Endophyll orients and organizes the early head region of the avian embryo.

By placing endophyll on the caudal area marginalis situated behind Rauber's sickle of avian unincubated blastoderms, we observed after using the quail-chick chimera system and culture the development of a (pre)neural plate or a miniature embryo, head-oriented towards this endophyll. A Rauber's sickle fragment placed in the same conditions gives no reaction. If we place endophyll close to Hensen's node (stage 4 Vakaet, 1962) on an isolated anti-sickle region of an avian unincubated blastoderm in vitro, a similar endophyll-oriented development takes place after culture. Under the same conditions, but in the absence of endophyll, a Hensen's node provokes a thickening of the upper layer in the immediate neighbourhood, eventually with formation of a neural axis, oriented according to the original caudocranial direction of the graft. Our study indicates that avian endophyll (from unincubated blastoderms) can induce in the upper layer a (pre)neural plate, with or without neural folds. By interaction with sickle endoblast coming from Rauber's sickle (the early gastrulation organizer: Callebaut and Van Nueten, 1994), or from Hensen's node (a later avian organizer: Waddington, 1932), it can orient or re-orient the head region and the caudocranial direction of an induced miniature embryo. The conclusions from our embryological experiments are in agreement with the results obtained by recent molecular biology studies.     

Competitive inhibition by Rauber's sickle of the primitive streak and/or (pre)neural plate inducing effects of sickle endoblast in avian blastoderms

When in unincubated chicken blastoderms the Rauber's sickle is (sub)totally mechanically removed by selective scraping, the further evolution of the blastoderm in culture is often profoundly disturbed, going from only expansion of the upper layer and preneural plate formation to the development of a slowly growing miniature embryo. Our results suggest that the developmental potencies of the embryo are related to the presence or absence of Rauber's sickle material left after its removal. This can be checked after culture by the presence or nonpresence of junctional endoblast (derived from Rauber's sickle) and the concomitant induction of blood islands in the immediate neighborhood. Our study thus indicates that without Rauber's sickle (in the cases of successful total selective removal), an avian blastoderm cannot develop normally, even in the presence of an intact caudal marginal zone. After placing a fragment of quail sickle endoblast on the anti-sickle region of unincubated chicken blastoderms from which the Rauber's sickle was (sub)totally removed, different developmental scenarios were seen, according to the degree of removal, both in the anti-sickle as in the sickle regions. 1) If Rauber's sickle activity is strongly reduced, then besides a centripetally directed miniature embryo, induced by the remnants of the autochthonous Rauber's sickle, an additional centripetally directed embryo or preneural plate (without accompanying blood islands) develops in the anti-sickle region under inductory influence of the apposed quail sickle endoblast. We make a distinction between a neural plate and a preneural plate. The latter consists of a thickening of the upper layer (with the same initial aspect as a neural plate) adjacent to endophyll or sickle endoblast in the absence of chordomesoblast and gastrulation phenomena. 2) If Rauber's sickle activity is totally absent, then the inducing power of the sickle endoblast fragment becomes maximal and, starting from the anti-sickle region, one single embryo (without blood islands) extending over the whole area centralis appears. 3) If much of the Rauber's sickle material has been left in the blastoderm, then the inducing activity of the sickle endoblast, placed on the anti-sickle region, will be totally suppressed (although the sickle endoblast remains intact) and neither a preneural plate nor a primitive streak was induced. After placing a fragment of quail sickle endoblast on the anti-sickle region of an unincubated chicken blastoderm from which the Rauber's sickle and surrounding tissues were completely excised, an embryo was always induced by the sickle endoblast in the adjacent upper layer of this anti-sickle region. In the absence of sickle endoblast, this never occurred. Thus, our experiments demonstrate that in the absence of the Rauber's sickle, a parent tissue (sickle endoblast) induces both gastrulation and neurulation phenomena, while in the full presence of Rauber's sickle these functions are totally suppressed. Moreover, Rauber's sickle not only organizes gastrulation and blood island formation by itself but also influences neurulation at a distance (in space and time) by part of its cell lineage (i.e., sickle endoblast). Our study suggests that the inhibitory effect of Rauber's sickle on its parent tissue (sickle endoblast) represents an early mechanism impairing polyembryony, so that only a single primary major organizer (Rauber's sickle) remains active in the young avian germinal disc.     

Avian sickle endoblast induces gastrulation or neurulation in the isolated area centralis or isolated anti-sickle region respectively

By the quail-chicken chimera technique, we studied, in culture, the inducing effect of sickle endoblast (derived from Rauber's sickle by centripetal and cranial migration) on the isolated Rauber's sickle-free central part of the area centralis or on the isolated Rauber's sickle-free anti-sickle region from unincubated chicken blastoderms. Just as Rauber's sickle, the flat one-cell-thick sickle endoblast (Stage 2-3, Hamburger & Hamilton, 1951) induces a primitive streak (PS) and a neural plate in the area centralis. If a vitelline membrane is interposed between the sickle endoblast and the area centralis, then a small primitive streak is still induced, suggesting the effect of a diffusible factor on PS formation. In the adjacent upper layer of an isolated anti-sickle region the apposed sickle endoblast induces only a (pre)neural plate. By contrast, this (pre)neural plate inducing effect is rapidly and totally suppressed after grafting on the anti-sickle region of whole unincubated blastoderms. This suggests dominating positional information phenomena emanating from Rauber's sickle over the whole blastoderm. After grafting sickle endoblast either on the isolated area centralis or on isolated anti-sickles, no junctional endoblast and no blood islands developed. This suggests that the differentiation of Rauber's sickle material into sickle endoblast is irreversible. Our results indicate that Rauber's sickle material under the form of sickle endoblast also influences early neurulation phenomena (at distance in space and time). The present study indicates the existence of a temporo-spatially bound cascade of gastrulation and neurulation phenomena and blood island formation in the avian blastoderm, starting from Rauber's sickle, the primary major organizer with inducing, inhibiting and dominating potencies. The latter not only plays a role by secretion of signalling molecules (positional information) but it also influences development by its cell lineages (junctional endoblast and sickle endoblast).     

In the absence of Rauber's sickle material,no blood islands are formed in the avian blastoderm

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast.     

Positional information by Rauber's sickle and a new look at the mechanisms of primitive streak initiation in avian blastoderms

The present experimental in vitro study suggests that a primitive streak (PS) in avian blastoderms is induced by diffusion of morphogenetic substances emanating from Rauber's sickle. Indeed, even without direct contact between a quail Rauber's sickle and the reacting upper layer (by interposition of a vitelline membrane), a PS can be induced in the isolated area centralis or antisickle region of unincubated chicken blastoderms. The so-formed PSs are localized below the vitelline membrane in the immediate neighborhood of the apposed Rauber's sickle material. This seems to indicate that Rauber's sickle organizes the formation of the avian PS according to the basic concept of "positional information." The morphogenetic substances seem to have an effect only on the formation of a PS. Each part of Rauber's sickle seems to have, point by point, the same thickening and PS-inducing effect on each corresponding part of the underlying upper layer (UL). By a mechanism of sliding over the basement membrane and fusion, this finally results in the formation of one single median PS. Our study shows that a PS can be induced in the total absence of hypoblast (sickle endoblast) or caudal marginal zone, by only the presence of Rauber's sickle material. In contrast, the differentiation of mesoblast into blood islands under the influence of Rauber's sickle and neural tissue development are impaired by the interposition of a vitelline membrane. The latter could be due to the absence of a normal interaction of Rauber's sickle-derived sickle endoblast with endophyll and/or upper layer and the absence of cranial migration of the mesoblast. Thus, earlier studies and the present study indicate the existence of a temporospatially bound cascade of gastrulation and neurulation phenomena and blood island formation in the avian blastoderm, starting from Rauber's sickle, the primary major organizer with inducing, inhibiting, and dominating potencies. The latter not only plays a role by secretion of signaling molecules, but also influences development by its cell lineages (junctional endoblast and sickle endoblast).     

Rauber's sickle and not the caudal marginal zone induces a primitive streak, blood vessels, blood cell formation and coelomic vesicles in avian blastoderms

By using the quail-chicken chimera technique, we studied the reactivity and the eventual developmental or inducing capacities of the avian caudal marginal zone (in comparison with Rauber's sickle), when associated in vitro with different avian blastoderm components. If a fragment of quail sickle endoblast is placed on the caudal marginal zone of a whole unincubated chicken blastoderm, then a secondary miniature embryo will develop in this caudal marginal zone. The primitive streak and accompanying neural plate of the secondary embryo are directed peripherally into the caudal germ wall, away from Rauber's sickle. Thus, the 'mirror image development' indicates that the upper layer of the caudal marginal zone can react in the same way as the upper layer of the area centralis, because of the presence of sickle endoblast. A quail Rauber's sickle fragment placed on an isolated anti-sickle region always induces a primitive streak directed centrally. After prolonged culture, blood vessels and associated coelomic vesicles are formed. By contrast if a quail caudal marginal zone is placed on an isolated chicken anti-sickle region, the primitive streak, blood vessels and coelomic vesicles do not form. Thus, in contrast to the inducing effect of Rauber's sickle, the caudal marginal zone has no inducing effect by itself, even in the absence of the dominating effect of Rauber's sickle.     

Interaction of central subgerminal ooplasm with the elementary tissues (endophyll, Rauber's sickle and upper layer) of unincubated avian blastoderms in culture

By placing a central subgerminal ooplasmic mass over isolated parts (alone or in association) of unincubated avian blastoderms and culture, we obtained an improvement in, or in some cases restoration of normal development. The evolution of small rectangular fragments (isolates) excised from different regions of the unincubated blastoderm was observed in association or not with subgerminal ooplasm. The only type of differentiation that was clearly distinguished in these isolates (taken from the caudocentral area centralis region) was a so-called 'primary neurula' formed by the endophyll and an associated thickened upper layer. In the present study, we also demonstrate that after removal of the area centralis from an unincubated caudal blastoderm quadrant, the upper layer (UL) and endophyll can no longer be restored from the associated subgerminal ooplasm (and form a miniature embryo), as is the case after removal of the endophyll alone. A deep layer (containing the endophyll) reformed during the migration of Rauber's sickle-derived cells into the neighbouring central subgerminal ooplasm only in the presence of the upper layer. This suggests that the upper layer has an early influence on the cells containing the original central deep ooplasm (delta ooplasm) to form the endophyll. The present study offers supplementary arguments in favour of the hypothesis that the endophyll is an inductor of preneurulation.     

The constituent oocytal layers of the avian germ and the origin of the primordial germ cell yolk

By radioactive or trypan blue induced fluorescence yolk labelling (used at certain developmental stages as intravital cytoplasmic markers), it can be demonstrated that the constituent yolk layers of quail blastoderms are formed when the precursor oocyte is growing from 3 to approximately 18 mm (rapid growth period). A previous study ( Callebaut , 1974) and the present study demonstrate that 2 cytoplasmic regions, each with a different constitution and behaviour, can be discerned in the avian germinal disc: 1) a deep and paraxial region, containing yolk that has been in contact with the t.i.c.o.s. (3H-thymidine incorporating cytoplasmic organelles) during oogenesis; 2) a superficial and peripheral region, which has not been in contact with the t.i.c.o. material and which penetrates into the first region along with the cleavage furrows. In the large blastomeres, the originally superficial ooplasm surrounds the deep ooplasm. The area centralis of the unincubated blastoderm must be considered as a heterogeneous cell population, containing both deep and superficial material in variable amounts. After laying and incubation, extra-embryonic tissues such as yolk endoderm and margin of overgrowth develop in the superficial and peripheral region. The embryonic mesoderm also develops from the latter. The yolk, which will be incorporated in the primordial germ cells (germinal yolk), derives only from the original deep and paraxial region of the oocytal germinal disc, i.e. from the region which has been in contact with the t.i.c.o.s. The germinal yolk plasm can be traced in the deep paraxial region of the oocytal germinal disc, in the central region of the unincubated blastoderm, in the endophyll (early primitive streak stage) and finally in the primordial germ cells (P.G.C.s.) at the moment of their separation from the endophyll wall (early somite stage). Thus our results provide evidence for the existence of a germ cell plasm in the avian postlampbrush oocyte.     

Induction and improved embryonic development by the nucleus of Pander in associated avian blastoderm parts: influence of delta or gamma ooplasm

After placing in vitro, central subgerminal ooplasm (containing a central nucleus of Pander) from a quail germ disc of a prelaid egg (before symmetrization) on the upper layer of an isolated chicken antisickle, we observed the induction of a radially oriented preneural plate (without interference of chordamesoblast). This observation suggests the primary existence during the period of symmetrization in utero of an until now unknown temporospatially linked "vertical" effect, emanating from the nucleus of Pander, on the parallel (pre)neural plate anlage forming part of the area centralis in the overlying blastoderm. For comparison, we "sandwiched" in vitro a quail sickle endoblast fragment between the deep side of the upper layer of an isolated chicken antisickle region and a central subgerminal ooplasmic mass. This resulted in a colonization of the subgerminal ooplasmic mass by quail sickle endoblast cells followed by improved neurulation and/or gastrulation phenomena. The latter never occurs in the absence of central subgerminal ooplasm. In both types of experiments there seems to exist a common link between the observed induction phenomena: the presence of delta ooplasm in the involved deep structures. Indeed, the nucleus of Pander contains delta ooplasm as well as the structures derived from it, i.e., endophyll with primordial germ cells and sickle endoblast-derived cells after colonization of the neighboring central ooplasm (present study). Therefore, we think that the preneural plate-inducing effect observed after placing a nucleus of Pander on the antisickle region is due to the presence of a factor in the delta ooplasm that diffuses in the neighborhood. The appearance of gastrulation phenomena in the second type of experiment seems to be due to colonization of the more peripheral part of the central subgerminal ooplasm containing the more superficial and peripheral gamma ooplasm in which Rauber's sickle material can develop. This suggests that the kind of involved ooplasm (delta or gamma) can predetermine the inductive activity of the deep structures that contain it: the central part of the nucleus of Pander and/or endophyll for preneurulation phenomena and sickle endoblast (in the presence of central subgerminal ooplasm) for gastrulation and/or neurulation phenomena.     

Induction of the avian coelom with associated vitelline blood circulation by Rauber's sickle derived junctional endoblast and its fundamental role in heart formation

In histological sections through chicken blastoderms of different ages we describe the temporospatial relationship between junctional endoblast, the formation of blood islands (appearing first from a peripherally migrating mesoblastic blastema), and the formation of coelomic vesicles developing later in/and from a more superficially extending mesoblastic blastema (coelomic mesoblast). After unilateral removal of the Rauber's sickle-derived junctional endoblast in early streak blastoderms (stage 2-4; Vakaet [1970] Arch Biol 81:387-426) and culture to stage 11 (Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed that the early formation of the coelomic cavity was locally or totally disturbed in the operated area. Besides the simultaneous absence of blood islands, the coelomic vesicles did not form normally. Instead of regularly aligned coelomic vesicles, progressively forming the coelomic cavity by fusion, some voluminous irregular cavities appeared. Thus, the extent of the coelomic cavity was greatly reduced and the operated side was considerably smaller than the unoperated side. Furthermore, in the youngest operated blastoderms the cranial portion of the involved coelomic cavity (hemipericardial cavity) exhibited rudimentary development and usually did not reach the region of the foregut endoderm. This resulted in the absence of the myoepicardium and associated endocardium at this side. In another experiment, after removal of the junctional endoblast at one side of the chicken blastoderm, a fragment of quail junctional endoblast was placed isotopically. This resulted, after further in vitro culture, in the restoration of the formation of coelomic vesicles and accompanying subjacent blood islands in the immediate neighborhood of the apposed quail junctional endoblast. Also, the pericardium and primary heart tube developed normally. Similarly, by using the quail-chicken chimera technique, we demonstrated that the splanchnic mesoderm cells of the pericardium develop in intimate association with the most cranial part of the junctional endoblast (derived from the Rauber's sickle horns). Our experiments indicate that the coelom and, in particular, the pericardium and primary heart tube form progressively (in time and space) under the inductory influence of Rauber's sickle and junctional endoblast.     

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.     

Determination of Erythroid Cells in Unincubated Chick Blastoderm

Portions the size of 1/6 to 1/32 part of unincubated blastoderm cultured in an albumen-salineagar solid medium form erythroid cells under conditions which suppress normal co-ordinated movements for mesoderm induction. Anterior and posterior regions of unincubated blastoderm have the same potentiality to form erythroid cells. The marginal zone seems to be the contributor of prospective erythroid cells. Portion 1/16 part of unincubated blastoderm forms morphologically distinct erythroid cells of the primitive and definitive lines as in normal development in ovo. It seems progenitor cell(s) is pre-programmed to a particular pattern of differentiation and/or includes differentiation to various erythroid cell types as an obligatory step. This system provides novel experimental possibilities in the study of erythroid cell determination and differentiation.     

Research of blastocyte-like structure in chicken

Embryonic stem (ES) cells known to be one of the most brilliant stars enlightening the biology science are a major tool to investigate embryonic development and differentiation, functional genomics and applied in medicine territory for cellular therapy. Even though some scientists have already established cultural sys-tems for selection and maintenance of putative avian ES cells, the study on them still drops behind the mammalian and proceeds slowly. The avian ES cells can be used to produce…     

Research of blastocyte-like structure in chicken

The chicken embryo is a classic model used to investigate embryonic development, gene expression, and tissue differentiation, and is also an important research tool in studying the animal functional genomics. The whole blastoderms of fresh unincubated eggs from White Leghorn chickens were collected with a paper ring, mechanically broken into small pieces and cultured in medium. Then the small pieces would develop into blastocyte-like structures (BLS), which could be facilitated by an addition of fetal bovine serum (FBS) to the primary culture and their diameter was nearly doubled from 12 to 24 h. The additional yolk had no positive effect on the development in the first 12 h but encouraged the BLSs attaching and inner cells differentiating instead in 24 h. The inner cells of the BLS showing a high alkaline-phosphatase activity similar to those in mouse embryonic stem (ES) cells and also expressing a large amount of the specific stage embryonic antigen-1 (SSEA-1) on the surface, which was known to be the characteristic of non-differentiated mouse and avian ES cells, could finally differentiate into nerve-like cells, fibroblast cells and so on in the medium. Leukemia inhibitory factor (LIF) facilitated the cells' proliferation and prevented differentiation in the suspended culture of the BLSs. So we drew the conclusion that the BLS obtained from broken blastoderm can be used to amplify avian ES cells so as to initiate a new method of producing transgenic chickens.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Activation of avian embryo formation by unfertilized quail germ discs: comparison with early amphibian development

In the present study we placed germ discs (or fragments containing the deep central part of it) from unfertilized laid or extracted quail eggs on the deep side of the upper layer of isolated anti-sickle regions from unincubated chicken blastoderms. After culture in vitro of associations where the central deep part of the germ discs was in contact with the deep side of the upper layer (UL), we observed in about 30% of the cases the onset of embryonic development. Both associated parts play a role in the final formation of an embryo. Our experimental results, suggest that the delta ooplasm of the nucleus of Pander influences the cranial upper layer to segregate an endophyll layer. The definitive embryonic structures i.e. mesoderm, epiblast and neural plate are derived from the chicken upper layer by respectively normal gastrulation and (pre)neurulation phenomena. Our experiments seem to have some homology with the association experiments of isolated cortices from various regions of unfertilized Xenopus eggs implanted into the ventroequatorial core of a recipient 8-cell Xenopus embryo.     

Early steps in neural development

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.     

Effect of gravity on the interaction between the avian germ and neighbouring ooplasm in inverted egg yolk balls

The developmental capacities of an avian germ (from before symmetrization to the moment of laying) are strongly diminished after inversion of its egg yolk ball followed by culture in egg white. Our present experiments show that even when the avian germ is completely horizontally inverted (without an upper or lower border) below its egg yolk ball before symmetrization, symmetrization and gastrulation phenomena take place. The germ grows slower and becomes smaller than after normal incubation. After culture of inverted unincubated germs, localized on freshly laid eggs, the closure of the neural tube is impaired and it remains open over a long distance. Although a primitive streak (PS) develops, mesoderm migration (mainly from the lateral part of the area pellucida) is also impaired. On sections through the germinal disc one can see the abnormal upward migration into the depth of the ooplasm and yolk of cells from the germ wall and the development of large cellular extensions encircling the yolk globules. Most prominent is the loss of contact between the superficial cell layers and the deep layer elements (junctional endoblast and yolk endoblast in the area opaca). Large areas without deep layer elements (even visible on surface micrographs) develop in the area vasculosa and area vitellina interna. The margin of overgrowth grows and extends normally over the egg yolk ball. An autoradiographic study after labelling of the yolk layers in inverted egg yolks reveals that mainly compression of the peripheral subgerminal and perigerminal ooplasm takes place. This suggests that the compression by the neighbouring yolk and upwards growth of cells are at the origin of the impaired development. After return to the normal upward orientation of the germ on the topmost part of the egg yolk ball, a more or less pronounced restoration to normal development takes place (depending on the duration of the inversion period and the age of the germ).     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

Ontogeny of lactate dehydrogenase isozymes in chicken-quail hybrid embryos

The ontogeny of lactate dehydrogenase (LDH) isozymes was examined in avian hybrids and compared with the isozyme patterns of the parental species. Hybrids were obtained by crossing female Japanese quali (Coturnix coturnix japonica) with male domestic chickens (Gallus gallus domesticus). By use of starch gel electrophoresis and an enzyme-specific stain, traces of embryonic paternally derived LDH were detected in unincubated hybrid eggs. It was concluded that the embryonic genes coding for the B subunits of LDH are activated during the hours between fertilization and oviposition. In early blastoderms, a great excess of maternally stored LDH is present. In the hybrid, the predominantly maternal pattern of isozymes shifts during embryogenesis to a predominantly paternal pattern. This was considered evidence for differential allelic regulation of LDH inactivation. A progressive trend toward the establishment of the adult distribution of isozymes in various tissues was also observed in the hybrid and quail, and found to be similar to chicken LDH isozyme ontogeny.