Of birds and mice: hematopoietic stem cell development

For many years it has been assumed that the ontogeny of the mammalian hematopoietic system involves sequential transfers of hematopoietic stem cells (HSCs) generated in the yolk sac blood islands, to successive hematopoietic organs as these become active in the embryo (fetal liver, thymus, spleen and eventually bone marrow). Very little was known about early events related to hematopoiesis that could take place during the 4.5 day gap separating the appearance of the yolk sac blood islands and the stage of a fully active fetal liver. Experiments performed in birds documented that the yolk sac only produce erythro-myeloid precursors that become extinct after the emergence of a second wave of intra-embryonic HSCs from the region neighbouring the dorsal aorta. The experimental approaches undertaken over the last ten years in the murine model, which are reviewed here, led to the conclusion that the rules governing avian hematopoietic development basically apply to higher vertebrates.     

The ontogeny of MHC class I expression in rainbow trout (Oncorhynchus mykiss)

In the present study, clonal rainbow trout (Oncorhynchus mykiss) embryos and larvae were assayed for the expression of key molecules involved in specific cell-mediated cytotoxicity using an anti-MHC class I monoclonal Ab and by RT-PCR using specific primers derived from classical MHC class I (class Ia), TCR and CD8. Whereas RT-PCR revealed that MHC class Ia and CD8 were expressed from at least 1 week after fertilisation (p.f.) on, TCR expression was detectable from 2 weeks p.f. Immunohistochemistry indicated an early and distinct expression of MHC class I protein in the thymus. Positive lymphoid, epithelial and endothelial cells were found in the pronephros, in the spleen and in the inner and outer epithelia at later stages. Whereas in older rainbow trout the intestine is counted among the organs of the highest class I expression, during ontogeny it was the last site (39 days after hatching) where such expression was detectable. Knowledge on the appearance of the assayed key molecules during fish development is relevant for the pathogenesis of infections as well as for early vaccine delivery. Besides such information regarding the development of the adaptive immune system, immunohistochemistry revealed that in early larvae MHC class I was expressed in neurons whereas in older rainbow trout this was not observed.     

The earliest stages in the development of the circulatory system of the Rainbow Trout Oncorhynchus mykiss

The older literature suggests that the development of the blood vascular system in teleosts differs from that of other vertebrates. The evidence, however, came mostly from studies of salmonid embryos beyond the stages when blood cells had begun to circulate, which overlooked earlier developmental stages. The development of the blood vessels of the rainbow trout are illustrated from the time of the first heartbeat to the stage of eye pigment formation. Unless they can be injected with a dye solution, the earliest vessels to develop remain invisible until blood flow makes them visible. By the time of the first heartbeat stage, the embryo has a dorsal aorta, caudal artery and vein, a few transverse vessels, and even the beginning of a vitelline network. One feature peculiar to teleosts is the development of the intermediate cell mass, from which the erythrocytes and a temporary capillary network are formed rather than from the yolk sac. Development of the early posterior cardinal and the subintestinal vein occurs much as in other vertebrates. Previous investigators missed these earliest phases of development because of the difficulty of making them visible. Early formation and transformation of the vascular system of the rainbow trout generally conforms to that seen in vertebrates, except as modified by the temporary presence of the intermediate cell mass and the specialized teleostean yolk mass. With the reduction of the intermediate cell mass, the primary circulatory system for yolk utilization is transformed into a secondary one for respiratory and metabolic functions, as happens usually among vertebrates. J. Morphol. 233:215–236, 1997. © 1997 Wiley-Liss, Inc.     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study

The ontogenetic development of IgM-containing cells is described as demonstrated by immunoperoxidase staining with a mouse anti-trout IgM monoclonal antibody and the differentiation of enzyme-histochemical markers in the non-lymphoid cells forming the stroma of the thymus, spleen and kidney of the rainbow trout. The first lymphoid cells staining with the monoclonal antibody occurred at day 4-5 after hatching in the renal lympho-haemopoietic tissue. By 1 month after hatching IgM-positive cells also appeared in the spleen and thymus. Enzyme-histochemical demonstration of the alkaline and acid phosphatase and non-specific σ-naphthyl acetate esterase enzymatic activities in the non-lymphoid cells indicated that a certain degree of maturation of the cellular stroma of the developing lymphoid organs of trout was reached before or at the time when IgM-expressing cells could be observed. The relationships of the stromal components of the various lymphoid organs to the development of IgM-positive cells, and the possible role of the renal lympho-haemopoietic tissue as a primary lymphoid organ for B-cell differentiation in the trout are discussed.     

Shifts in δ15N signature following the onset of exogenous feeding in rainbow trout Oncorhynchus mykiss: importance of combining length and age data

The δ¹⁵N isotopic change of recently emerged rainbow trout Oncorhynchus mykiss due to diet shift from yolk sac to exogenous feeding was evaluated in a field study. The fit of a general model including both fish length and age in days as co‐variables indicates that the specific δ¹⁵N of individual fish at any given time along the ontogeny is determined by its growth trajectory. The results suggest that estimations based on fish size alone could bias data interpretation and maternal origin determinations in partially migratory salmonids.     

Phylogeny and ontogeny of fish leucocytes

In contrast to higher vertebrates, most fish species hatch at the embryonic stage of life. Consequently, they have to defend against a variety of micro-organisms living in their aquatic environment. This paper is focussed on the development of leucocytes functioning within this early innate system and later on in the acquired immune system (B and T cells). Most of the data are derived from cyprinid fish (zebrafish, carp), which are excellent models to study early ontogeny. Attention is also paid to the phylogeny of leucocytes, with special attention to early chordates. It is clear that young fish use innate mechanisms during the first weeks/months of their development. In zebrafish, a variety of hematopoietic genes have been sequenced which allow a detailed picture of the development of the distinct leucocytes and their precursors. In cyprinids and sea bass, the thymus is the first lymphoid organ and T cells appear to be selected there much earlier than the first detection of T cell-dependent antibody responses. The first B cells are most probably generated in head kidney. Although T cells are selected earlier than B cells, T cell independent responses occur earlier than the T cell-dependent responses. The very early (pre-thymic) appearance of T-like cells in gut of sea bass and carp suggests an extra-thymic origin of these cells. However, B cells populate the GALT much later than spleen or kidney, indicating a rather late appearance of mucosal immunity. The first plasma cells are found long after the intake of food in cyprinids, but in many marine fish they appear around the first food intake. In general, acquired immunity is not correlated to food intake.     

Patterns of angiogenic and hematopoietic gene expression during brown trout embryogenesis

In this article, whole mount in situ hybridization is used to examine early blood vessel and blood cell development in the embryos of the brown trout Salmo trutta lacustris. cDNAs encoding for the angiogenic markers fli1 and flk1, and for the hematopoietic markers gata1 and gata2, were identified from an expressed sequence tag library of rainbow trout. Results show that fli1, flk1 and gata2 are activated in bilateral bands of the lateral trunk mesoderm before the onset of somitogenesis, shortly followed by gata1. These bands then converge toward the ventral midline to form the intermediate cell mass (ICM) (anterior ICM). Subsequent axial vasculogenesis and initial blood cell formation involve a clear spatial separation of fli1 and gata gene expression. Fli1 staining is most intense within the axial vessel (dorsal aorta, posterior cardinal vein) forming and lateral ICM cells, whereas binding of gata1 and gata2 probes becomes confined to the central portion of ICM cells beneath the dorsal aorta. This is followed by a first wave of angiogenesis, indicated by expression of fli1 and flk1. This gives rise to the intersegmental, dorsal longitudinal anastomotic and intestinal vessels. Further angiogenesis and hematopoiesis are activated in the "posterior ICM" of the tail. Here, the absence of gata1 indicates that hematopoiesis in this tissue generates myeloid rather than erythroid cells. The results supplement and validate previous, now historical morphological work in salmonids, thus aiding the elucidation of a comprehensive general scheme of angiogenic and hematopoietic development in the teleost embryo.     

Ontogeny of hemopoietic and lymphopoietic tissues in the lizard Chalcides ocellatus (Reptilia,Sauna, Scincidae)

The first and major blood-forming organ to develop in the viviparous lizard Chalcides ocellatus is the yolk sac, which exhibits prominent erythropoietic activity from as early as stage 21 through birth (stage 41). Myeloid cells and megakaryocytes are produced in the yolk sac from stage 23 onward. During lizard embryogenesis hemopoietic activity is also observed in spleen and bone marrow but in neither kidney nor liver. Cells capable of giving rise to lymphocytes both in vivo and in vitro are first found in the thymus at stage 35. Active lymphopolesis in thymus and spleen begins at stages 36 and 39, respectively. In contrast, the gut-associated lymphoid aggregates are not evident before birth.     

Anserine and free amino acid contents in the egg and fry stages of developing rainbow trout (Oncorhynchus mykiss).

1. Anserine and free amino acid contents were examined in rainbow trout eggs, yolk sac and yolk-free fry before feeding. 2. Free amino acid levels showed little change in eggs and yolk sac and had a tendency to increase in yolk-free fry. 3. No anserine was detected in the eggs and yolk sac, but yolk-free fry significantly increased its concentration after hatching. This continuous increment of anserine was discussed in relation to buffering capacity of adult fish muscle.     

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.     

鱼类Fc受体研究

Fc受体是免疫细胞表面一种重要受体分子,通过与免疫球蛋白Fc段结合触发多种生物学功能,是联系体液免疫和细胞免疫的桥梁。部分硬骨鱼中已经发现了Fc受体,在斑马鱼、斑点又尾鲴和鲤鱼中都克隆到了Fc受体的γ亚基,在鲨鱼和大西洋鲑中证明有能够与免疫球蛋白结合的Fc受体存在,并在斑点叉尾鲴、河豚和虹鳟中存在着类似α亚基的Fc受体。对鱼类Fc受体的发现和研究必将为了解鱼类的免疫机制及免疫进化提供重要的资料。     

An in vitro morphological study of the mouse visceral yolk sac and possible yolk sac immunocyte precursors

Summary Mouse visceral yolk sac has been organ cultured from 9 days of gestation, a time prior to the thymus being lymphoid, until 12 days of gestation, a time after which the thymus is lymphoid. During the culture period the endodermal epithelial cells survived well, erythropoiesis diminished, endothelial-lined cavities formed in the mesodermal mass, and cells developed which have been classified as large, medium and small immunocyte precursors. The cytoplasm of the immunocyte precursors contains polysomes, spherical mitochondria, a few profiles of rough endoplasmic reticulum, occasional granules and a large Golgi complex. This study offers morphological support for the yolk sac origin of immunocyte precursors in the mouse which may seed the thymus and liver.Supported by NIH Grant AI 13486-01     

The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules

Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss). A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2. The extracellular domain of this allomorph was expressed in E. coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein. In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells. The level of Onmy-UBA*501 expression in erythrocytes was very low. Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells. Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs. No significant expression was observed in muscle fibres, hepatocytes or neurons. These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates.     

Blood-forming potential of vascular endothelium in the human embryo

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.