Upstream Enhancer Elements of Shh Regulate Oral and Dental Patterning.

Sonic hedgehog ( Shh) is important in pattern formation during development. Shh transcription is modulated by a long-range regulatory mechanism containing a number of enhancers, which are spread over nearly 850 kb in the mouse genome. Shh enhancers in the nervous system have been found between intron and 430 kb upstream of Shh. Enhancers in the oral cavity, pharynx, lung, gut, and limbs have been discovered between 610 kb and 850 kb upstream of Shh. However, the intergenic region ranging from 430 to 610 kb upstream of Shh remains to be elucidated. In the present study, we found a novel long-range enhancer located 558 kb upstream of Shh. The enhancer showed in vivo activity in oral cavity and whiskers. A targeted deletion from the novel enhancer to mammal reptile conserved sequence 1 (MRCS1), which is a known enhancer of Shh in oral cavity, resulted in supernumerary molar formation, confirming the essential role of this intergenic region for Shh transcription in teeth. Furthermore, we clarified the binding of Lef1/Tcfs to the new enhancer and MRCS1, suggesting that Wnt/ß-catenin signaling regulates Shh signaling in the oral cavity via these enhancers.

Cutting edge: homologous recombination of the MHC class I K region defines new MHC-linked diabetogenic susceptibility gene(s) in nonobese diabetic mice.

To localize the MHC-linked diabetogenic genes in the nonobese diabetic (NOD) mouse, a recombinational hotspot from the B10.A(R209) mouse was introduced to the region between the MHC class I K and class II A of the NOD mouse with the recombinational site centromeric to the Lmp2/Tap1 complex by breeding the two strains. Replacement of the NOD region centromeric to the recombinational site with the same region in R209 mice prevented the development of diabetes (from 71 to 3%) and insulitis (from 61 to 15%) in the N7 intra-MHC recombinant NOD mice. Similarly, the replacement of the NOD class II A, E and class I D region with the same region in R209 mice prevented the diseases (diabetes, from 71 to 0%; insulitis, from 61 to 3%). In addition to the MHC class II genes, there are at least two MHC-linked diabetogenic genes in the region centromeric to Lmp2.

The genomic organization of type I keratin genes in mice.

We isolated two new keratin cDNAs by screening a cDNA library constructed from poly(A)+ RNA of the dorsal and abdominal skin of C57BL/10J mice with a probe of human KRT14. Due to its high sequence homology to human keratin 17 cDNA, one full-length cDNA is most likely to be mouse keratin 17 (Krt1-17) cDNA. The other is the putative full-length cDNA of a novel type I keratin gene, designated Krt1-c29. These two keratin genes were mapped to the distal portion of Chromosome 11, where the mouse keratin gene complex-1 (Krt1) is localized. To elucidate the genomic organization of Krt1 in mice, we carried out genetic and physical analyses of Krt1. A large-scale linkage analysis using intersubspecific backcrosses suggested that there are two major clusters in Krt1, one containing Krt1-c29, Krt1-10, and Krt1-12 and the other containing Krt1-14, -15, -17, and -19. Truncation experiments with two yeast artificial chromosome clones containing the two clusters above have revealed that the gene order of Krt1 is centromere-Krt1-c29-Krt1-10-Krt1-12-Krt1-13-K rt1-15-Krt1-19-Krt1-14-K rt1-17-telomere. Finally, we analyzed sequence divergence between the genes belonging to the Krt1 complex. The results clearly indicated that genes are classified into two major groups with respect to phylogenetic relationship. Each group consists of the respective gene cluster demonstrated by genetic and physical analyses in this study, suggesting that the physical organization of the Krt1 complex reflects the evolutionary process of gene duplication of this complex.

rim2 (recombination-induced mutation 2) is a new allele of pearl and a mouse model of human Hermansky-Pudlak syndrome (HPS): genetic and physical mapping.

A mouse mutation, rim2, is one of a series of spontaneous mutations that arose from the intra-MHC recombinants between Japanese wild mouse-derived wm7 and laboratory MHC haplotypes. This mutation is single recessive and characterized by diluted coat color and hypo-pigmentation of the eyes. We mapped the rim2 gene close to an old coat color mutation, pearl (pe), on Chromosome (Chr) 13 by the high-density linkage analysis. The pearl mutant is known to have abnormalities similar to Hermansky-Pudlak syndrome (HPS), a human hemorrhagic disorder, characterized by albinism and storage pool deficiency (SPD) of dense granules in platelets. A mating cross of C57BL10/Slc-rim2/rim2 and C57BL/6J-pe/pe showed no complementation of coat color. Additionally, characteristics similar to SPD were also observed in rim2. Thus, rim2 appeared to be a new allele of the pe locus and serves as a mouse model for human HPS. We have made a YAC contig covering the rim2/pe locus toward positional cloning of the causative gene.

A new inbred strain JF1 established from Japanese fancy mouse carrying the classic piebald allele.

A new inbred strain JF1 (Japanese Fancy Mouse 1) was established from a strain of fancy mouse. Morphological and genetical analysis indicated that the mouse originated from the Japanese wild mouse, Mus musculus molossinus. JF1 has characteristic coat color, black spots on the white coat, with black eyes. The mutation appeared to be linked to an old mutation piebald (s). Characterization of the causative gene for piebald, endothelin receptor type B (ednrb), demonstrated that the allele in JF1 is same as that of classic piebald allele, suggesting an identical origin of these two mutants. Possibly, classic piebald mutation was introduced from the Japanese tame mouse, which was already reported at the end of the 1700s. We showed that JF1 is a useful strain for mapping of mutant genes on laboratory strains owing to a high level of polymorphisms in microsatellite markers between JF1 and laboratory strains. The clarified genotypes of JF1 for coat color are "aa BB CC DD ss".

A new mutation Rim3 resembling Re(den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromosome 11.

A new mouse mutation, recombination-induced mutation 3 (Rim3), arose spontaneously in our mouse facility. This mutation exhibits corneal opacity as well as abnormal skin and hair development resembling rex denuded (Re(den)) and bareskin (Bsk). Large-scale linkage analysis with two kinds of intersubspecific backcrosses revealed that Rim3 is mapped to the distal portion of Chromosome (Chr) 11, in which Re(den) and Bsk have been located, and is very close to the retinoic acid receptor, alpha (Rara). The genes, keratin gene complex-1, acidic, gene 10, 12 (Krt1-10, 12), granulin (Grn), junctional plakoglobin (Jup) and Rara, all of which regulate growth and differentiation of epithelial cells, are genetically excluded as candidate genes for Rim3, but are clustered in the short segment on mouse Chr 11.

Multigenic control of the localization of the zone of polarizing activity in limb morphogenesis in the mouse.

We report here that in three preaxial polydactylous mutants in the mouse, namely, 1st, 1x, and Xp1, ectopic expression of the Shh and Fgf-4 genes can be detected at the anterior margin of limb buds. These and three other mutants, namely, Rim4, Hx, and Xt1, which we described in our previous study, all appeared to form a duplicated zone of polarizing activity (ZPA) at the anterior margin of the limb bud. We studied the spatial and temporal pattern of expression of the Gli3 gene, which is affected in a loss-of-function type of mutation, Xt1. The expression domain of Gli3 appeared to be complementary to the ZPA region and the gene was expressed prior to Shh. The results support the hypothesis that GLI3 functions in the anterior portion of limb mesoderm to suppress the expression of Shh. In Drosophila, the gene ci, the fly homologue of Gli, functions to repress hh, suggesting that the negative regulation of the expression of hedgehog by genes belonging to the GLI-kruppel family has been conserved from flies to mice. Finally, we found that the polydactylous phenotype of the mutants Rim4, Xt, 1st, and 1x could be abrogated by the crossing with an inbred strain derived from wild mouse, MSM, whereas the phenotype of Xp1 could not. These results indicate the presence of a modifier gene(s) that can influence the mutant phenotype and also that the mutations could be classified into two categories with regard to the mode of interaction with the modifier gene(s). Thus, this study revealed a multigenic control in the establishment of the anteroposterior axis in mouse limb development.

Molecular analysis of a recombinational hotspot adjacent to Lmp2 gene in the mouse MHC: fine location and chromatin structure.

Meiotic recombinations in the proximal region of the mouse major histocompatibility complex (MHC) are clustered within certain segments of chromosome, known as hotspots. In this study, we found that one of such hotspots, previously mapped between the Pb and Ob genes, is located very close to the 3' end of the Lmp2 gene, which encodes a subunit of a proteolytic proteasome. To analyze the molecular basis of the site specificity of hotspots, we examined the structure of the chromatin around this Lmp2 hotspot and another one located in the MHC class II Eb gene, by monitoring DNase I-hypersensitive sites (DHSSs) of the chromatin. DHSSs were detected at the both hotspots in the somatic cells. In the meiotic cells, DHSS was detected within the Eb hotspot, as previously reported, but not in the Lmp2 hotspot. Thus, open structure of chromatin during meiosis, as monitored by hypersensitivity to DNase I, is not a general feature of mouse recombinational hotspots, contrasting the case of the lower eukaryote, S. cerevisiae, in which hotspots are always associated with DHSSs.

A duplicated zone of polarizing activity in polydactylous mouse mutants.

The positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at the posterior margin. Recently, it was established that the Sonic hedgehog (Shh) mediates ZPA activity. Here we report that a new mouse mutant, Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes (Hx) and Extra toes (Xt), exhibit mirror-image duplications of the skeletal pattern of the digits. In situ hybridization of the embryos of these mutants revealed ectopic expression of Shh and fibroblast growth factor-4 (Fgf-4) genes at the anterior margin of limb buds. The new mutation, Rim4, was mapped to chromosome 6 with linkage to HoxAbut segregated from HoxA. No linkage to other known polydactylous mutations was detected. In this mutant, ectopic expression of the Hoxd-11 gene, thought to be downstream of ZPA, was also observed at the anterior margin of the limb buds. All results indicate the presence of an additional ZPA at the anterior margin of limb buds in these mutants. Thus, it appears that multiple endogenous genes regulate the spatial localization of the ZPA in the developing mouse limb bud.

Allele-dependent recombination frequency: homology requirement in meiotic recombination at the hot spot in the mouse major histocompatibility complex.

Meiotic recombination break joints in the mouse major histocompatibility complex (MHC) are clustered within short segments known as hot spots. We systematically investigated the requirement for sequence homology between two chromosomes for recombination activity at the hot spot next to the Lmp2 gene. The results indicated that a high rate of recombination required a high degree of similarity of overall genome structure at the hot spot. In particular, the same copy number of repetitive sequences within the hot spot was essential for a high frequency of recombination, suggesting that recombination in mouse meiosis is more sensitive to heterozygous deletion or insertion of DNA than to mismatches of single-base substitutions.

Hotspots of homologous recombination in mouse meiosis.

The molecular mapping of recombinational breakpoints in the proximal region of the mouse MHC has revealed four hotspots at which breakpoints are clustered. A direct comparison of the nucleotide sequences of two independent hotspots revealed common molecular elements: a consensus sequence of the middle-repetitive MT-family, a repeat of tetramer sequences and a sequence homologous to a solitary LTR of mouse retroviruses. Extremely high frequency of recombination is observed at these hotspots when particular MHC haplotypes are used in genetic crosses. Wild mouse-derived wm7 haplotype instigates recombination at the hotspot located at the 3'-end of the Lmp-2 gene only during female meiosis. Fine genetic analysis demonstrated that the wm7 haplotype carries a genetic factor to instigate recombination and another factor to suppress recombination specifically during male meiosis. In addition, there is no dose effect of the hotspot on frequency of recombination. Finally, we described an attempt to establish an efficient in vitro assay system for monitoring recombination using plasmid DNAs that contain the Lmp-2 hotspot and nuclear extracts prepared from mouse testis.

No dosage effect of recombinational hotspots in the mouse major histocompatibility complex.

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment.

Recombination in the class III region of the mouse major histocompatibility complex.

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped recombinational breakpoints of 79 Ab:H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order of Eb-61.1-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13+ ++-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were confined within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotspot within the Int3:Tnx interval.

Hotspots of meiotic recombination in the mouse major histocompatibility complex.

Meiotic recombination is not random in the proximal region of the mouse major histocompatibility complex (MHC). It is clustered at four restricted positions, so-called hotspots. Some of the MHC haplotypes derived from Asian wild mice enhance recombination at the hotspots in genetic crosses with standard MHC haplotypes of laboratory mouse strains. In particular, the wm7 haplotype derived from Japanese wild mouse indicated an approximately 2% recombination frequency within a 1.2 kb fragment of DNA in the interval between the Pb and Ob genes. Interestingly, this enhancement of recombination was observed only in female meiosis but not in male meiosis. Mating experiments demonstrated that the wm7 haplotype carries a genetic factor in the region proximal to the hotspot, which instigates recombination. In addition, the wm7 haplotype has a genetic factor located in the region distal to the hotspot, which suppresses recombination. From the molecular characterization of the two hotspots located in the Eb gene and the Pb-Ob interval, it appeared that there are several common molecular elements, the consensus of the middle repetitive MT-family, TCTG or CCTG tetramer repeats, and the solitary long terminal repeat (LTR) of mouse retrovirus.

Genetic control of sex-dependent meiotic recombination in the major histocompatibility complex of the mouse.

Meiotic recombination within the proximal region of the major histocompatibility complex (MHC) of the mouse is not random but occurs in clusters at certain restricted sites, so-called recombinational hotspots. The wm7 haplotype of the MHC, derived from the wild mouse, enhances recombination specifically during female meiosis within a fragment of 1.3 kb of DNA located between the A beta 3 and A beta 2 genes in genetic crosses with laboratory haplotypes. Previous studies revealed no significant strain differences in nucleotide sequences around the hotspot, irrespective of the ability of the strain to enhance the recombination. It appeared that a distant genetic element might, therefore, control the rate of recombination. In the present study, original recombinants whose breakpoints were defined by direct sequencing of PCR-amplified DNAs were tested for the rate of secondary recombination in the crosses with laboratory strains in order to determine the location of such a genetic element. The results clearly demonstrated that the chromosomal segment proximal to the hotspot is essential for enhancement of recombination. Moreover, the male recombination is suppressed by a segment distal to the hotspot.

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex.

The wm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouse Mus musculus molossinus, enhances recombination specific to female meiosis in the K/A beta interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of the wm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between the A beta 3 and A beta 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in the Mus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from the wm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A beta 3/A beta 2 hotspot with a previously characterized hotspot in the E beta gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.

New evidence for trans-species evolution of the H-2 class I polymorphism.

A serological survey using alloantisera specific for the H-2 class I antigens in Japanese wild mice. Mus musculus molossinus, revealed a high frequency of the H-2Kf antigen. This antigen has also been found in European wild mice, M. m. domesticus and M. m. musculus. In this survey, the H-2Kf antigen was characterized through the use of ten newly isolated monoclonal antibodies raised against cells of a Japanese wild mouse, and by Southern blot analysis using an H-2K locus-specific probe which hybridizes with the 3' end of the gene. The serologically identified H-2Kf antigens revealed several minor variations in reactivities to the monoclonal antibodies. However, all the antigens examined could be clearly separated into two types with respect to the restriction fragment length polymorphism (RFLP) pattern. The first type, found together with a single, characteristic RFLP pattern, was always associated with the presence of reactivity to one particular monoclonal antibody MS54. The second type, found to represent different RFLP patterns, is associated with the absence of reactivity to MS54. This concordance between the presence of an antigenic determinant and a particular RFLP was observed not only within Mus musculus subspecies but also in a different species: M. spretus, carrying the same antigenic determinant, gave an identical RFLP to that of the other MS54-positive Mus musculus subspecies. The data suggest that the antigenic determinant specific for MS54 is an ancient polymorphic structure which has survived the long period of diversification of Mus species (approximately 2-3 million years) without alteration, and is associated with a stable DNA structure at the 3' end of the H-2K gene.

Steroid 21-hydroxylase deficiency in mice.

The enzyme steroid 21-hydroxylase (21-OHase) plays a key role in adrenal steroidogenesis. Defects in this enzyme are responsible for one of the most common inborn errors of metabolism in humans. Duplicated genes for the enzyme are located in the class III region of the major histocompatibility complex (MHC), HLA. In the mouse, the genes encoding 21-OHase have been mapped to the homologous region of the H-2 complex. We previously described an H-2 recombinant haplotype aw18, in which the gene for the complement component C4 and one of the two genes for 21-OHase in the H-2 class III region have been deleted. We now report that newborn aw18 homozygous mice are deficient in 21-OHase activity, and that homozygosity for the aw18 haplotype directly causes death at the early postnatal stage. Morphological changes in the adrenal glands of newborn aw18 homozygotes are also observed. The aw18 recombinant haplotype is expected to serve as a useful and, thus far, unique experimental system to study adrenal steroidogenesis in vivo and as an animal model for the inherited human disease of congenital adrenal hyperplasia.

Lethal deletion of the complement component C4 and steroid 21-hydroxylase genes in the mouse H-2 class III region, caused by meiotic recombination.

A recombinant H-2 haplotype, designated aw18, was produced that underwent meiotic recombination in the E alpha (I-E alpha chain)--Slp (sex-limited protein) interval of the H-2 class III region between B10.A (H-2a) and wild-derived B10.MOL-SGR (H-2wm7) strains. It appeared that the H-2aw18 haplotype has a single, recessive, lethal mutation, since homozygotes for H-2aw18 were not detected in progeny generated from the intercross of mice that were heterozygous for this H-2 haplotype. Nine newly established recombinant H-2 haplotypes, which arose from the heterozygous mice that resulted from a cross between common inbred H-2 haplotypes and the aw18 haplotype, allowed us to map the lethal gene to the class III region of the H-2 complex. Southern blot analysis indicated that the aw18 haplotype has a deletion of the C4 gene and a deletion of one of the steroid 21-hydroxylase genes. This result was confirmed by an immunodiffusion test that demonstrated the absence of production of the C4 protein in mice of haplotype H-2aw18. All data that were obtained supported the hypothesis that the meiotic, presumably unequal, recombination between homologous chromosomes of the H-2a and H-2wm7 haplotypes caused the deletion of the C4 and the 21-hydroxylase genes.