The murine situs inversus viscerum (iv) gene responsible for visceral asymmetry is linked tightly to the Igh-C cluster on chromosome 12

The iv gene controls left-right determination during murine organogenesis. To map this gene, we analyzed backcross progeny produced by mating (C57BL/6J X MEV/Ty)F1-iv/+heterozygotes to C57BL/6J-iv homozygotes. Hybridization of a murine ecotropic virus probe and several homeotic box gene probes coupled with analysis of dominant visible markers enabled us to exclude the iv locus from much of the mouse genome. Spurred by a recent report that mapped the iv gene to mouse chromosome 12 which was not excluded by our previous work, we used the polymerase chain reaction on our larger cohort to determine that the iv gene is indeed linked tightly to the Igh-C locus on this chromosome: we observed 0/156 recombinants between the iv and Igh-C loci. Combining data from the two studies demonstrates that the murine iv gene is close (1/201 recombinants) to the Igh-C cluster on chromosome 12.     

The development of handed asymmetry in aggregation chimeras of situs inversus mutant and wild-type mouse embryo

Mutant iv/iv mice develop as if they have no sense of left and right, so the development of asymmetry is random: half normal, half as a mirror-image of normal, situs inversus. We have made aggregation chimeras of 8-cell stage iv/iv and +/+ embryos, transferred them into pseudopregnant mice, and examined their phenotype on day 10 of gestation. The contribution of mutant and wild-type cells to tissues of the embryo was estimated by strain-specific isozyme (GPI-1) analysis. We have also performed reciprocal embryo transfers, iv/iv blastocysts into +/+ mice, and vice versa. These transfers show that the development of handed asymmetry is determined by embryonic genotype, and is unaffected by the maternal environment (at least after day 3), or by the procedures of embryo collection, culture and transfer. Our observations on the development of 21 viable chimeric embryos show that neither iv/iv nor +/+ cells are dominant. All embryos (12) with less than 50% contribution of iv/iv cells to the heart developed with normal situs. Of 9 embryos with greater than 50% iv/iv cells, only 2 developed with inverted situs. These findings suggests that there was partial 'rescue' of embryos by some influence of normal over mutant cells. However, we cannot, statistically, exclude an alternative interpretation that cells are behaving autonomously. Interestingly, the embryos that developed with inverted situs were unique in having greater than two thirds contribution of iv/iv cells to both the heart and the visceral yolk-sac.     

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.     

Two populations of node monocilia initiate left-right asymmetry in the mouse

The vertebrate body plan has conserved handed left-right (LR) asymmetry that is manifested in the heart, lungs, and gut. Leftward flow of extracellular fluid at the node (nodal flow) is critical for normal LR axis determination in the mouse. Nodal flow is generated by motile node cell monocilia and requires the axonemal dynein, left-right dynein (lrd). In the absence of lrd, LR determination becomes random. The cation channel polycystin-2 is also required to establish LR asymmetry. We show that lrd localizes to a centrally located subset of node monocilia, while polycystin-2 is found in all node monocilia. Asymmetric calcium signaling appears at the left margin of the node coincident with nodal flow. These observations suggest that LR asymmetry is established by an entirely ciliary mechanism: motile, lrd-containing monocilia generate nodal flow, and nonmotile polycystin-2 containing cilia sense nodal flow initiating an asymmetric calcium signal at the left border of the node.     

The development of asymmetry: the sidedness of drug-induced limb abnormalities is reversed in situs inversus mice

We are studying the development of handedness, in particular the relationships between handed structures with bilateral symmetry, for example the limbs, and those with lateral asymmetry, such as the heart, lungs and gut. Asymmetric (unilateral) developmental limb abnormalities can be induced by chemical treatment of mouse embryos, either in utero by acetazolamide, or in culture by misonidazole. We have examined these effects in mice homozygous for the iv gene. The development of bilateral symmetry in iv/iv mice is normal, but the control of asymmetry appears to be random, that is 50% develop normally (situs solitus), 50% with laterally inverted viscera (situs inversus). We find that the handedness of induced asymmetric limb defects is highly correlated with embryonic visceral situs. Right limb defects are induced in situs solitus embryos, left-sided defects in situs inversus. This suggests that the mechanism of induction of asymmetric defects is not related to any intrinsic difference between the development of left and right limbs, but is connected to visceral asymmetry. In addition, the high correlation of limb defects with situs was observed in culture as well as in utero suggesting that the maternal environment plays no role in the development of asymmetry.     

Generation of robust left-right asymmetry in the mouse embryo requires a self-enhancement and lateral-inhibition system

The bilateral symmetry of the mouse embryo is broken by leftward fluid flow in the node. However, it is unclear how this directional flow is then translated into the robust, left side-specific Nodal gene expression that determines and coordinates left-right situs throughout the embryo. While manipulating Nodal and Lefty gene expression, we have observed phenomena that are indicative of the involvement of a self-enhancement and lateral-inhibition (SELI) system. We constructed a mathematical SELI model that not only simulates, but also predicts, experimental data. As predicted by the model, Nodal expression initiates even on the right side. These results indicate that directional flow represents an initial small difference between the left and right sides of the embryo, but is insufficient to determine embryonic situs. Nodal and Lefty are deployed as a SELI system required to amplify this initial bias and convert it into robust asymmetry.     

cDNA cloning and mapping of mouse pleckstrin (Plek), a gene upregulated in transformation-resistant cells

Changes that occur during tumor promotion, the rate-limiting phase of multistep carcinogenesis, may offer the best targets for prevention of cancer or reversal of early disease. The murine epidermal JB6 promotion-sensitive (P+) and -resistant (P-) cell lines provide a cell culture model for tumor promoter-induced neoplastic transformation ideally suited to the identification of molecular events that mediate or inhibit transformation. A differential display comparison of P+ and P- cell mRNAs yielded seven differentially expressed sequences. One of the sequences preferentially expressed in P- cells identified an approximately 3. 6-kb message that was induced to higher levels in P- cells following exposure to the tumor promoter 12-O-tetradecanoylphorbol acetate than in P+ cells. The message was detected in mRNA from heart, lung, and spleen. cDNA cloning of the P- preferential sequence revealed a high degree of identity to human pleckstrin (PLEK), the major PKC substrate in platelets (Tyers et al., 1988, Nature 333: 470). We report the complete mouse cDNA sequence of pleckstrin and the localization of the gene to chromosome 11, its expression in a nonhematopoetic cell line, and its potential role in blocking neoplastic transformation.     

Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.

Determination of the left-right position (situs) of visceral organs involves lefty, nodal and Pitx2 genes that are specifically expressed on the left side of the embryo. We demonstrate that the expression of these genes is prevented by the addition of a retinoic acid receptor pan-antagonist to cultured headfold stage mouse embryos, whereas addition of excess retinoic acid leads to their symmetrical expression. Interestingly, both treatments lead to randomization of heart looping and to defects in heart anteroposterior patterning. A time course analysis indicates that only the newly formed mesoderm at the headfold-presomite stage is competent for these retinoid effects. We conclude that retinoic acid, the active derivative of vitamin A, is essential for heart situs determination and morphogenesis.     

Genetic mapping of a mouse modifier gene that can prevent ALS onset

Mutations in the cytoplasmic Cu/Zn superoxide dismutase (SOD1) gene on human chromosome 21q22.1 cause 10-20% of familial amyotrophic lateral sclerosis (ALS) cases. The expression of the ALS phenotype in mice carrying the murine G86R SOD1 mutation is highly dependent upon the mouse genetic background. This is similar to the phenotypic variation observed in ALS patients containing identical SOD1 mutations. In the FVB/N background, mice expressing mG86R SOD1 develop an ALS phenotype at approximately 100 days. However, when these mice were bred into a mixed background of C57Bl6/129Sv, the onset of the ALS phenotype was delayed (143 days to >2 years). Using 129 polymorphic autosomal markers in a whole genome scan, we have identified a major genetic modifier locus with a maximum lod score of 5.07 on mouse chromosome 13 between D13mit36 and D13mit76. This 5- to 8-cM interval contains the spinal muscular atrophy (SMA)-associated gene Smn (survival motor neuron) and seven copies of Naip (neuronal apoptosis inhibitory protein), suggesting a potential link between SMA and ALS.     

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy)

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.     

Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli.

the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.     

Chromosomal mapping of the peroneal muscular atrophy (pma) gene in the mouse.

We conducted chromosomal mapping of the pma gene that is a causative gene in the peroneal muscular atrophy mouse, which shows a club foot at birth and unusual gait due to a dropped foot in the adult. Linkage analyses using backcross progeny revealed a significant linkage between the pma gene and three microsatellite markers, D5Mit263 at 73 cM, D5Mit141 and D5Mit97 at 74 cM on Chr 5. The gene order was determined as follows: centromere-D5Mit263-[2.65 cM]-D5Mit141-[2.56 cM]-pma-[5.13 cM]-D5Mit97-telomere.     

Genomic structure and chromosomal mapping of the mouse hic-5 gene that encodes a focal adhesion protein

The hic-5 gene encodes a focal adhesion protein that has striking similarity to paxillin. Genomic clones of the mouse hic-5 gene were isolated, and included 10 exons that covered the whole mouse mRNA sequence. Comparison of the sequence with those in the expressed sequence tag database suggested that the hic-5 gene contained an extra exon (named exon 1') located about 1kb upstream of exon 1, and mouse cells seemed to express two alternatively spliced forms of mRNA. All the exon-intron boundaries followed the GT/AG rule. Physical mapping and fluorescent in situ hybridization analysis indicated that the hic-5 gene is located on mouse chromosome 7, 60. 0cM from the centromere.