Bruce M. Cattanach

Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.

A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.

The gene mutated in bare patches and striated mice encodes a novel 3β-hydroxysteroid dehydrogenase

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa ) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Mapping the murine Xce locus with (CA)n repeats

The X Chromosome (Chr) controlling element locus (Xce) in the mouse has been shown to influence the X inactivation process. Xce maps to the central region of the X Chr, which also contains the Xist sequence, itself possibly implicated in the X inactivation process. Three microsatellite markers spanning the Xist locus have been isolated from an Xist containing YAC. All three microsatellite markers showed complete linkage with Xce in recombinants for the central span of the mouse X Chr between Ta and Moblo and strong linkage disequilibrium with Xce in all but one of the inbred mouse strains tested. In the standard Xceb typing strain JU/Ct, the two microsatellites most closely flanking Xist fail to carry the allelic forms expected if Xist and Xce are synonymous. Alternative explanations for this finding are presented in the context of our search for understanding the relation between Xist and Xce.

A candidate model for Angelman syndrome in the mouse

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15ql 1-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated.

Disruption of ferroportin 1 regulation causes dynamic alterations in iron homeostasis and erythropoiesis in polycythaemia mice

Coding region mutations in the principal basolateral iron transporter of the duodenal enterocyte, ferroportin 1 (FPN1), lead to autosomal dominant reticuloendothelial iron overload in humans. We report the positional cloning of a hypermorphic, regulatory mutation in Fpn1 from radiation-induced polycythaemia (Pcm) mice. A 58 bp microdeletion in the Fpn1 promoter region alters transcription start sites and eliminates the iron responsive element (IRE) in the 5′ untranslated region, resulting in increased duodenal and hepatic Fpn1 protein levels during early postnatal development. Pcm mutants, which are iron deficient at birth, exhibited increased Fpn1-mediated iron uptake and reticuloendothelial iron overload as young adult mice. Additionally, Pcm mutants displayed an erythropoietin (Epo)-dependent polycythemia in heterozygotes and a hypochromic, microcytic anemia in homozygotes. Interestingly, both defects in erythropoiesis were transient, correcting by young adulthood. Delayed upregulation of the negative hormonal regulator of iron homeostasis, hepcidin (Hamp), during postnatal development correlates strongly with profound increases in Fpn1 protein levels and polycythemia in Pcm heterozygotes. Thus, our data suggest that a Hamp-mediated regulatory interference alleviates the defects in iron homeostasis and transient alterations in erythropoiesis caused by a regulatory mutation in Fpn1.

Ubiquitous expression and imprinting of Snrpn in the mouse

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.

Canine homolog of the T-box transcription factor T; failure of the protein to bind to its DNA target leads to a short-tail phenotype

Abstract. Domestic dog breeds show a wide variety of morphologies and offer excellent opportunities to study the molecular genetics of phenotypic traits. We are interested in exploring this potential and have begun by investigating the genetic basis of a short-tail trait. Our focus has been on the T gene, which encodes a T-box transcription factor important for normal posterior mesoderm development. Haploinsufficiency of T protein underlies a short-tail phenotype in mice that is inherited in an autosomal dominant fashion. We have cloned the dog homolog of T and mapped the locus to canine Chromosome (Chr) 1q23. Full sequence analysis of the T gene from a number of different dog breeds identified several polymorphisms and a unique missense mutation in a bob-tailed dog and its bob-tailed descendants. This mutation is situated in a highly conserved region of the T-box domain and alters the ability of the T protein to bind to its consensus DNA target. Analysis of offspring from several independent bobtail × bobtail crosses indicates that the homozygous phenotype is embryonic lethal.

Translocation yield from the immature mouse testis and the nature of spermatogonial stem cell heterogeneity

Abstract The response of the immature mouse testis to X-irradiation was found to differ from the adult in three respects. 1. (1) Treatment of 5-day old mice with 300 R or 500 R produced no clear sterile period and even with a 1000 R exposure some animals became fertile at maturity. However, others showed a temporary sterile period or were permanently sterilized. 10-day old mice showed a response which more clearly approximated that of the adult. Comparison with histological observations on rats irradiated when immature suggested that the spermatogonia present at this age are not more radioresistant than those of the adult testis but that surviving cells can procede directly into spermatogenesis rather than first repopulating their numbers as in the adult. 2. (2) Testis weights on reaching adulthood were severely reduced and the degree of effect was found to be dependent upon both radiation dose and age at time of treatment. This was attributed largely to the failure of compensatory repopulation in the immature testis but greater cell killing or lower cell numbers in the immature testis could be contributory factors. 3. (3) Translocation yields from mice irradiated at 5 days of age approximated half that of the adult yield with both 300 R and 500 R treatments. This agrees well with Selbys specific locus mutation data from immature mice. Yields from animals exposed to 1000 R at 3 or 5 days of age were extremely low. Translocation yield from these immature mice thus showed a humped dose-response curve as in the adult. The response from 10-day old mice generally approximated that of the adult, this also agreeing with Selbys findings. It is concluded that the low yields are attributable to the presence of proliferating stem cell populations. As in the adult several days after radiation exposure, the stem cells may have a short cell cycle, be sensitive to radiation killing and yield low levels of genetic damage. A model to account for the heterogeneity in radio-sensitive of spermatogonial stem cell populations both in the adult and immature testis is proposed, this based on Smith and Martins concept of the cell cycle. The heterogeneity is attributed to a variation in cell cycle times within a single stem cell type. Those in a long G 1 (A state) would be the most radioresistant having greater time available for repair of pre-mutational or pre-breakage lesions before the onset of S, those with a shorter A state, leaving the A state, or in other stages of the cell cycle (SG 2 M) would be more radiosensitive and contribute most to the total yield of genetic damage at lower ( 1 and this will account for most of the genetic damage recovered. However, proliferating cells with a much shorter cell cycle must spend a much higher proportion of their time in S and G 2 and the recovery of both translocations and point mutations from these stages should approximate only one-half of that from G 1 due to chromatid segregation. Yields from short cycling cells such as in the immature testis or in the adult following earlier acute (or chronic) radiation exposure will be lower.

Time of initiation and site of action of the mouse chromosome 11 imprinting effects

Previous studies have shown that mice with paternal disomy for chromosome 11 are consistently larger at birth than their normal sibs, whereas mice with the maternal disomy are consistently smaller. An imprinting effect with monoallelic expression of some gene/s affecting growth was indicated. Here we show that the size differences become established prior to birth and are only maintained subsequently, indicating that the gene repression is limited to prenatal development. Fetal analysis was limited to 12.5-17.5 days post coitum. However by extrapolating the data backwards it could be calculated that both the maternal and paternal size effects might commence as early as 7 days post coitum, although possibly slightly later. It may be deduced that initiation of expression of the gene/s responsible may occur at about this time in development. The two disomy growth rates were mirror-images of each other, suggesting that expressed gene dosage is the underlying cause. Differential growth of the placentas of the two disomies was also found, and extrapolation of these data backwards suggested that the placental size differences were initiated later in development than those for the fetuses. The differential placental growth of the maternal and paternal disomies may therefore have developed independently or emerged as a consequence of the differential fetal growth. In either event it would seem that the expression of the responsible gene occurs in the fetus itself to cause the anomalies of growth. The data therefore provide information on the temporal and tissue specificity of the gene/s responsible for the chromosome 11 imprinting effects. Possible candidate genes are discussed.