Contribution of Cells Derived from the Area Pellucida to Extraembryonic Mesodermal Cell Lineages in Heterospecific Quail Chick Blastodermal Chimeras

Key Words : Development · Area pellucida · Blastoderm · Yolk sac · Allantois · Chorion · Amnion · Hematopoietic cell · Endothelial cell · Quail chick chimera

Abstract

The current study has two main objectives: first, to deter- mine if cells derived from the area pellucida are able to pop- ulate extraembryonic membranes, and second, to deter- mine if donor cells have the potential to differentiate to en- dothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes function- ing as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the un- incubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting puta- tive chimeras was examined using quail cell-specific anti- body against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN+ donor cells was examined using QH-1, a present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembry- onic membranes of resulting heterospecific quail chick chi- meras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages de- rived from the posterior mesoderm both in the yolk sac and allantois.

Introduction

Depending on the species [Sellier et al., 2006], the freshly laid egg often contains a stage X–XII embryo ac- cording to the staging system established by Eyal-Giladi and Kochav [1976] (EG&K). At this stage of development, the avian blastoderm is divided into two distinct regions, the area opaca, a peripheral ring of cells attached to the yolk, and the area pellucida, which encompasses the cen- ter of the embryo. The embryo proper develops from the area pellucida, whereas the area opaca gives rise to the yolk sac [Bellairs, 1993; Bellairs and Osmond, 2005]. It is now well established that, when introduced into the sub- germinal cavity of a recipient embryo, cells obtained from the areae pellucidae contribute to both the germ line and somatic tissues derived from the ectoderm, mesoderm, and endoderm [Marzullo, 1970; Petitte et al., 1990; Naito et al., 1991; Watanabe et al., 1992; Carscience et al., 1993; Thoraval et al., 1994; Kagami et al., 1995; Etches et al., 1996]. Although, apart from the melanocyte lineage, in- formation regarding the specific lineages these cells give rise to in somatic tissues is lacking for the most part, it is generally accepted that at least a subpopulation of cells within the area pellucida behave as pluripotent cells when introduced into the subgerminal cavity of a recipient em- bryo. Nevertheless, the contribution of cells derived from the area pellucida to extraembryonic membranes has not been demonstrated. Among the extraembryonic mem- branes, the yolk sac and allantois are of particular impor- tance as they both function as hematopoietic organs ca- pable of producing hematopoietic (HC), endothelial (EC), and circulating endothelial (CEC) cells [Moore and Owen, 1967; Dieterlen-Lievre, 1975; Caprioli et al., 1998; Pardanaud and Eichman, 2011]. To the best of our knowl- edge, however, whether or not cells derived from the area pellucida have the potential to differentiate to EC and HC in the yolk sac and allantois of the recipient chick embryo remains, to date, an open question. In an attempt to ad- dress these issues, quail chick blastodermal chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X–XII (EG&K) quail em- bryo into the subgerminal cavity of the unincubated chick blastoderm and the distribution of quail cells in ex- traembryonic membranes of resulting putative chimeras, namely the yolk sac, allantois, amnion and chorion, were examined using quail cell-specific antibody against a perinuclear antigen (QCPN).

The presence of EC, HC, and smooth muscle cells among the QCPN+ donor cells was also examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin (α-SMA) antibody. Evi- dence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes and, most importantly, give rise to HC, EC, and/or smooth muscle cells, all of the three mesodermal lineages derived from the posterior 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. The study was approved by the institutional Animal Ethics Committee of Adnan Menderes University. Donor cells were obtained from areae pel- lucidae of stage X–XII [Eyal-Giladi and Kochav, 1976; Sellier et al., 2006] quail blastoderms. Freshly laid, unincubated quail eggs were cracked open and the blastoderms were removed by adher- ence 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) and streptomycin (100 µg/ml). The blastoderms were cleaned off the adhering yolk and the areae pellucidae were dissected out from the surrounding area opaca. The areae pellucidae of stage X–XII (EG&K) embryos were pooled in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (DMEM-FBS) and they were dissoci- ated enzymatically using 0.0125% trypsin and 0.005% 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 areae pellucidae were dispersed further to obtain a single cell suspension by gentle titration 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 (190%, by trypan blue exclu- sion).

Recipient blastoderms were prepared as described previously [Petitte et al., 1990; Karagenç and Sandikci, 2010]. Briefly, freshly laid eggs were swabbed with 70% alcohol and a 1-cm window was made in the equatorial plane of the egg shell directly over the blas- toderm. Approximately 2,000 cells were injected into the subger- minal cavity in 2–4 µl of medium using a finely drawn micropi- pette with 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% until day 6 of embryonic development, with a rocking motion through a 90° angle at hourly intervals.

Immunohistochemistry

In order to detect donor cells of quail origin in extraembry- onic membranes of the resulting putative chimeras, the allantois, amnion, chorion, and yolk sac were isolated separately in sterile PBS (pH 7.4) under a dissecting microscope equipped with a cold light source. Extraembryonic membranes were cut into pieces with a diameter of approximately 0.5–1 cm. All tissue samples were fixed in 5% formal-acetic at 4 °C overnight and washed sev- eral times in PBS (pH 7.4) supplemented with 1% Triton X-100, 0.2% bovine serum albumin, and 0.1% sodium azide (PBT). All

subsequent steps involved in immunohistochemical staining of extraembryonic membranes as whole mounts were carried out in round-bottomed polypropylene tubes on a rocking platform. In order to inactivate potential endogenous peroxidase, membranes were bleached in 3% H2O2 in PBT for 10 min. After rinsing in PBT three times for 10 min each, samples were washed in PBT three times for 30 min each and blocked in normal goat serum in PBT for 1 h at room temperature. The supernatant of QCPN hybrid- oma cells grown in DMEM supplemented with 20% FBS were used for differential diagnosis of quail cells. Samples of extraem- bryonic membranes were incubated in undiluted QCPN superna- tant supplemented with 0.1% Triton X-100 for 48 h at room tem- perature. After rinsing 3 times in PBT for 10 min each, samples were washed in PBT 3 times for 30 min each. Membranes were then incubated in universal biotinylated link (Dako Cytomation LSAB+ system-HRP) supplemented with 0.1% Triton X-100, pen- icillin (100 IU/ml) and streptomycin (100 µg/ml) for 48 h at room temperature, rinsed and washed in PBT as above, and then incu- bated in streptavidin HRP (Dako Cytomation LSAB+ system- HRP) supplemented with 0.1% Triton X-100, penicillin (100 IU/ ml), and streptomycin (100 µg/ml) for 48 h at room temperature. Samples were then rinsed 3 times in PBT for 10 min each, followed by 3 washes (1 h each) in Tris-HCl saline buffer (pH 7.6). Cells expressing the antigen were detected using 3, 3′ diaminobenzi- dine tetrahydrochloride (DAB) solution (3 mg/ml in Tris-HCl, pH 7.6 with 3% H2O2). The reaction was stopped by washing the membranes in PBS several times. Extraembryonic membranes determined to contain immune-positive cells upon stereomicro- scopic examination were placed on a slide in a minimum amount of 70% glycerol in PBS and flattened under a coverslip supported with spots of silicone grease at each corner. Once representative pictures had been taken, samples having QCPN-positive cells were frozen in OCT and 10-µm sections were taken in a cryostat. Cryosections taken from samples of the extraembryonic membranes determined to contain quail cells were further exam- ined using the monoclonal antibodies QH-1, anti-α-SMA, GRL-2, and BEN. The monoclonal antibody QH-1 is specific for HC and EC of the quail species [Pardanaud et al., 1987]. Visualization of cells expressing QH-1 (undiluted hybridoma supernatant) was carried out using Alexa-455 goat anti-mouse IgG1 (Invitrogen). The monoclonal antibody α-SMA (Sigma) was diluted 1/500 in PBS (pH 7.4) and its expression was revealed using Alexa-488 goat anti-mouse IgG2a (Invitrogen). QH-1/αSMA double staining was performed according to standard procedures using Alexa-455 and Alexa-488-conjugated secondary antibodies, respectively. GRL-2 is an avian specific monoclonal antibody recognizing thrombocytes, myelocytes, myeloid progenitors, and a subpopu- lation of erythroid progenitors [Thomas et al., 1993]. Visualiza- tion of cells expressing GRL-2 (undiluted hybridoma superna- tant) was carried out using Alexa-488 goat anti-mouse IgG1 (In- vitrogen). In order to determine the presence of definitive erythrocytes among QCPN+ donor cells, some sections were also stained with benzidine according to standard procedures. BEN monoclonal antibody stains quail and chick peripheral projecting neurons and hematopoietic precursors [Pourquié et al., 1992]. The monoclonal antibody BEN (Developmental Studies and Hybrid- oma Bank; DSHB) was diluted 1/100 in PBS (pH 7.4) and its ex- pression was revealed using Alexa-488 goat anti-mouse IgG1 (In- vitrogen). Cell nuclei were counterstained with DAPI (Invitrogen).

Distribution and Relative Numbers of Donor Cells within the Recipient Tissues

In order to determine the distribution and relative numbers of quail cells of donor origin in recipient tissues, images were ac- quired from sections double stained with QCPN and DAPI. A minimum of 1,000 cells were counted manually in at least 10 dif- ferent sections for each extraembryonic membrane. The relative number of quail cells was determined as the proportion of QCPN+ cells to the total number of cells.

Results

One of the aims of the present study was to determine if cells derived from the areae pellucidae are able to pop- ulate extraembryonic membranes, namely the yolk sac, allantois, amnion, and chorion, when injected into the subgerminal cavity of an unincubated blastoderm. To this end, a total of 30 quail chick chimeras were con- structed by transferring dissociated cells from the areae pellucidae of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blasto- derm. A representative picture of a stage X (EG&K) quail blastoderm along with the blastodermal cell suspension and a schematic drawing showing the injection proce- dure is given in figure 1. Among the putative chimeras, only 23 survived to day 6 of embryonic development and were examined for the presence of quail cells in the al- lantois, yolk sac, amnion, and chorion. Twenty out of 23 putative chimeras were demonstrated to be definite chi- meras by the presence of QCPN+ donor cells in at least one of the extraembryonic membranes examined. Im- munohistochemical staining of extraembryonic mem- branes as whole mounts revealed the presence of quail cells in all of the extraembryonic membranes. The fre- quency of chimerism along with relative numbers of QCPN+ donor cells observed in each extraembryonic membrane are given in table 1. In general, the frequency of chimerism observed in the amnion, allantois, and cho- rion was higher than that observed in the yolk sac. The extent of the quail cell contribution as well as the distri- bution of donor cells in recipient tissues varied greatly between specimens.
Quail cells of donor origin could readily be detected in the allantoic membrane (fig. 2a, b), with the majority of QCPN-positive cells being in close proximity to the blood vessels. Histological evaluation of immune-positive sam- ples in tissue sections confirmed the presence of quail cells in the mesodermal layer rich in blood vessels (fig. 2c) as well as among cells forming the endoderm of the al- lantois (fig. 2d).

In addition to the allantois, immune-positive cells were also detected in the yolk sac (fig. 3a). Descendants of donor cells were observed within extraembryonic me- soderm of the yolk sac among EC lining the blood vessels and among blood cells of the recipient chick embryo (fig. 3b). Extraembryonic ectoderm (fig. 3c) and endo- derm (fig. 3d) layers of the yolk sac also contained im- mune-positive cells.

Amniotic membrane surrounding the embryo proper (fig. 4a, b) as well as the chorion (fig. 4 c, d) also contained quail cells of donor origin. Immune-positive cells ap- peared to scatter throughout the amniotic membrane of the recipient embryo. In one case, a dome-like structure consisting entirely of quail cells was observed (data not shown).

In order to determine whether or not donor cells of quail origin contributing to the extraembryonic membranes, especially the yolk sac and allantois of the recipi- ent chick embryo, were able to differentiate to EC, HC, and smooth muscle cells, QH-1 monoclonal antibody, specific for HC and EC of the quail species, along with anti-α-SMA, were used. QH-1+ EC and HC were ob- served in both the yolk sac (fig. 5) and allantois (fig. 6) among EC lining the blood vessels and blood cells of the recipient chick embryo. It was noted that not all quail cells detected in the yolk sac and allantois expressed QH-1. Double staining of sections for QH-1 and αSMA re- vealed the presence of smooth muscle cells along with QH-1+ EC sometimes within the same vessel wall in both the yolk sac (fig. 5) and allantois (fig. 6). QH-1+ HC were also observed outside the vessel wall either as aggregates of cells in the yolk sac (fig. 5) or as single cells in both the yolk sac (fig. 5) and allantois (fig. 6). Histological analyses of the chorion also revealed the presence of smooth mus- cle cells and QH-1+ HC within this extraembryonic membrane (fig. 7). Unlike the yolk sac, allantois, and chorion, QH-1 expression was not observed in any of the QCPN+ cells detected in the amnion. On the other hand, some of the smooth muscle cells in the amnion were determined to be of quail origin (fig. 7). Histological analyses of the yolk sac revealed that neither BEN nor GRL-2 was ex- pressed by any donor cells observed among blood cells of the recipient chick embryo. Similarly, no benzidine-pos- itive cells were observed among the QCPN+ donor cells.

Discussion

There is overwhelming evidence demonstrating that, when introduced into the subgerminal cavity of a recipi- ent embryo, cells obtained from the areae pellucidae con- tribute to both somatic tissues and the germ line [Mar- zullo,1970; Petitte et al. 1990; Naito et al., 1991; Watanabe et al., 1992; Carscience et al., 1993; Thoraval et al., 1994; Kagami et al., 1995; Etches et al., 1996]. However, the con- tribution of blastodermal cells obtained from the area pellucida to the extraembryonic membranes in such blas- todermal chimeras has remained for the most part un- examined. Among the extraembryonic membranes, the yolk sac and allantois are of particular importance as they both function as hematopoietic organs capable of pro- ducing HC, EC, and CEC [Moore and Owen, 1967; Die- terlen-Lievre, 1975; Caprioli et al., 1998; Pardanaud and Eichmann, 2011]. However, to the best of our knowledge, whether or not cells derived from the area pellucida have the potential to differentiate to EC and HC in the yolk sac and allantois of the recipient chick embryo has not been demonstrated previously. Evidence gathered in the pres- ent study demonstrates that, when introduced into the subgerminal cavity of a recipient chick embryo, quail blastodermal cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of the recipient chick embryo and, most importantly, give rise to the three main mesodermal lineages, namely the HC, EC, and/or smooth muscle cells.

Although donor cells of quail origin were observed in all of the extraembryonic membranes, the frequency of chimerism observed for the yolk sac (39.1%) was lower than that detected for the allantois (73.9%), amnion (69.6%), and chorion (63.6%). The relatively low level of chimerism observed in the yolk sac could simply be due to a very large surface area of this extraembryonic mem- brane. Alternatively, cells introduced into the subgermi- nal cavity are preferentially integrated among cells of the recipient blastoderm destined to give rise to the allantois, amnion, and chorion rather than the yolk sac. However, it is impossible from the data gathered in the present study to distinguish between these two possibilities as it appears that, once introduced into the subgerminal cav- ity, donor cells integrate into the recipient blastoderm in a rather random fashion depending on the site of injec- tion [Watanabe et al., 1992] and how far the micropipette penetrates through the subgerminal cavity during the operation [Maeda et al., 1997].
The yolk sac, one of the extraembryonic membranes examined in the present study, is formed from the area opaca and consists initially of a thin epithelial ectoderm underlain by a layer of tall endodermal cells containing many intracellular yolk droplets [Bellairs and Osmond, 2005]. Mesodermal cells ingressing through the primi- tive streak are then joined to this structure at about stage 5 [Hamburger and Hamilton, 1951]. This extraembryon- ic mesodermal layer is continuous with the lateral plate mesoderm of the embryo proper and similarly arranged into somatic and splanchnic layers. Of the two, the extra- embryonic somatic mesoderm forms the amnion and chorion along with the extraembryonic ectoderm. On their migration towards the yolk sac, mesodermal cells passing through the posterior part (1/2 to 1/3) of the primitive streak first form aggregates in the splanchnic mesodermal layer of the yolk sac. The peripheral cells of these aggregates, referred to in the literature as blood is- lands, angioblasts, or hemangioblasts [Murray, 1932; Sheng, 2010], subsequently flatten and differentiate to EC, whereas the centrally located cells differentiate to HC [Dieterlen-Lievre and Le Douarin, 2004; Eichmann et al., 2005; Jafredo et al., 2005). Based on observations demon- strating simultaneous emergence of EC and HC in blood islands, it was proposed that these two cell lineages derive from a common precursor, the hemangioblast [Murray, 1932].

Results of the present study indicate that cells derived from the areae pellucidae are able to populate all layers of the yolk sac. Histological analyses of sections using the monoclonal antibody QH-1 clearly demonstrate that quail blastodermal cells can give rise to both HC and EC within the splanchnic mesodermal layer of the yolk sac. The data gathered in the present study, however, does not provide any evidence of whether or not these cells are de- rived from a common hemangioblastic cell. However, the presence of QH-1+ clusters of cells along with EC and HC suggest that this would indeed be the case. Whether or not blastodermal cells introduced into a recipient embryo could give rise to functional hemangioblasts warrants further investigations.

The yolk sac also serves as the main source of cells for primitive erythropoiesis [Sheng, 2010]. In an attempt to determine the specific lineage of QCPN+ cells observed among blood cells of the recipient chick embryo, sections of the yolk sac were analyzed using monoclonal antibody GRL-2, recognizing thrombocytes, myelocytes, myeloid progenitors and a subpopulation of erythroid progenitors and BEN, a marker for hematopoietic precursors. Al- though chick cells expressing GRL-2 could readily be identified (data not shown), neither BEN nor GRL-2 was expressed by any donor cells. Our failure to detect QCPN+ with a covering of splanchnic mesoderm [Bellairs and Osmond, 2005]. Before its fusion with the inner layer of the chorion, the allantois consists of two layers, namely the splanchnic mesoderm and endoderm. Evidence pro- vided in the present study clearly indicates that donor cells were able to populate both layers of the allantois. The presence of immune positive cells especially within the splanchnic mesoderm is particularly interesting as the avian allantois, like the yolk sac and the ventral wall of the dorsal aorta, serves as a site capable of producing HCs and ECs [Caprioli et al., 1998, 2001]. Histological analysis of sections using the monoclonal antibody QH-1 clearly demonstrates that quail blastodermal cells can give rise to both HCs and ECs within the splanchnic mesodermal layer of the allantois. As is the case for the yolk sac, the data gathered in the present study, however, does not pro- vide any evidence of whether or not these cells are derived from a common hemangioblastic cell.
It is important to note that, apart from the HCs and ECs, both the yolk sac and the allantois are able to gener- ate CEC [Caprioli et al., 1998; Pardanaud and Eichmann, 2011). In light of the fact that CEC, like EC and HC, ex- press QH-1, the possibility that at least some of the QH-1+ cells observed in the yolk sac and allantois are CEC should also be taken into consideration.

The evidence provided in the present study also dem- onstrates that cells derived from the area pellucidae are able to populate the chorion and amnion of the recipient chick embryo. Both chorion and amnion are bilayered tissues of dual origin, composed of extraembryonic ecto- derm and extraembryonic somatic mesoderm. Whereas both QH-1+ and α-SMA+ cells were observed in the cho- rion, only smooth muscle cells were detected in the am- nion, with no cells expressing the hemangioblastic mark- er QH-1. This is in line with the fact that amnion never develops blood vessels [Bellairs and Osmond, 2005]. The existence of QH-1+ cells in the chorion, on the other hand, is a particularly interesting finding as this extraembry- onic structure exhibits hematopoietic potential in the mammalian embryo [Zeigler et al., 2006]. To the best of our knowledge, there are no studies demonstrating that the chorion plays a similar role in the avian embryo. Based on the presence of a population of QH-1+ HC, it is tempting to speculate that, like the yolk sac and allantois, the chorion may also have hematopoietic potential in the avian embryo.

Particular details concerning cell-cell interactions and molecular mechanisms governing the fate of each blasto- dermal cell introduced into the subgerminal cavity of a recipient embryo remain, for the most part, unknown. Evidence gathered in the present study indicates that not all donor cells observed in recipient tissues differentiated to any of the mesodermal lineages examined, suggesting a possible random incorporation of these cells into re- spective tissues. However, evidence demonstrating dif- ferentiation of donor cells to HC, EC, and/or smooth muscle cells in all of the extraembryonic membranes ar- gues against random incorporation of at least some of the donor cells. In this context, it is important to note that determination and differentiation of EC can take place in the absence of gastrulation [Christ et al., 1991]. However, evidence demonstrating differentiation of donor cells to all of the three main mesodermal cell lineages simultane- ously, especially in the yolk sac and allantois, suggests that at least some of the donor cells introduced into the subgerminal cavity of the recipient chick embryo entered through the posterior primitive streak in an orderly fash- ion and were first committed to a common lateral/poste- rior mesodermal cell lineage before their segregation to smooth muscle and blood/endothelial progenitors [Shin et al., 2009].

Taken together, data gathered in the present study demonstrate for the first time that, when introduced into the subgerminal cavity of a recipient chick embryo, quail cells derived from the areae pellucidae are able to popu- late all of the extraembryonic membranes of the recipient chick embryo and, most importantly, give rise to the three main mesodermal lineages, namely the HC, EC, and/or smooth muscle cell lineages. The quail chick blas- todermal chimera model used in the present study can provide an experimental scheme for examining develop- mental processes governing lineage specification within the extraembryonic mesoderm. It will be important to determine if avian embryonic stem cells [Pain et al., 1996; Petitte et al., 2004; van de Lavoir et al., 2006] Purmorphamine are endowed with a similar developmental potential.