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 Xl-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.

Chimeric animals have proved very useful for investigating aspects of development, including cell lineage, cell migration and cell-cell interactions, because the progenies of one cell type may be identified by their own specific marker.

Chimeric mice had been produced by aggregation methods (Tarkowski, 1961; Mintz, 1962) and by blastocyst injection methods (Gardner, 1968). Various markers have been developed to analyze the chimerism of mice. For example, pigmentation in the coat (Mintz, 1967) and in the retinal pigment epithelium (Tarkowski, 1964), species-specific antigen in rat-mouse interspecific chimeras (Gardner and Johnson, 1973), satellite DNA sequences in interspecific chimeras between Mus caroli and Mus musculus (Rossant et al., 1983), and strainspecific antigen in mouse-mouse chimeras (Kusakabe et al., 1988) have been used as markers. The /5-galactosidase (-gal) gene, introduced to embryonic stem cells, was recently used as a cell marker for cell lineage analysis (Kadokawa et al., 1990).

In Aves, it is well known that the quail cell has an in situ cell marker, which can be used in quail-chick chimeras to investigate developmental processes (Le Douarin, 1973). Most chimeras are constructed by transplanting differentiated organs and their chimerisms are limited to those organs. In the first attempt to produce chick chimeras with unrestricted chimerism, Marzullo (1970) produced chick chimeras between White Leghorn and Barred Plymouth Rock or Rhode Island Red breeds by injecting blastodermal cells of one breed into the other. None of the chimeras lived to hatch.

Recently, one live chick chimera was produced by transferring blastodermal cells (Petitte et al., 1990), and, using an interspecific combination, we have succeeded in hatching seven quail-chick blastodermal chimeras (Naito et al., 1991). It was demonstrated that the transferred cells differentiated to melanocytes shown by feather pigmentation (Marzullo, 1970; Petitte et al., 1990; Naito et al., 1991) and to erythrocytes and germ cells using DNA fingerprints (Petitte et al., 1990).

We present a detailed analysis of the contribution of transferred cells to host tissues in blastodermal chimeras constructed between quail and chick. These results may be helpful in establishing an effective blastodermal chimera production method.

Construction of blastodermal chimeras

Fertilized quail (Coturnix coturnix) and chick (Gallus gallus) eggs from commercial sources were used in these experiments. Donor cells were obtained from area pellucida of stage XI-XIII (Eyal-Giladi and Kochav, 1976) quail blastoderm. Stage XI-2 (stage 2; Hamburger and Hamilton, 1951) chick blastoderms were used as recipients. Quail blastoderms were removed from the egg and placed in Tyrode’s solution supplemented with penicillin (100 i.u./ml). The blastoderms were cleaned of adhering yolk. Area opaca and vitelline membrane were discarded. Then the blastoderms were dispersed by repeatedly aspirating the solution into a Pasteur pipette (pipetting), or by digesting with 0.25% trypsin/0.04% EDTA. After dissociation, the cells were washed, collected by centrifugation and resuspended in 0.5 ml of the solution. Prior to injection, the viability of cell suspension was checked by trypan blue exclusion. A 1 cm window was made in the equatorial plane of the recipient egg shell directly over the blastoderm. A micropipette containing the cell suspension was held in a micromanipulator (Narishige MM-333, Tokyo) and inserted through the albumen capsule and the vitelline membrane into the center of stage XI-XIII (pre-primitive streak stage) blastoderm (group 1), into the center (group 2), and the posterior edge (group 3) of stage XIV-2 (primitive streak formation stage) blastoderm (Fig. 1). The micropipettes were made by drawing out 1.0 mm siliconized glass capillary tubes. The tips were bevelled down (20°) to an outer diameter of about 70–100 μm. Approximately 700–2000 cells in 1–3 μl of the solution were injected into the subgerminal cavity of a chick embryo using a microinjector (Narishige IM-4B, Tokyo). The windows were tightly sealed with transparent adhesive tape (Scotch 800). The eggs were placed pointed end down in an incubator. They were incubated at 38°C in relative humidity (RH) of 60%, with a rocking motion through a 90° angle at hourly intervals. These manipulations were achieved under semisterile conditions.

Fig. 1.

Experimental designs for constructing blastodermal chimeras. The quail cells are injected into the chick blastoderm using a glass micropipette. (A) In group 1, the quail cells are injected in the center, beneath the epiblast, of the stage XI-XIII blastoderm, which has an incomplete hypoblast layer. (B) In group 2, the quail cells are injected in the center of the stage XIV-2 blastoderm, which has a complete hypoblast layer. (C) In group 3, the quail cells are injected to the posterior edge of the forming primitive streak, epi, epiblast; hypo, hypoblast; ps, primitive streak; r, rostral; c, caudal.

Fig. 1.

Experimental designs for constructing blastodermal chimeras. The quail cells are injected into the chick blastoderm using a glass micropipette. (A) In group 1, the quail cells are injected in the center, beneath the epiblast, of the stage XI-XIII blastoderm, which has an incomplete hypoblast layer. (B) In group 2, the quail cells are injected in the center of the stage XIV-2 blastoderm, which has a complete hypoblast layer. (C) In group 3, the quail cells are injected to the posterior edge of the forming primitive streak, epi, epiblast; hypo, hypoblast; ps, primitive streak; r, rostral; c, caudal.

We analyzed the contribution of the injected quail cells in the quail-chick chimeras.

Some of the quail cells injected into the unincubated chick blastoderm were incorporated into the host epiblast after 12 hours incubation though many quail cells still remained as a single mass (Fig. 2); others were in the hypoblast and were scattered in the yolk. To investigate the efficiency of incorporation of injected quail cells into the hosts, three different groups of blastodermal chimeras were constructed as described below and in Fig. 1.

Fig. 2.

Feulgen-stained section of blastoderm after 12 hours of quail cell grafting. Some of the quail cells have been incorporated into the host epiblast layer (arrows). The quail cells were distinguished from chick cells by the presence of densely stained nucleolus-associated heterochromatin. Q, injected quail cell mass still remaining. Scale bar, 40 μm.

Fig. 2.

Feulgen-stained section of blastoderm after 12 hours of quail cell grafting. Some of the quail cells have been incorporated into the host epiblast layer (arrows). The quail cells were distinguished from chick cells by the presence of densely stained nucleolus-associated heterochromatin. Q, injected quail cell mass still remaining. Scale bar, 40 μm.

As shown in Table 1, in all experimental groups, more than 50 percent of the operated embryos were histologically chimeric as observed in 100 μ m serial sections. The percentage of chimeric embryos was not significantly different among the groups (χ2=2.227, P>0.25). The extent of quail cell contribution to a chimeric embryo varied between specimens. A complete quantitative analysis was not conducted.

Table 1.

Number of chimeric embryos

Number of chimeric embryos
Number of chimeric embryos

The results of the investigation where quail cells contributed to a chimeric embryo are summarized in Table 2. In Table 2, “+” symbolizes that the quail cells contributed to the chimeric embryo and “−” symbo-

Table 2.

Distribution of quail cells

Distribution of quail cells
Distribution of quail cells

Histological procedures

After 6 days incubation, to detect of the descendants of the donor quail cells that had been incorporated into the host chick embryos, the embryos were removed from extraembryonic membrane and fixed in Zenker’s solution for 2 days. Then, they were dehydrated with a graded series of ethanols, embedded in paraffin and serially sectioned at 6 μm. Sections were stained with Feulgen-Rossenbeck’s reaction and were counterstained with Light green SF. Quail cells were distinguished from chick cells since their nuclei have a large nucleolus-associated heterochromatin mass which is positive to the Feulgen reaction (Le Douarin, 1973). Chick and quail germ cell nuclei are less different than somatic cell nuclei. Therefore, the germ cells from chick and quail were distinguished using the criteria of Hajji et al. (1988); namely, chick germ cells have many heterochromatin granules and quail germ cells have two to four discrete nucleolus-associated heterochromatin masses (ref. Fig. 6 insets). General chimerism of presumptive chimeric embryos was analyzed by light microscopic observations of the sections at about 100 μm intervals. lizes the observed absence of a contribution. In all serial sections of the thymus and the gonad of 26 definite chimeras, the quail cell contribution to the thymus epithelium and to the primordial germ cells (PGCs) were thoroughly examined. These results are also shown in Table 2. The quail cell contribution to the blood cells and to the extraembryonic membranes have not yet been examined.

The results of quail cell contribution are described below in detail according to each experimental group.

Group 1

The quail cells were injected into the subgerminal cavity in the center of stage XI-XIII chick blastoderm, having a developing hypoblast layer (Fig. 1A). The blastoderm had no apparent positional mark at this time. For the reproducibility of the distribution analysis, a micropipette was put into the central area of blastoderm.

Of the 26 operated embryos, 13 (50.0%) embryos (Table 1) had well-differentiated quail cells. The injected cells contributed to the ectodermal tissues of the head, for example, the forebrain, the midbrain (Fig. 3A), the hindbrain, the neural crest derivatives, such as the Schwann’s sheath cell, the epidermis (Fig. 3B), the lens (Fig. 3C) and the glandular pituitary lobe (Fig. 3D). No quail cells contributed to the ectodermal tissues of the trunk (Table 2). The quail cells were seen among the mesenchyme of the head. In the cranial and cervical region, most of the mesenchyme originated from the cephalic neural crest cells. It was difficult to distinguish between cells from the mesoderm and cells from the neural crest originating from the ectoderm. We concluded that no quail cells contributed to the mesodermal mesenchyme of the head because, whenever the quail cells were seen among the mesenchyme, they contributed other neural crest derivatives. Quail cells were not seen in either the heart or the mesodermal tissues of the trunk in any of the 13 chimeras. Thus, the contribution of quail cells to the ectodermal tissues was limited to the head region.

Fig. 3.

Feulgen-stained sections of operated embryos on the 6th day after grafting showing ectodermal chimerism. The quail cells contributed to the midbrain in ICh-2346 (A), the facial epidermis in ICh-2338 (B), the lens in ICh-2330 (C) and the glandular lobe of the pituitary in ICh-2337 (D). V in (A), ventricle. Scale bar, 20 μ m.

Fig. 3.

Feulgen-stained sections of operated embryos on the 6th day after grafting showing ectodermal chimerism. The quail cells contributed to the midbrain in ICh-2346 (A), the facial epidermis in ICh-2338 (B), the lens in ICh-2330 (C) and the glandular lobe of the pituitary in ICh-2337 (D). V in (A), ventricle. Scale bar, 20 μ m.

Of the 13 chimeric embryos, six (46.2%) showed endodermal chimerism (Table 3). The quail ceils contributed to the endodermal epithelium of the digestive tract, such as of the rectum (Fig. 5). The area of these chimerisms varied from the foregut to the hindgut among specimens. In another endodermal chimerism, the quail cells contributed to the thymus primordium in two chimeric embryos (ICh-2350, 2366).

Table 3.

Number of restricted chimera

Number of restricted chimera
Number of restricted chimera

Germ cells were easily identified by their nuclei, which are large and clear. It was observed that the quail cells contributed to the primordial germ cells in the gonad (Fig. 6). Of the 13 chimeras, six (46.2%) were germline chimeras (Table 3). The quail primordial germ cells were found in the extragonadal tissues too (data not shown).

In group 1, the quail cell contribution to the ectodermal tissue was limited to the head region, and no quail cells contributed to the mesodermal tissues.

Group 2

The quail cells were injected into stage XIV-2 (primitive streak formation stage) blastoderm, having a complete hypoblast layer (Fig. IB). In order to compare the distribution analysis results obtained with those from group 1, the quail cells were injected into the central area.

Of the six operated embryos, five (83.3%) were chimeric (Table 1). The quail cells contributed to the ectodermal and/or the mesodermal tissues on the anterior trunk but, with the exception of ICh-2451, not the tissues on the head. The quail cells contributed to a patch of the palate epidermis on ICh-2451. Assuming the patch was an anomaly, the contribution of quail cells with respect to the ectodermal and the mesodermal tissues was limited to the anterior trunk region.

Of the five chimeras, only one (ICh-2430) showed the endodermal chimerism (Table 3). The quail cells contributed to the pharynx, the thymus and the cloaca. Of the five chimeras, four (80.0%) were germline chimeras having quail primordial germ cells (Table 3). Thus, in group 2, the injected quail cells tended to contribute to the trunk region.

Group 3

The quail cells were injected into the posterior edge of the primitive streak cell mass at stage XIV-2 blastoderm (Fig. 1C). Of the 15 operated embryos, eight (53.3%) were chimeras (Table 1). The quail cells contributed to the mesenchymal cells of the limbs (Fig. 4). The injected cells widely contributed to the ectodermal and/or mesodermal tissues of the trunk. In all chimeras, except for ICh-2285, the injected cells did not contribute to those of head region. In ICh-2285, the quail cells contributed to the ectodermal tissues of the head but not to the ectodermal tissues on the trunk or the mesodermal tissues as was the case in group 1. Thus, assuming that ICh-2285 was an anomaly, the quail cell contribution to the ectodermal and the mesodermal tissues was limited to the trunk region (Table 2).

Figs 4-6.

Feulgen-stained sections of operated embryos on the 6th day after grafting. Fig. 4. Mesodermal chimerism. The quail cells contribute to the mesenchymal cells of the right leg in ICh-2279 (arrowheads). Fig. 5. Endodermal chimerism. The quail cells contribute to the epithelium of the rectum in ICh-2321. Fig. 6. Germline chimerism. The stromal cells and the germ cells in a gonad are easily distinguished by the nuclei; the nucleus of germ cells is large and clear. The quail primordial germ cell (arrow) is present in the chick gonad in ICh-2338. Arrowhead indicates the chick PGC. The insets show normal chick (left) and normal quail (right) PGCs at E6. Fig. 7. Feulgen-stained section after 24 hours of grafting. The quail cells contribute to the marginal epiblast layer (arrow) and the germ wall (arrowheads). In Figs 4–7, scale bar, 20 μ m.

Figs 4-6.

Feulgen-stained sections of operated embryos on the 6th day after grafting. Fig. 4. Mesodermal chimerism. The quail cells contribute to the mesenchymal cells of the right leg in ICh-2279 (arrowheads). Fig. 5. Endodermal chimerism. The quail cells contribute to the epithelium of the rectum in ICh-2321. Fig. 6. Germline chimerism. The stromal cells and the germ cells in a gonad are easily distinguished by the nuclei; the nucleus of germ cells is large and clear. The quail primordial germ cell (arrow) is present in the chick gonad in ICh-2338. Arrowhead indicates the chick PGC. The insets show normal chick (left) and normal quail (right) PGCs at E6. Fig. 7. Feulgen-stained section after 24 hours of grafting. The quail cells contribute to the marginal epiblast layer (arrow) and the germ wall (arrowheads). In Figs 4–7, scale bar, 20 μ m.

Of the eight chimeric embryos, six (75.0%) showed endodermal chimerism (Table 3). The percentàge of endodermal chimera was higher in group 3 than that in group 2. The quail cells widely and largely contributed to the epithelium of the digestive tract in four chimeras (ICh-2440, 2441, 2442, 2446) and to the thymus in one chimera (ICh-2279). Of the eight chimeras, six (75.0%) had quail primordial germ cells (Table 3). Thus, in group 3, the injected cells tended to contribute to the trunk ectodermal and mesodermal tissues, as in group 2.

Analysis showed that contribution of injected cells to the ectodermal and/or the mesodermal tissues of the head or of the trunk was significantly restricted according to the stage of host (Table 3, χ2=22.48, P<0.01). With regard to the endodermal organs and the primordial germ cells, the quail cells contributed without any significant difference in all experimental groups (endoderm: χ2=3.877, P>0.10, PGCs: p=2.633, P>0.20).

In the present study, quail cell contributions to the blood cells and to the extraembryonic membrane, such as amnion and allantochorion, have not been examined. It is probable that injected quail cells have contributed to these components. Because, as shown in Fig. 7, some of the quail cells contributed to the marginal epiblast layer and to the germ wall by 24 hours after grafting.

Another remarkable phenomenon was found in the present study. In group 2 and group 3, when the quail cells were injected into the stage XIV-2 blastoderm, ectopic epidermal cysts were discovered among the mesenchyme of the cranial region of one chimera (ICh-2282; Fig. 8) and of the cervical region of three chimeras (ICh-2428, 2430, 2450). The epidermal cysts were composed of quail cells only and were differentiated just like the sinus. The sinusoids were not joined with any host blood vessels and had no contents in their cavity.

Fig. 8.

Feulgen-stained section of the ectopic epidermal cyst. This is found among chick mesenchymal cells in the head region (ICh-2282). Scale bar, 20 μm.

Fig. 8.

Feulgen-stained section of the ectopic epidermal cyst. This is found among chick mesenchymal cells in the head region (ICh-2282). Scale bar, 20 μm.

More than 50 percent of operated embryos were histologically identified as definite chimeras in the present study. We consider the method devised by Marzullo (1970), that of injecting donor cells to another blastoderm, is accessible to all for the production of blastodermal chimeras. In previous studies, the percentage of chimeras that survived to the feather formation stage was 15.8% (Marzullo, 1970), at least 24.0% (Petitte et al., 1990) and 9.5% in our quail-chick chimera study (Naito et al., 1991), judging from the feather color. The values, however, are the proportion of melanocytic chimeras and do not always reflect the proportion of real chimeras. Our results prove that “silent” chimeras, without feather chimerism, are present among the blastodermal chimeras.

Two remarkable phenomena were induced by varying the site and the stage of injection: the restriction on the spatial arrangement of donor quail cells and the ectopic epidermal cyst.

Ectodermal chimerism was limited to the head region when the quail cells were injected into the center of the stage XI-XIII blastoderm (group 1). Some of the injected cells adhered and incorporated into epiblast while others were caught by a hypoblast sheet being formed or scattered in yolk. According to the classical fate map (Rudnick, 1948), many cells destined to form the brain are located about the center of stage XI-XIII blastoderm. It is possible that the cells that are incorporated into epiblast in this area contribute to the ectodermal tissues of the head as a result of normal morphogenesis.

In contrast to the distribution pattern in group 1, no quail cells contributed to the ectodermal tissues of the head in either group 2 or group 3 though there were a few anomalies. Such a pattern in these groups may have resulted from the movement of the destined area. That is, the injected cells may have been unable to form the ectodermal tissues on the head because the destined area had already departed from the center at stage XIV2 as a result of active cell movement in epiblast.

Ectodermal and mesodermal chimerism was widely visible in the trunk in group 2 and group 3 while no mesodermal chimerism was apparent in group 1. At stage XIV-2, the cell density at the posterior side of the blastoderm increases because the primitive streak starts to form in the posterior cellular bridge (Eyal-Giladi and Kochav, 1976). It is likely that some of quail cells are incorporated into the epiblast and others into a group of mesodermal cells, which have been invaginated through the primitive streak, when they are injected into the center of stage XIV-2 blastoderm (group 2). When they are injected into the posterior edge of stage XIV-2 blastoderm (group 3), most of them mix in and are incorporated into the primitive streak cell mass. The injected cells, therefore, probably widely contributed to the mesodermal tissues of the trunk in both group 2 and group 3. In group 1, the injected cells may have had little chance of contributing to any mesodermal tissues. The present results suggest that it is possible to obtain a chimera with chimerism limited to a particular germ layer and a certain region by varying the site and stage of donor cell injection.

Histological analysis showed that the injected quail cells contributed to the primordial germ cells. Avian primordial germ cells are of epiblastic origin (Eyal-Giladi et al., 1981), and they begin to migrate from the epiblast to the developing hypoblast at stage XII. They are carried to the germinal crescent by hypoblast and mesoderm. This process continues until stage 6 (Ginsburg and Eyal-Giladi, 1986). The cells that are destined to become PGCs may already be determined by stage X (Ginsburg and Eyal-Giladi, 1987). It is possible that either the donor cells incorporated into the epiblast first and then differentiated to PGCs or the destined or differentiated PGCs are injected and actively contributed to host germline.

The other remarkable phenomenon in the present study was the emergence of the ectopic epidermal cyst. The cysts were discovered among mesenchyme when the quail cells were injected at stage XIV-2 and at those stages in which the cell density at the posterior side of blastoderm became higher as mentioned above. They seem to have been formed by the independent development of a cluster of quail cells which remained unincorporated among host cells or by a direct introduction of cells that are not yet destined to be mesoderm into the integrated mesoderm. Although the epiblast at stage XIII had been believed to be pluripotent (Eyal-Giladi, 1984), Stem and Canning (1990) recently demonstrated that epiblast at stage XIII contains two distinct populations of cells with different developmental fates; one gives rise to ectoderm and the other gives rise to mesoderm and endoderm.

Our results are insufficient to answer the question of whether the somatic chimerism and the germline chimerism in the present study are due to the random incorporation and differentiation of pluripotent cells or due to the selective incorporation of committed cells. Further studies are now in progress in this regard. The quail-chick blastodermal chimera will be useful in clarifying aspects of development, for example, cell lineage, size of precursor cells and cell-cell interaction, by utilizing the quail cell as a marker.

Our results show that the injected quail cells contribute to the thymus primordium. The quail-chick blastodermal chimeras are able to hatch (Naito et al., 1991). These interspecific chimeras could therefore be used for investigating immunological tolerance by comparison with results from the quail-chick spinal cord chimeras (Kinutani et al, 1986, 1989) and the quailchick thymic epithelium chimeras (Ohki et al, 1987, 1988).

Germline chimera could be useful in the development of transgenic animals. Transgenic mice were obtained from the germline chimeras, constructed using the genetically modified embryonic stem cells (Gossler et al., 1986; Robertson et al., 1986). In Aves, the method using the cell as a vehicle may be more effective to introduce foreign DNA than direct gene introduction, such as retroviral infection (Salter et al., 1987; Bosselman et al., 1989) and microinjection (Sang and Perry, 1989). Our results show that some germline chimeras can be obtained by blastodermal cell transfer. The quail-chick blastodermal chimera thus could be useful in the production of transgenic chicken.

We thank Dr K. Tan for her helpful discussion. This work was supported by the Special Coordination Funds of Science and Technology Agency of the Japanese Government.

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