Blastoderm Cells Research Articles

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture.

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a 60Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germline transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally.

External-anion-dependent anionic current in blastoderm cells of early medaka fish embryos.

1. Anionic current was studied by a whole-cell variation of the patch-clamp technique in blastoderm cells dissociated from medaka (Oryzias latipes) embryos at the early blastula stage. The blastoderm cells were mechanically dissociated without using proteolytic enzymes. 2. The anionic current was deactivated by hyperpolarizing steps. The steady-state current-voltage (I-V) relationship of the anion current was accurately represented by the Boltzmann relation with z = 1.01 +/- 0.02 (+/- S.E.M., n = 4) for voltage-dependent activation when internal calcium ions were buffered at 100 nM by BAPTA-Ca2+ buffer. 3. When the internal calcium concentration was reduced to 10 nM, this anionic current became an external-calcium-dependent current and was remarkably decreased by removal of external calcium ions. Furthermore, this anionic current was almost abolished when the internal calcium concentration was greatly reduced by chelating Ca2+ ions with 10 mM BAPTA. 4. This current was not affected by replacing external Na+ with TEA+ or N-methyl-D-glucamine. The reversal potentials shifted by +58.6 mV for a 10-fold decrease in the external Cl-concentration. External Cl-ions were substituted with various anions and respective current-reversal potentials were measured. The order of permeability was I- > Br- > Cl- > F-. Both the outward and inward currents almost disappeared in external Cl(-)-free solutions. 5. The voltage dependency of the anionic current shifted in a positive direction with the reduction in the external Cl- concentration, the potentials at which half the channels were activated (V 1/2) being -59.8 +/- 1.2 mV (n = 4), -40.7 +/- 0.8 mV (n = 4), -14.9 +/- 0.7 mV (n = 4), and -0.7 +/- 0.6 mV (n = 4) for 135, 90, 67, and 45 mM Cl-, respectively. This implies that the inward current, brought by efflux of internal anions, decreases with reductions in the external Cl- concentration. In the presence of external 135 mM Br-, the voltage dependency of the anionic current shifted in the negative direction (V 1/2 = -84.5 +/- 1.0 mV and z = 0.91 +/- 0.02 (n = 3)) in comparison with that in the external Cl- medium. It further shifted in the negative direction in the presence of 135 mM I- (V 1/2 = -93.8 +/- 1.5 mV and z = 0.85 +/- 0.03 (n = 3)). The inward current of the anion channel is considered to be affected by both the type and the concentration of external halide ions. 6. At the beginning of the blastula stage, the blastocoel cavity is formed for the first time. A mechanism is necessary to transport ions to the cavity without losing them to pond water outside the embryos. Since this anion channel is external-anion dependent, the current cannot be activated in the membrane facing the pond water, where concentrations of anions are much lower than those of the intracavernous solution. Only after blastocoel formation are blastoderm cells first exposed to the extracellular fluid with high ionic concentrations. Then, the anion channel would become effective for maintaining the Cl- equilibrium potential.

Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.

Pollux, a novel Drosophila adhesion molecule, belongs to a family of proteins expressed in plants, yeast, nematodes, and man.

Adhesion molecules have pivotal roles in cellular processes critical to the development and maintenance of multicellular organisms. Here we describe a new member of the adhesive repertoire encoded by the Drosophila pollux (plx) gene. Marked by a novel 74-amino-acid domain, Plx belongs to a highly conserved family with members in plants, yeast, nematodes, and man, including the human oncoprotein TRE17. Essential for viability, plx mutant analysis indicates that larval death is attributable to asphyxiation brought on by fluid-congested tracheal tubes. Ultrastructural examination of mutant tracheae reveals defects in cell-extracellular matrix contacts. During embryogenesis, Plx uniformly covers the apical surface of cellular blastoderm cells. It is later found regionally concentrated along subsets of central nervous system axon pathways and on the apical surface of the trachea's tubular epithelium. Cell attachment assays demonstrate that Plx can serve as a ligand for cell surface integrins. Plx also contains a motor neuron-selective adhesive site, multiple proteoglycan-binding motifs, and a leucine zipper: all suggest possible associations with additional components of the adhesion complex.

Origin of primordial germ cells in the prestreak chick embryo.

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII-XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vitelline membranes at stages VII-IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX-XIV with and without on STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII-IX. When individual stage IX-XIV embryos were dispersed and cultured without a feeder layer, 25-45 PGCs/embryo were detected only with stage X-XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX-XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI-XIV.

Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321-337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only by male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25-33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific, DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W-chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ "oogonia" were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm.

Response of avian nuclei to mammalian maturation promoting factor (MPF)

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

Monoclonal antibody raised against murine IL-1 ? peptide cross-reacts with a 60-kDa antigen in early Drosophila melanogaster embryo

Whole-mounts of Drosophila embryos were stained with the monoclonal antibody Vmp 18, raised against the peptide 199-208 of murine interleukin 1/ alpha. Immunofluorescence observations showed that the antibody cross-reacted with an antigenic determinant that changed in localization during Drosophila development. In syncytial Drosophila embryos, the antibody recognized an epitope localized on the nuclear envelope throughout mitotic division. As cellularization occurred, the fluorescence was mainly concentrated in the apical region of the blastoderm cells. Western blot analysis of whole Drosophila embryo extracts showed that the antibody recognized a 60-kDa protein in syncytial embryos and during germ band elongation. This suggests that the 60-kDa antigen undergoes dynamic redistribution during embryogenesis.

The Organizer-Associated Chick Homeobox Gene, Gnot1, Is Expressed before Gastrulation and Regulated Synergistically by Activin and Retinoic Acid

Gnot1, a Not (for notochord) family homeobox gene, is expressed in the chick pregastrulation blastoderm. Gnot1 expression in the epiblast is upregulated as the posteriorly derived hypoblast moves forward anteriorly to form a layer beneath it, which is of particular interest considering the known inductive role of the hypoblast in axis formation in the chick. Both activin and retinoic acid are able to activate Gnot1 expression in cultured blastodermal cells and show a strong synergistic effect when applied in combination. Strong superinduction of Gnot1 transcripts in the presence of cycloheximide also indicates the presence of a potent and labile intracellular inhibitor capable of modulating Gnot1 expression. During gastrulation, Gnot1 transcripts become localized specifically to tissues associated with "organizer" function (Hensen's node, head process, notochord). The expression data and the response to mesoderm inducing factors and axial "caudalizing" signals suggest that Gnot1 may be involved in specification of the embryonic body axis and could play a part in regulating features of the trunk/tail organizer in the chick embryo.

Virofection: A One-Step Procedure for Using Replication-Defective Retrovirus Vectors

Virofection is a simple new way to use replication-defective retrovirus vectors. It consists of the cotransfection of two plasmids: one plasmid bearing the genome of the replication-defective retrovirus vector and a second "helper" plasmid expressing the gag, pol, and env retrovirus sequences. It provides stable integration into the chromosome and long-term expression of only vector-borne sequences. We present here several helper plasmids derived from avian leukosis viruses, which we have used for the virofection of the lacZ reporter gene in chicken cell cultures. Expression can be stabilized at a very high rate in both chicken fibroblasts and blastoderm cells, without giving rise to replication-competent virus.

Order and coherence in the fate map of the zebrafish nervous system.

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns.

Early embryonic development, including germ-disk stage, in the theridiid spider Achaearanea japonica (Bös. et Str.).

Early embryonic development, from the first cleavage to the germ-disk stage, in the theridiid spider Achaearanea japonica was examined by light and electron microscopy. The eggs are syncytial during the first four cleavages, and then invaginations of cell membranes fuse to generate the blastomeres at the sixteen-nucleus stage. The cleavage pattern is a modified type of total cleavage. It appears that radial bundles of microtubules that radiate from the perinuclear cytoplasm may participate in the migration of cleavage nuclei for the formation of the blastoderm. The large yolk granules are sequestered by cell membranes from the blastomeres or blastoderm cells into the interior of the embryo together with various organelles and glycogen granules. Most of the blastoderm cells converge in the upper hemisphere to form the germ disk, whereas a few cells remain in the lower hemisphere. The embryo at the germ-disk stage contains many spherical germ-disk cells. Almost no large yolk granules are found in these cells, but the flat remaining cells each contain several large yolk granules. These remaining cells may preserve a flat shape to cover the surface of the embryo that does not include the germ disk. © 1995 Wiley-Liss, Inc.

Production of Quail Chimeras by Transfer of Early Blastoderm Cells: Plumage Chimeras and a Germline Chimera without Plumage Mixture.

Dispersed blastoderm cells taken from the unincubated quail eggs were microinjected into the blastoderms of the other unincubated quail eggs. The transfer was performed with the following four kinds of strain combinations, i.e., (1) from the wildtype plumage (WP) to the sex-linked albino (AL), (2) from WP to the black (D), (3) from AL to WP and (4) from D to WP strains. The embryos thus treated were cultured in vitro with the previously reported protocol (ONO et al., Jpn. Poult. Sci., 31: 119-129, 1994). In the WP to AL combination 50 hatchlings were obtained out of 193 treated embryos (hatchability: 25.9%). Among them 3 were somatic chimeras as judged by the pigmentation patterns of down feathers. In addition, 13 dead embryos were also plumage chimeras, therefore, 16 out of all treated embryos (8.3%; 16/193) became plumage chimeras. However, the other three combinations failed to produce any plumage chimeras (0/191). In the D to WP combination 27 hatchlings were obtained out of 83 treated embryos (hatchability: 32.5%). Among them 14survived to maturity and were progeny-tested by mating with WP quail, one male yielded 50 heterozygous and 67 wild-type plumageoffsprings, revealing itself as a germline chimera.

Manipulation of Gap Junctional Communication Between the Yolk Syncytial Layer and Blastoderm Cells and Its Effect On Embryonic Development of Cyprinus Carpio

In an attempt to block gap junctional communication during early development antibodies against connexins were injected into the YSL of carp embryos at the 30% epiboly stage. No effect on development in terms of survival or specific aberrations was observed. It appeared that no antibodies could be demonstrated in the YSL bordering the blastoderm. Connexin translation was aimed to be interrupted by antisense connexin RNA injection into the yolk just beneath the blastoderm cells of the 2- to 16-cell stage carp embryos. Preliminary results indicate a relatively small body volume in a number of treated embryos, which might indicate a disturbance of cell convergence during early gastrulation. The overexpression of connexin RNA by the injection of sense connexin RNA formed another approach to study gap junctional communication. This treatment resulted in aberrant development, which most seriously affected embryos after injection at the 2- to 8-cell stage.

Somatic and Germline Chicken Chimeras Obtained from Brown and White Leghorns by Transfer of Early Blastodermal Cells

Stage × blastodermal cells were isolated from freshly laid unincubated Brown Leghorn chicken eggs. Five hundred cells from Stage × Brown Leghorn embryos were injected into the subgerminal cavity of White Leghorn unincubated embryos exposed to 550 rad of γ irradiation from a cesium-137 source. Of 712 White Leghorn embryos that were irradiated and injected with Brown Leghorn blastodermal cells, 52 (7.3%) survived to hatching. Somatic chimerism was examined in the melanocyte population and erythroid lineage. The presence of brown feathers indicating donor cell contribution to melanocyte pigmentation was observed in 23 (44%) out of the 52 hatched chicks. Analysis of blood DNA was performed using a probe that revealed an endogenous retroviral gag fragment specific for the donor genome. Three out of these 23 chimeric chickens exhibited the gag-specific fragment. To test germline chimerism, chickens that reached sexual maturity were mated with Brown Leghorns. Three somatically chimeric hens produced Brown Leghorn progeny at a rate of 30.7, 9.2, and 2.9% respectively, thus proving donor cell contribution to the germline differentiation. Chimeric chickens obtained after injection of nonirradiated embryos exhibited a lower extent of chimerism at the feather level and did not show any chimerism in the erythroid lineage and the germline, thus demonstrating the value of the use of compromised recipient embryos to produce chimeras in chickens. Nevertheless, the extent of somatic chimerism could not be used to predict the germline chimerism.

Changes in the shapes of cells during formation of the germ disk in the theridiid spiderEnoplognatha transversifoveata(Bös.etStr.)

Summary Formation of the germ disk in the theridiid spider Enoplognatha transversifoveata was examined by light and electron microscopy. Cytoplasmic regions of blastoderm cells were flat and regions with many large yolk granules protruded into the blastocoel. Most of the blastoderm cells converged in one hemisphere of the egg to form the germ disk while only a few cells remained in the other hemisphere. The embryo at the germ-disk stage contained many spherical germ-disk cells. Almost no large yolk granules were found in these cells while the flat remaining cells each contained several large yolk granules. Cytoskeletal microtubules along the cell membrane were observed in the peripheral regions of germ-disk cells and at the periphery, apart from the thin frontier region, of the remaining cells. After microinjection of colchicine (10−4 M, 2 nl) into the blastoderm at the early blastula stage, large cells with a flattened cytoplasmic region formed the germ disk. By contrast, the microinjection of cytochalas...

Morphogenesis in the Early Embryo of the Lepidopteran Bombyx mori

The regional patterns of nuclear and cell division and the changes in size and shape of the developing Bombyx mori embryo were monitored from just prior to blastoderm formation until the onset of gastrulation. Nuclear invasion of the periphery and subsequent cellularization occurs with a marked anterior to posterior gradient. Consequently, there is no syncytial blastoderm stage in which all nuclei simultaneously rest at the periphery. In addition, the posteriormost blastoderm cells do not form pole cells. Prior to gastrulation, the Bombyx blastoderm cells undergo several unusual types of cell behaviors, indicated in part by the modifications and specializations of their lateral edges. Subsequent to the completion of cellularization, localized regions of mitosis can be found within the germ anlage, but at the stage monitored, just prior to the onset of gastrulation, these regions do not appear to be the equivalent of the Drosophila mitotic domains. The implications of these findings for modeling how segmental fates are established in insects are discussed. This work confirms and extends the work of earlier histological investigations of Bombyx development.

Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages.

A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.

Development of the germ cells and reproductive primordia in male and female embryos of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Newly deposited eggs of Rhodnius prolixus lack a visible pole plasm and require 14 days to develop at 27 °C and 70% RH. The first germ cells originate at 9% of embryogenesis by asynchronous mitosis of blastodermal cells behind the germ Anlage at the posterior pole of the egg. From 9 to 17%, these proliferate to a mean of 270 cells and, from 13 to 18%, migrate forward over the dorsal surface of the mesoderm and lodge in abdominal segments 3–7. Between 22 and 30%, they shift laterally and segregate into three or four paired clumps between segments 3 and 4, 4 and 5, 5 and 6, and, sometimes, 6 and 7 and, from 30 to 37%, gradually assemble into a continuous longitudinal mass on either side of segments 3–6, where they begin to associate with mesodermal cells. Between 37 and 46%, these collect between (males) and around the germ cells to form the rudiments of the terminal filaments (females), inner and outer gonadal sheaths, interstitial cells (males), and primary exit ducts. Dorsally situated sheath cells then invaginate ventrally into each gonadal rudiment, partitioning it into seven compartments, each containing a mean of 15 oogonia or 16 spermatogonia. These seem to fuse into a rosette, at least in females, but do not begin to divide again until after hatch. Excluded germ cells lodge within the rudiments of one or both exit ducts. The evolutionary and functional aspects of our findings are addressed and new observations are presented on the mechanism of anatrepsis.

Production of Quail Chimera by Transfer of Early Blastodermal Cells and its Use for Transgenesis.

Dispersed blastodermal cells taken from the eggs of the wild-type plumage quail were microinjected into the blastoderms of unicubated fertilized eggs of sex-linked albino quail. The embryos thus treated were transferred to another quail egg shell beds and cultured for 52-53 hours at 37.5°C while being rocked at an angle of 90 degrees at 30 minutes intervals. Then, the embryos were transferred to the chicken egg shell beds and cultured at 37.5°C with rocking at a 30 degree angle until moved to hatcher just before hatching. Forty-one out of 184 injected embryos hatched and the chimeric plumage was observed in 6 hatchlings. The plumage chimerism was also found in 5 dead embryos. For transgenesis, dispersed blastodermal cells were co-cultured in vitro with a plasmid construct containing β-actin-lacZ hybrid gene (pMiwZ) and a cationic liposome (Lipofectin) for gene transfection, and then injected into the host blastoderm. The embryos were cultured in vitro for 52-53 hours. Ten out of 30 survived embryos exhibited lacZ expression in embryonic tissues as detected by histochemistry. The other 10 exhibited lacZ expression only in exrtaembryonic tissues, and the remaining 10 failed to show any sign of gene transfection. The technique described here should be highly applicable for the production of transgenic avian animals.