Marie Aimee Teillet Catherine Ziller and Nicole M Le Douarin 1 Introduction

The understanding of several mechanisms that are essential for embryonic development has greatly benefited from cell-marking techniques that allow tracing of definite cells and their progeny, and thus, the study of their behavior and fate. A cell marker must be precise and stable; it must not interfere with normal development. The quail-chick labeling technique meets these requirements perfectly.

The principle of the method (1) is based on the observation that in all embryonic and adult cells of the quail (Coturnix coturnix japonica), the het-erochromatin is condensed in one (sometimes two or more, depending on the cell types) large mass(es) associated with the nucleolus, thus making this organelle strongly stained after DNA staining, e.g., the Feulgen and Rossenbeck staining (2). When quail cells are combined with cells of the chick (Gallus gallus) which possess, like most of the animal cells, only small chromocenters dispersed in the nucleoplasm, they are readily recognizable by the structure of their nucleus, which thus provides a permanent natural marker (Fig. 1A).

The main purpose of constructing quail-chick chimeras is to follow the fate of definite embryonic territories during development and, in this way, to discover the place and time of origin of the different groups of cells constituting certain organs. The investigations carried out on the neural crest (3) and the mapping of the neural primordium at different stages (4,5) provide good examples of the possible uses of the quail-chick chimera system for studying developmental problems. This type of study implies that the developmental processes unfold in the chimeras as they do in normal embryos. To achieve this, transplantations of quail tissues into chick embryos (or vice versa) are performed in ovo and do not consist of adding the grafts to normal embryos,

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

Fig. 1. Quail cells are differentially identified in 5-pm paraffin sections of a chimeric cerebellum (see refs. 16,21,22). (A) With the Feulgen and Rossenbeck staining (F&R), quail neuronal and glial cells show a strongly stained nucleolus (see arrowheads), whereas chick cells present almost homogeneously stained nuclei. (B) With the QCPN MAb, nucleolus of quail neurons and glial cells are more violently stained, but chick nuclei are not labeled. (C) The SMP probe hybridizes specifically with the quail oligodendrocytes. Medial and high magnifications of each staining are presented. A1, B1, C1: bar = 70 pm; A2, B2, C2: bar = 30 pm.

Fig. 1. Quail cells are differentially identified in 5-pm paraffin sections of a chimeric cerebellum (see refs. 16,21,22). (A) With the Feulgen and Rossenbeck staining (F&R), quail neuronal and glial cells show a strongly stained nucleolus (see arrowheads), whereas chick cells present almost homogeneously stained nuclei. (B) With the QCPN MAb, nucleolus of quail neurons and glial cells are more violently stained, but chick nuclei are not labeled. (C) The SMP probe hybridizes specifically with the quail oligodendrocytes. Medial and high magnifications of each staining are presented. A1, B1, C1: bar = 70 pm; A2, B2, C2: bar = 30 pm.

but of removing a given territory in the host and replacing it as precisely as possible by the equivalent region of the donor, which must be at the same developmental stage. Quail and chick are closely related in taxonomy, although they differ by the duration of their incubation time (17 d for the quail, and 21 d for the chick) and their size at birth (about 10 g for the quail and 30 g for the chick). However, during the first days of incubation, when most of the important events in embryogenesis take place, the size of the quail and chick embryos and the chronology of their development differ only slightly. Obviously, it is safe to carry out the grafts not only from quail to chick, but also from chick to quail, and to perform control chronological studies in order to discard any bias owing to differential development processes between chick and quail embryos.

Quail-chick chimeras constructed according to the above principles can hatch and survive in good health for certain time; time of survival is limited by the appearance of an immunological reaction developed by the host. Although no immune reaction against the graft takes place during embryogenesis, when the immune system is immature, the transplant triggers its own rejection, which occurs at various times after birth. For strictly neural grafts, a long delay (1-2 mo) is observed between the onset of immune maturity and the rejection (6). For neural grafts associated with other tissue grafts and for grafts of any nonneural tissue, rejection occurs as soon as maturation of the immune system is achieved (7,8).

Isochronic and isotopic grafts are not the only type of grafts used to construct quail-chick embryonic chimeras. Certain developmental processes can be studied by heterotopic, and heterochronic grafts with or without previous ablation of tissues. This was instrumental in testing the degree of determination of the neural crest cells and their derivatives (3,9) and in demonstrating the precise periodicity of the colonization of the primary lymphoid organ rudiments (thymus and bursa of Fabricius) by hemopoietic cells in birds (10).

For many years, the analysis of the quail-chick chimeras was based on the differential staining of the nucleus by either the Feulgen and Rossenbeck nucleal reaction (Fig. 1A) or any other method revealing specifically the DNA profiles in light or electron microscopy. Recently, significant progress was accomplished when species-specific antibodies recognizing either quail or chick cells of one or several types were prepared (see Subheading 3.4.3. and Fig. 1B). Nowadays, several specific quail or chick nuclear probes are also available, allowing, at the single-cell level, specific gene activities to be distinguished (see Subheading 3.4.4. and Fig 1C).

This chapter will describe the protocols of several representative examples of neural quail-chick chimeras. These particular protocols can be adapted to the graft of any other type of tissue.

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