Implants of quail thymic epithelium generate permanent tolerance in embryonically constructed quail/chick chimeras

In situ implantation of a quail wing bud into a chick embryo at 4 days of incubation (E4) regularly results in the normal development of the implant followed by its acute rejection starting within two weeks post-hatching. If the epithelial thymic rudiments of the quail donor are implanted into the branchial arch area of the chick recipient after partial removal of its own thymic primordia, a chimeric thymus develops in the chick host and this induces tolerance to the quail wing by the chick recipient. The species identity of cells in chimeric thymuses was mapped using Feulgen-Rossenbeck' staining and immunolabelling with monoclonal antibodies directed against quail or chick B-L antigens. Certain lobes contained only chick cells both at the stromal and hemopoietic cell levels. Others had a quail epithelial stroma containing host hemopoietically derived cells. Only chimeras in which at least one third of the thymic lobes were chimeric showed permanent tolerance to the grafted wing. Since the two species exhibit distinct developmental rates, we decided to study the kinetics of thymic involution after birth. Although the changes in thymus weight and histological structure are fundamentally similar in quail and chick, those in the quail start about 7-8 weeks earlier. In the chimeric thymuses, the lobes whose epithelial cells were quail involuted at the rate of control quail showing no influence of the hemopoietic thymic compartment in this process. Tolerance induced by the thymic epithelium during embryogenesis and in early postnatal life was maintained after a profound involution of the quail thymic graft had occurred.     

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.     

Sexual phenotype of avian chimeric gonads with germinal and stromal cells of opposite genetic sexes

The respective roles of germinal and stromal cells in determining the sexual phenotype of the gonad were analyzed in chimeric gonads obtained by surgical recombination between young avian blastodiscs in ovo. Equivalent territories were exchanged between two blastodisc, in order that the germinal crescent and the gonad territory had a different origin (fig. 3). Embryos used for these experiments carried a sex linked pigment mutation, that made it possible to diagnose the genetic sexes of germ cells and stroma at the time when the gonad was retrieved for examination. On the basis of species, three types of combination were performed: chick germ cells in chick or quail stroma, quail germ cells in chick stroma. In each chimera, the genetic sexes of the two gonadal cell populations could be identical or opposite. However it appeared that the germ cell population was not always homogeneous. In some grafting schemes, ectopic germ cells, located outside the germinal crescent, contributed to the colonization of the experimental gonad. These germ cells were from the same territory as the stroma element of the gonad, i.e., they were of the same species and the same genetic sex. Whatever the case, in 87 chimeras that were studied, the sex phenotype of the gonads always corresponded to the genetic sex of the stroma. Thus the genetic sex of germ cells has no role in the sexual differentiation of the gonadal rudiments.     

Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells

The origin of prospective M cells, which are median neuroepithelial cells that become wedge-shaped during bending of the neural plate and eventually form the midline floor of the neural tube, was determined by constructing quail/chick chimeras and using the quail nucleolar marker to identify quail donor cells in chick host blastoderms. Two possible sites of prospective M-cell origin in the epiblast were examined: a single, midline rudiment located just rostral to Hensen's node and paired rudiments flanking the cranial part of the primitive streak. Our results suggest that M cells arise exclusively from the midline, prenodal rudiment. From this rudiment, M cells extend caudally throughout the entire length of the neuroepithelium. This new information on the origin of prospective M cells will aid in the analysis of their role in neurulation.     

Seeding of the 10-day mouse embryo thymic rudiment by lymphocyte precursors in vitro

The thymic rudiment was removed from the mouse embryo at 10 days of gestation, while it was still included in the 3rd branchial arch. When cultured alone, either in vitro or on the chick chorioallantoic membrane (CAM), it failed to develop as a lymphopoietic organ and remained in an epithelial state. If it was associated in transfilter culture with various types of hemopoietic organs from either embryonic or adult mice (e.g. yolk sac, fetal liver, thymus, bone marrow), it became seeded by lymphoid precursor cells and underwent a normal histogenetic process. If the donor and the receptor explants belonged to different strains of mice, the thymus that developed in culture was chimeric: thymic stroma cells (i.e., epithelial and connective cells) were of the receptor explant type, whereas the lymphoid population was of the donor type. Two genetic markers were used to label the thymic cell types, the Thy-1-1-Thy-1-2 system and the isozymes of the glucose phosphate isomerase.     

Avian thymic accessory cells

On the basis of morphologic criteria and ingestion of latex particles, two basic types of accessory cells can be identified from quail and chick thymuses, dendritic cells, and macrophages. By using embryonic grafting techniques, we show that cells of this lineage enter the thymus during the initial colonization of the epithelial thymic rudiment by hemopoietic cells, and within a few days differentiate into cells exhibiting properties of glass adherence, Ia expression, and formation of rosettes with thymocytes. It appears that the precursors of this lineage undergo extensive, but finite, proliferation and are eventually replaced by further influx of the accessory cell lineage. In chimeric grafts, quail thymocytes were seen forming rosettes with chick accessory cells, and vice versa, indicating, as in the interaction between the epithelial cells and thymocytes, that the molecules involved in thymocyte-accessory cell association can interact across species barriers in our system.     

Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.     

Avian spinal cord chimeras. I. Hatching ability and posthatching survival in homo- and heterospecific chimeras

Quail-chick spinal cord chimeras were constructed by grafting isotopically, at the brachial level, the neural tube of a quail embryo into a chick of the same developmental stage. The chimeras were allowed to hatch and their behavior and survival after birth were observed. We found that if white Leghorns of the rapid-feathering strain were taken as hosts, the ability of the operated embryos to hatch was higher than in the slow-feathering wild-type chickens. The important point arising from this study is that the establishment of the neuronal circuits and of the connexions of the grafted neurons to their peripheral and central targets occurs between cells of two different species in such a way that normal behavior of the chimera is ensured. These animals can stand, walk, and fly as normal chickens do. Moreover, the size reached by the fragment of quail spinal cord implanted into the chick axial structures is larger than it would have been in the donor at the same age. This results in perfectly normal morphogenesis of the vertebrae which develop from the chick somites at the level of the graft. The pigment pattern of the chick feathers colonized by quail melanoblasts of graft origin is very close to that of the quail, albeit somewhat different, probably due to the different size of the feathers in the two species. Normality of the chimeras is only transient. During the second month of their life they develop a neurological syndrome characterized first by the paralysis of the wings and later by their inability to stand. In strong contrast, spinal cord chimeras constructed between two histoincompatible chickens, remain healthy and seem to develop a complete tolerance to the graft. What seems to be the development of an immune rejection of the grafted neural tube in the quail-chick spinal cord chimeras is now under investigation.     

Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo.

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macrophage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macrophage-like cells were mostly distributed in the mesenchyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.     

Chemical and immunological characterization of proteoglycans of embryonic chick calvaria

We have previously demonstrated in quail embryos grafted on chick yolk sacs the existence of intraembryonic stem cells responsible for definitive hemopoiesis. In order to determine the origin of these cells, we now examine the diffuse hemopoietic processes within the avian embryo's mesoderm. At 4–5 days of incubation in the two species, basophilic cells were found throughout the dorsal mesentery. At 6–8 days these cells became very numerous and built up dense foci at the level of branching of the anterior and posterior cardinal veins. These cells often infiltrated the wall of lymph spaces and channels and were also present in the lumen of blood vessels. Such locations support the interpretation that these basophilic cells represent early stages of hemopoietic differentiation. At 8–10 days, erythropoiesis or granulopoiesis was seen in the foci, which then regressed rapidly. The foci maximal development coincided with the period of colonization of the intraembryonic organ rudiments. In “yolk sac chimeras,” the foci were always constituted by quail cells, indicating their intraembryonic origin. The primordial origin of the intramesodermal cells remains to be determined. A likely source might be the ventral wall of the aorta which appeared to shed cells into the lumen and into the mesentery in the 3-day embryo.     

Split tolerance induced by chick embryo thymic epithelium allografted to embryonic recipients

To test the capacity of the epithelial component of the chick embryo thymus to induce tolerance to major histocompatibility complex (MHC) antigens, pre-colonized thymic rudiments were grafted into chick embryonic recipients. Semi-allogeneic or allogeneic transplantations were done between two lines of chickens histocompatible at the MHC locus. Approximately 10% of these thymic chimeras hatched and were studied 3 mo after hatching. Thymic grafts were not rejected by the allogeneic host. The tolerance of chimeric chickens to thymus donor MHC antigens was tested by using a skin graft rejection test and a graft-vs-host (GvH) assay. Chimeric chickens that received an MHC-incompatible thymic graft during the embryonic life tolerated skin graft with the MHC haplotype of the thymus donor. Nevertheless, the lymphocytes within the thymic graft, the host thymus, and the blood were tolerant to the host MHC antigens but were alloreactive in GvH reaction for the MHC antigens of the thymic graft type. These results suggest that the epithelial component of the thymus when taken before the starting of the colonization by hemopoietic precursors and grafted into an early chick embryonic host can induce a tolerance for the MHC determinants involved in allograft rejection but not in the GvH reaction.     

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation

Summary A method of in vitro culture for uterine quail blastoderms has been developed, which allows them to develop from cleavage throughout gastrulation and further: stages 4–10 of Hamburger and Hamilton (1951). The method consists of cultivating the blastoderms on egg albumen in a vertical position; this permits about 50% of the blastoderms explanted before area pellucida formation to develop bilateral symmetry and to form normal primitive streak, somites and head structures. Development of the blastoderms explanted after their area pellucida was already formed, occurred normally and was not influenced by their spatial position in the culture.This work was performed as part of project no. 09.7.1.5.2 of the Polish Academy of Sciences     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. II. Immune response of bursectomized chicks and chimeras and post-natal rejection of the grafted quail bursas

Two methods to bursectomize chick embryos before hemopoietic cell seeding of the bursa of Fabricius were compared in this work: section of the tail region at E3 including the presumptive bursal territory, and selective removal of the bursa at E5. Hatching ability is better with the former method, but survival rate and effectiveness of bursectomy are favored with the second, novel technique. Moreover, selective removal of the bursa at E5 can be followed by in situ engraftment of a quail bursa and construction of quail-chick bursal chimeras. The immune response of bursaless birds and bursal chimeras has been studied. Total absence of the bursa does not prevent a few B cells from differentiating and nonspecific Ig (IgM and/or IgG) from being secreted. As reported previously, bursaless birds, however, are unable to mount an immune response by producing specific antibodies. This immune function is restored by the graft of a quail bursa. The microenvironment of the bursa, although heterospecific, allows the expansion of the B cell population and generates the repertoire of the B cell antigen receptors. This process takes place during late embryonic and early postnatal life because the grafted quail bursal stroma is subjected to immune rejection from 2 to 3 wk after birth in all chimeras, which are, however, perfectly immunocompetent.     

Development of hematopietic and lymphoid tissues in conjoint bone marrow and thymus transplants]

The model of heterotopic transplantation of the mixture of bone marrow and thymus fragments was used to study the interaction of hemopoietic and lymphoid tissues under their direct contact. The bone marrow and thymus fragments of adult mice F1 (CBAXXC57BL) were transplanted separately or in the mixture under the kidney capsule of mice of the same strain. During the whole period of observation (from 10 days up to 14 months), the development of bone marrow and thymus fragments in the joint transplants proceeded independently, no "mixed" stroma appeared, and the stroma of each organ ensured the differentiation characteristic of its organ. The development of joint transplants somewhat differs from that of isolated transplants: on the 10th day a greater amount of hemopoietic tissues was noted in the former; the bone marrow component increases continuously up to 6 months (vs. 1--2 months in the isolated transplants); the bone and hemopoietic tissues predominate in the joint transplants by 14 months, the amount of thymic tissue markedly decreases but it does not disappear completely.     

Extraembryonic origin of circulating endothelial cells

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.     

The Claude Bernard lecture, 1987. Embryonic chimeras: a tool for studying the development of the nervous and immune systems

Chimeras have been constructed in the avian embryo following the observation of the particular structure of the interphase nucleus in the Japanese quail (Coturnix coturnix japonica). In all embryonic and adult cell types of this species a large amount of heterochromatin is associated with the nucleolus, making quail cells readily distinguishable from those of the chick where the constitutive heterochromatin is evenly dispersed in the nucleus. These structural differences have been used to devise a cell-marking technique through which cell migrations and cell interactions during embryogenesis can be followed in the embryo in ovo by grafting quail cells into chick embryos or vice versa. This method was applied to the ontogeny of the neural crest and of the immune system. Recently quail-chick chimeras have been allowed to hatch and the immunological status of the embryonic grafts after birth scrutinized. Xenogeneic tissue grafts made in the embryo are rejected after birth with a more or less prolonged delay according to the nature of the graft. However, rejection can be prevented and a permanent state of tolerance induced for the embryonic tissue grafts by isotopically implanting the thymic epithelium from the same quail donor.     

SENSITIVITY OF EARLY EMBRYONIC THYMIC LYMPHOID DEVELOPMENT TO 3H-THYMIDINE OF HIGH SPECIFIC ACTIVITY

The pulse technique, using high specific activity ³H-TdR to selectively kill cells in cell cycle, was applied to the thymic anlagen of chick embryos. With optimal specific and total ³H-TdR activities and pulse times of 2–4 hr the subsequent lymphoid development in organ culture of the thymic anlagen of 10-day-old chick embryos could be almost completely inhibited. The most important effect of the ³H-TdR was on the lymphoid precursor cells of the anlagen. The thymic epithelium appeared more resistant to ³H-TdR and allowed a lymphoid development of pulsed anlagen grafted to the chorioallantoic membrane of chick embryos when new lymphoid precursor cells were provided. The lymphoid precursor cells of the thymic anlagen of 10-day-old chick embryos therefore appeared to be in cell cycle with short generation time. The thymic anlagen of 8-, 9- and 10-day-old but not 7-day-old embryos showed a lymphoid development in organ culture. They did not differ with respect to the sensitivity to hot pulses of ³H-TdR. Thus no evidence of a lag in the onset of lymphoid precursor cell proliferation during the development of the early embryonic chick thymus was noted.