The effect of splenectomy on bone marrow haematopoiesis in mice.

Splenectomy was performed in strain H mice. Erythrocyte macrocytosis and an increase in the reticulocyte, leucocyte and thrombocyte count were found in the peripheral blood of splenectomized animals; only the erythrocyte count fell in the first 3 weeks after splenectomy. Changes in the myelogram during the first weeks after splenectomy were characterized by an increase in the proportion of cells of the erythrocytic and lymphocytic series. The stem cell count in the bone marrow (determined after Till and McCulloch) was slightly elevated on the 8th day after splenectomy, but in subsequent weeks was rather lower than the control group values. Whatever the post-splenectomy interval at which bone marrow was taken from splenectomized mice, there was no significant difference in the transplantation effect of bone marrow cells on white and thrombocyte haematopoiesis. Bone marrow transplantation was found have a stimulant effect only on the reticulocyte count and the sooner bone marrow was collected after splenectomy, the more pronounced the effect. Changes in the myelogram and splenogram of irradiated mice to which the bone marrow cells of splenectomized mice had been transplanted were relatively small.     

Protein-energy malnutrition alters histological and ultrastructural characteristics of the bone marrow and decreases haematopoiesis in adult mice

Protein-energy malnutrition (PEM) decreases resistance to infection by impairing a number of physiological processes, including haematopoiesis. The aim of this study was to evaluate the microanatomical aspects of bone marrow (BM) in mice that were subjected to PEM, in particular, with respect to the components of the local extracellular matrix and the proliferative activity of haematopoietic cells. For this, histological, histochemical, immunohistochemical and ultrastructural techniques were used. Two-month old male Swiss mice were fed with a low-protein diet containing 4% protein and control mice fed a 20% protein diet. When the experimental group had attained a 25% loss of their original body weight, we collected the different biological samples. Malnourished mice had presented severe BM atrophy as well as a reduction in proliferating cell nuclear antigen and gelatinous degeneration. The malnourished mice had more fibronectin accretion in paratrabecular and endosteal regions and more laminin deposition in perisinusal sites than controls. Endosteal cell activation and hyperplasia were found, suggesting their participation in the process. Additionally, we have observed a decrease in the capacity of malnourished haematopoietic stroma to support the growth of haematopoietic stem cells (CD34+) in vitro. These findings point to a structural impairment of the haematopoietic microenvironments in mice with PEM, possibly hampering the interactions between cells and cellular signalling.     

Selective role of calcineurin in haematopoiesis and lymphopoiesis

The calcineurin/NFAT (nuclear factor of activated T-cells) signalling pathway is essential for many aspects of vertebrate development and is the target of the widely used immunosuppressive drugs FK506 and cyclosporine A. The basis for the therapeutic specificity of these drugs has remained unclear, as calcineurin is expressed ubiquitously. By inactivating calcineurin during haematopoietic development, we found that although this signalling pathway has an important, non-redundant role in the regulation of lymphocyte developmental checkpoints, it is not essential for the development of blood myeloid lineages. These studies have shown that the specificity of calcineurin inhibitors arises from the selective use of calcineurin at distinct developmental stages. The requirement for calcineurin/NFAT in the development of the adaptive but not of the innate immune system is consistent with the idea that the evolutionary appearance of this pathway was involved in the emergence of vertebrates.     

Rac1 and Rac2 GTPases in haematopoiesis

The highly homologous Rac1 and Rac2 GTPases are co-expressed in cells of haematopoietic origin and are likely to show some functional redundancy. While disruption of the Rac2 gene in mice has provided insight into some of its functions, Rac1 null mice are embryonic lethal and only recently has conditional gene disruption been possible. Consequently, two articles1,2 have recently elucidated some overlapping and unique key roles of Rac1 and Rac2 in haematopoietic processes including specialized roles in innate and humoral immunity.     

Enhanced bone formation in lipodystrophic PPARgamma(hyp/hyp) mice relocates haematopoiesis to the spleen

The peroxisome proliferator-activated receptor gamma (PPARgamma) controls adipogenesis and metabolism. We demonstrate here that the absence of PPARgamma in fat has potent osteogenic activities, which affect haematopoiesis. The congenital absence of PPARgamma in fat of lipodystrophic PPARgamma(hyp/hyp) mice, strongly enhanced bone mass and consequentially reduced the bone-marrow cavity. Consistent with this, PPARgamma(hyp/hyp) mice had a significant decrease in bone marrow cellularity and resorted to extramedullary haematopoiesis in the spleen to maintain haematopoiesis. Our data indicate that antagonizing PPARgamma activity in fat could be an effective way to combat osteoporosis and suggest that haematopoietic function should be scrutinized in lipodystrophic subjects.     

Human haematopoiesis in steady state and following intense perturbations

Haematopoiesis is comprised of multiple stages, originating from pluripotent stem cells through intermediate progenitors to mature differentiated cells. Consequently, during the development of blood cells numerous sites are potentially exposed to the intense perturbations induced by anticancer chemotherapy. However, little is known about human haematopoietic stem cell kinetics in health and following cytotoxic perturbations. Here we reconstruct the complex in vivo dynamics of haematopoietic populations, including the elusive pluripotent stem cells, with a detailed mathematical representation of the marrow biology. The bone marrow kinetic parameters were estimated by using white blood cell counts routinely collected in patients during high dose chemotherapy (HDCT) followed by autologous peripheral blood stem cell transplantation and granulocyte colony stimulating factor (G-CSF) injections. Studying the model performance under a wide variety of parameter values reveals that bone marrow is surprisingly robust in the physiologically feasible parameter space. We infer that the human haematopoietic pluripotent stem cell density is approximately 1 in 2 · 10⁵ mononuclear cells and that most of these cells are quiescent, dividing once in 3–4 weeks. Our results suggest that the re-infused stem cell content is relatively high (10⁴ kg⁻¹ or 1/300 of CD34+ cells) which contributes to both the long-term marrow re-population as well as to short-term support. This study implies that, in most patients, the pluripotent population recovers within 4 months following HDCT. The proposed model accurately predicts the bone marrow dynamics over a wide range of perturbations caused by clinical interventions. It provides valuable insights about the haematopoietic regeneration capacity, predicts the effect of G-CSF manipulation and of ex vivo graft expansion in improving transplantation procedures, and may have implications for effective stem cell gene therapy.     

Extramedullary haematopoiesis in thrombotic thrombocytopenic purpura.

Extramedullary hematopoiesis was detected in routine sections of spleen and liver from two patients with thrombotic thrombocytopenic purpura (TTP). In patients with other types of hemolytic anemia and in normal persons, extramedullary hematopoiesis was not found in spleen or liver. The results of this study raise the possibility that TTP may fit within the myeloproliferative category, perhaps representing an acute variant of Di-Guglielmo Syndrome.     

The MLL/SET family and haematopoiesis

As demonstrated through early work in Drosophila, members of the MLL/SET family play essential roles during embryonic development through their participation in large protein complexes that are central to epigenetic regulation of gene expression. One of its members, MLL1, has additionally received a lot of attention as it is a potent oncogenic driver in different types of leukaemia when aberrantly fused to a large variety of partners as a result of chromosomal translocations. Its exclusive association with cancers of the haematopoietic system has prompted a large number of investigations into the role of MLL/SET proteins in haematopoiesis, a summary of which was attempted in this review. Interestingly, MLL-rearranged leukaemias are particularly prominent in infant and paediatric leukaemia, which commonly initiate in utero. This, together with the known function of MLL/SET proteins in embryonic development, has focussed research efforts in recent years on understanding the role of this protein family in developmental haematopoiesis and how this may be subverted by MLL oncofusions in infant leukaemia. A detailed understanding of these prenatal events is essential for the development of new treatments that improve the survival specifically of this very young patient group.     

Promiscuous and lineage-specific roles of cell cycle regulators in haematopoiesis

DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.     

Radiation induction of mutations affecting sperm morphology in mice

To investigate the basis of the sperm abnormality assay, studies have been made of the frequency of sperm abnormalities in the genital tracts of the progeny of irradiated males. Male C57BL/6 mice were irradiated (75-600 rad X-rays to the testes) and were then bred in the pre-sterile period to untreated C57BL/6 females. The sperm of their male progeny were examined for the frequency of sperm abnormalities. Variant males with clearly elevated levels of sperm abnormalities were more frequently seen amongst the progeny of irradiated fathers than with the progeny of sham-irradiated controls (10 in 170 compared with 2 in 188; P less than 0.02). Although no clear dose-response relation could be discerned with the number of animals studied, similar differences were observed with irradiated male SWR, C3H/He in inbred crosses and with C57BL/6 in hybrid crosses with C3H/He females. In contrast, matings of males made at longer times following irradiation did not lead to a significant increase in the number of affected progeny for the number tested. Breeding experiments with the affected F1 males showed that the sperm morphology defect could be transmitted in 7 of 19 cases. Parallel cytogenetic studies showed that 3 of the initial affected progeny had detectable reciprocal translocations and that in 2 of these cases the translocation was transmitted with the sperm defect. The studies thus showed that radiation can induce mutations that affect the levels of sperm abnormalities and that these mutations can be associated with reciprocal translocations.     

The ontogeny of haematopoiesis in the marine teleost Leiostomus xanthurus and a comparison of the site of initial haematopoiesis with Opsanus tau

The ontogeny of haematopoiesis in the perciform fish, spot Leiostomus xanthurus , differed from that reported as the norm for fishes, as exemplified by the cypriniform zebrafish Danio rerio , and observed in the batrachoidiform oyster toadfish Opsanus tau . Erythropoiesis in spot was first evident in the head kidney of yolk‐sac larvae 3 days after hatching (DAH). No embryonic intermediate cell mass (ICM) of primitive stem cells or blood islands on the yolk were apparent within embryos. Erythrocytes were first evident in circulation near the completion of yolk absorption, c . 5 DAH, when larvae were c . 2·0 mm notochord length ( L _N). Erythrocyte abundance increased rapidly with larval development for c . 14 to 16 DAH, then became highly variable following changes in cardiac chamber morphology and volume. Erythrocytic haemoglobin (Hb) was not detected within whole larvae until they were 12 DAH or c . 3·1 mm L _N, well after yolk and oil‐globule absorption. The Hb was not quantified until larvae were >47 DAH or >7 mm standard length. The delayed appearance of erythrocytes and Hb in spot was similar to that reported for other marine fishes with small embryos and larvae. In oyster toadfish, a marine teleost that exhibits large embryos and larvae, the ICM and Hb were first evident in two bilateral slips of erythropoietic tissue in the embryos, c . 5 days after fertilization. Soon thereafter, erythrocytes were evident in the heart, and peripheral and vitelline circulation. Initial haematopoiesis in oyster toadfish conformed with that described for zebrafish. While the genes that code for the development of haematopoiesis are conserved among vertebrates, gene expression lacks phylogenetic pattern among fishes and appears to conform more closely with phenotypic expression related to physiological and ecological influences of overall body size and environmental oxygen availability.     

Fgf21 is essential for haematopoiesis in zebrafish

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21-knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo-erythroid progenitor cell fate in vivo.     

WNT signalling and haematopoiesis: a WNT-WNT situation

The evolutionarily conserved WNT-signalling pathway has pivotal roles during the development of many organ systems, and dysregulated WNT signalling is a key factor in the initiation of various tumours. Recent studies have implicated a role for WNT signal transduction at several stages of lymphocyte development and in the self-renewal of haematopoietic stem cells. Here, we outline new insights into the WNT-signalling pathway, review its role in the self-renewal of haematopoietic stem cells and in the development of T and B cells, and discuss controversies and future developments with regard to WNT signalling in the thymus.     

Principal signalling complexes in haematopoiesis: structural aspects and mimetic discovery

Blood production is a highly regulated process involving multiple inhibitory and stimulatory cytokines present in the haematopoietic stem cell niche. Small molecules mimics of these signalling molecules have substantial potential as drugs and in the development of bioreactors to generate blood products. We review the structural biology of the extracellular signalling domains of five of the most important cytokines, analyze their structure-property relationships, and summarize the progress in developing small molecule mimics using the molecular information from structural biology and mutation studies.     

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.     

Production of presumptive humoral haematopoietic regulators in canine cyclic haematopoiesis

Abstract. Conditioned media (CM) were prepared according to previously published techniques from the bone marrow of dogs with cyclic haematopoiesis (CH). CM prepared from day 9 marrows inhibited mouse bone marrow CFU-s proliferation rate while CM from day 10 marrows were stimulatory and also contained an erythroid stimulating factor which appeared to be erythropoietin. In addition a highly significant trend from CM containing CFU-s inhibitory materials to media with CFU-s stimulatory activity was observed through cycles day 1 to 8. These studies further support the concept that CH is due to a defect in factors controlling stem cell proliferation and suggest that a major event occurs in CH dog marrow on days 9 and/or 10 of the cycle. Bone marrow transplantation studies (Dale & Graw, 1974; Weiden et al., 1974; Jones et al., 1975b) have indicated that canine cyclic haematopoiesis (CH) is probably due to a disorder in the multipotential stem cells. Morphological evidence (Scott et al., 1973) and the almost synchronous cycling of CFU-e, CFU-c and diffusion chamber progenitor cells (DCPC) (DUM et al., 1977, 1978a, b) lend support to such a theory. However, efforts to identify the mechanisms controlliig multipotential stem cell proliferation in dogs have been handicapped by the lack of suitable techniques to study these cells in the canine. Recently, Wright and co-workers (Wright & Lord, 1978, 1979; Wright et al., 1979; Lord et al., 1979), on the basis of previous observations (Frindel et al., 1976; Frindel & Guigon, 1977), described the preparation of species non-specific, bone marrow conditioned media (CM) which are capable of influencing the proliferation rate of murine colony forming units-spleen (CFU-s). The studies now reported were designed to determine if CM prepared from canine CH marrow would influence the proliferation rate of murine bone marrow CFU-s. The results indicate that a major event, possibly related to the in vivo control of stem cell proliferation in dogs with CH, occurs on days 9–10 of the cycle; day 1 being the first day when the peripheral blood neutrophil count falls below-1600 mm³.