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We describe how to prepare unincubated avian eggs to obtain a greater number of clearly visible Rauber's sickles for experimental embryology. After hemi-sectioning of unincubated chicken (Gallus domesticus) blastoderms and cultivating both halves in vitro, two kinds of development can be discerned: (1) when the unincubated blastoderms were hemi-sectioned according to the plane of bilateral symmetry, going through the middle region of Rauber's sickle, we obtained two hemi-embryos (a left and a right one). Each contained a half primitive streak, localized at the cut edge (starting from the most median part of Rauber's sickle) giving rise to a half mesoblast mantle and half area vasculosa, thus indicating mosaic development (each part of the whole fertilized egg would be able to form independently on its own). (2) When the unincubated blastoderm is hemi-sectioned more obliquely, going through a more lateral part of Rauber's sickle (sickle horn), two complete bilaterally symmetrically miniature embryos will form, indicating the so-called regulation phenomena. We demonstrate that these two types of development are in reality due to the different spreading and concentration of Rauber's sickle tissue (containing gamma ooplasm) around the area centralis. Embryonic regulation thus must not be considered as a kind of totipotent regeneration capacity of isolated parts of the unincubated avian blastoderm, but depends on the spatial distribution of a kind of extraembryonic tissue (Rauber's sickle) built up by the oblique uptake of gamma ooplasm (ooplasmic mosaicism) at the moment of bilateral symmetrization (Callebaut [1994] Eur Arch Biol 105:111-123; Callebaut [2005] Dev Dyn 233:1194-1216).

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., ((c) 2006 Wiley-Liss, Inc.)

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., (Copyright 2004 Wiley-Liss, Inc.)

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., (Copyright 2003 Wiley-Liss, Inc.)

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)., (Copyright 2003 Wiley-Liss, Inc.)

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.

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., (Copyright 2002 Wiley-Liss, Inc.)

An upper layer (epiblast) fragment taken laterally from the Anlage fields of neural plate or chordamesoderm of a quail blastoderm, labelled with 3H-glucosamine, was grafted isotopically (in a similar region), isochronically (at the similar stage of development) and isotropically (with the same caudocranial and dorsoventral polarity) in the epiblast of a mesoblast free area of a chicken blastoderm (St 4-5 Vakaet, 1970: full grown primitive streak). On the autoradiographs of the sections through such cultured blastoderms with fully integrated quail grafts, we observed a labelling of the basement membrane laterally and slightly cranially from the labelled graft in its final position. Since only the epiblast and its basement membrane are involved, the pattern of the observed labelling indicates that the grafted and integrated quail epiblast fragment glides in toto over the mediocaudally localized basement membrane, leaving behind a track of radioactivity. Sliding of whole groups of epiblast cells over the basement membrane seems thus to be a normal phenomenon during avian gastrulation.

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.

In the present study, we demonstrate that quail junctional endoblast fragments have powerful embryo-inducing potencies when placed on the deep side of the upper layer (UL) of the anti-sickle region of unincubated chicken blastoderms. Moreover, in most cases the inducing potencies of the autochthonous Rauber's sickle-endophyll complex were inhibited at distance. Also, junctional endoblast still in situ in an early streak embryo strongly inhibits the inducing capacities of a Rauber's sickle - endophyll complex apposed on the deep side of the upper layer of the cranial part of the area pellucida. So junctional endoblast, besides embryo inducing potencies also seems to dominate and to inhibit at distance the gastrulation potencies of an ectopically placed or autochthonous Rauber's sickle - endophyll complex.

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.

From unincubated quail blastoderms, we have excised, caudal marginal zones (caudally from Rauber's sickle), upper layer fragments covering Rauber's sickle or Rauber's sickle fragments alone (as controls), and placed them on the ventral side of the cranial quadrant of unincubated chicken blastoderms. Also, quail Rauber's sickle fragments, all or not associated with quail endophyll, were placed on the ventral side of isolated central upper layer discs of prestreak chicken blastoderms from which the deep layer was previously removed. Only the Rauber's sickle-derived cells (sickle endoblast cells), placed on unincubated or shortly incubated blastoderms induce, after culture, a primitive streak (PS) and a normal embryo. This indicates, together with previous experimental evidence, that even in the presence of endophyll, neither the deep part of the caudal marginal zone nor the upper layer above it can induce a primitive streak. This experimental study affords further evidence that the function of the avian Rauber's sickle is homologous to the function (mesoderm induction) of the vegetal dorsalizing cells (Nieuwkoop centre) in amphibian blastulas.

By isotopic quail-chicken chimera experiments of the Rauber's sickle or by radioactive labelling and isotopically replacing of the caudal endophyllic sheet in unincubated avian blastoderms, followed by culture, we demonstrated that the displacement of the endophyll by the cranially extending sickle endoblast is not exclusively a mechanical phenomenon, as suggested by earlier studies (Vakaet, 1962 a, b). Indeed, our study suggests that the sickle endoblast also migrates centripetally very soon (already after 5 h) in and through the caudal endophyll before ingression of upper layer cells takes place. We also describe the early spatial relationship between the three elementary tissues (endophyll, Rauber's sickle, upper layer) (Callebaut et al, 1996a) and the induction phenomena between quail sickle endoblast and chicken upper layer (UL) during the formation of the primitive groove. The latter already develops before ingression occurs. We found no evidence for an endophyllic origin of avian primordial germ cells.

In the present study, we removed the whole area marginalis, Rauber's sickle and the peripheral part of the area centralis from unincubated chicken blastoderms (st IV, Vakaet, 1962a). By placing a fragment of a quail Rauber's sickle (functioning as early gastrulation organizer: Callebaut and Van Nueten, 1994) at different places and oriented in different directions on the remaining central part of the area centralis, we observed, after in vitro culture, a normal embryonic development. This indicates that the area marginalis itself is not indispensable for gastrulation and neurulation. Our study also indicates that none of the three elementary tissues (Rauber's sickle, endophyll and upper layer) of the avian unincubated blastoderm present an irreversible functional polarity.

By excision at different sites of rectangular fragments from unincubated chicken blastoderms and replacement by isotopic fragments from unincubated quail blastoderms, we could make the first complete map of the Anlage fields in the freshly laid avian blastoderm. All the Anlage fields (Fig. 11) are found in the upper layer (UL) of the caudal half of the area centralis (bordered by the Rauber-Koller's sickle). In the UL of the area marginalis, peripheral to Rauber-Koller's sickle, neither gastrulation nor neurulation phenomena could be observed. Similar heterotopic replacement experiments indicate that before incubation, the different parts of the UL of the area centralis are still uncommitted or reversibly committed. The Anlage fields of chordamesoblast and definitive endoderm (gut endoderm) in unincubated avian blastoderms appeared to be disposed caudally in the caudal half of the area centralis. As far as we know we are the first to demonstrate that the Anlage field of the definitive gut endoderm (which is derived from the upper layer: Hunt, 1937; Vakaet, 1962b) is localized in the most caudal upper layer part of the area centralis just centrally to the Rauber-Koller's sickle. The Anlage field of the neural plate is localized in the upper layer over the more cranial endophyll. The Anlage of the brain is shield-shaped, whilst the other Anlage fields are sickle-shaped, parallel with the Rauber-Koller's sickle. Their general hemicircular disposition and form still seem to reflect (together with the Rauber-Koller's sickle) the original ooplasmic radial symmetry (Callebaut, 1972) combined with the eccentricity of the deep layer components, which was observed during early symmetrization by gravitational orientation of the egg yolk (Callebaut, 1993a,b). The Rauber-Koller's sickle might be homologous with the vegetal dorsalizing cells or centre of Nieuwkoop (1973) in amphibian blastulas.

The closure of experimental excision-wounds in the upper layer of the gastrulating chick blastoderm was studied by time-lapse videography and videomicrography and by light microscopy, transmission electron microscopy and scanning electron microscopy. One experimental excision-wound was made in the upper layer of stages 4V to 6V blastoderms (Vakaet, Arch. Biol. (Liège) 81:387-426, 1970), in the proamnion where no middle layer cells are present. The deep layer was previously discarded, so that the wounds were made in the bare upper layer. They closed within 2 to 6 hours and further development was normal by in vitro standards. With videography, global movements of the upper layer towards the wound were observed. With videomicrography, the wound submarginal region cells were seen to move like sheep in a flock: individual cells in different directions, the whole flock towards the wound. During closure the shape of the wound edge was irregular. The structure of the epithelium of the wound submarginal region was unchanged throughout closure: a pseudostratified columnar epithelium in which cell divisions occur at its dorsal side and are parallel to its surface. The basal lamina was absent below the edge of the wounds. We propose that the cells of the upper layer are mobile against one another and are not confined to a specific part of the basal lamina. During wound closure the movements of the cells on the basal lamina would be driven by mitotic pressure. This is the horizontal pressure exerted by the addition of daughter cells and their parting during anaphase and telophase.(ABSTRACT TRUNCATED AT 250 WORDS)

Connecting cords are elongated telophase bridges persisting between separating daughter cells. We have studied them with Scanning Electron Microscopy in the upper cell layer of the quail blastoderm where a high mitotic activity accompanied by interkinetic nuclear migration coincides with morphogenetic movements. The predominant orientation of the connecting cords is parallel to the direction of the morphogenetic movements.

We have used immunohistochemical techniques to study laminin in quail blastoderms milked from the oviduct and the distribution of laminin in laid chicken and quail blastoderms. Laminin is a constituent of the basement membrane in both chicken and quail blastoderms. It is found at the ventral side of the upper layer cells. Laminin is first observed under individual upper layer cells in prelaid quail blastoderms 15 h post-ovulation, but is absent at the ingression site of endophyll cells. The presence of a continuous laminin layer coincides with the epithelialization of the epiblast after 5-10 h incubation. The laminin layer is discontinuous at the primitive streak and at Hensen's node. It is thinner and partly discontinuous at the median part of the neural plate. By induction, either of an ectopic primitive streak or a neural plate, we have demonstrated, using the chicken-quail nucleolar marker technique, that at these sites the laminin layer is interrupted. A laminin layer might confer rigidity onto the epiblast, whereas disruption of a laminin layer seems to be correlated with ingression of cells or bending of the neural plate.

An upper layer (epiblast) fragment taken laterallyfrom the Anlage fields of neural plate or chordamesoderm of a quail blastoderm,labelled with [sub 3] H-glucosamine, was grafted isotopically (in a similarregion), isochronically (at the similar stage of development) and isotropically(with the same caudocranial and dorsoventral polarity) in the epiblast ofa mesoblast free area of a chicken blastoderm (St 4–5 Vakaet, 1970:full grown primitive streak). On the autoradiographs of the sections throughsuch cultured blastoderms with fully integrated quail grafts, we observeda labelling of the basement membrane laterally and slightly cranially fromthe labelled graft in its final position. Since only the epiblast and its basement membrane are involved, the pattern of the observed labelling indicatesthat the grafted and integrated quail epiblast fragment glides in toto over the mediocaudally localized basement membrane, leaving behind atrack of radioactivity. Sliding of whole groups of epiblast cells over thebasement membrane seems thus to be a normal phenomenon during avian gastrulation. [ABSTRACT FROM AUTHOR]