A search for radiosensitive mouse mutants by use of the micronucleus technique

In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel (Slcon), scabby (scb), viable dominant spotting (Wv), quaking (qk), fidget (fi) and postaxial hemimelia (px), heterozygous lurcher (Lc), hemizygous gyro (Gy), the compounds Slcon/grizzle-belly (SlgbH) and Wv/rump-white (Rw) and MS/Ae mice, were regarded as radiosensitive, with Slcon/Slcon the highest in rank order. Homozygous wabbler-lethal (wl) and wasted (wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.     

Haemopoietic stem cell proliferation in the bone marrow of S1/S1d mice

In marrow from Sl/Sld mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sld marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sld marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sld CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment. Abnormalities in Sl/Sld CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sld donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sld CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sld or +/+ marrow. Sl/Sld mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Sl/Sld mice have an increased number of gap junctions in their bone marrow stromal cells

To study the defect of the hematopoietic inductive microenvironment (HIM) in Sl/Sld mice, femoral bone marrow tissue of 10 of each mutant, (Sl/Sld and W/Wv) their normal littermates (Sl+/Sl+ and W+/W+), and 20 normal C57BL mice were examined by electron microscopy using morphometric and statistical methods. Gap junctions were observed in all strains of mice, in the following stromal cell types: 1) reticular cells, 2) between reticular cells and periarterial adventitial cells, and 3) between periarterial adventitial cells. The frequency of gap junctions in bone marrow stromal cells of Sl/Sld mice (mean = 2.2/9.4 X 10(-3) mm2) was significantly higher than in control mice. It is suggested that there is a relationship between the increased numbers of gap junctions in bone marrow stromal cells of Sl/Sld mice and the defect in HIM function in these genetically anemic animals.     

Regulatory mechanisms of mast cell differentiation

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.     

Infertility due to growth arrest of ovarian follicles in Sl/Slt mice

Sl, Sld, and Slt are mutant alleles at the steel locus. All Sl/Sld and most Sl/Slt female mice are infertile, but the cause of the infertility is different. Germ cells are absent in Sl/Sld ovaries but present in Sl/Slt ovaries. The infertility of Sl/Slt female mice was attributed to the growth arrest of ovarian follicles, and the mechanism was analyzed by producing aggregation chimeras between Sl/Slt and +/+ embryos. Sl/Slt oocytes were ovulated and fertilized in Sl/Slt----+/+ chimeras. We investigated the origin of granulosa cells in the growing follicles and that of granulosa-derived luteal cells in the chimeras by using the electrophoretic pattern of phosphoglycerate kinase-1 and the histochemical activity of beta-glucuronidase as markers. Granulosa cells of Sl/Slt genotype developed and constituted pregnant corpora lutea in Sl/Slt----+/+ chimeras. Therefore, the growth arrest of Sl/Slt ovarian follicles may not be due to an intrinsic defect in granulosa cells but may instead be due to an intrinsic defect in ovarian stromal cells. This suggests that normal stromal cells are essential for the development of ovarian follicles.     

Hemopoietic precursor cell defects in nonanemic but stem cell-deficient W44/W44 mice

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.     

A role for mast cells and the vasoactive amine serotonin in T cell-dependent immunity to tumors

The involvement of mast cells in anti-tumor resistance was studied by employing 2 strains of mast cell deficient but otherwise immunocompetent mice on a C57BL/6 (H-2b) background (W/Wv and Sl/Sld) and their respective normal +/+ littermate controls. Sensitization of control mice with irradiated semisyngeneic B16 melanoma cells (H-2b) resulted in protection against subsequent challenge with viable B16 cells, in contrast to sensitization of either W/Wv or Sl/Sld mice. The involvement of serotonin in antitumor resistance was studied by employing 2 serotonin active drugs: reserpine, that depletes mast cells of serotonin; and methysergide, a serotonin antagonist. Sensitization of BDF1 mice with irradiated B16 cells and sensitization of DBA/2 mice (H-2d) with irradiated SL2 cells (H-2d) resulted in protection against subsequent challenge with viable B16 cells and viable SL2 cells, respectively. Treatment with either reserpine or methysergide resulted in a decreased protection. Delayed-type hypersensitivity (DTH) footpad responses to allogeneic L5178Y (H-2d) tumor cells in C57BL/6 mice showed a biphasic reaction pattern, similar to that found in DTH responses to simple reactive haptens, such as picryl chloride. Moreover, the early swelling responses were also dependent on T cells and on mast cells. BDF1 mice carrying a semisyngeneic L5178Y tumor on the chest showed an early swelling response after footpad challenge but no late response, possibly indicating that selective down regulation of the late component of DTH was associated with progressive tumor growth in these animals. The biphasic patterns of DTH to both tumor cells and picryl chloride and the T cell and mast cell dependence of both antitumor resistance and DTH to tumor cells suggest that T cell-dependent activation of mast cells to allow entry of mononuclear leukocytes into sites of tumor growth is similar to the mechanism that occurs in DTH.     

A comparative morphometric study on the ultrastructure of adherent cells in long-term bone marrow culture from normal and congenitally anemic mice

The relationship between structure and function of bone marrow stromal tissue in adherent layers of long-term bone marrow cultures (LTBMCs) from normal and congenital anemic mice (C57BL, Sl/Sld, Sl+/Sl+, W/Wv, and W+/W+) was investigated. Many previously reported features were confirmed. However, in LTBMC from all strains of mice examined, isolated cilia with the axonemal structure of a 9 + O pattern with obvious dynein arms were observed in the blanket cells. The frequency of cilia was approximately 2%-5% of total number of profiles of blanket cells examined. Crystalloid inclusions (CI) were observed in cultured macrophages similar to those reported in vivo in all strains of murine LTBMC. The CI could be classified into four types according to their structure in the same way as in vivo (type A to type D), with a predominance of type A in the cultures. Viral particles were also apparent in adherent cells of all strains (except W/Wv and W+/W+), which were compatible with a type C retrovirus. Gap junctions occurred regularly between the adherent cells of LTBMC, particularly between blanket cells and preadipocytes. The most frequent appearance of gap junctions was found in Sl/Sld cultures. The phenomena of normal and abnormal hematopoiesis appear to be accurately reproduced in culture, thus retaining the same relationship between function and structure as occurs in vivo. The surface of isolated cilia of blanket cells, CI of macrophages, viral particles among adherent cells, and gap junctions between blanket cells and preadipocytes is discussed.     

Characterization of spleen colonies derived from mice with mutations at the W locus.

Mice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU-S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony-derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI-1). Donor GPI-1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post-transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post-injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non-erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte-committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Maintenance of granulopoiesis in long-term bone marrow cultures from W/Wv mice and effects of lipopolysaccharide on granulopoiesis in culture

An attempt was made to establish long-term cultures of marrow cells from genetically anaemic W/Wv mice. Two batches of horse sera were used. One batch of horse serum (HS-lot A) supported long-term maintenance (up to 20 weeks) of granulopoiesis in vitro. The number of suspension cells in W/Wv marrow culture was maintained at the same level as that in the control +/+ culture, but the number of granulocyte-macrophage progenitor cells (GM-CFC) and the ratio of immature to mature granulocytes were at a lower level than those in +/+ culture. These data suggest that haemopoietic progenitors in W/Wv cultures maintain a higher level of differentiation, and hence an increased self-renewal than those in +/+ cultures. Another batch of horse serum (HS-lot B) was less effective in the maintenance of the cultures, and the cultures deteriorated within 10 weeks. Addition of bacterial lipopolysaccharide (LPS) induced increased granulopoiesis in +/+ cultures, whereas such treatment resulted in the depletion of suspension cells in W/Wv cultures. The results suggest that haemopoietic cells of W/Wv mouse cannot cope with the strong stimulus for differentiation that occurs after the administration of LPS, although the cells can continue a moderately increased self-renewal and differentiation, as indicated by the results in the culture with HS-lot A.     

Functionally abnormal stromal cells and megakaryocyte size, ploidy, and ultrastructure in Sl/Sld mice

The first goal of the present studies was to determine if Sl/Sld megakaryocytes have features in common with the macrocytic megakaryocytes that genetically normal mice produce in response to acute platelet depletion. The second was to test the hypothesis that megakaryocyte abnormalities in Sl/Sld mice are due to genetically determined hemopoietic stromal cell abnormalities. Sizes and ploidies of mature Sl/Sld megakaryocytes were measured. Macrocytosis and a shift to higher ploidy values were found compared with normal. Within ploidy groups 16N-64N, Sl/Sld megakaryocytes were larger than normal megakaryocytes of the same ploidy. Transmission electron microscopy revealed that Sl/Sld megakaryocyte nuclei contain more and larger nucleoli, and the chromatin was more dispersed than in normal megakaryocyte nuclei of comparable maturity. Asynchronous megakaryocyte cytoplasmic maturation was found. Sl/Sld macrophages were also ultrastructurally abnormal. Megakaryocytic macrocytosis was reproduced in long-term bone marrow cultures in which the adherent layer was formed by Sl/Sld cells. It was the same if cultures were recharged with Sl/Sld or +/+ hemopoietic cells. Previously reported ambiguities in mixed cell cultures were avoided by recharging the adherent layers with only a million cells. These results were correlated with previously published observations. Sl/Sld megakaryocytes have features in common with megakaryocytes from acutely thrombocytopenic animals. One feature, macrocytosis, appears to be due to abnormal Sl/Sld stromal cells that are reproduced as adherent layer cells in long-term cultures. The responsible stromal cells in Sl/Sld mice may be counterparts of megakaryocytopoietic regulatory cells in the marrow stroma of normal animals.     

Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

On the origin of hematopoietic stem cells after local marrow extirpation.

The early hematopoietic regeneration in a depopulated segment of femur shaft is compared in +/+ and W/Wv mice and in W/Wv mice previously treated with +/+ marrow. Since the W/Wv mouse has an intrinsic CFU deficiency on spleen colony assay and since immigrant cells play a negligible role in the onset of regeneration after marrow extirpation, the W/Wv(+/+) chimera provides a model for evaluation of the contribution of residual cells to the regenerative program. There was little difference in the relative recovery of CFU in +/+, W/Wv, and W/Wv-(+/+), Moreover, +/+ derived CFU were responsible for nearly all of the CFU repopulation in chimeric mice. Thus, recovery of hemic cellularity must be due to residual stem cells rather than to stem cells derived by transformation of more primitive mesenchymal elements. The residual CFU are probably intimately associated with bone, most likely within the endosteum and haversian system.     

Differentiation of germ cells in seminiferous tubules transplanted to testes of germ cell-deficient mice of W/Wv and Sl/Sld genotypes

(WB X C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice and (WC X C57BL/6)F1-Sl/Sld (hereafter, WCB6F1-Sl/Sld) mice are sterile due to the deficient spermatogenesis in the testes. The cause of deficient spermatogenesis in WBB6F1-W/Wv mice is considered to be a defect in germ cells themselves, whereas that in WCB6F1-Sl/Sld mice is considered to be a defect in tissue environment necessary for differentiation of germ cells. Seminiferous tubules isolated from cryptorchid testes of C57BL/6- +/+ mice were transplanted into the testes of WBB6F1-W/Wv and WCB6F1-Sl/Sld mice to clarify that the extratubular environment of these mice was intact or not. Type A spermatogonia in the transplanted tubules normally differentiated into spermatids, suggesting that the extratubular environment is intact in both WBB6F1-W/Wv and WCB6F1-Sl/Sld mice.     

Mast cells in spotted mutant mice (W Ph mi)

Mast cells derived from haematopoietic tissue are deficient in numbers in spleen, stomach and skin of Harwell mice doubly mutant at the spotting W locus: seven viable combinations of four mutants. Most combinations have variably impaired viability, anaemia and infertility; but homozygous WshWsh are normal in these respects yet still lack mast cells. The effect of the W gene on mast cells acts in recessive fashion. Effects of doubly mutant W genes on mast cells and coat colour, the latter usually regarded as dominant, appear more closely related than other pleiotropic effects. The spotting gene Ph, closely linked to W, has but marginal effects on mast cells, whereas mi, another spotting gene, quite unrelated to W affects mast cells in the spleen in a dominant way. Thus, splenic mast cells may be a special category of a heterogeneous population. Peptic ulceration, recorded in W/Wv mice of Jackson stock, was not seen in Harwell mice. We suggest that this lesion is due to genetic complementation or environmental causes.