to a shortening of the mouse tail by resorption of distal structures and tissues in the caudal axis during development. Regressing Brachyury tail tissues of embryos prepared for conventional electron microscopy contained cells with numerous lysosome-like organelles and pseudopodal cytoplasmic extensions, but nonregressing tail tissues had few comparable structures. Such morphological evidence, when correlated with observations of high levels of acid phosphatase activity detected in resorbing Brachyury tail tissues with light microscope histochemistry, suggests that tail regression is mediated by considerable autophagy and/or phagocytosis.     

The Mammalian Cervical Vertebrae Blueprint Depends on the T (brachyury) Gene

A key common feature of all but three known mammalian genera is the strict seven cervical vertebrae blueprint, suggesting the involvement of strong conserving selection forces during mammalian radiation. This is further supported by reports indicating that children with cervical ribs die before they reach reproductive age. Hypotheses were put up, associating cervical ribs (homeotic transformations) to embryonal cancer (e.g., neuroblastoma) or ascribing the constraint in cervical vertebral count to the development of the mammalian diaphragm. Here, we describe a spontaneous mutation c.196A > G in the Bos taurus T gene (also known as brachyury) associated with a cervical vertebral homeotic transformation that violates the fundamental mammalian cervical blueprint, but does not preclude reproduction of the affected individual. Genome-wide mapping, haplotype tracking within a large pedigree, resequencing of target genome regions, and bioinformatic analyses unambiguously confirmed the mutant c.196G allele as causal for this previously unknown defect termed vertebral and spinal dysplasia (VSD) by providing evidence for the mutation event. The nonsynonymous VSD mutation is located within the highly conserved T box of the T gene, which plays a fundamental role in eumetazoan body organization and vertebral development. To our knowledge, VSD is the first unequivocally approved spontaneous mutation decreasing cervical vertebrae number in a large mammal. The spontaneous VSD mutation in the bovine T gene is the first in vivo evidence for the hypothesis that the T protein is directly involved in the maintenance of the mammalian seven-cervical vertebra blueprint. It therefore furthers our knowledge of the T-protein function and early mammalian notochord development.     

Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer

Early detection of lung cancer is challenging due to a lack of adequate biomarkers. To discover novel tumor suppressor genes (TSGs) silenced by aberrant promoter methylation, we analyzed the gene expression profiles of two lung adenocarcinoma cell lines using pharmacologic-unmasking and subsequent microarray-analysis. Among 617 genes upregulated, we selected 30 genes and investigated the methylation status of their promoters by bisulfite sequencing analysis. Aberrant methylation was detected in four genes (CRABP2, NOEY2, T, MAP2K3) in at least one lung adenocarcinoma cell lines. Furthermore, the T promoter was methylated in 60% of primary lung adenocarcinomas versus 13% of non-malignant lung tissues. Conversely, RT-PCR analysis revealed T expression was low in lung tumors, while high in normal tissues. In addition, no non-synonymous mutations related to gene silencing were found. While further analysis is warranted, our results suggest that T has the potential to be a novel candidate TSG in lung cancer.     

Fertility of male and female mice heterozygous for the reciprocal translocation T(7;17)3BKM.

The present paper describes the fertility of male and female mice heterozygous for the reciprocal translocation T(7;17)3BKM. This translocation was induced by gamma rays in the spermatozoa of an irradiated parent. It is characterized by "asymmetrical" localization of the breakpoints, distally in Chromosome 7 (7F5) and proximally in Chromosome 17 (17B1). The data presented here relate only those matings in which, for both partners, heterozygosity or normality could be confirmed cytogenetically. The results indicate that both male and female translocation heterozygotes are fertile, their mean litter size being reduced to about 50% of that of normal littermates. This leads to the conclusion that the multivalents mainly undergo either alternate or adjacent-1 2:2 segregation. No viable tertiary trisomics were observed among the progeny of the translocation carriers. Analysis of the frequency of the different types of multivalents in diakinesis-metaphase I spermatocytes showed a significant predominance of chain-type figures (CIV and CIII+I), with chains of four elements (CIV) being more frequent than other configurations. This demonstrates that the small marker chromosome remains attached by one of its segments to the tetravalent.     

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.     

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.     

Cytogenetic study of synapsis and crossing-over in male mice heterozygous for reciprocal translocation T(14; 15)6Ca

Synapsis and crossing over in male mice heterozygous for reciprocal translocation T (14; 15)6Ca were studied. The translocated multivalent undergoes the synaptic adjustment in the course of meiotic prophase. Translocated distal region of the 14th chromosome forms inproportionally long lateral element of synaptonemal complex. The number of chiasmata in the 14th chromosome increases from 1.02 0.02 in normal karyotype to 1.41 0.03 in heterozygous mice. The density of chiasmata in translocated, distal region is ten times higher than in the other part of the 14th chromosome.     

The induction of tail malformations in trisomy 16 mouse fetuses heterozygous for the curly tail recessive gene

The mouse mutant curly tail is thought to be inherited as an autosomal recessive (ct/ct) with incomplete penetrance so that approximately 60% of ct/ct individuals exhibit the curly tail (CT) phenotype. By outcrossing ct/ct with mouse stock carrying specific heterozygous combinations of Robertsonian (Rb) chromosomes, trisomy 16 (Ts16) and Ts19 mouse fetuses (and their chromosomally balanced littermates) were derived which were heterozygous for the ct gene. All of the Ts16 (ct/Rb;Rb) fetuses, studied between days 14-19 gestation had tail malformations, 86% of which were tail flexion defects (TFD) apparently very similar to the curly tail phenotype. Neither Ts19 nor any of the chromosomally balanced (ct/Rb) littermates from both experimental crosses showed any type of tail or other spinal malformation. At the 27-29 somite stage of development, Ts16 (ct/Rb;Rb) fetuses did not show any significant delay in the closure of the posterior neuropore (PNP) compared with their littermate controls, suggesting that the tail malformation observed in Ts16 (ct/Rb;Rb) occur as a result of mechanisms which differ significantly from those thought to be responsible to causing the curly tail malformation.     

Steel (Sl) mutation in mice: Identification of mutant embryos early in development

A method is described for the reliable identification of Steel mutant mouse embryos in segregating litters as early as Day 11 of gestation based on the color of hair developed in embryonic skin explants.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.     

Genetic analysis of jumbled spine and ribs (Jsr) mutation affecting the vertebral development in mice

The jumbled spine and ribs (Jsr) mouse was derived from a spontaneous mutation. As the phenotype, a shortened trunk and kinky tail are characteristic Jsr traits. In this study, on high resolution mapping it was found that Lunatic fringe (Lfng) mapped at the same position as Jsr. Lfng was identified as the candidate gene for Jsr, but sequence analysis of this gene revealed no substitution in the coding region of cDNA. Therefore, we adopted the strategy of positional cloning for Jsr using a mouse bacterial artificial chromosome (BAC) library. A BAC contig was constructed from three BAC clones showing positive signals of Lfng and 11MMHAP75FRD8.seq near the Jsr locus on chromosome 5. Based on the genetic mapping of both T7 and sp6 ends of a clone of BAC382-O-7 (BAC382), the Jsr gene was considered to exist in BAC382 and to be positioned near the sp6 side.