The somitic level of origin of embryonic chick hindlimb muscles

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.     

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.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ

Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments, where also differences between cell types were noted. We asked whether similar differences between species and between cell types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to 7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in vivo. Accepted: 5 January 2000     

Pathogenesis of persistent truncus arteriosus induced by nimustine hydrochloride in chick embryos

Nimustine hydrochloride (ACNU) is a nitrosourea derivative anticancer agent which has been shown to cause persistent truncus arteriosus in chick embryos. The objective of this study was to confirm the teratogenic effects of ACNU on the cardiovascular system of chick embryos and to determine whether ACNU induces persistent truncus arteriosus by interfering with neural crest cells. Various doses of ACNU ranging from 10 to 200 micrograms were injected under the chorioallantoic membrane of chick embryos on the third day of incubation. Saline solution was used as the control. After 10 to 11 days of incubation, 242 (46%) survivors of the 524 treated eggs were obtained. The survival rates of the embryos and the frequencies of cardiovascular anomalies were dose dependent. Of 146 embryos with cardiovascular anomalies, 104 (71%) had persistent truncus arteriosus. Ventricular septal defect and double-outlet right ventricle were seen in 37 (25%) and one (1%), respectively. Aortic arch anomalies were seen in 116 embryos (79%). Quail-chick chimeras (chick embryos with quail cardiac neural crest) were treated with 50 micrograms of ACNU and examined histologically 24 hours later. These chimeras showed dying neural crest cells in the pharyngeal arches. Dying cells were also noted in the neural tube, cranial ganglia, retina, and otocyst. These results suggest that persistent truncus arteriosus in chick embryos treated with ACNU is induced by neural crest cell death.     

Demonstration of the neural crest origin of type I (APUD) cells in the avian carotid body,using a cytochemical marker system

Summary The biogenic amines present in the carotid body Type 1 cells of two avian species (Japanese quail and chicken) were identified, by microspectrofluorometry of formaldehyde-induced fluorescence, as dopamine and 5-hydroxytryptamine respectively. These and other cytochemical properties establish the cells as members of the APUD series.Grafts of the neural rhombencephalic primordium from 6 to 10-somite quail embryos were implanted in the appropriate region of chick embryos of the same age. After up to 11 days incubation the carotid bodies of the host were freeze-dried and treated with hot formaldehyde vapour. The carotid body Type 1 cells in the chick host were identified, by the presence of dopamine and the absence of 5-HT, as cells from the quail neural crest.The dopamine phenotype in cells of quail origin thus provides a cytochemical marker which may be used for other allograft experiments. The present work confirms earlier findings, using a biological (nuclear chromatin) marker, which showed the avian carotid body to be of neural crest 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.     

The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.     

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.     

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.     

Interspecific cell markers and cell lineage in birds

A cell marking technique based on the structural differences existing between the interphase nucleus in two closely related species of birds, the chick and the Japanese quail, is described. In all embryonic and adult cell types of the quail, a large mass of heterochromatin is associated with the nucleolus making quail and chick cells easy to identify at the single cell level after application of any DNA-specific staining procedure and also at the electron microscope level. This method has been largely used to construct chimeras in ovo and to study dynamic processes such as cell migrations or cell lineage segregation during ontogeny. Recently monoclonal antibodies specific for either quail or chick antigenic determinants (for example, class II MHC antigens) have been prepared, increasing the interest of the quail-chick chimera system as an experimental model.     

A study of the development of the hemopoietic system using quail-chick chimeras obtained by blastoderm recombination

The anterior part of the area pellucida from quail blastoderms extending to the 10th or the 17th somite level was substituted for the corresponding region of chick blastoderms in ovo. Reciprocal grafts were also carried out. In external appearance the resulting chimeras had a composite body, one species contributing the head and neck or the head, neck, and wing regions while the other species provided the remainder. The chimeras were always grafted on a chick yolk sac. The cellular composition of hemopoietic organs according to species was analyzed by means of the quail-chick nuclear difference, in 39 viable chimeras at 11–13 days of incubation. The thymus composition depended on the level of the boundary between the two species. In chimeras in which the quail contributed head and neck, the thymic epithelial stroma was made of quail cells while the lymphoid population was of chick origin. In contrast, when the quail contribution also extended to the wings, both thymic stroma and lymphoid cells were of quail origin. In reciprocal combinations, in which head and neck were of chicken origin on a quail body, a different result was obtained: no lymphoid cells were present in the thymus which was reduced to its epithelial component and this was of chick origin. On the other hand, if the chick contribution extended to the wings, as in the reciprocal combination, all thymus components were of chick origin. The spleen and the bursa of Fabricius in most instances did not differ in their cellular composition from the surrounding tissues; however in some chimeras a minor admixture of cells of the other species was also found. Overall these results suggest that hemopoietic stem cells destined to colonize intraembryonic organs arise in territories derived from the whole area pellucida excluding the prospective head-neck region. Furthermore, each hemopoietic organ rudiment appears to be colonized by precursors derived from adjacent territories.     

The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos.

We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, alpha Enhb-1, which recognizes chick En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71% of integrated grafts had lost En expression (n = 17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n = 14). In addition, in 86% of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1-positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.     

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.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method to obtain avian germ-line chimaeras using isolated primordial germ cells.

Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0.048% PGCs and the concentrated fraction contained 3.9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38 degrees C for 24 h, and then fixed. Serial sections were stained with the periodic acid-Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63.6 +/- 5.3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39.3 +/- 4.5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.