The Wilms tumor suppressor gene wt1 is required for development of the spleen.

The Wilms tumor suppressor gene WT1 (wt1 in mouse) is unique among tumor suppressors because, in addition to its involvement in cancer [1] [2] and various other diseases [3] [4] [5] [6], it has an essential role in the development of certain organs. This is revealed by the phenotype of mice with inactivated wt1 alleles [7]. These animals exhibit a complete failure of kidney and gonad development as well as abnormalities of the heart and mesothelial structures. On a C57BL/6 genetic background, wt1(-/-) animals die between day 13.5 (E13.5) and 15.5 (E15.5) of embryonic development [7]. We report here that crossing of the wt1 mutation onto different mouse backgrounds delayed embryonic lethality until birth. In wt1(-/-) mice on these different genetic backgrounds, we observed a dramatic failure of spleen development, in addition to the well characterized phenotypic abnormalities. The spleen anlage formed at around E12 to E13 and involuted by the E15 stage, before the invasion of hematopoietic cells. The absence of proper spleen development in these wt1(-/-) embryos correlated with enhanced apoptosis in the primordial spleen cells. The expression of hox11, a gene that also controls development of the spleen [8] [9], was not altered by the inactivation of wt1. In situ hybridization revealed that the two genes are regulated independently. These findings demonstrate that the penetrance of the wt1(-/-) phenotype depends on the existence of one or more modifier gene(s) and that wt1 plays a pivotal role in the development of the spleen, thereby extending its role in organogenesis.     

Dental lamina develops even within the mouse diastema

In embryos of albino mice of ICR strain, collected between days 13 and 15, the epithelial lining within the future upper maxillary diastema was studied using frontal histological sections stained with hematoxylin-eosin and PAS methods. In embryos harvested on the 12th hr of day 13 (stage 13/12), a continuous epithelial rudiment of dentition was found in the anterior extension of the epithelial anlage of the first upper molar, up to the level of the lower anterior margin of the primary choana. In this stage the rudiment acquired, in the most anterior region of the future diastema, an arrangement typical for the dental lamina. In its dorsal extension there was found a distinct tooth anlage at the transitory stage lamina bud, which further (at stage 13/24) disintegrated into several segments. Starting with the day 14 (stage 14/12), the epithelial rudiment of dentition within the future upper diastema began to regress. From the stage 14/24 on, the anlage persisted only in its posterior terminal part where it merged with the epithelial lamina extending anteriorly from the anlage of the first upper molar. The existence of the dentition rudiment within the future mouse diastema constitutes the ontogenetic evidence that the diastema originates only secondarily--by regression. In some mutant strains of mice (tabby, crooked, sleek), the regression appears incomplete. The odontogenic potency of mouse diastema tissues should be considered when interpreting the results of in vitro experiments investigating the odontogenic inductive tissue interactions in mouse.     

The transplantability of bone marrow and spleen cells after filtration through silon tissue]

Investigations were carried out on the separation of haematopoietic stem cells from suspensions of the bone-marrow and spleen by means of filtration with silon tissue. The presence of stem cells in the filtrates was determined by the spleen colony test according to the method of Till and McCulloch in irradiated mice. The investigations revealed that a selective separation of haematopoietic stem cells could not be achieved when proceeding in this way. From the results of further test series, in which suspensions were also used which had been gained from haematopoietic tissues of hypersplenic mice, the conclusion could be drawn that the haematopoietic stem cells obtained by filtrating the bone-marrow will have another affinity to the spleen tissue of irradiated mice than the haematopoietic stem cells gained by filtrating the spleen tissue.     

Radiation and haematopoiesis in Harwell steel mice

Haematological information on steel (Sl) mice is limited largely to Sl/Sld mice of Bar Harbor stock (WC.B6 F1). Therefore, two Harwell alleles, SlgbH and Slcon, were investigated. In the steady state both heterozygotes were modestly anaemic, homozygous Slcon and compound Slcon/SlgbH more so. On perturbation by X-irradiation Slcon/SlgbH showed a decrease in median lethal dose (MLD)--6.5 Gy, Slcon/+ and Slcon/Slcon slightly less change (7.5 Gy) compared with +/+, 8 Gy. In recovery from sublethal doses single heterozygotes, double heterozygotes with Wv, and compounds showed no delay in restoration of the count of red blood corpuscles (RBC) such as that seen in typical W mice (e.g. Wv/+, W/Wv). Effects on Slcon/Slcon and Slcon/SlgbH differ from those reported for Sl/Sld in that they show normal growth of spleen colonies when used as lethally irradiated recipients of bone marrow, they support growth of implanted bone marrow to form radiation chimaeras. When Harwell steel mice are donors of bone marrow to lethally irradiated +/+ mice the chimaeras ultimately are not anaemic; when lethally irradiated Harwell steel mice are recipients of +/+ marrow they remain macrocytically anaemic. One deduces that, for normal development and production of normal RBC in the steady state, the erythron requires intrinsic factors determined by wild type alleles at the W locus and extrinsic factors determined by wild type alleles at the Sl locus. Mutant alleles at either locus may determine macrocytosis. Two mutant alleles at either locus are still more deleterious, often lethal. Whereas mutant W alleles may also influence the pluripotent haematopoietic stem cell (HSC) leading to reduced MLD on X-irradiation, a similarly reduced MLD for Sl mutants may represent an increased need for and consumption of products of the haematopoietic stem cells rather than truly increased radiosensitivity, since the Do for spleen colony-forming units is the same for Slcon/SlgbH as +/+ mice.     

The Development of Haematopoietic Cells Is Biased in Embryonic Stem Cell Chimaeras

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using β-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in the organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for β-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.     

Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.

We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(-/-) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845-1854).     

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.     

Obesity and Hepatic Steatosis Are Associated with Elevated Serum Amyloid Beta in Metabolically Stressed APPswe/PS1dE9 Mice

Diabesity-associated metabolic stresses modulate the development of Alzheimer’s disease (AD). For further insights into the underlying mechanisms, we examine whether the genetic background of APPswe/PS1dE9 at the prodromal stage of AD affects peripheral metabolism in the context of diabesity. We characterized APPswe/PS1dE9 transgenic mice treated with a combination of high-fat diet with streptozotocin (HFSTZ) in the early stage of AD. HFSTZ-treated APPswe/PS1dE9 transgenic mice exhibited worse metabolic stresses related to diabesity, while serum β-amyloid levels were elevated and hepatic steatosis became apparent. Importantly, two-way analysis of variance shows a significant interaction between HFSTZ and genetic background of AD, indicating that APPswe/PS1dE9 transgenic mice are more vulnerable to HFSTZ treatment. In addition, body weight gain, high hepatic triglyceride, and hyperglycemia were positively associated with serum β-amyloid, as validated by Pearson’s correlation analysis. Our data suggests that the interplay between genetic background of AD and HFSTZ-induced metabolic stresses contributes to the development of obesity and hepatic steatosis. Alleviating metabolic stresses including dysglycemia, obesity, and hepatic steatosis could be critical to prevent peripheral β-amyloid accumulation at the early stage of AD.     

Morphology of interactions of the haematopoietic microenvironment with the haematopoietic cells in erythroid spleen colonies

The morphology of the interactions of the cells of the haematopoietic microenvironment with haematopoietic cells was studied in exogenous erythroid spleen colonies formed in mice, on the fifth and eighth day after their irradiation with a lethal dose of gamma rays and bone marrow transplantation. The characteristic type of stromal cell interacting with less mature cells of the erythroid series was a dark, branching reticular cell. The typical structural interaction of the reticular cells with erythroblasts was the formation of very long, fine cytoplasmic processes by the reticular cells. The processes were in close contact with the erythroblasts and formed a three-dimensional network stretching long distances from the nucleus of the reticular cells. Other cells of the haematopoietic microenvironment, in contact chiefly with poly- and ortochromic erythroblasts, were macrophages and the two together formed typical erythroblastic islands. In places, the macrophages and erythroblasts formed close, firm contacts by means of their cytoplasmic membranes. These morphological observations support the conception that close functional cooperation exists between the cells of the haematopoietic microenvironment and the haematopoietic cells in differentiation and proliferation processes in the haematopoietic tissue of spleen colonies.     

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse.

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.     

Association of B-1 B cells with follicular dendritic cells in spleen

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.     

Embryonic origins of spleen asymmetry

The spleen is a vertebrate organ that has both hematopoietic and immunologic function. The embryonic origins of the spleen are obscure, with most studies describing the earliest rudiment of the spleen as a condensation of mesodermal mesenchyme on the left side of the dorsal mesogastrium. The development of spleen handedness has not been described previously, presumably because of the difficulty in assaying spleen position in the embryo and the lack of early, organ-specific molecular markers. Here we show that expression of the homeobox gene Nkx2-5 serves as a marker for spleen precursor tissue. Pre-splenic tissue is initially located in symmetric domains on both sides of the embryo but, during subsequent development, only the left side goes on to form the mature spleen. Therefore, the final location of the spleen on the left side of the body axis appears to result from preferential development of the spleen precursor cells on the left side of the embryo. Our studies indicate that the spleen and heart become asymmetric via different cellular mechanisms. Nkx2-5 may function locally as part of the laterality cascade, downstream of nodal and Pitx2, or it may direct asymmetric morphogenesis after laterality has been determined.     

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.     

Myelopoiesis in spleen-producing distinct dendritic-like cells

Dendritic cells (DC) represent a heterogeneous class of antigen presenting cells (APC). Previously we reported a distinct myeloid dendritic-like cell present in spleen, as an in vivo counterpart to cells produced in murine spleen long-term cultures (LTC-DC). These cells, named 'L-DC', were found to be functionally and phenotypically distinct from conventional (c)DC, plasmacytoid (p)DC and monocytes. These results suggested that spleen may represent a niche for development of L-DC from endogenous progenitors. Adult murine spleen has now been investigated for the presence of L-DC progenitors. Lineage-negative (Lin)(-) ckit(lo) and Lin(-) ckit(hi) progenitor subsets were identified as candidate populations, and tested for ability to produce L-DC; in vitro upon co-culture with the spleen stromal line STX3, and in vivo after adoptive therapy into mice. Both subsets colonized STX3 stroma in vitro for L-DC production, indicating that they contained either a common or two distinct progenitors for L-DC. However, only the Lin(-) ckit(hi) subset gave progeny cells after adoptive transfer into lethally irradiated mice. In vivo development was however multilineage and not restricted to L-DC development. Multilineage reconstitution reflects long-term reconstituting haematopoietic stem cells (LT-HSC), suggesting a close relationship between L-DC progenitors and LT-HSC. L-DC were however produced in vivo in much higher number than monocytes/macrophages and cDC, indicating the presence of a specific L-DC progenitor within the Lin(-) ckit(hi) subset. A model is advanced for development of L-DC directly from haematopoietic progenitors in spleen and dependent on the spleen microenvironment.     

Specific partial depletion of graft-vs-host activity by incubation and centrifugation of mouse spleen cells on allogeneic spleen cell monolayers.

Spleen cells (from BALB/c mice immunized with the C57BL/6 lymphoma EL4, or from non-immune BALB/c) were incubated on monolayers of [C57BL/6 times BALB/cF1 (B6CF1) spleen cells on polylysine-coated polystyrene Petri plate, for 1/2 hr or for 1 hr at 37 degrees C followed by centrifugation of the monolayers for 5 min at 70 times G to 110 times G at 34 to 37 degrees C. Control monolayers were BALB/c spleen cells. As measured by the Simonsen spleen weight assay in neonatal mice, graft-vs-host (GVH) activity was partially depleted in cell populations nonadherent to B6CF1 monolayers. Residual GVH activity of these nonadherent cells was about half that of cells incubated on the control syngeneic monolayers (the mean of eight experiments was 49% +/- 11% S.D.). Two or three consecutive cycles of incubation and centrifugation did not significantly diminish the residual GVH activity, suggesting that spleen cells with GVH activity are heterogeneous with respect to binding to allogeneic target cells under the above conditions. Cell populations nonadherent to third-part [A times AL]F1 monolayers retained full activity, and cell populations partially depleted of GVH activity in B6CF1 neonates had full activity in third-party [BALB/c times AL]F1 neonates.