Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites.

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/ aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

Hematopoiesis: progenitors and their genetic program.

Recent advances in our understanding of blood cell development have included the identification of a hematopoietic progenitor derived from endothelial cells and the possibility that the embryonic and fetal environment can reprogram gene expression in adult hematopoietic stem cells.

Tumorigenic effects mediated in the avian embryo by one or more oncogenes associated with v-myc.

We have previously shown that introduction of the v-myc oncogene in chick or quail embryos at E3 induces rapidly growing heart rhabdomyomas. We now report that a retrovirus containing one or two other oncogenes induces additional pathologies specified by the v-myc-associated oncogene. The v-mil/myc combination introduced at E3 induces, in addition to heart rhabdomyomas, tumors of proliferating cells aggregated onto the luminal aspect of vessels in both chick and quail embryos. In the quail these cells react positively with the quail-specific mAb QH1, which recognizes endothelial and most hemopoietic cells, while chick intravascular cells do not react with the chick-specific mAb VIA2 that recognizes hemopoietic cells. Thus the v-mil/myc tumors appear to be of endothelial origin. The v-myb-ets/myc combination injected at E3 induces cardiorhabdomyomas and aggressive VIA2-positive hemopoietic tumors in chick embryos, but only the v-myc-induced cardiorhabdomyomas in quail embryos. When injected into hatched animals, v-myc alone transforms hemopoietic and perhaps endothelial cells, but not cardiac cells. Thus the developmental stage at which a cell type can be transformed by v-myc and another associated oncogene depends on as yet undefined species-specific factors. More importantly, several examples of oncogene cooperation in vivo are adduced by these experiments. The type of cell transformed is specified by the viral oncogene combination.

Cooperative effect of v-myc and v-erbA in the chick embryo.

We show that a construct designated as MAHEVA, which encodes oncogenes v-myc from MH2 virus and v-erbA from AEV under the control of the LTR of MH2, induces rapidly growing heart rhabdomyosarcomas, when it is injected in E3 but not E5 chick embryos. A similar pathology has previously been observed with MC29, within the same limited time frame. The tumors, which expressed P61-63myc, P75gag-erbA and Pr76gag proteins were detectable from E14 onwards. Compared with MC29, MAHEVA induced a secondary anomaly, not detectable prior to E17. This is the appearance of cartilage nodules within the heart rhabdomyosarcomas. The constant location of these nodules inside the rhabdomyosarcomas and their delayed appearance suggests that the chondrocytes originate from myoblasts prevented from differentiating by the expression of the v-myc product. This interpretation is supported by the appearance of chondrocytes in E3 heart muscle cells infected in vitro with MAHEVA.

An identical effect mediated by thyroid deficiency or oncogene v-erbA in the chick embryo.

We have shown earlier that the association of v-myc and v-erbA (MAHEVA construct) is responsible for the appearance of a specific phenotype in chick embryos inoculated at E3. This phenotype comprises rapidly growing heart rhabdomyomas (induced by v-myc alone) and within these tumors secondarily appearing cartilage nodules (Bachnou et al., Oncogene 6: 1041-1047, 1991). Here we report that v-erbA can be replaced by thyroid deficiency. When decapitated embryos were inoculated with virus MC29 (v-myc alone) or when v-myc inoculated embryos were treated with thiourea, 100% of the embryos reaching E17 to E19 displayed tumoral hearts bearing cartilage nodules. We thus report in vivo evidence that v-erbA acts by antagonizing the effects of thyroid hormones. Remarkably, thyroid deficiency rendered embryos more sensitive to the effect of v-myc, since 100% developed heart rhabdomyomas and cartilage nodules, versus about 70% affected when either v-myc or MAHEVA were inoculated. Thyroid deficiency did not alter the species-specific character of transdifferentiation, since only chick but not quail embryos developed cartilage nodules after thyroidectomy or MAHEVA infection.

Oncogenes and avian development.

In order to detect signs of oncogene activity and elucidate their possible role in avian ontogeny we implemented two different strategies. One was to detect either the protein product or messenger RNA in situ at various stages of development. The other was to try and disturb development with retroviruses carrying one or several oncogenes in their activated forms. Time- and tissue-specific expression of c-myc was apparently not related to particular phases of cell evolution, such as population amplification. Rather the presence of c-myc immunoreactive product at particular stages appeared to depend on cell types. c-myb and c-ets messenger RNAs were found expressed preferentially in the blood system, respectively in hemopoietic and differentiating endothelial cells. The developing embryo heart was found to be uniquely sensitive to the effect of retroviruses provided that two conditions were respected. The first was the injection of the virus or construct prior to E3.5. The second was the presence of the v-myc gene, whether alone or associated with one or several other v-onc. In such cases a large proportion (70%) of chick and all quail embryos developed multiple heart rhabdomyosarcomas within 10 days. In chickens the association of a second v-onc or of two others induced the formation of secondary tumors, whose type was determined by the nature of the other oncogene(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the compartments of the immune system in the avian embryo.

Because of the easily traceable quail-chick marker, the early development of the immune system, in particular the connections between the differentiating organs, has been extensively studied in birds. These earlier studies clarified the origin, movements and potentialities of progenitors, and the colonization of primary lymphoid organs. With the advent of an array of similar markers in mammals, it will now be possible to identify the interactions between the stromal components of lymphoid organs and the progenitors, which give rise to the effector cells of the immune system. Rules established over the last 20 years, such as extrinsic origin of stem cells, periodic colonization of the thymus, unique colonization of the bursal rudiment, are now extended by new cellular and molecular acquisitions. Relationships between vasculogenesis, angiogenesis and hemopoiesis, acquisition of xenogeneic tolerance by thymic epithelium grafting, avian-specific strategy of immunoglobulin diversification, and identification of thymotaxin, the polypeptide responsible for progenitor attraction to the thymus, are reviewed.

A comparative study of the GVH-R in the chick embryo: effects of various allogeneic cells and various administration routes.

Allogeneic cells inoculated into immunologically immature chick embryos induce a Graft-Versus-Host Reaction (GVH-R), one of the manifestations of which is splenic enlargement. Splenomegaly varies with the composition of allogeneic cell suspensions, route of inoculation and age of recipient embryos at inoculation and autopsy. We have compared the splenomegaly induced by lymphoid cells from spleen or bursa of Fabricius with that induced by peripheral blood lymphocytes, after these cell preparations were either grafted on the chorioallantoic membrane (CAM) at 9 or 13 days or injected intravenously at 13 days. We confirm our previous findings that bursa cells grafted on the 9-day CAM induced splenomegaly as efficiently as other cell preparations. On the other hand, bursa cells did not mediate such an effect after they were injected at 13 days. On the whole, a hierarchy of decreasing efficiency is observed from PBL to spleen and finally to bursal cells. It appears that, as the recipient embryo ages, this hierarchy becomes more marked.

Functional capacities of chick embryo thymocytes in the mixed lymphocyte reaction.

Thymocytes from chick embryos homozygous for the B19 haplotype of the major histocompatibility locus were tested in a one way MLR either as responder or stimulator cells, against adult peripheral blood lymphocytes from B14/14 or B19/19 strains. 13-day embryonic thymocytes were strongly stimulated by adult allogeneic PBL. By contrast 16-day thymocytes were unresponsive. This difference might be related to the rhythmic waves of stem cell entry and multiplication which characterize the ontogeny of the avian thymus. Mitomycin-treated thymocytes from 13-day or 16-day embryos were both efficient in stimulating thymidine uptake by adult allogeneic PBL.

Serial transfer of GVH-R splenomegaly in chicken embryos.

Using histocompatible chicken strains B14 and B19, we demonstrate that the capacity to induce a GVH-R in an embryo could be induced precociously by the reaction itself. While naive chickens display this capacity around 3 to 4 weeks posthatching, embryos engrafted with adult allogeneic cells at E13 or E8 became endowed with this capacity at E20 and E15, respectively. Furthermore, this acceleration could be obtained by serial transfer of splenic cells through a sequence of three embryos undergoing the GVH-R. The highest efficiency in transfer was realized by regularly alternating the MHC haplotype at each transfer. It is concluded that the original cells from the adult donor may be partially responsible for the transfer, but that cells from the successive embryos are also certainly involved. Thus, maturation of the embryonic immune system appears accelerated by a GVH-R.

Ontogeny of the GVH-R inducing capacity, in conventional and germ-free chickens.

Three strains of MHC homozygous chickens were used to study the ontogeny of the GVH-R. It was found that this function of the immune system was acquired with a delay in animals raised in germ-free conditions. In our previous work, we had shown that, contrary to expectation, bursal cells were capable of mounting a GVH-R against embryos, and actually were particularly efficient in this regard when compared to spleen cells. The present experiments have disclosed that splenomegaly induction may be dissociated from the toxic effect that bursal cells also exert on recipient embryos.

Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites.

Two hemogenic sites are present in mouse embryos before the onset of fetal liver hemopoiesis. While the yolk sac provides for immediate erythropoiesis, an intraembryonic region encompassing the dorsal aorta produces definitive hematopoietic stem cells, as shown experimentally. At early developmental stages this region, that we named paraaortic splanchnopleura, produces multipotent progenitors. At the time of fetal liver colonisation, the paraaortic splanchnopleura further evolves into aorta, gonads and mesonephros (AGM) and contains progenitors capable of long term multilineage reconstitution. Only then are cytologically identifiable collections of early hemopoietic cells present in various arteries and in the mesentery. The present report focuses on the antigenic characterisation of immature hemopoietic progenitors in order to trace back the intraembryonic precursors at earlier developmental stages. CD34, an antigen expressed by immature progenitors and endothelial cells, labels all potential hemopoietic sites. Markers, supposed to counterstain endothelial cells and spare CD34+ hemopoietic cells, also stain various hemopoietic cells. The meaning of these shared antigenic expressions between cells of the endothelial and hemopoietic lineages in the early embryo is discussed.

Intraembryonic hematopoietic stem cells.

Intraembryonic hematopoietic stem cells (HSC) were first detected in avian chimeras associating an embryo with a yolk sac (YS). Cell markers were used to construct chimeras. The results showed that YS blood precursors undergo primitive erythropoiesis and become extinct, whereas intraembryonic precursors colonize rudiments of blood-forming organs and settle in the bone marrow as self-renewable HSC. The model is valid in the mouse as shown by in vitro cultures of cells obtained from embryo structures or YS separated prior to circulation. This approach, as well as restoration of irradiated adults, demonstrates that YS precursors have a limited potential compared with embryo precursors. The emergence of hematopoietic precursors in both YS and embryos is closely linked to the emergence of the endothelial network and is restricted to the mesoderm layer associated with endoderm.

Emergence of endothelial and hemopoietic cells in the avian embryo.

During organogenesis, endothelial cells develop through two different mechanisms: differentiation of intrinsic precursors in organ rudiments constituted of mesoderm associated with endoderm, and colonization by extrinsic precursors in organs constituted of mesoderm associated with ectoderm (Pardanaud et al. 1989). On the other hand, both types of rudiment are colonized by extrinsic hemopoietic stem cells. In the present work we extend our former study by investigating the hemangioblastic (i.e. hemopoietic and angioblastic) potentialities of primordial germ layers in the area pellucida during the morphogenetic period. By means of interspecific grafts between quail and chick embryos, we show that splanchnopleural mesoderm gives rise to abundant endothelial cells, and to numerous hemopoietic cells in a permissive microenvironment, while somatopleural mesoderm produces very few cells belonging to these lineages, or none. Thus we confirm that the angioblastic capacities of the mesoderm differ radically, depending on its association with ectoderm or endoderm. Furthermore, at this embryonic period, both endothelial and hemopoietic potentialities are displayed by splanchnopleural mesoderm. However the site of emergence of intraembryonic hemopoietic stem cells appears spatially restricted by comparison to more widespread angioblastic capacities.

Does the paraxial mesoderm of the avian embryo have hemangioblastic capacity?

In a previous study of the hemangioblastic capacity of lateral plate mesoderm, we showed that the endoderm-associated splanchnopleural layer is capable of giving rise to both endothelial and hemopoietic cells while the ectoderm-associated somatopleural layer is not (Pardanaud and Dieterlen-Lièvre 1993a). In order to complete the inventory of territories able to produce these two cell lineages, we assayed the paraxial mesoderm, and report the results here. Quail somites or segmental plates were treated with mab QH1+complement in order to eliminate attached aortic endothelial cells, which cling to the ventral aspects of these structures. They were grafted in the limb bud or the coelom of chick host, since these sites promote the differentiation of endothelial and hemopoietic cells, respectively. Vascular development and hemopoietic cell emergence were analyzed using QH1 immunocytology. Segmental plate and somites both produced abundant endothelial cells. In addition, the segmental plate gave rise to small groups of hemopoietic cells when grafted in the coelom.

Potential intraembryonic hemogenic sites at pre-liver stages in the mouse.

In the course of a previous experimental study on the early development of the mouse embryo hemopoietic system, we found that, at the 10-25 pairs of somite stages, the para-aortic splanchnopleure contains hemopoietic progenitors. Trying to discover a structural basis for this potentiality, we have looked for cytological signs of hemopoiesis in the embryo proper between 8.5 and 12 days post-coitum, i.e. prior to full-blown fetal liver hemopoiesis. Two suggestive findings are reported: (1) intra-arterial hemopoietic cells aggregates are present in the omphalomesenteric and umbilical arteries and to a lesser degree in the dorsal aorta; (2) cells groups resembling yolk sac blood islands are observed in the mesentery. The intra-arterial aggregates are strikingly similar to the intra-aortic clusters of avian embryos. These cytological aspects provide the anatomical basis underlying recent functional data that revealed the hemogenic capacity of the para-aortic splanchnopleure.

The c-ets1 protooncogene is expressed in human trophoblast during the first trimester of pregnancy.

Expression of the c-Ets1 protooncogene which codes for a transcription factor is associated with neovascularization and invasive processes. In order to determine c-Ets1 expression at the mRNA level, during the process of implantation during the first trimester of human pregnancy, samples of trophoblast were retrieved at the time of legal abortion and processed for in situ hybridization. We found that c-Ets1 mRNAs are transcribed in the endothelial cells of villous trophoblast and in the extravillous trophoblastic cells invading the uterine vessels. However, no transcript was found in maternal endothelial cells. We conclude that c-Ets1 plays a role in angiogenesis occurring in the development of the villous tree and is involved during the invasive process of the endometrium and maternal vessels by trophoblastic cells; this latter physiological event is crucial for a normal development of the fetus, its failure leading to pathological cases. We suggest that the role of the c-Ets1 protooncogene is related to the regulation of metalloproteinase genes transcription, a gene family which is known to be a target for Ets protein.

Ontogeny of the endothelial system in the avian model.

The avian model provides an experimental approach for dissecting the origin, migrations and differentiation of cell lineages in early embryos. In this model, the endothelial network was shown to take place through two processes depending on the origin of endothelial precursors: vasculogenesis when angioblasts emerge in situ, angiogenesis when angioblasts are extrinsic. Two different mesodermal territories produce angioblasts, the somite which only gives rise to endothelial cells and the splanchnopleural mesoderm which also produces hemopoietic stem cells. Potentialities of the mesoderm are determined by a positive influence from the endoderm and a negative control from the ectoderm. The presence of circulating endothelial precursors in the embryonic blood stream is also detected.

Migration of erythropoietic and prebursal stem cells from the early chicken embryo to the yolk sac.

Lymphocyte development and ontogenetic changes in erythroid cells have been studied in chick-chick yolk sac-embryo chimeras constructed of histoincompatible partners. The results obtained indicate that the early chick yolk sac produces transiently erythroid stem cells whereas definitive erythrocytes are derived from the intraembryonic stem cells. Such a change from the yolk sac-derived cells into embryo-derived cells is not observed in the lymphocytes which are exclusively derived from the embryo-borne stem cells. Experiments with cell transfers from the chimeric yolk sacs demonstrate that erythropoietic and prebursal stem cells migrate from the early embryo to the yolk sac during the second to the seventh day of incubation. The results obtained also exclude the de novo generation of prebursal stem cells in the yolk sac.

Avian models in developmental biology.

The avian embryo is uniquely amenable to experimental manipulation. The most widely used models are chimeras resulting from heterotopic or orthotopic exchanges of rudiments between chick and quail embryos, according to Le Douarin's technique (1969). Cell migrations and fates are traced in these chimeras either through the identification of quail cell nuclei stained for DNA or by means of monoclonal antibodies that recognize a particular lineage in only one of the two species. The ontogeny of the hemopoietic and endothelial lineages, as enlightened through appropriately designed chimeras, is reviewed in the present article. Homologies recently disclosed in mouse and human embryo are emphasized. Finally, the possibilities afforded by retroviral somatic transgenesis in the avian embryo will be envisaged.