Avian spinal cord chimeras. Further studies on the neurological syndrome affecting the chimeras after birth

These experiments bring new information concerning the immunological status after birth of quail → chick spinal cord chimeras. Such birds have been produced using recipient flocks of chickens different from those in our previous experiments. The breakdown of tolerance after hatching has been recorded and found to vary with the origin of the embryos. Chickens of broiler JA 657 strain and of a white leghorn strain raised in Japan started to exhibit signs of neural graft rejection later in life than the white leghorn chickens from a French breeder used in our previous studies. As previously described, in two animals, long-term tolerance was observed only for allogeneic chick → chick neural tube grafts. In one chimera the neurological syndrome resulting from rejection was reversible, and no signs of immune attack of the grafted central nervous tissue could be detected at sacrifice. This and other observations reported in this article strongly support the contention that the host immune response to foreign neural tissues starts in peripheral nerves and ganglia where no blood-brain barrier exists rather than in the spinal cord. A humoral response of the host against non-polymorphic quail antigens present on fibroblasts was observed in all birds at the time of rejection.     

Postnatal development of a demyelinating disease in avian spinal cord chimeras

Xenogeneic spinal cord chimeras were constructed by grafting fragments of quail neural primordium into chick embryos at 2 days of incubation. Hatched birds displayed normal motor behavior for about 5 to 7 weeks, whereupon they developed a neurological syndrome; in the grafted spinal cord the pathological signs of the disease were very similar to those of the active plaques of multiple sclerosis and of the lesions of experimental allergic encephalomyelitis and neuritis, including Ia expression by brain capillary endothelia, rupture of the blood-brain barrier, leukocytic infiltration in the nervous tissue, and demyelination. In the animals at the most advanced stage of the disease an autoimmune attack occurred on the host's nervous system with the same histopathological signs.     

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.     

A new marker for identifying quail cells in embryonic avian chimeras: a quail-specific antiserum

The characterization of cell behavior in quail chick chimeras has greatly increased our knowledge of the ontogeny of embryonic cell populations and the role of cell-cell interactions in development. We sought to extend the value of avian chimeras by producing a marker that would recognize cell surface components and that could be used instead of the traditional nuclear marker to identify quail cells within chimeras. We describe here a quail-specific antiserum produced by injecting chickens with a membrane fraction of 6-10-day quail embryos. By use of peroxidase coupling of a second antibody, serum reactivity was tested in tissue sections of normal quail and chick embryos and of somitic mesoderm and neural tube chimeras. The primary time period examined was 6-10 days of development. At these stages, the antiserum recognizes only quail cells and stains both plasma membrane-associated and cytoplasmic cell components. The latter characteristics allow the identification of quail axons in chimeras and facilitate visualization of quail cells at low magnification. We show that antiserum staining can also be used to identify quail cells in culture and can be combined with orthograde HRP labeling of neurons.     

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.     

Pigment patterns in neural crest chimeras constructed from quail and guinea fowl embryos

The pattern of pigmentation in bird embryos is determined by the spatial organization of melanocyte differentiation. Some of the results from recent, neural crest transplantation experiments support a model based on a prepattern in the feathers; others could be interpreted in terms of a nonspecific pattern resulting from a failure of the crest cells to read the positional values in another species. To distinguish between these possibilities, the crucial test is to construct chimeras from two species with different pigment patterns. We have examined the wing plumage of quail and guinea fowl embryos. The quail has a characteristic pattern of pigmented and unpigmented feather papillae, whereas the guinea fowl shows uniform pigmentation. Chimeras were constructed by grafting wing buds isotopically between embryos. The wing buds were transplanted before they had become invaded by neural crest cells. Quail wing buds grafted to the guinea fowl developed, in most cases, a pigment pattern resembling that of the quail and not that of the guinea fowl. A few cases became uniformly pigmented and appeared to represent nonspecific patterns. The reciprocal grafts (guinea fowl wing buds grafted to the quail) became pigmented all over. We found evidence that the timing of melanocyte differentiation is controlled by cues in the feather papillae. Some cases developed a severe inflammatory response. The model which best accounts for these findings--and which can account for inconsistencies in previous reports--is the following. A prepattern is present in the feathers and this can control the differentiation of melanoblasts, even if they come from a different species. The local cues which constitute the prepattern are not positional values. In some chimeras melanoblasts fail to respond to the prepattern and so a nonspecific pattern of uniform pigmentation is produced.     

Mouse-chick neural chimeras

Embryonic chimera production was used to study the developmental processes of the mouse nervous system. The difficulty of performing in situ transplantation experiments of neural primordium of mouse embryo was overcome by isotopic and isochronic grafting of mouse neural tube fragments into chick embryo. Mouse neural tube cells differentiated perfectly in ovo and neural crest cells associated with the grafted neural tube were able to migrate and reach the normal arrest sites of host neural crests. Cranial neural crest cells penetrated into chick facial areas and entered into the development of dental bud structures, participating in vibrissa formation. Depending on graft level, in ovo implanted mouse neural crest cells formed different components of the peripheral nervous system. At trunk level, they located in spinal ganglia and orthosympathetic chains and gave rise to Schwann cells lining the nerves. When implanted into the lumbosacral region, they penetrated into the enteric nervous system. At the precise 18-24 somite level, they colonized host adrenal gland. Mouse neural tube was involved in the mechanisms required to maintain myogenesis in host somites. Furthermore in ovo grafts of mouse cells from genetically modified embryos, in which many mutations induce early death, are particularly useful to investigate cellular events involved in the development of the nervous system and to identify molecular events of embryogenesis.     

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.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

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.     

Mitogenic effect of muscle on the neuroepithelium of the developing spinal cord

A previous study revealed that segments of bowel grafted between the neural tube and somites of a younger chick host embryo would induce a unilateral increase in cellularity of the host's neural tube. The current experiments were done to test the hypotheses that muscle tissue in the wall of the gut is responsible for this growth-promoting effect and that the spinal cord enlargement is the result of a mitogenic action on the neuroepithelium. Fragments of skeletal (E8-15) or cardiac muscle (E4-14) were removed from quail embryos and grafted between the neural tube and somites of chick host embryos (E2). Both skeletal and cardiac muscle grafts mimicked the effect of bowel and induced an increase in cell number as well as a unilateral enlargement of the region of the host's neural tube immediately adjacent to the grafts. The growth-promoting effect of muscle-containing grafts was restricted to the neural tube itself and was not seen in proximate dorsal root or sympathetic ganglia. The action of the grafts of muscle was neither species- nor class-specific, since enlargement of the neural tube was observed following implantation of fetal mouse skeletal muscle into quail hosts. Grafts of skeletal muscle or gut increased the number of cells taking up [3H]thymidine in the host's neuroepithelium as early as 9 h following implantation of a graft. The increase in the number of cells entering the S phase of the cell cycle preceded the increase in cell number. These observations demonstrate that muscle-containing tissues can increase the rate of proliferation of neuroepithelial cells when these tissues are experimentally placed together.     

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.     

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.     

Intestinal coelomic transplants: a novel method for studying enteric nervous system development

Normal development of the enteric nervous system (ENS) requires the coordinated activity of multiple proteins to regulate the migration, proliferation, and differentiation of enteric neural crest cells. Much of our current knowledge of the molecular regulation of ENS development has been gained from transgenic mouse models and cultured neural crest cells. We have developed a method for studying the molecular basis of ENS formation complementing these techniques. Aneural quail or mouse hindgut, isolated prior to the arrival of neural crest cells, was transplanted into the coelomic cavity of a host chick embryo. Neural crest cells from the chick host migrated to and colonized the grafted hindgut. Thorough characterization of the resulting intestinal chimeras was performed by using immunohistochemistry and vital dye labeling to determine the origin of the host-derived cells, their pattern of migration, and their capacity to differentiate. The formation of the ENS in the intestinal chimeras was found to recapitulate many aspects of normal ENS development. The host-derived cells arose from the vagal neural crest and populated the graft in a rostral-to-caudal wave of migration, with the submucosal plexus being colonized first. These crest-derived cells differentiated into neurons and glial cells, forming ganglionated plexuses grossly indistinguishable from normal ENS. The resulting plexuses were specific to the grafted hindgut, with quail grafts developing two ganglionated plexuses, but mouse grafts developing only a single myenteric plexus. We discuss the advantages of intestinal coelomic transplants for studying ENS development. This work was supported by NIH K08HD46655 (to A.M.G.).     

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.     

Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Differentiation of peptidergic neurones in quail-chick chimaeric embryos

The problem raised in this work was whether peptidergic neurones with vasoactive intestinal peptide (VIP)-and substance P-like immunoreactivity could develop in chimaeric embryos in which quail neural crest cells had been implanted into chick at an early developmental stage. Differentiation of peptide-containing nerve somas was looked for in different situations: i) when the quail neural primordium had been grafted orthotopically and isochronically into the chick host either at the adrenomedullary (level of somites 18-24) or at the vagal (level of somites 1-7) levels of the neural axis; ii) when the quail adrenomedullary neural primordium had been heterotopically implanted at the vagal level of the chick host. In all conditions, VIP- and substance P-like immunoreactivity were observed in a number of quail neurones located either in the peripheral ganglia of the trunk at the level of the graft (in orthotopic grafts of the adrenomedullary neural primordium) or in the enteric ganglia of the chick gut (in the other types of grafts). The developmental stage at which the first neurones become detectable in the host conforms to the genetic characteristics of the effector cells, i.e. they differentiate at the same stage in normal quail neuroblasts and in quail neuroblasts transplanted into the chick host. In contrast, the distribution of the peptidergic neurones in the host depends on the tissue into which the neural crest cells migrate and not on their origin in the neural axis and their fate in normal development.     

Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern

We are using a monoclonal antibody, QH-1, as a label for angioblasts in quail embryos to study vascular development. Our previous experiments showed that major embryonic blood vessels, such as the dorsal aortae and posterior cardinal veins, develop from angioblasts of mesodermal origin that appear in the body of the embryo proper (Coffin and Poole: Development, 102:735-748, '88). We theorized that there are two separate processes for blood vessel development that occur in quail embryos. One mechanism termed "vasculogenesis" forms blood vessels in place by the aggregation of angioblasts into a cord. The other mechanism, termed "angiogenesis," is the formation of new vessels by sprouting of capillaries from existing vessels. Here we report the results of microsurgical transplantation experiments designed to determine the extent of cell migration taking place during blood vessel formation. Comparison of the chimeras to normal embryos suggests that the vascular pattern develops, in part, from the normally restricted points of entry of angioblasts into the head from the ventral and dorsal aortae. Transplantations of quail mesoderm (1-15 somite stage) into the head of 5-15 somite chick hosts resulted in extensive sprouting and in migration of single and small groups of angioblasts away from the graft sites. Transplantations into the trunk resulted in incorporation of the graft into the normal vascular pattern of the host. Lateral plate mesoderm was incorporated into the dorsal aortae and individual sprouts grew between somites and along the neural tube to contribute to the intersomitic and vertebral arteries, respectively.     

The distribution of neural crest-derived Schwann cells from subsets of brachial spinal segments into the peripheral nerves innervating the chick forelimb.

Neural crest cells from brachial levels of the neural tube populate the ventral roots, spinal nerves, and peripheral nerves of the chick forelimb where they give rise to Schwann cells. The distribution of neural crest cells in the developing forelimb was examined using homotopic and heterotopic chick-quail chimeras to label neural crest cells from subsets of the brachial spinal segments. Neural crest cells from particular regions of the spinal cord populated ventral roots and spinal nerves adjacent to or immediately posterior to the graft. Crest cells also populated the brachial plexus in accord with their segmental origins. In the forelimb, neural crest cells populated muscle nerves with anterior brachial spinal segments populating nerves to anterior musculature of the forelimb and posterior brachial spinal segments populating nerves to posterior musculature. Similar patterns were seen following both homotopic and heterotopic transplantation. In both types of grafts, the distribution of neural crest cells largely matched the sensory and motor projection pattern from the same spinal segmental level. This suggests that neural crest-derived Schwann cells from a particular spinal segment may use sensory and motor fibers emerging from the same segmental level as substrates to guide their migration into the periphery.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.