Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci.

Mutations in either the dominant white-spotting (W) or Steel (Sl) loci of the mouse lead to coat color, primordial germ cell and hematopoietic defects. Consistent with the cell autonomous and microenvironmental nature of W and Sl mutations, respectively, it has recently been shown that W encodes the c-kit receptor tyrosine kinase while Sl encodes a ligand for this receptor. Previous in situ hybridization analysis has shown that both c-kit and steel are expressed in the embryo in anatomical sites known to be affected by W and Sl mutations and in various tissues in which no corresponding phenotype has been described. To investigate the possible involvement of the Kit transduction pathway in developmental processes, we compared the patterns of expression of c-kit and steel in wild-type embryos and in embryos homozygous for severe (lethal) and mild (viable) alleles at the W and Sl loci. In addition, we analyzed the patterns of expression of both genes in adult wild-type and mutant gonads and brain. Both c-kit and steel are contiguously expressed in a wide variety of anatomical locations in both the developing embryo and in the adult. In adult gonads, steel is expressed in the follicular cells of the ovary and in Sertoli cells of the testis, the layers that immediately surround the c-kit expressing germ cells. In adult brain, the complementary patterns are particularly striking in the olfactory bulb, cerebral cortex, hippocampus region and cerebellum. steel expression in brain is probably restricted to neurons in certain areas, while c-kit is expressed in neurons and in some glial cells. Severe mutations in the W or Sl loci result in dramatic reduction or absence of c-kit positive cells in lineages known to be affected by these mutations. In contrast, these mutations do not affect the number or histological organization of c-kit positive cells in the embryonic peripheral or central nervous systems, nor is the number or organization of c-kit positive cells detectably altered in Wv/Wv or Sld/Sld adult brain. Taken together, these results suggest that the Kit signaling pathway is not obligatory for the viability and/or migration of most c-kit expressing cells either because of functional redundancy with another signaling pathway or because the Kit pathway is involved in post-developmental processes of mature cells.     

Epigenetic heterogeneity at imprinted loci in normal populations

Genomic imprinting is the phenomenon by which the two alleles of certain genes are differentially expressed according to their parental origin. Extensive analysis of allelic expression at multiple imprinted loci in a normal population has not performed so far. In the present study, we examined the allelic expression pattern of three imprinted genes in a panel of 262 Japanese normal individuals. We observed differences in the extent of maintenance of allele-specific expression of the three genes. The allelic expression of small nuclear ribonucleoprotein N (SNRPN) was stringently regulated while that of multimembrane-spanning polyspecific transporter-like gene 1 (IMPT1) showed a large degree of variation. Significant biallelic expression of insulin-like growth factor II (IGF2) was observed in about 10% of normal individuals. Our findings add to the accumulating evidence for variable allelic expression at multiple loci in a normal human population. This epigenetic heterogeneity can be a stable trait and potentially influence individual phenotypes.     

Adhesion of mouse mast cells to fibroblasts: adverse effects of steel (Sl) mutation

Mouse bone marrow-derived cultured mast cells proliferate on +/+ mouse embryo-derived 3T3 fibroblasts, but not on Sl/Sld mouse embryo-derived 3T3 fibroblasts, in the absence of IL-3 and IL-4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888-2891, 1989). To further characterize the mast cell-fibroblast interactions and the effects of Sl mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 10(3) to 5 x 10(5) cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited by an RGD-containing peptide, an anti-LFA-1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from Sl/Sld mouse embryos. Adhesion to +/+ mouse spleen-derived fibroblasts lacking mast cell-supporting activity was comparable to that to Sl/Sld/3T3 cells. The failure of mast cells to adhere to fibroblasts with the Sl mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type Sl gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.     

Close linkage of a transgene insertion site to the steel (Sl) locus on mouse chromosome 10.

The characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome. We have found that the transgene insertion site in the transgenic line TG.EB is tightly linked with the Steel (Sl) locus on mouse chromosome 10. In a backcross between doubly heterozygous transgenic Sl (Tg.EB +/+ Sl) mice and wild-type mice, only one recombinant was found in 135 progeny (recombination percentage = 0.7 +/- 0.7). The recombination frequency of the transgene with marker loci known to flank Sl was consistent with a map position close to Sl. Genomic sequences that are adjacent to the transgene insertion site were cloned and found to be tightly linked with the Sl locus in interspecific crosses using nontransgenic mice. Recombination analysis utilizing the transgene insertion site locus was used to show that a recently identified hematopoietic growth factor is encoded at Sl. The cloned sequences from the transgene insertion site are polymorphic in inbred strains of mice and can be utilized to determine the genotype at Sl during early embryonic development. Further, they may be useful in characterizing the genomic region near Sl that is affected in Sl deletion mutants.     

Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse

Growth hormone (GH) cells were analyzed by means of ultrastructural morphometry in the pars distalis of pituitary glands from male adult and immature normal (C57BL) and homozygous little (lit/lit) mutant mice. Thin sections were exposed to anti-GH serum and processed immunocytochemically with the colloidal-gold technique. In the pars distalis of adult lit/lit mice, the mean volume density of GH cells/total tissue was 24% of the normal value, granules/GH cells was 58% of normal, and granules/total tissue was only 12% of normal. Deficits in all of these parameters likewise occurred in immature glands, though to a lesser extent than in the adults. The results indicate that the GH deficiency in this mutant reflects quantitative deficits in both the secretory granule content of GH cells, as well as the GH cell content of the gland, with the latter being the more severely affected.     

Prostacyclin biosynthesis in activated, stimulated and normal mouse peritoneal cell populations

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of “immunomodulators”. The levels of enhancement can be monitored by following function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH₂ was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI₂) when incubated with PGH₂. Homogenates of PCs from mice injected with the immunomodulators . , levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI₂. Reductions ranged from 73% for . to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage “activation” and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

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.     

Immunohistochemical study of protein 4.1B in the normal and W/W(v) mouse seminiferous epithelium.

Cell-cell adhesion is crucial not only for mechanical adhesion but also for tissue morphogenesis. Protein 4.1B, a member of the protein 4.1 family named from an erythrocyte membrane protein, is a potential organizer of an adherens system. In adult mouse seminiferous tubules, protein 4.1B localized in the basal compartment, especially in the attaching region of spermatogonia and Sertoli cells. Protein 4.1B localization and appearance were not different in each spermatogenic stage. Developmentally, protein 4.1B was not detected at postnatal day 3 (P3), was diffusely localized at P15, and was found in the basal compartment during the third week. By double staining for protein 4.1B and F-actin, their localizations were shown to be different, indicating that protein 4.1B was localized in a region lower than the basal ectoplasmic specialization that formed the Sertoli-Sertoli junction. By electron microscopy, immunoreactive products were seen mainly on the membranes of Sertoli cells. In the W/W(v) mutant mouse, the seminiferous epithelium had few germ cells. Protein 4.1B and beta-catenin were not detected, although the basal ectoplasmic specialization was retained. These results indicate that protein 4.1B may be related to the adhesion between Sertoli cells and germ cells, especially the spermatogonium.     

Abnormal development of genetically normal fetal hematopoietic stem cells in steel mutant mouse fetuses

The role and possible transplantability of the early hematopoietic microenvironment was investigated by transplacental inoculation of fetal liver cells from normal donors into steel mutant early fetuses. Donor hematopoietic stem cells were able to lodge in the livers of recipients and to progress to the bone marrow postnatally. However, self-renewal of stem cells and production of differentiated blood cells was very limited in extent and duration after transplantation into mildly anemic steel as compared with Wv/+ heterozygotes. The microenvironmental defect known to exist (albeit undefined) in steel and not in W mutants thus adversely affects proliferation and differentiation of stem cells from the very inception of hepatic hematopoiesis and is not correctable by introducing normal stromal cells under the conditions of the experiment.     

Prostacyclin biosynthesis in activated, stimulated and normal mouse peritoneal cell populations.

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of "immunomodulators". The levels of enhancement can be monitored by following in vitro function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH2 was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI2) when incubated with PGH2. Homogenates of PCs from mice injected with the immunomodulators C. parvum, levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI2. Reductions ranged from 73% for C. parvum to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage "activation" and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.     

Histological studies of normal and mutant mouse embryo hearts (a glycogen content study)

Mutant tail-short (Ts/+) embryos are developmentally retarded compared with normal +/+ litter mates. The development of the heart of Ts/+ embryos is severely affected if the tail-short gene is transferred to a new genetic (50% A/Gr) background. The aim of the present study was to investigate the glycogen content of the sinus muscle, the cushion and the atrial and ventricular walls of the heart. In normal embryos the sinus muscle is well developed by the 15th day post coitum (d.p.c.) and is crowded with glycogen granules. In Ts/+ mutant embryos, on the other hand, the development of this muscle is retarded and it contains only a little, diffusely distributed glycogen. The atrial and ventricular walls of embryos with a normal heart are well trabeculated and contain a large quantity of glycogen granules, while in mutant embryos they are less well trabeculated and contain only a little glycogen in a diffuse of finely granular form.     

Interactions between imprinting effects in the mouse

Mice with uniparental partial or complete disomies for any one of 11 identified chromosomes show abnormal phenotypes. The abnormalities, or imprinting effects, can be attributable to an incorrect dosage of maternal or paternal copies of imprinted gene(s) located within the regions involved. Here we show that combinations of partial disomies may result in interactions between imprinting effects that seemingly independently affect fetal and/or placental growth in different ways or modify neonatal and postnatal imprinting effects. Candidate genes within the regions have been identified. The findings are generally in accord with the "conflict hypothesis" for the evolution of genomic imprinting but do not clearly demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that represses or limits embryonic/fetal growth to the advantage of the mother--by any developmental means--will have been subject to evolutionary selection for paternal allele repression. Likewise, any gene that favors embryonic/fetal development at consequent cost to the mother--by any developmental means--will have faced selection for maternal allele repression. The classical Igf2-Igf2r axis may therefore be unique. The findings involve reinterpretation of older imprinting data and consequently revision of the mouse imprinting map.     

Diversity and genetic structure in populations of Pseudotsuga menziesii (Pinaceae) at chloroplast microsatellite loci.

Genetic variation was compared between uniparentally-inherited (chloroplast simple sequence repeats, cpSSRs) vs. biparentally-inherited (isozyme and random amplified polymorphic DNA, RAPD) genetic markers in Douglas-fir (Pseudotsuga mensiezii) from British Columbia. Three-hundred twenty-three individuals from 11 populations were assayed. In Douglas-fir, the cpSSR primer sites were well-conserved relative to Pinus thunbergii (11 of 17 loci clearly amplified), but only 3 loci were appreciably polymorphic. At these cpSSR loci, we found an unexpectedly low level of polymorphism within populations, and no genetic differentiation among populations. By contrast, the nuclear markers showed variation typical of conifers, with significant among-population differentiation. This difference is likely the outcome of both historical factors and high pollen dispersal.