C.V. Beechey

Peg1/Mest locates distal to the currently defined imprinting region on mouse proximal chromosome 6 and identifies a new imprinting region affecting growth

Mice with maternal duplication for proximal chromosome 6 (Chr 6) die in utero before 11.5 dpc, an effect that can be attributed to genomic imprinting. Previous studies have defined the region of Chr 6 responsible as lying proximal to the T6Ad translocation breakpoint in G-band 6B3. Evidence presented here with a new Chr 6 translocation T77H has substantially reduced the size of the imprinting region, locating it between G-band 6A3.2 and the centromere. The paternally expressed imprinted gene Mest had been mapped within the original imprinting region and was therefore a candidate for the early embryonic lethality. FISH has shown that Mest locates distal to T77H and therefore outside the redefined imprinting region. This evidence confirms that Mest is not a candidate for the early embryonic lethality found with two maternal copies of proximal Chr 6. Furthermore mice with maternal duplication for Ch 6 distal to T77H (MatDp.dist6) were found to be growth retarded at birth, the weight reduction remaining similar until adulthood. It can be concluded that the growth retardation is established in utero and is maintained at a similar level from birth to adulthood. Therefore Mest locates in a new imprinting region, distal to G-band 6A3.2 which affects growth. A targeted mutation of Mest has been reported that exhibits growth retardation, reduced postnatal survival and abnormal maternal behaviour. Here the phenotype of MatDp.dist6 mice is compared to that of Mest-deficient mutant mice. Unlike the latter, MatDp.dist6 mice have good survival rates and females have normal maternal behaviour. Possible reasons for these differences are discussed.

A male-sterile insertion in the mouse

Is(7;1)40H was found in the daughter of a male mouse given spermatozoal X-irradiation. It is a non-inverted insertion of about half of chromosome 7 into chromosome 1, generating a long somatic marker chromosome. Breakpoints are in bands 1B, 7B1, and 7F1; linkage tests show that these breakpoints are about midway between fz and In on the 1, and 0.2 units distal to ru-2 and 12 units proximal to fr on the 7. Female carriers had litters of about one-third normal size and showed some decline in length of reproductive life. Males were sterile, with testis weights only 30% of normal and with abrupt cessation of spermatogenesis in pachytene at stage IV of the seminiferous epithelial cycle. Positive sex-vesicle contact with the insertional configuration was found in only 40% of pachytene spermatocytes, which suggested that other factors may be involved in the spermatogonial breakdown. In oocytes at metaphase I 76% of insertion configurations were multivalent, because of one or more chiasmata in the inserted segment, as were 79% of synaptonemal complex configurations in male pachytenes. Karyotyping at 12.5 to 14.5 days of gestation showed that all embryos with duplications of the inserted segment were exencephalic, and the only example of a corresponding deficiency was retarded. Analysis of the consequences of heterozygosity for the insertion shows that the insertion length should be correlated with the frequency of unbalanced offspring and thus with the amount of F1 lethality. The genetic length of 36 cM estimated in this way from data on liveborn offspring is in reasonable agreement with estimates from cytological measurements and meiotic configurations but rather higher than that from linkage tests.

A reciprocal translocation induced in an oocyte and affecting fertility in male mice.

The reciprocal translocation T(5;12)31H, induced by irradiation of oocytes, causes sterility in most heterozygous male carriers, a tenfold reduction in mean sperm count, and a high frequency of abnormal sperm. Chromosome breakpoints are in bands 5B and 12F1, leading to a long (125) and a short (512) marker chromosome. Ninety five percent of germ cells showed chain quadrivalents or trivalents at meiotic metaphase I (MI). The order of loci on chromosome 5 is centromere—T31H—Rw—go with a recombination frequency of 31 + 7% between T31H and Rw. No linkage was found between T31H and linkage group XVI genes previously assigned to chromosome 12; it was concluded that they are located elsewhere. Viable tertiary monosomic and trisomic young (lacking the 512 region or with it in excess) were generated with post-natal frequencies of 4.5% and 14.6%, respectively, from outcrosses of heterozygous females. A deficiency of monosomics was also found in 11½ to 14½ day foetuses, when abnormal phenotypes or severe retardation produced by other unbalanced karyotypes were found. There was also a marked deficiency of monosomic offspring from outcrossed monosomic mothers, only 16.1% being found, whereas trisomic mothers gave 52.8% trisomic young. Surviving monosomics of both sexes were smaller than trisomics at birth and tended to have unusual skeletal fusions. Both types of aneuploid male were sterile, but tertiary monosomics were less severely affected, as judged by testis mass, sperm counts, and the appearance of testis sections. Both tertiary monosomic and trisomic females have smaller litters than normal females. Possible reasons for these findings are discussed, as well as recent observations throwing light on mechanisms of sterility in this type of translocation.

A reassessment of imprinting regions and phenotypes on mouse chromosome 6: Nap1l5 locates within the currently defined sub-proximal imprinting region

Previous studies (Beechey, 2000) have shown that mouse proximal chromosome (Chr) 6 has two imprinting regions. An early embryonic lethality is associated with two maternal copies of the more proximal imprinting region, while mice with two maternal copies of the sub-proximal imprinting region are growth retarded at birth, the weight reduction remaining similar to adulthood. No detectable postnatal imprinting phenotype was seen in these earlier studies with two paternal copies of either region. The sub-proximal imprinting region locates distal to the T77H reciprocal translocation breakpoint in G-band 6A3.2 and results reported here show that it does not extend beyond the breakpoint of the more distal T6Ad translocation in 6C2. It has been confirmed that the postnatal growth retardation observed with two maternal copies of the sub-proximal region is established in utero, although placental size was normal. A new finding is that 16.5–18.5-dpc embryos, with two paternal copies of the sub-proximal imprinting region, were larger than their normal sibs, although placental size was normal. As no postnatal growth differences have been observed in these mice, the fetal overgrowth must normalize by birth. The imprinted genes Peg1/Mest, Copg2, Copg2as and Mit1/Lb9 map to the sub-proximal imprinting region and are thus candidates for the observed imprinting phenotypes. Another candidate is the recently reported imprinted gene Nap1l5. Expression studies of Nap1l5 in mice with two maternal or two paternal copies of different regions of Chr 6 have demonstrated that the gene locates within the sub-proximal imprinting region. FISH has mapped Nap1l5 to G-band 6C1, within the sub-proximal imprinting region but several G-bands distal to the Peg1/Mest cluster. This location, and the 30-Mb separation of these loci on the sequence map, makes it probable that Nap1l5 defines a new imprinting domain within the currently defined sub-proximal imprinting region.

FISH mapping of the mouse Ret oncogene to the junction of G-bands E3/F1 on Chromosome 6 indicates a need for reassessment of the physical and consensus maps

The RET proto-oncogene encodes a cell-surface glycoprotein with cytoplasmic tyrosine kinase activity (Takahashi et al., 1989). Mutations in RET have been associated with four human disease syndromes: Hirschprung’s disease, familial medullary thyroid carcinoma, and two forms of multiple endocrine neoplasia, type 2A and 2B (van Heyningen, 1994). The mouse Ret gene has been cloned (Iwamoto et al., 1993) and is expressed in normal mouse spinal cord and in Fas and Fas1 lymphadenopathy (Takahashi et al., 1988). Mice homozygous for the targeted knockout of Ret develop to term, but die soon after birth with renal agenesis/dysgenesis (Schuchardt et al., 1995). They also lack enteric neurons throughout the digestive tract. Thus the Ret gene product may be a receptor for a factor involved in development of a number of neural cell lineages, and in kidney organogenesis (Pachnis et al., 1993). Ret was assigned by interspecific backcross analysis to 53.2 cM on Chr 6, 1.1 cM distal to Raf1 (Elliott and Moore 1998, Li et al., 1995). The mapping of loci in this region on the physical map appears contradictory in that Raf1 has been positioned in G-band 6C3 by in situ hybridisation (Tailor and Martin-DeLeon, 1989), but Pparg and Sdf1, only 0.5 cM distal to Raf1 on the consensus map, have been assigned by FISH to bands E3/ F1 and F1 respectively (Zhu et al., 1995, Nomura et al., 1996). The close linkage of these loci is incompatible with these alleged G-band locations since, according to the Mouse Chromosome Atlas (Lyon and Kirby, 1996), G-bands 6C3 and F1 could be up to 10 cM apart. Del(6)Ums26H (hereafter referred to as Del(6)Ums) is a deletion mutant found by screening progeny of spermatogonially X-irradiated male mice, carriers having umbrous coats, low viability and small body size (Cattanach et al., 1993 ). The deleted chromosome segment of Chr 6 was initially thought to include all of G-band C3. If the FISH assignment of Raf1 to C3 is correct then the Ret locus may be located within the deleted region, or at either of its two breakpoints. If this is the case, mice carrying the Del(6)Ums deletion could provide potential haplo-insufficient models for the human disease syndromes associated with mutations at the RET locus. Therefore, to determine the unequivocal location of the mouse homologue within or outside the deletion Del(6)Ums, and to resolve the existing ambiguity caused by the in situ and FISH mapping of loci in this region, we have positioned Ret directly by FISH using mitotic cells from Del(6)Ums heterozygous mice, carrying both a deleted and normal Chr 6.