Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras

Authors

  • Levent Karagenç,

    1. Department of Histology and Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Bati Kampusu, Isikli, Aydin, Turkey
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  • Mustafa Sandikci

    1. Department of Histology and Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Bati Kampusu, Isikli, Aydin, Turkey
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Levent Karagenç, Department of Histology and Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Bati Kampusu, Isikli, Aydin, Turkey. E: lkaragenc@adu.edu.tr; leventkaragenc@yahoo.com

Abstract

The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-μm intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm.

Introduction

Experimental chimeras have been used by developmental biologists to investigate a range of questions important in developmental biology (Papaioannou & Dieterlen-Lievre, 1984). Among these, quail-chick interspecies chimeras generated through the transplantation of tissue grafts from one species to another at early stages of development have been particularly instrumental in examining the developmental origin of various tissues, including the thymus, hematopoietic stem cells, craniofacial mesenchyme and neural-crest derivatives (Papaioannou & Dieterlen-Lievre, 1984; Le Douarin et al. 1996, 2008). In these heterospecific chimeras, quail cells are distinguished from the chick cells using either Feulgen-Rossenback staining (Le Douarin, 1973) or quail-specific antibodies such as the monoclonal antibody QCPN (Le Douarin et al. 1996, 2008).

In addition to these well-established approaches in avian tissue grafting, avian chimeras can also be generated by transferring cells from one embryo to another at pre-streak stages of development. The first attempt to produce what is now referred to as blastodermal chimeras was made by Marzullo (1970), who transferred clumps of cells from unincubated blastoderm to a recipient embryo and demonstrated that chimeric embryos can be obtained between White Leghorn and Barred Plymouth Rock or Rhode Island Red breeds. These studies, however, did not culminate in the production of any live chicks. This was accomplished later in a landmark study by Petitte et al. (1990) with the transfer of dissociated blastodermal cells obtained from stage X (Eyal-Giladi & Kochav, 1976) Barred Plymouth Rock embryos into the subgerminal cavity of recipient White Leghorn embryos at about the same developmental stage. Petitte et al. (1990) also demonstrated that apart from melanocytes derived from the neural crest, donor cells could also give rise to erythrocytes and most importantly to germ cells in the recipient embryo. In a similar study, Naito et al. (1991) demonstrated that blastodermal cells from unincubated quail embryos could be introduced into stage X (EG&K) chicken embryos to form interspecies chimeras. The expression of quail-like feather pigmentation in recipient embryos indicated that quail cells differentiated into cells of the melanocyte lineage in the recipient chicken embryo (Naito et al. 1991).

These pioneering studies clearly demonstrated that blastodermal cells obtained from a stage X embryo (EG&K) could contribute to both somatic and germ cell lineages, but fell short in analyzing the contribution of donor cells to various tissues of the recipient. In an attempt to determine the fate of blastodermal cells introduced into the subgerminal cavity of a recipient embryo, Watanabe et al. (1992) constructed quail-chick blastodermal chimeras. These authors transferred blastodermal cells obtained from the area pellucidae of stage XI–XIII embryos (EG&K) into the subgerminal cavity of stage XI–2 (EG&K, Hamburger & Hamilton, 1951) chick blastoderms and analyzed the distribution of donor cells in resulting chimeras using Feulgen-Rossenback staining to identify cells of quail origin (Le Douarin, 1973). Quail cells were detected in subsequent stages of development in all tissues deriving from the three germ layers, including forebrain, midbrain, hindbrain, neural crest derivatives, the epidermis, lens, glandular pituitary lobe, epithelium of the digestive tract and thymic primordium (Watanabe et al. 1992).

It is important to note that in all studies conducted to date in generating blastodermal chimeras, only cells from the area pellucida (Petitte et al. 1990; Naito et al. 1991; Watanabe et al. 1992) were used, with the exception of the work described by Petitte et al. (1993). In an attempt to examine the potential of cells from the area opaca to form chimeras, Petitte et al. (1993) established that cells from the area opaca have the potential to form melanocytes in the recipient embryo, although less frequently than cells of the area pellucida. These results suggest that at least a subpopulation of cells present within the area opaca has the potential to differentiate into various cell lineages when introduced into the subgerminal cavity of a recipient embryo. However, the question remains as to the extent of the incorporation of cells from the area opaca to embryonic as well as to extra-embryonic tissues of the recipient embryo. In an attempt to address this issue, quail-chick chimeras were constructed in the present study by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm and the distribution of quail cells were examined using the QCPN monoclonal antibody. Evidence gathered in the present study indicates that cells of the area opaca injected into the subgerminal cavity of a recipient chick embryo can be identified not only in extra-embryonic tissues, but also in various embryonic structures derived from the ectoderm and mesoderm.

Materials and methods

Construction of blastodermal chimeras

Fertilized quail (Coturnix coturnix) and white Leghorn chick (Gallus gallus) eggs were used in the present study. Donor cells were obtained from area opaca of stage X–XII (Eyal-Giladi & Kochav, 1976; Sellier et al. 2006) quail blastoderm. Freshly laid, unincubated quail eggs were cracked open and the blastoderms were removed by adherence to filter-paper rings (Petitte et al. 1990) and placed in a Petri dish with 20 mL of sterile phosphate-buffered saline (PBS, pH 7.4) supplemented with penicillin (100 IU mL−1) and streptomycin (100 μg mL−1). The blastoderms were cleaned off the adhering yolk. The area opacae of stage X–XII (EG&K) embryos was pooled in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM–FBS) and were dissociated enzymatically using 0.0125% trypsin and 0.005% ethylenediaminotetraacetic acid (EDTA) in PBS at 37 °C for 10 min. The trypsin/EDTA solution was gently removed and the cells were resuspended in 500 μL DMEM–FBS. The fragmented area opacae were dispersed further to obtain a single cell suspension by gentle trituration using a 200-μL pipette tip. The cells were centrifuged at 311 g for 5 min and resuspended in fresh DMEM–FBS. Prior to injection, a sample of cell suspension was used to determine viability (> 90%, by trypan blue exclusion).

To prepare recipient blastoderms, freshly laid eggs were swabbed with 70% alcohol and a 1-cm window was made in the equatorial plane of the eggshell directly over the blastoderm. Approximately 2000 cells were injected into the subgerminal cavity in 2–4 μL of medium using a finely drawn micropipette. The micropipettes were made by drawing out 1.0-mm glass capillary tubes. The tips were then beveled down to an outer diameter of about 80 μm. The windows were tightly sealed using parafilm. The eggs were placed pointed end down in an incubator and were incubated at 37.5 °C in relative humidity of 60%, with a rocking motion through a 90° angle at hourly intervals.

Histological procedures

To detect descendants of donor quail cells in resulting quail-chick blastodermal chimeras, the embryos along with the surrounding amniotic membrane were removed after 6 days of incubation. Samples of the yolk sac from each embryo were also isolated. Tissue samples and embryos were fixed in 5% formal-acetic for 48 h at room temperature, dehydrated with a graded series of ethanols and embedded in paraplast X-tra (Sigma). To determine the distribution of donor cells in presumptive chimeric embryos, three 7-μm serial sections were taken at 100-μm intervals. Sections were bleached in 3% H2O2 for 10 min, washed in PBS (pH 7.4) twice for 5 min each, and blocked by 5% normal goat serum in PBS (pH 7.4) for 15 min. The monoclonal antibody QCPN (DSHB) was used for differential diagnosis of quail cells. To this end, supernatant of QCPN hybridoma cells grown in DMEM supplemented with 20% FBS was applied to sections overnight at 4 °C. Sections were rinsed once in PBS and then washed two times in PBS at room temperature for 10 min each. Sections were then incubated in universal biotinylated link (Dako Cytomation LSAB+system-HRP) for 1 h at room temperature, rinsed and washed in PBS as above and then incubated in streptavidin HRP (Dako Cytomation LSAB+system-HRP) for 1 h at room temperature. After a final rinse and wash in PBS, cells expressing the antigen were detected using 3,3′ diaminobenzidine tetrahydrochloride (DAB) solution (3 mg mL−1 in Tris–HCl, pH 7.6 with 3% H2O2). The reaction was stopped by washing the sections in PBS.

Results

The aim of the present study was to analyze the tissue distribution of cells in quail-chick blastodermal chimeras constructed by transferring dissociated cells obtained from the area opaca of stage X–XII quail embryos into the subgerminal cavity of the unincubated chick blastoderm. To this end, a total of 10 chimeric embryos were constructed, among which only seven survived to day 6 of embryonic development and were included in the study. Five of seven putative chimeras were demonstrated to be definite chimeras by the presence of QCPN-positive cells in various tissues. The extent of the quail cell contribution to chimeric embryos varied between specimens. A complete quantitative analysis, however, was not conducted. Contributions of quail cells to blood cells and to cells of the amniotic fluid were not examined.

Quail cells of donor origin were detected in various tissues of the recipient chick embryo derived from the ectoderm and mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in the diencephalon and the optic cup (tissues derived from the neural ectoderm), as well as in tissues derived from the surface ectoderm, as is the case for the epidermis and the lens. Apart from tissues derived from the ectoderm and mesoderm, quail cells were also observed in the yolk sac and the amniotic membrane of the resulting chimeras. Table 1 shows a complete list of chimeric tissues. No cells of quail origin were observed in tissues derived from the endoderm. In general, the frequency of chimerism for tissues derived from the ectoderm was higher than that determined for tissues derived from the mesoderm (Table 1).

Table 1.   Distribution of quail cells in the 6-day-old recipient chick embryo.
Germ layerRecipient tissueCode number of chimerasFrequency of chimerism (%)
1234567
EctodermDiencephalon++++80
Optic cup++++80
Lens+++60
Epidermis+++++100
MesodermSomitic mesoderm+20
Heart++40
Extra-embryonic ectoderm and endodermYolk sac++++80
Amniotic membrane++++80

Optic cup and diencephalon were among the chimeric tissues derived from the neural ectoderm. Both the sensory and the pigment layers of the optic cup contained cells of the quail origin (Fig. 1A,D). Although a quantitative analysis was not performed, the number of quail cells within the sensory layer appeared to be higher than that detected in the pigment layer. Surface ectoderm overlying the sensory layer of the optic cup (Fig. 1B,C) also contained cells of quail origin. Quail cells were also observed in the diencephalon located between the two optic cups in four of five chimeric embryos (Fig. 2A,B). The extent of the contribution of quail cells to this tissue varied between specimens.

Figure 1.

 Distribution of quail cells in the optic cup and the lens in 6-day-old quail-chick blastodermal chimeras. Chimeric embryos were generated by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. Sections were stained using the monoclonal antibody QCPN, specific for quail cells. Immune-positive cells appear brown in color. Please note the presence of QCPN-positive cells (arrowheads) in the sensory layer (sl) and the pigment layer (pl) of the optic cup (A), the surface ectoderm overlying the sensory layer of the optic cup (B) and the lens (C). Arrow in C indicates the surface ectoderm. Please also note that quail cells are present both in the outer epithelial layer and the inner layer of the lens (C). (D) Quail cells within the sensory layer of the optic cup at high magnification.

Figure 2.

 Distribution of quail cells in various tissues of 6-day-old quail-chick blastodermal chimeras. Chimeric embryos were generated by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. Sections were stained using the monoclonal antibody QCPN, specific for quail cells. Immune-positive cells appear brown in color. Please note the presence of QCPN-positive cells (arrowheads) in representative pictures of the diencephalon (A,B) near the optic cup, the surface ectoderm overlying the appendage bud (C,D), the trunk (E), the heart (F), somitic mesoderm close to the neural tube (G) and the amniotic membrane (H). A few immune positive cells are also present in the yolk sac (I). oc, optic cup; nt, neural tube.

Quail cells of the donor origin were also detected in the lens, a tissue derived from the head ectoderm. In the lens, immune-positive cells were observed both in the outer epithelial layer and within the inner layer of highly elongated cells, the lens fibers (Fig. 1C). Surface ectoderm overlying the lens (Fig. 1C) also contained cells of quail origin. In all of the chimeras, quail cells made a contribution to the epidermis. QCPN- positive cells were detected in the epidermis overlying the appendage buds (Fig. 2C,D), the trunk (Fig. 2E) and the lens (Fig. 1C).

Apart from tissues derived from the ectoderm, two of the definite chimeras contained cells of quail origin in the heart (Fig. 2F) and in the mesenchymal tissue surrounding the diencephalon. In one embryo, a few quail cells were also observed in the somitic mesoderm (Fig. 2G).

Examination of extra-embryonic membranes of the recipient chick embryos revealed the presence of quail cells in the amnion (Fig. 2H) and the yolk sac (Fig. 2I) in the majority of chimeric embryos.

Discussion

Depending on the species (Sellier et al. 2006), the freshly laid egg often contains a stage X–XII embryo according to the staging system established by Eyal-Giladi & Kochav (1976). At this stage of development, the blastoderm is divided into two distinct regions, area opaca, a peripheral ring of cells attached to the yolk, and area pellucida, which encompasses the centre of the embryo. The embryo proper develops from the area pellucida, whereas the area opaca gives rise to the yolk sac (Bellairs, 1993; Bellairs & Osmond, 2005).

It is now well established that cells obtained from the area pellucida have the potential to differentiate into various cell lineages, including the germ cell lineage even in heterospecific chimeras generated by introduction of blastodermal cells into the subgerminal cavity of a recipient embryo (Marzullo, 1970; Petitte et al. 1990, 1993; Naito et al. 1991; Watanabe et al. 1992; Eames & Schneider, 2005). In accordance with these observations, the area pellucida also harbors a population of embryonic stem (ES) cells that have the potential to differentiate into various cell types, including cells from the ectodermic, mesodermic and endodermic lineages (Pain et al. 1996; Petitte et al. 2004). On the other hand, there are only a few studies addressing the developmental potential of cells obtained from the area opaca.

Area opaca, consisting of an upper layer of epiblast and a lower layer of endoderm, is not considered essential for embryonic development and does not have the potential to form any sort of axial structures in explant cultures (Khaner et al. 1985). As it is, the area opaca generally has been associated with the pulling and stretching of the blastoderm as it grows radially (Bellairs et al. 1969). This is supported by the observation that some cells of the area opaca are detached at the leading edge of the embryo (Watt et al. 1993). Cells at the periphery of the area opaca, referred to as the margin of overgrowth, migrate circumferentially over the surface of the yolk as a membrane consisting of extra-embryonic ectoderm and endoderm to form the yolk sac at later stages of development (Stern, 2005). However, it appears that the developmental potential of at least a subpopulation of cells residing within the area opaca is not restricted to the extra-embryonic ectoderm and endoderm of the yolk sac. For example, studies performed by Petitte et al. (1993) indicate that when dissociated cells from the area opaca are introduced into the subgerminal cavity of unincubated embryos, cells of the donor origin are able to give rise to melanocytes in the recipient embryo. There is also evidence suggesting that cells derived from the area opaca have the potential to give rise to primordial germ cells in mixed-sex chimeric chickens (Naito et al. 2001). Although the frequency of chimerism is low in both cases, these data would suggest that the area opaca harbors a population of cells that have the potential to give rise to various cell lineages, including the germ cell lineage, when introduced into the subgerminal cavity of embryos at pre-streak stages of development. At the same time, these results have left the question concerning the extent of the incorporation of cells from the area opaca open. Specifically, it remained to be determined whether cells from the area opaca make any contributions to embryonic as well as to extra-embryonic tissues of the recipient embryo. With this objective in mind, a number of quail-chick chimeras were generated in the present study to determine the distribution of quail cells in embryonic as well as in extra-embryonic tissues of the recipient chick embryo. A single cell suspension derived from the area opaca of the stage X–XII (EG&K) quail embryo was injected into the subgerminal cavity of the unincubated chick blastoderm. Cells of the quail origin were identified using the quail-specific monoclonal antibody QCPN. To the best of our knowledge, this is the first study analyzing the contribution of cells from the area opaca to various embryonic and extra-embryonic tissues using the quail-chick chimera model.

Although the number of chimeric embryos generated in the present study is low to reach to a definite conclusion, the frequency of chimerism for tissues derived from the ectoderm was higher than that determined for tissues derived from the mesoderm. It is important to note in this context that injection of quail blastodermal cells into the subgerminal cavity of stage XI–XII (EG&K) chick blastoderms yields no mesodermal chimerism (Watanabe et al. 1992). Underlying causes of the incorporation of donor cells mainly into tissues derived from the ectoderm remain unknown. However, it should be noted that unlike cells destined to give rise to somites, intermediate mesoderm and heart, cells contributing to surface ectoderm, neural ectoderm and extra-embryonic tissues (amnion, yolk sac and allantois) are more widely distributed over the entire surface of the pre-streak blastoderm (Hatada & Stern, 1994). Therefore, it is possible that quail blastodermal cells introduced into the subgerminal cavity of a recipient chick blastoderm are more likely to be incorporated among cells destined to give rise to the surface ectoderm, neuroectoderm and the extra-embryonic tissues, the end result of which is a relatively high frequency of chimerism in tissues derived from these structures.

Ectodermal chimerism observed in the present study was confined mainly to the head region. Interestingly, Watanabe et al. (1992) also observed that when cells derived from the area opaca at stage XI–XII (EG&K) are introduced into the subgerminal cavity of the stage XI–XIV (EG&K) chick blastoderm, ectodermal chimerism is limited to the head region. Similarly, Petitte et al. (1990) observed in chimeric embryos generated between a line of Barred Plymouth Rock (donor) and a line of White Leghorn (recipient) that in all but one case of phenotypic chimerism, black feathers were observed in the head region. Cellular/molecular mechanisms responsible for the confinement of ectodermal chimerism specifically to the head region remain to be determined.

Evidence gathered in the present study indicates that cells derived from the area opaca do contribute to extra-embryonic tissues, namely the yolk sac and the amnion. The yolk sac initially consists of a thin epithelial ectoderm and a tall layer of endodermal cells derived from the area opaca (Bellairs & Osmond, 2005). Mesoderm cells ingressing through the primitive streak are then joined to this structure at about stage 5 (Hamburger & Hamilton, 1951). Like the yolk sac, the amniotic membrane is also composed of cells derived from the ectoderm, endoderm and mesoderm (Bellairs & Osmond, 2005). However, it is impossible from the evidence gathered in the present study to characterize the quail cells detected in the yolk sac and the amniotic membranes of the recipient chick embryo.

The most interesting finding of the present study was the detection of quail cells in tissues derived from the neural ectoderm and the surface ectoderm. As the area opaca is destined to give rise to extra-embryonic ectoderm and endoderm of the yolk sac (Bellairs, 1993; Bellairs & Osmond, 2005), these results raise the possibility that the area opaca harbors cells that have the potential to produce various cell types in tissues derived from the neural ectoderm and surface ectoderm, including the optic cup, diencephalon and the lens. Alternatively, the presence of quail cells in chimeric tissues is simply due to random incorporation of the transplanted cells to the respective tissues rather than the incorporation of donor cells to prospective organ-forming areas of the recipient chick blastoderm and their subsequent differentiation to various cell lineages. It is impossible from the data gathered in the present study to distinguish between these two possibilities.

Optic cup, an embryonic structure of the vertebrate eye originating from the lateral walls of the diencephalon, was among the tissues that were populated by quail cells of donor origin. As diencephalon is derived from the neural ectoderm, these results would suggest that cells obtained from the area opaca have the potential to give rise to functional neuroectodermal cells in the recipient embryo. The evidence demonstrating the presence of quail cells in the diencephalon of recipient chick embryos is in support of this hypothesis. It might also be important to note that both the sensory and the pigment layers of the optic cup contained cells of quail origin. The neuroepithelium of the sensory layer of the optic cup gives rise to a variety of glia, ganglion neurons, interneurons, and light-sensitive photoreceptor neurons (Gilbert, 1997). On the other hand, cells of the pigment layer produce pigment and ultimately become the pigmented retina. In light of the evidence gathered in the present study, it will be interesting to see whether cells obtained from the area opaca have the potential to give rise to any of the specific cell types of the sensory and pigment layers of the optic cup.

Arguably the most significant finding of the present study was the demonstration of quail cells in the lens of the recipient chick embryo. Cells of quail origin were detected both in the outer epithelial layer and the inner layer of highly elongated cells, the lens fibers. Unlike the optic cup, the lens of the eye arises from the superficial ectoderm of the head under the inductive signals emanating from the optic vesicle (Reza & Yasuda, 2004). In this context, it is important to note that cells of quail origin were also detected in the superficial ectoderm overlying the lens and the sensory layer of the optic cup. These data along with the evidence demonstrating the presence of quail cells in the optic cup would suggest that immune-positive cells of quail origin detected in the lens epithelium and within the inner portion of the lens are direct descendants of cells residing in the surface ectoderm of the head. It also remains to be determined whether quail cells which are incorporated into the surface ectoderm of the head are permissive to lens-inducing signals emanating from the optic vesicle of the recipient chick embryo.

Data gathered in the present study demonstrate for the first time that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently from the mesoderm. The evidence demonstrating the presence of donor cells in tissues such as the optic cup, diencephalon and the lens is particularly interesting and suggests that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types derived from the surface ectoderm and neural ectoderm.

Acknowledgements

The QCPN monoclonal antibody developed by Bruce M. Carlson and Jean A. Carlson was obtained from the Developmental Studies and Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA.

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