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).

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.

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).

Autoradiographic evidence for the sliding of the upper layer over the basement membrane in chicken blastoderms during gastrulation.

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.

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.

Avian junctional endoblast has strong embryo-inducing and -dominating potencies.

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.

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.

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.

Only the endophyll-Rauber's sickle complex and not cells derived from the caudal marginal zone induce a primitive streak in the upper layer of avian blastoderms.

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.

Induction of (pre) gastrulation and/or (pre) neurulation by subgerminal ooplasm and Rauber's sickle in cultured anti-sickle regions of avian unincubated blastoderms.

Rauber's sickle fragments from unincubated quail blastoderms, associated or not with chicken central subgerminal ooplasm, were placed on the deep side of the upper layer (UL) of the isolated anti-sickle region of unincubated chicken blastoderms and cultured in vitro. When only a Rauber's sickle fragment was placed, we observed always a pronounced thickening of the UL (pregastrulation) in the immediate neighbourhood. Some times a primitive streak (PS) developed. When the Rauber's sickle fragment was "sandwiched" between the UL and a central subgerminal ooplasmic mass [containing the nucleus of Pander (1817)], always a (pre)neural plate accompanied by endophyll developed, not or well associated with a primitive streak. In the latter case a complete miniature embryo developed. De novo formation of endophyll was observed. As it contained quail nuclei it was derived from Rauber's sickle cells which colonized the subgerminal ooplasm. Our experiments indicate that the uncommitted upper layer (UL) of the anti-sickle of unincubated blastoderms constitutes an excellent reactor tissue for inductions. The thickening induced in the UL of the anti-sickle region by Rauber's sickle initiates (pre)gastrulation and the thickening induced by endophyll initiates (pre)neurulation. In the beginning, pregastrulation and preneurulation seem to be independent phenomena. It is only later, when both become correctly linked at the right place and time, that a normal embryo will develop.

Comparison between oogenesis and related ovarian structures in a reptile, Pseudemys scripta elegans (turtle) and in a bird Coturnix coturnix japonica (quail).

The aspect of the oogonia during their premitotic DNA synthesis and of the premeiocytes during their premeiotic DNA synthesis was studied in turtles by autoradiography, after injection of 3H-thymidine. As in the adult laying quail, the intrafollicular oocytes of the adult turtle go through three successive stages: prelampbrush, lampbrush and postlampbrush. During the prelampbrush and lampbrush stage two kinds of nucleoli exist: peripheral and central. In contrast to avian yolk, during its final rapid growth, no polyhedric protein yolk units were found in turtle yolk. As in the yellow yolk of quail, highly osmiophilic alcohol insoluble satellite yolk (egg oil) accumulates between the protein globular yolk of Pseudemys. Turtle yolk globules increase in volume by fusion. The penetration of peripherally assembled yolk in the turtle germinal disc is analogous to what we have described in the quail. Also in postlampbrush germinal discs subcortical ooplasmic organelles are present. Below the turtle germinal disc no structure comparable to the avian nucleus of Pander could be observed. No pyriform cells (as in squamate reptiles) and no pyriform-like cells (as in birds: Callebaut, 1991 b) were found in the chelonian ovarian granulosa layer. We could not demonstrate functional lacunoperitoneal communications via openings in the hilus ovarii of the turtle as is the case in birds.

Spatial relationship between endophyll, primordial germ cells, sickle endoblast and upper layer in cultured avian blastoderms.

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.

Avian gastrulation and neurulation are not impaired by the removal of the marginal zone at the unincubated blastoderm stage.

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.

Map of the Anlage fields in the avian unincubated blastoderm.

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.

Immunohistochemical localization of transforming growth factor-beta 1 beta 2 during folliculogenesis in the quail ovary.

Immunohistochemical methods were used to show the presence and distribution of transforming growth factor-beta 1 and beta 2 during folliculogenesis in quail ovarian tissues. The results indicated that both transforming growth factor-beta subtypes are present. Immunolabelling for transforming growth factor-beta 1 demonstrated that prelampbrush oocytes are immunoreactive in the Balbiani complex, and developing and pre-ovulatory oocytes in the zona radiata. Immunolabelling was also associated with granulosa cells. The number of stained granulosa cells decreased during folliculogenesis. In the pre-ovulatory follicles, immunolabelling was found predominantly in the theca interna. Immunolabelling for transforming growth factor-beta 2 was associated with the zona radiata of developing and pre-ovulatory follicles, and with stromal interstitial cells. Moderate immunoreactivity was found in the Balbiani complex of prelampbrush oocytes. Weak immunolabelling was localized in the granulosa cells of prelampbrush follicles, and in a few cells of the theca interna of pre-ovulatory follicles. The immunolocalization of transforming growth factor-beta 1 and -beta 2 in the quail ovary supports their autocrine and/or paracrine role in avian ovarian processes.

Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.

Immunohistochemical localization of epidermal growth factor in the ovary of the adult Japanese quail.

The present study focuses on the immunohistochemical localization of epidermal growth factor in the ovary of the adult Japanese quail. Immunoreactivity was predominantly found in the smooth muscle cells of blood vessels and of chordae, in granulosa cells of pre-lampbrush follicles, in interstitial cells, in the Balbiani complex of pre-lampbrush oocytes, and in ganglia. In developing follicles, immunoreactivity was also detected in some granulosa and thecal cells, in the zona radiata, and especially in cell clusters localized in the thecal periphery. The number of immunostained cells in the granulosa decreased during folliculogenesis, and increased after ovulation. In the ooplasm of oocytes, immunoreactivity was shifted from the Balbiani complex to the zona radiata during development. These observations support the hypothesis that epidermal growth factors acts primarily on less differentiated follicles. It is also suggested that epidermal growth factor can modulate ovarian contractility. Finally, in one ovary, we detected immunostained bodies in the ooplasm of small developing oocytes.

Persistence after hypothalamectomy of a tamoxifen-like effect in the ovary of quail embryos exposed cyclically in ovo to low incubation temperatures.

Three days old quail embryos were hypothalamectomized in ovo by the decapitation technique according to Callebaut (1993). Half of these embryos was exposed to day cyclically repeated prolonged low subnormal incubation temperatures (25 degrees C). The other half of these decapitated embryos was normally continuously incubated. As is the case for not decapitated embryos the ovaries of the first group presented a tamoxifen like effect: cortical rim atrophy with proportional hypertrophy of the medulla and a pronounced decrease in the number of oogonia and oocytes. By contrast the ovaries of the second group of decapitated embryos presented a normal aspect without sterilization of the ovigerous sex cords. It was concluded that: 1. the hypophysis, the hypothalamus or other prosencephalic tissues play no intermediary role in the establishment of a tamoxifen like effect in the ovary; 2. the early embryonic development of the quail ovary during the considered period takes place without the intervention of the hypothalamus.

Gastrulation inducing potencies of endophyll and Rauber's sickle in isolated caudocranially oriented prestreak avian blastoderm quadrants (or fragments) in vitro.

Unincubated quail and prestreak chicken blastoderms were sectioned into cranial, caudal and lateral quadrants and cultured in vitro. Whilst in the caudal and lateral quadrants (containing both endophyll and Rauber's sickle material) a primitive streak (PS) developed, in the cranial quadrants (where merely central endophyll is present) only an ectoblastic thickening (early gastrula: phase I of gastrulation) appeared. If, however, sickle material was grafted on a cranial quadrant then a PS developed (phase II of gastrulation). In the anti-sickle region (lacking sickle material and endophyll) no gastrular differentiation occurred spontaneously. The grafting of endophyll and/or Rauber's sickle evokes here also phase I or II of gastrulation. When the endophyll was removed from the caudal quadrant no PS appeared. If, however, endophyll was replaced, a PS and neurula developed. Our study demonstrates that the presence of endophyll is necessary to obtain an early gastrular development (phase I of gastrulation). It seems also to have an influence on the formation of the future neurectodermal region by induction of a thickening of the cranial upper layer. Only when both endophyll and sickle material were present, a PS formed in caudal quadrants. In the caudal quadrant, the endophyll (which seems to represent the vegetative pole of the avian blastula) does not regenerate, neither from the upper layer, nor from Rauber's sickle (containing blastoporal material: Callebaut and Van Nueten: 1994).