Chapter 2 Quail–Chick Transplantations

Publisher Summary

This chapter provides methods for quail–chick transplantations. The method is based on the observation that all embryonic and adult cells of the quail possess condensed heterochromatin in one large mass in the center of the nucleus, associated with the nucleolus. When combined with chick cells, quail cells can readily be recognized by the structure of their nucleus, thus providing a permanent genetic marker. The transformation from the early neural ectoderm to the mature brain involves an enormous complexity that comes about via differential growth of various regions of the neuroepithelium, extensive cell migrations, and assembly of the very complicated wiring taking place between the neurons of the central nervous system. Quail–chick chimeras provide a means to unveil some of the mechanisms underlying these complex processes. Quail and chick are closely related in taxonomy, although they differ by their size at birth and by the duration of their incubation period. For many years, analysis of chimeras relied on the differential staining of the nucleus by either the Feulgen–Rossenbeck reaction or other DNA staining methods, such as acridine orange or bizbenzimide, which could be combined with immunocytochemistry. Subsequently, species-specific antibodies that recognize either quail or chick cells have been developed. In addition, cell type-specific reagents are available either as monoclonal antibodies or as nuclear probes that distinguish, at the single resolution, whether a cell produces a particular product and if it belongs to the host or the donor.

Introduction

Because it is easily accessible to experimentation, the avian embryo has long been a popular subject of study for embryologists. The combination of cells or rudiments from two related avian species (quail, Coturnix coturnix japonica, and chick, Gallus gallus), adapted as a means to identify cells that migrate during embryogenesis (Le Douarin, 1973a), has served as a useful tool for the study of many developmental biology problems. The use of quail–chick grafts was motivated by the need to selectively label defined groups of cells in order to follow their pathways of migration and identify interactions during prolonged period encompassing morphogenesis and organogenesis.

The method is based on the observation (Le Douarin, 1969) that all embryonic and adult cells of the quail possess condensed heterochromatin in one (sometimes two or three) large mass(es) in the center of the nucleus, associated with the nucleolus. As a consequence, this organelle was strongly stained with the reaction by Feulgen and Rossenbeck (1924). When combined with chick cells, quail cells can readily be recognized by the structure of their nucleus, thus providing a permanent genetic marker (Fig. 1).

The main purpose in constructing quail–chick chimeras was to follow the fate of defined embryonic territories not only to their ultimate destinations and fates in the mature bird but also at intermediate time points during embryonic development. The investigations carried out on the neural crest provide a good example of the utility of employing the quail–chick chimera system, as they have uncovered the nature of the tissues and organs derived from this structure as well as showing the migratory pathways taken by neural crest cells (NCCs) en route to their destination (Le Douarin, 1982, Le Douarin and Kalcheim, 1999)—another clear example stems from the mapping of the neural primordium (Le Douarin, 1993). The transformation from the early neural ectoderm to the mature brain involves an enormous complexity that comes about via differential growth of various regions of the neuroepithelium, extensive cell migrations, and assembly of the very complicated wiring taking place between the neurons of the central nervous system (CNS). As will be shown in this chapter, quail–chick chimeras provide a means to unveil some of the mechanisms underlying these complex processes.

This type of study assumes that the developmental processes occur in the chimeras as they do in the normal embryo. To achieve this, transplantations of quail tissues into chick embryo (or vice versa) do not consist of adding a graft to an otherwise normal embryo but rather in removing a given territory in the recipient and replacing it as precisely as possible by the equivalent region of the donor of the same developmental stage.

Quail and chick are closely related in taxonomy, although they differ by their size at birth (the quail weight is about 10 g and that of the chick is 30 g) and by the duration of their incubation period (17 days for the quail and 21 days for the chick). However, during the first week of incubation, when most of the important events take place in embryogenesis, the size of the embryos and the chronology of their development differ only slightly. When the dynamics of development of a given organ are to be studied by the quail–chick substitution method, the exact chronology of development of this organ in each species must first be established. This is a prerequisite to choose the exact stage of donor and host embryos at operation time as well as for later interpretation of the results. An example of this requirement can be found in the study that was performed on the origin of the calcitonin‐producing cells that develop in the ultimobranchial body and of the enteric nervous system (see Le Douarin, 1982, and references therein).

In order to rule out possible differences in developmental mechanisms between the two species, it is beneficial not only to carry out the grafts from quail to chick (the most often performed because it is easier to recognize one isolated quail cell within chick tissues than the other way around) but also to perform reciprocal control experiments from chick to quail.

In addition to isochronic–isotopic substitutions, grafts can be placed into normal embryos without previous extirpation of the corresponding territory to study certain developmental processes. For example, this was instrumental in demonstrating the colonization of the primary lymphoid organ rudiments (thymus and bursa of Fabricius) by hemopoietic cells and in showing that this process occurs according to a cyclic periodicity (Le Douarin et al., 1984, and references therein).

Quail–chick chimeras generated by isotopic–isochronic grafts of embryonic territories appear to develop normally. To further verify this, the chimeras were examined after hatching and postnatal survival. This was tested in a variety of experimental designs: neural chimeras in which parts of the CNS (including the brain) or the peripheral nervous system of chick were replaced by their quail counterpart (Kinutani et al., 1986, Le Douarin, 1993, and references therein) or by immunological chimeras in which the thymus rudiment of the chick was replaced by that of the quail (Ohki et al., 1987). Neural chimeras are able to hatch and exhibit an apparently normal sensory motor behavior even when their brain is chimeric. Because quail and chick exhibit species‐specific behavioral characteristics, these chimeras can be analyzed to determine whether a particular trait is linked to a specific area of the neuroepithelium, as demonstrated for certain songs of quail and chick (Balaban et al., 1988).

However, the analysis of quail–chick chimeras after birth is time‐limited. Although there is no immune rejection during embryogenesis, when the immune system is immature, the transplant is rejected at various times after birth. For neural grafts, a long delay is observed between the onset of immune maturity and rejection due to the relative isolation of the CNS from circulating lymphocytes by the blood–brain barrier as well as the low immunogenicity of the neural cells. This delay, which may be more than a month, allows behavioral studies to be carried out in early postnatal life.

The immune rejection of the implant raised a series of interesting problems concerning the mechanisms of self–nonself discrimination. This demonstrated an unexpected role of the epithelial component of the thymus in tolerance to self (Belo et al., 1989, Martin, 1990, Ohki et al., 1987, Ohki et al., 1988). In allogeneic (chick–chick) grafts, it was found that embryonic neural grafts between major histocompatibility complex (MHC)‐mismatched chick species trigger little or no immune response from the host. This led to identification of brain areas responsible for an autosomic form of genetic epilepsy (Fadlallah et al., 1995, Guy et al., 1992, Guy et al., 1993, Teillet et al., 1991).

For many years, analysis of chimeras relied on the differential staining of the nucleus by either the Feulgen–Rossenbeck reaction or other DNA staining methods such as acridine orange or bizbenzimide (Hoechst 33258, Serva, Heidelberg) which could be combined with immunocytochemistry (see, for example, Fontaine‐Perus et al., 1985, Nataf et al., 1993). Subsequently, species‐specific antibodies that recognize either quail or chick cells have been developed. In addition, cell type‐specific reagents are available either as monoclonal antibodies (MAbs) or as nuclear probes that distinguish, at the single resolution, whether a cell produces a particular product and if it belongs to the host or the donor.

Section II reviews the technical requirements and experimental procedures for production and analysis of quail–chick chimeras.