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.     

Immunohistochemistry in the analysis of mouse aggregation chimaeras

Summary We have used cellular mosaicism in chimaeric mice to study the clonal organization of normal tissues. The mosaicism has been demonstrated in sections and in whole mounts of intestinal epithelium, aortic endothelium and retinal pigment epithelium using H2 antigens and a carbohydrate polymorphism recognized byDolichos biflorus lectin as strain-specific markers.The results show that the epithelium of each adult intestinal crypt is derived from a single progenitor cell. Because crypts of differing genotype may contribute cells to the same villus, the pathways of cell migration up the villi can be demonstrated. The ability to stain mosaic patches in two dimensions in large intact sheets of epithelium has permitted a more satisfactory analysis in terms of clonal development than was previously possible with data from tissue sections. We have adapted statistical procedures from plant ecology to examine the scale of clustering of patches of like genotype, and thence to recognize descendent clones, i.e. groups of cells which are not contiguous, but are related by descent from a common ancestor in embryogenesis.The 1985Histochemical Journal Lecture given by Dr Ponder at York on 10 July, 1985 at the invitation of the Royal Microscopical Society.     

Prenatal fate of parthenogenetic cells in mouse aggregation chimaeras

Parthenogenetically activated BCF1 and fertilized BALB/c embryos were aggregated to form chimaeras. The fate of the parthenogenetic component was followed in the conceptus during the second half of gestation. The results indicate an early strong selection against parthenogenetic cells in the extra-embryonal part, which is presumably complete by term, and a weaker selective process in the embryo. During early development, parthenogenetic cells have nearly normal developmental potency in the embryo, which allows their balanced contribution in the chimaeras on day 12. Later, this contribution declines significantly resulting in an unbalanced relation to the advantage of the fertilized counterpart. From the results, we suggest that gametic imprinting may play a role not only in the key steps of preimplantation and early postimplantation development, but later in cell and tissue differentiation.     

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.     

Polyclonal origin of pancreatic islets in aggregation mouse chimaeras.

In the present study, we have examined the origin and growth pattern of the beta cells in pancreatic islets, to determine whether a single progenitor cell gave rise to all the precursors of the islets, or if each of a few progenitor cells is the founder of a different islet, or if each islet is a mixture of cells originating from a pool of progenitor cells. Aggregation mouse chimaeras where the pancreatic beta cells derived from each embryo can be identified in the islets on histological sections were analyzed. In two chimaeras, all the islets contained cells from both the aggregated embryo. This clearly demonstrates that each islet resulted from several independent cells. In addition, the beta cells derived from either embryo component were in very small clusters in the islets, suggesting that in situ cell division did not account significantly for islet growth.     

Development of T-cell function in relation to T-cell set diversification in nu/nu mice

The differentiation pattern of splenic T-cell populations in germ- and pathogen-free nu/nu mice, as compared to nu/+ littermates, is characterized by two abnormal features: the expression of TL determinants on peripheral T cells and the delayed onset of their differentiation from the predominant Lyt-123:TL+ set into TL- cells of Lyt-1+ and Lyt-123+ phenotype, which, in these mice, does not occur until 10 weeks of age. We report here that the delayed onset of mitogen- or alloantigen-induced interleukin-2 synthesis and T-cell proliferation as well as the development of cytotoxic T-lymphocyte activity of enriched T-cell populations is strictly correlated with the time point of T-cell subset diversification in nu/nu mice and depends in particular on the presence of the Lyt-1 (TL-:Lyt-2-) T-cell set which is lacking in splenic T-cell populations of germ-free young nu/nu mice.     

Characterization of a CD4-positive T-cell line derived from an athymic (nu/nu) mouse.

We have isolated a Thy-1+, CD3+, CD4+ T-cell line from the spleen of a 12-week-old nu/nu (nude) BALB/c mouse. The cell line is clonal, and it expresses an alpha beta T-cell antigen receptor. Upon activation, these cells secrete IL-2 but not IL-4, putting them in the Th1 category. The cells can be triggered to proliferate and secrete lymphokines in the presence of irradiated syngeneic or allogeneic splenic feeder cells that express a variety of MHC haplotypes. This response is MHC class II-specific, because it can be blocked by either anti-Ia or anti-CD4 antibodies. From the response pattern of this T-cell line, we conclude that it recognizes a common determinant on class II MHC antigens. This nude mouse T-lymphocyte presumably has not undergone thymic selection. Therefore its unique specificity may reflect both the bias of T-cell antigen receptor genes for encoding receptors that recognize MHC molecules and the requirement for functional thymic epithelial cells for the efficient education of a self-MHC-restricted repertoire.     

Quantitative analysis of mid-gestation mouse aggregation chimaeras: non-random composition of the placenta

Mouse chimaeras were produced by aggregating eight-cell embryos from two different F₂ matings, abbreviated to AF₂ and BF₂ respectively: (C57BL/ OIa.AKR-Gpi-1s ^a, c/Ws female × BALB/c male)F₂ and (C57BL/Ws female × CBA/Ca male)F₂. Quantitative electrophoresis of glucose phosphate isomerase (GPI-1) was used to estimate the proportions of the two cell populations in different tissues of the 12 day chimàeric conceptuses, with the % GPI-1A indicating the percentage of cells derived from the AF₂ embryos. The % GPI-1A was found to be highly positively correlated within the primitive ectoderm lineage (between the fetus, amnion and yolk sac mesoderm) and within the primitive endoderm lineage (between the yolk sac endoderm and the parietal endoderm) but no correlation (either positive or negative) was seen between the two lineages. This confirms the results of a previous,study of chimaeras made between partially congenic strains and suggests the original conclusions have general validity. The % GPI-1A in the placenta was corrected for the expected contribution of maternal GPI-1, based on control experiments involving transfer of homozygous Gpi-1s ^b /Gpi-1s ^b embryos to the uteri of Gpi-1s ^a /Gpi-1s ^a pseudopregnant females. The corrected % GPI- lA in the placenta was positively correlated with that in each of the three primitive ectoderm derivatives. This suggests either (1) exchange of cells between the polar trophectoderm and the underlying part of the inner cell mass that forms the primitive ectoderm or (2) cells are incompletely mixed in the chimaeric blastocyst and patches of AF₂ and BF₂ cells straddle the boundary between the polar trophectoderm and the underlying primitive ectoderm. The second explanation does not imply the existence of shared developmental lineages between trophectoderm and primitive ectoderm in non-chimaeric embryos. Unlike that of any other tissue, the distribution of placental GPI-1A was U-shaped; in 17/28 placenta samples the proportion of the minor component was 10% or less. This suggests that the placental trophoblast is derived from a small number of coherenct clones of polar trophectoderm cells (either a small number of polar trophectoderm cells or a larger number if the two cell populations are not finely intermingled). Thus, although as a population the placentas of chimaeric conceptuses are balanced with respect to the % GPI-1A (mean close to 50%), individually most placentas are extremely unbalanced in their chimaeric composition (< 10% or > 90% GPI-IA). This non-random composition of the chimaeric placentas is in contrast to the widely held assumption that the distribution of cells in chimaeric conceptuses is normally random. Correspondence to: J.D. West     

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Rearrangement patterns of T-cell receptor genes in the spleen of athymic (nu/nu) young mice

Although the athymic nude mouse is grossly deficient in peripheral T cells, the number of lymphocytes bearing T-cell markers (L3T4, LyT2) and the or T-cell receptor (Tcr) increases steadily with age. The anatomical site(s) where cells arise are unkown. Splenocytes from 3–5-week-old C57BL/6 (nu/nu) mice contain 2%–5% Pro-T cell progenitors identified with the Joro 37-5 and Joro 75 antibodies, but not mature T cells. To study Tcr gene rearrangement outside the thymus, we fused splenocytes from 3–5-week-old C57BL/6 nude mice with the T-cell lymphoma BW100.129. Of 22 hybrids that grew stably in culture, four had Tcrd-VD1-D2-J1, two had Tcrd-VD2-J1, and seven had Tcrd-D1-D2 types of rearrangement. Eight hybrids had rearranged the Tcrg-2 gene cluster, but none had rearranged Tcrg-1, -3, or -4. None of the hybrids had rearranged the Tcrd gene cluster and 13 contained DJ rearrangements at the Igh locus. We conclude that the spleen is one of the extrathymic sites where T-cell progenitors can rearranged Tcrd and Tcrg genes. However, there was no evidence for Tcrb gene rearrangements in this organ. Furthermore, the analysis of this limited number of hybrids suggests that extrathymic Tcr gene rearrangements seem to be distinct and much less diverse than those found in the developing thymocytes.     

Cisplatin sensitivity in Hmbg1-/- and Hmbg1+/+ mouse cells

The study presented here investigates the effect of HMGB1 knockout on the sensitivity of mouse embryonic fibroblasts treated with the anticancer drug cisplatin. We evaluated both the growth inhibition by cisplatin and cisplatin-induced cell death in the Hmgb1(-/-) cells and its wild-type counterpart. No significant differences were observed in the responses of these cells to cisplatin, indicating that HMGB1 does not play a significant role in modulating the cellular responses to cisplatin in this context. Since HMGB1 significantly enhances the cytotoxicity of cisplatin in other cells, these results illustrate the importance of cell type in determining the ability of this and probably other cisplatin-DNA-binding proteins to influence the efficacy of the drug.     

Viability of trisomy 12 cells in mouse chimaeras

Summary Mouse aggregation chimaeras consisting of trisomy 12 and normal euploid cells were produced. The analysis of one trisomy 12euploid chimaera, using biochemical and cytological markers, showed that the trisomic cells were able to participate in the formation of most tissues including the ovary. On the other hand, no trisomy 12 cells were found in lymphocyte populations, which is most likely due to early selection in this particular cell lineage. The viability of two adult trisomy 12 chimaeras demonstrates that trisomy 12 cells are able to develop beyond the fetal stage which is not observed in completely trisomic fetuses.Furthermore, these chimaeras did not show any sign of a trisomy 12 syndrome, indicating that the trisomy 12 cells were functionally integrated and participated normally in the differentiation of the various tissues. Our results suggest that trisomy 12 in the mouse is not autonomously cell lethal but can be rescued and is perfectly viable in the presence of normal diploid cells.This article is dedicated to the memory of Prof. A. Gropp     

Tolerance in early embryo aggregation-derived mouse chimaeras.

Epidermal cell suspensions of 90% average viability prepared from adult mouse tail skin by trypsinization and glass wool filtration were compared with leucocytes for their capacity to induce and reveal cell-mediated cytotoxicity to histocompatibility antigens in an H-2 different strain combination. As targets in short-term chromium release assays, epidermal cells incorporated ten times more ⁵¹Cr than normal lymph node cells yet released a lower proportion spontaneously. Although they were more resistant to lysis than lymph node cells, they registered high levels of cytotoxicity when particularly active attacker cells were used, even at low attacker-to-target cell ratios. In mixed cell cultures, irradiated epidermal cells were as effective as spleen cells in boosting immunity induced in vivo by skin allografts. Epidermal cells also were effective primary immunogens in vitro, but at higher responder-to-stimulator cell ratios than required for spleen cells. The specificity of epidermal cells as both targets and immunogens fully paralleled that of leucocytes from the same donors.     

Lymphocyte function in experimental African trypanosomiasis. IV. Immunosuppression and suppressor cells in the athymic nu/nu mouse

The role of T cells in the development and expression of antigen-nonspecific immunosuppression in experimental African trypanosomiasis was addressed. Nude ($\text{nu}\text{nu}$) C57BL/ 6 NIH mice and their thymus-bearing ($\text{nu}\text{+}$) littermates were infected with Trypanosoma rhodesiense and examined for suppression of splenic B-cell responses in vitro to the mitogen LPS. All animals developed splenic unresponsiveness to LPS. Further, both nu/nu and nu/ + infected mice displayed suppressor cell activity in their spleen cell populations upon transfer to normal uninfected mouse spleen cell cultures. On the basis of these findings we suggest that both the generalized immunosuppression and the development of suppressor cell activity in the spleens of mice infected with T. rhodesiense are T-independent processes.     

Histochemical studies of 3 beta- and 20alpha-hydroxysteroiddehydrogenase in the adrenals and ovaries of the nu/nu mouse.

Adrenal glands and ovaries of nu/+ and nu/nu female mice were examined histochemically throughout the postnatal life for changes in distribution, intensity and appearance of activities of 3 steroid-converting enzymes. While the 3beta-OH-steroid- and pregnenolone-dehydrogenase did not reveal any difference, the 20alpha-hydroxysteroid-dehydrogenase (20alpha-OH SDH) showed a constant behaviour: the onset of the enzyme activity was detectable in the adrenals of nu/+ mice after the second week of age, in nu/nu mice 3--4 weeks later. The localization of 20alpha-OH-SDH was strictly confined to the zone of small cells, a zone lying between the x-zone and the zona fasciculata. In nu/+ mice, after reached sexual maturity at about 6--8 weeks of age, the enzyme progressively disappeared from the adrenals and simultaneously appeared in the ovaries. In nu/+nu mice, which had been kept separated from mating animals, the 20alpha-OH SDH persisted in adrenals even in adult stages. After pregnancy, both--nu/nu and nu/+, showed 20alpha-OH SDH-activity in ovaries and none in adrenals. In both, the enzyme was localized in involuting corpora lutea, whilst in nu/nu it was also present in the intersitial cells. This study indicates no disturbance in the formation of progesterone, but the process of its degradation, as indicated by 20alpha-OH SDH-activity, is definitely different in nu/+ and nu/nu.     

Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.

Spleens and lymph nodes of nu/nu, congenitally athymic, mice have 16–32% and 72–76%, respectively, of large, brain-associated-T (BAT) antigen positive cells. These BAT positive large cells were isolated from nu/nu spleens with a fluorescence-activated cell sorter (FACS-II) and then cultured on thymic reticuloepithelial cell (TRC) monolayers. Both concanavalin A and mixed lymphocyte reactivity was induced during this culture period. We concluded that these BAT positive cells are pre-T cells which can he induced to at least some T cell functions by an inductive factor produced by TRC.