鳜鱼头肾的组织发生及成鱼头肾B淋巴细胞的分布

通过整体连续切片,研究了鳜鱼不同发育时期的头肾结构,并利用原位PCR方法检测了B淋巴细胞在鳜鱼头肾中的分布。在孵化后第1d观察到了肾组织,主要由肾小管组成。尔后头肾的发育经历了三个结构和功能的转变。第一个阶段为孵化后第1d到第7d,头肾作为滤过性器官存在,由肾小管及少量淋巴细胞组成。第二个阶段从第8d到第36d,是一个功能混合型阶段,头肾中既有肾小管,又有造血组织;随时间推移,肾小管数量减少,淋巴细胞数量剧增。紧接着进入第三个阶段：肾小管完全消失,头肾中开始出现大量的嗜铬细胞,头肾作为淋巴-肾上腺组织而存在。肾上腺首先出现在头肾前端,随发育成熟,集中分布于头肾门静脉周围。IgM在鳜头肾中大量表达,IgM分泌细胞分布于整个头肾组织,在血管周围有集中趋势[动物学报51(3)：440—446,20051。     

Line-restricted hemoglobin synthesis in chick embryonic erythrocytes

The presence of embryonic hemoglobin in early definitive erythrocytes was checked by indirect immunofluorescence assay, using specific antibodies raised against embryonic Hb P. As positive control we used anti-Hb A which reacted with the alpha A chain shared by the minor embryonic Hb E and the adult Hb A. The assay was performed using blood smears from embryos between 6 and 15 days of incubation and yolk sac sections from embryos between 4 and 6 days. Hb P was never detected in the definitive line in circulating erythrocytes or in maturing erythroblasts still sequestered in the blood islands of the yolk sac. The expression of the 'specific' embryonic genes is thus restricted to the primitive line (as the 'specific' adult beta gene is restricted to the definitive line), and the hemoglobin switch is the result of the progressive substitution of the primitive line by the definitive one.     

人卵黄囊造血的探讨

采用卵黄囊组织切片、涂片的形态学、细胞化学染色、造血干/祖细胞体外培养及CD_(34)单克隆抗体免疫荧光检测等方法研究表明:人卵黄囊中存在造血岛,造血岛内由于造血微环境的特点致使此期造血主要向红系分化。血岛中检测出CD_(34)~ 细胞,比例高于胎肝及成人骨髓,干/祖细胞于体外培养形成红系集落。结论:人胚胎期造血源于卵黄囊。     

Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Embryonic macrophages of early rat yolk sac: Immunohistochemistry and ultrastructure with reference to endodermal cell layer

Macrophages are multifunctional cells that participate in numerous biological processes; they actively phagocytose foreign particles and cell debris. Embryonic tissue macrophages are present at early stages of mammalian development; their ontogeny and function is still under investigation. Our study used immunohistochemistry and electron microscopy to investigate early rat yolk sac macrophages using mouse antirat macrophage monoclonal antibodies (mAb) Mar 1 and Mar 3 produced by our laboratory. Mar 3 mAb revealed the first emergence of immature macrophages in the rat yolk sac at fetal day nine coinciding with the beginning of yolk sac haemopoiesis that consisted mainly of erythropoiesis, while Mar 1 mAb detected specifically rat yolk sac macrophages at about the 13th to 14th day of gestation. Immunoreactivity against Mar mAbs was mainly located in the yolk sac endodermal cell layer, which may signify endodermal origin of the yolk sac macrophages. Ultrastructurally mature yolk sac macrophages contained numerous endocytic vesicles or vacuoles, well-developed Golgi saccules and many electron dense granules in their cytoplasm and a number of microvillous projections from the cell surface. After establishment of the circulation between yolk sac and embryo, Mar 3 positive cells were also demonstrated inside fetal undifferentiated mesenchymal tissue at fetal day 12. The study demonstrated the first emergence of immature yolk sac macrophages being among the earliest haemopoietic cells formed in mammalian development. Thus, Mar mAbs managed to detect macrophage differentiation antigens through their development early in the rat yolk sac.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

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.     

Ontogeny of the immune system inSebastiscus marmoratus: Histogenesis of the lymphoid organs and effects of thymectomy

Synopsis The ontogenetic development of the immune system in a marine teleostSebastiscus marmoratus was studied by histological examination and removal of the thymus. The pronephros and the spleen had been differentiated at the time of birth and contained small numbers of haemopoietic cells. In contrast to most vertebrates, the rudiments of the thymus were first visible 1 week post-birth in the dorsoposterior part of the pharynx, the same location as in the adults. However, small lymphocytes first appeared in the thymus of fish at 3 weeks of age, followed by the pronephros at 4 weeks and the spleen at 6 weeks. Complete or partial suppression of the antibody response to sheep red blood cells (SRBC) occurred in fish that were thymectomized at 1.5 months of age and immunized 2 weeks later, and a marked decrease in lymphocytes was observed in the pronephros and spleen. The thymectomy of adult fish also caused reduced serum antibody titres in fish immunized 1 month after the operation. These results suggest that the thymus plays an essential role in the development of the immune system and its functions continue into adult life.     

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.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos I. Influence of the embryo preparation method

Summary Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode’s solution containing [³H]amino acids (mixture) at a concentration of 27 μCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo “naked”); d) naked embryos cut randomly into pieces (Day 10 mebryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked mebryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first methods (see above) fulfilled the conditions required at the best. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic Analysis of Cell Proteins).     

Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands

Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.     

Chick erythrocyte membrane antigens: Their expression in irradiated chicks grafted with haematopoietic tissues from different origins

Three erythrocyte populations (E, EA, A) were characterized during normal chick development by presence on cells of the embryonic (E) or adult (A) antigen or both (EA). Embryonic and adult stem cells were grafted into irradiated animals in order to distinguish the respective influence of stem cell origin and physiological conditions in the production of antigens. Adult marrow stem cells produce A erythrocytes. Embryonic stem cells (from 6- or 11-day-old embryo yolk sac) give rise first to E, then to EA populations. These results confirm the existence of adult stem cells with their own properties. It was not possible to decide whether the E and EA populations arise from a unique embryonic stem cell or from the existence of two stem cell populations.     

ONTOGENESIS OF HAEMATOPOIETIC SITES IN BRACHYDANIO RERIO (HAMILTON-BUCHANAN) (TELEOSTEI)*

The first haematopoietic sites in Brachydanio rerio are formed by the “intermediate cell mass”, situated between somite and lateral plate. The sequential sites are in the endocardium, pro- and mesonephros. The results are compared with those of Pterophyllum scalare, which has its first blood anlage on the yolk sac.     

Alternatively Spliced Tissue Factor Is Not Sufficient for Embryonic Development

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.     

Haemoglobin-oxygen affinity in developing embryonic erythroid cells of the mouse

Summary Haemoglobin-oxygen equilibrium curves have been measured in developing embryonic mouse erythroid cells. At 11.5 days gestation, shortly after blood islands from the yolk sac have formed but before the placenta is complete, erythrocytes have a high affinity for oxygen and a reverse Bohr effect below pH 7.0 (Figs. 1, 2, 3). By 13.5 days both the Bohr shift and the shape of the equilibrium curves are similar to those obtained from adult mice. From 13.5 days onwards, 2,3-diphosphoglycerate plays an important role in regulating oxygen affinity (Figs. 2, 4). It is suggested that the oxygen equilibrium properties of embryonic erythrocytes are adapted to service the developing embryo with oxygen under hypoxic and hypercapnic conditions in the intra-uterine environment up until the time when the embryonic circulation makes contact with the placenta.     

Distinct origins of adult and embryonic blood in Xenopus

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.     

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.     

Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a(-/-)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.