Formation of Embryo Thromboblasts in Chick Blastoderm: Morphology, Site of Production and Time of Emergence in the Blood

Thrombocytes in the blood of chick embryos (termed embryo thrombocytes by L ucas and J amroz ) have PAS-positive granules in their cytoplasm. Electron microscopic observations reveal that the embryo thrombocytes contain glycogen granules present singly or in clumps. The presence of these inclusions and other morphological characteristics were used as specific markers to distinguish embryo thrombocytes from primitive erythroid cells. These markers also made it possible to determine the time at which the immature thromboblasts first emerge in blood vessels, and the period of their continued presence in the circulation. In this way we found that thromboblasts were detectable in embryos as early as stage 10⁺ of H amburger and H amilton (after 35 hr incubation) and that the thromboblasts were present in the circulation until day 4 of incubation (stage 23). In ovo and in vitro culture of de-embryonated blastoderm demonstrated that thromboblasts were formed in the area opeca vasculosa. The present observations suggest that embryo thromboblasts are formed at the same time and in the same area as the primitive cells of erythroid line.     

The pluripotency of the extraembryonic mesodermal cells of the early chick blastoderm: effects of the AP and AOV environments

This study investigates the developmental potential of the extraembryonic mesodermal cells of the early chick blastoderm. [3H]Thymidine-labeled mesodermal fragments from the extraembryonic area pellucida (AP) and area opaca vasculosa (AOV) were transplanted into the AP or AOV of nonlabeled host blastoderms in culture, and their fate followed autoradiographically. All the homotopically transplanted mesodermal cells differentiated in accordance with their normal fates. However, not all the heterotopically transplanted mesodermal cells did so, for some of the stage 8 AP extraembryonic mesodermal cells (normally nonerythropoietic) gave rise to blood cells when transplanted into the AOV. We also observed that the stage 4-5 AOV mesoderm continues to migrate peripherally when heterotopically transplanted into the AP, at a time when the AP mesodermal cells are nonmigratory. In support of our premise that the stage 8-9 AP extraembryonic mesoderm has the potential to form blood, we observed a clear-cut production of hemoglobin when the latter mesoderm was co-cultured on coverslips with stage 4 AOV endoderm.     

Immunohistological localisation of monoclonal antibody R 24-recognized ganglioside Gilac2* in early chick embryos

The spatio-temporal cellular expression and biosynthesis of ganglioside Glac2 was investigated in early chick embryogenesis. For demonstration of embryonic Glac2-biosynthesis, chick embryos of stage 0 and of stages 4-5 were incubated in vitro in the presence of radioactive sugar precursors. It was found that chick embryos synthesize Glac2 as early as at the blastula stage as well as at the gastrula stage, both within the area pellucida and the area opaca. In contrast to the biosynthetical findings immunohistochemical staining of the chick embryos at various stages by aid of the mouse monoclonal antibody (mAb) R 24, specific for the immunoepitope NeuAc alpha, 8NeuAc alpha, 3Gal beta less than, as present on the ganglioside Glac2, revealed a spatio-temporal cellular pattern of expression of this ganglioside in early chick embryos. Immunohistochemical staining of the chick embryo at stage 0 shows that all cells of the embryo, the extraembryonic epiblast and the yolk endoderm included, are mAb R 24-positive. At the intermediate streak stage (stage 3), the cranial part of the deep layer, the so-called endophyll, is strongly mAb R 24-positive, whereas at the end of gastrulation (stage 5), mAb R 24-recognized epitopes appear to be restricted to a narrow band of deep-layer cells in the endophyllic crescent and to the yolk endoderm of the area opaca. At this stage, no labelling by the antibody is observed in cell layers of the future embryo. The beginning of neurulation (stage 7) is characterized by the expression of the mAb R 24-recognized epitope in the notochord, whilst the deep layer in the cranial part of the neural fold still expresses this epitope. No ecto- or mesodermal structures are stained by the antibody at this developmental stage. During further development (stage 12 and 13), mAb R 24-reactivity is restricted to the cranial part of the embryo with a preferential staining of cells of endodermal origin. At these stages, the notochord expresses mAb R 24 binding sites only in its cranial region. The spatial and temporal correlation between the presence of mAb R 24-recognized epitopes and the morphogenetic positioning of tissues may be indicative for a possible role of the ganglioside Glac2 in corresponding cellular interactions.     

A study of the expansion of the chick area vasculosa

The radial expansion of the area vasculosa of the chick embryo was studied with regard to the location of the cells which generate the mesodermal movement. The expansion of stage 12 area vasculosae was shown to be autonomous from either continued blastoderm expansion or the continued presence of the embryo. Small glass rods were used as barriers to area vasculosa expansion. When placed peripheral to the terminal sinus, glass rods blocked the vascular expansion; however, when placed just central to the terminal sinus, glass rods had no effect on vascular expansion. In addition, removal of large amounts of tissue within the area vasculosa had no significant effect on vascular expansion. We conclude that the majority of the mesoderm within the boundaries of the terminal sinus plays no essential role in the expansion of the area vasculosa, and that the cells which generate the force for expansion are located at or very near the terminal sinus. A histological study of the area vasculosa and adjacent blastoderm was performed with light and electron microscopy. This survey showed that, as the terminal sinus and a group of mesenchymal cells just peripheral to it (“edge cells”) move out into the blastoderm, morphological changes occur in the epiblast overlying the terminal sinus and edge cells. Three major changes in the epiblast cell layer were observed: (1) The cells change from a squamous, monolayered arrangement to a cuboidal or columnar, bilayered arrangement; (2) the epiblast basal lamina is thrown into convoluted folds; (3) an electron-dense extracellular matrix becomes associated with both the epiblast basal lamina and mesenchymal edge cells. Histochemical staining (Alcian blue at pH 2.5 and 1.0 and two-step PAS) shows positive reactions at the epithelial-mesenchymal interface located just over the terminal sinus and edge cells. These results suggest that glycosaminoglycans are present in relatively large amounts at the advancing mesoderm edge.     

Fibronectin in the area opaca of the young chick embryo

Summary Distribution of fibronectin-like immunoreactivity was studied in the area opaca of the young chick embryo (stages 4–6 HH) by use of the immunofluorescence and protein A-coupled to colloidal gold techniques. Fibronectin, associated to the basement membrane, formed a fibrillar network, the pattern of which changed from the centre to the periphery of the area opaca. At the ultrastructural level, differences in fibronectin distribution were found between non-moving and moving cells. The epithelial-like cells presented fibronectin staining exclusively on their basal side. Actively migrating cells (edge and mesodermal cells) showed immunoreactive material localized around their entire surface and within the cytoplasm. The fibronectin distribution is discussed in relation to three important phenomena taking place during the early growth of the area opaca: (i) anchorage and migration of the edge cells, (ii) modification of cell shape in relation to mechanical tension, and (iii) expansion of the area vasculosa.     

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5–7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens. Mol. Reprod. Dev. 48:501–510, 1997. © 1997 Wiley-Liss, Inc.     

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

Summary Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[³⁵S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.     

Hepten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo

The early chick blastoderm expresses two endogenous galactose-bindinglectins of 14 kDa and 16 kDa. We have studied the effect thelectin hapten inhibitors thiodigalactoside and the syntheticneoglycoprotein lactosyl-bovine serum albumin as well as polyclonalanti-lectin antibodies on the development of early chick embryoscultured in a defined medium. Controls consisted of maltose,maltosyl bovine serum albumin and rabbit IgG. Embryos treatedat the onset of cell migration during early gastrulation underwentblastoderm retraction with decrease in surface area. In addition,they exhibited a lack of demarcation between the presumptiveembryonic area (area pellucida) and the presumptive extraembryonicarea (area opaca). These blastoderms also lacked a primitivestreak, that is, the structure that forms in the area pellucidaduring gastrulation as cell migrate to form the endodermal andmesodermal layers of the embryo. Embryos treated at later stagesof gastrulation showed development similar to that of controlsin that they were able to undergo early organogenesis. The resultssuggest that lectin mediated mechanisms are essential for themigratory movements of early gastrulation and that, at lategastrulation, other mechanisms exist in the embryo to compensatefor lectin function. blastoderm chick embryo galectin     

CHANGES IN SUSCEPTIBILITY OF CHICK EMBRYONIC BLOOD CELLS TO NEWCASTLE DISEASE VIRUS

To analyse the age-dependent changes in susceptibility of chick embryonic cells to viral infection, observations were made on the blood cells after the inoculation of Newcastle disease virus. A lethal dose of Sato strain of Newcastle disease virus was introduced into chick embryos via injection of inoculum into the blood vessel and allantoic sac. Observations of blood cells by immunofluorescent technique revealed two types of cells, susceptible and resistant to the virus infection. Erythroblasts of both primitive and definitive lines, embryonic thromboblasts and thrombocytes were of the former type and mid- and late-polychromatic erythrocytes and mature ones were of the latter. Erythroblasts gradually decrease in their viral susceptibility as they differentiate into polychromatic erythrocytes and finally become resistant to the virus at the mid-polychromatic erythrocyte stage or later. Thromboblasts, on the other hand, retain high susceptibility to the virus throughout the course of their differentiation to mature thrombocytes. The change in the viral susceptibility of erythroid cells with differentiation is discussed in relation to the structural and functional alterations during the cell specialization.     

Induction of multiple embryos with NaHCO3 or calcium free sea water in the horseshoe crab

Summary When horseshoe crab embryos were treated with NaHCO₃ at the developmental stage when the germ disc appears, multiple embryos were formed. NaHCO₃ may effect the formation of multiple embryos by binding Ca²⁺ ions of the embryo since multiple embryos were also formed by treatment with Ca²⁺ free sea water.The treatment caused the blastoderm layer to tear. When the embryos were returned to normal sea water after the treatment, the blastoderm recovered. Some cell masses, probably derived from the germ disc or its prospective cells, formed during the process of the recovery. Each cell mass developed into an embryo.Contributions from the Shimoda Marine Research Center, Nor. 348     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

The incorporation of 5-bromodeoxyuridine into DNA of the area opaca vasculosa of chick embryos

The incorporation of 5-bromodeoxyuridine (5-BrdUrd) into DNA of the area opaca vasculosa (AOV) of chick embryos during organ culture was measured. The AOV from blastoderms of the definitive primitive streak stage will not form red cells in the presence of BrdUrd while the AOV of 1–3 somite blastoderms is unaffected by the presence of 5-BrdUrd. About 90% of the original non-density labeled DNA can replicate in the presence of 5-BrdUrd if the tissues come from the younger sensitive embryos, but only 65% of the original DNA will replicate from tissues of older insensitive embryos. Tissues from embryos of both ages replace about 80% of the thymidine by BrdUrd in each newly synthesized strand of DNA; tissues from embryos of both ages will form DNA of hybrid density after one cell generation, and will also form double-heavy DNA after longer periods of culture in the presence of 5-BrdUrd. During recovery from 5-BrdUrd inhibition during a thymidine chase, the density-labeled DNA is replicated so that the new DNA of normal density is formed, but the original heavy 5-BrdUrd containing strands are conserved. It is suggested that inhibition of red cell formation by 5-BrdUrd may occur by incorporation of 5-BrdUrd into DNA of endoderm cells, rather than by acting only directly on red cell precursors.     

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF_2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0-3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.     

The L5 epitope: an early marker for neural induction in the chick embryo and its involvement in inductive interactions.

The pattern of expression of the carbohydrate epitope L5 was studied during early development of the chick neuroepithelium. Immunoreactivity first appears during gastrulation, at mid-primitive streak stage, and persists until at least 3.5 days of development. The epitope is expressed on all the components of the developing nervous system, both central and peripheral. In immunoblots, the antibody recognises a major component of about Mr 500,000 and several more minor components of lower molecular mass. If a Hensen's node from a donor embryo is transplanted into the area opaca of a host embryo, L5 immunoreactivity appears in the epiblast surrounding the graft. If hybridoma cells secreting the antibody are grafted together with Hensen's node into a host chick embryo, the induction of a supernumerary nervous system is inhibited. We suggest that the L5 epitope is an early and general marker for neural induction and that it may be involved directly in inductive interactions.     

Endothelins participate in the central and peripheral regulation of submandibular gland secretion in the rat

We previously reported that endothelins (ETs) are involved in the rat central and peripheral regulation of bile secretion. In this study we sought to establish whether ET-1 and ET-3 modulated submandibular gland secretion when locally or centrally applied. Animals were prepared with gland duct cannulation to collect saliva samples and jugular cannulation to administer sialogogues. ETs were given either into the submandibular gland or brain lateral ventricle. Intraglandularly administered ETs failed to elicit salivation per se. However, ET-1, but not ET-3, potentiated both cholinergic- and adrenergic-evoked salivation through ET(A) receptors. ET-1 decreased cAMP content but increased phosphoinositide hydrolysis, whereas ET-3 attenuated both intracellular pathways. The expression of ET(A) and ET(B) receptor mRNAs as well as that of ETs was revealed in the submandibular gland by RT-PCR. Immunohistochemical studies showed that ET(A) receptor staining was localized around the interlobular ducts and acini, compatible with the myoepithelial cells' location, whereas ET(B) receptor staining was restricted to small blood vessels. When applied to the brain, both ETs induced no salivation but enhanced cholinergic- and adrenergic-evoked salivary secretion through parasympathetic pathways. ET-1 response was mediated by brain ET(A) receptors, whereas that of ET-3 was presumably through nonconventional ET receptors. Present findings show that ETs are involved in the brain regulation of cholinergic- and adrenergic-stimulated submandibular gland secretion through the activation of distinct brain ET receptors and parasympathetic pathways. However, when ETs were administered into the gland, only ET-1 enhanced cholinergic and adrenergic salivation likely through myopithelial cell contraction by activating ET(A) receptors coupled to phospholipase C. The presence of ETs and ET receptors suggests the existence of an endothelinergic system in the submandibular gland.     

A New Oxidative Stress Model, 2,2-Azobis(2-Amidinopropane) Dihydrochloride Induces Cardiovascular Damages in Chicken Embryo

It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD₅₀ value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.     

Post-nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.