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Keywords:

  • α, β, γ, δ ooplasm;
  • avian germ disc;
  • Rauber's sickle;
  • gastrulation;
  • blood islands;
  • neurulation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

In the avian oocytal germ disc region, at the end of oogenesis, we discerned four ooplasms (α, β, γ, δ) presenting an onion-peel distribution (from peripheral and superficial to central and deep. Their fate was followed during early embryonic development. The most superficial and peripheral α ooplasm plays a fundamental role during cleavage. The β ooplasm, originally localized in the peripheral region of the blastodisc, becomes mainly concentrated in the primitive streak. At the moment of bilateral symmetrization, a spatially oblique, sickle-shaped uptake of γ and δ ooplasms occurs so that γ and δ ooplasms become incorporated into the deeper part of the avian blastoderm. These ooplasms seem to contain ooplasmic determinants that initiate either early neurulation or gastrulation events. The early neural plate-inducing structure that forms a deep part of the blastoderm is the δ ooplasm-containing endophyll (primary hypoblast). Together with the primordial germ cells, it is derived from the superficial centrocaudal part of the nucleus of Pander, which also contains δ ooplasm. The other structure (γ ooplasm) that is incorporated into the caudolateral deep part of the blastoderm forms Rauber's sickle. It induces gastrulation in the concavity of Rauber's sickle and blood island formation exterior to Rauber's sickle. Rauber's sickle develops by ingrowth of blastodermal cells into the γ ooplasm, which surrounds the nucleus of Pander. Rauber's sickle constitutes the primary major organizer of the avian blastoderm and generates only extraembryonic tissues (junctional and sickle endoblast). By imparting positional information, it organizes and dominates the whole blastoderm (controlling gastrulation, neurulation, and coelom and cardiovascular system formation). Fragments of the horns of Rauber's sickle extend far cranially into the lateral quadrants of the unincubated blastoderm, so that often Rauber's sickle material forms three quarters of a circle. This finding explains the regulative capacities of isolated blastoderm parts, with the exception of the anti-sickle region and central blastoderm region, where no Rauber's sickle material is present. In avian blastoderms, there exists a competitive inhibition by Rauber's sickle on the primitive streak and neural plate-inducing effects of sickle endoblast. Avian primordial germ cells contain δ ooplasm derived from the superficial part of the nucleus of Pander. Their original deep and central ooplasmic localization has been confirmed by the use of a chicken vasa homologue. We conclude that the unincubated blastoderm consists of three elementary tissues: upper layer mainly containing β ooplasm, endophyll containing δ ooplasm, and Rauber's sickle containing γ ooplasm). These elementary tissues form before the three classic germ layers have developed. Developmental Dynamics 233:1194–1216, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Spemann and Mangold (1924) discovered the so-called “Spemann organizer” in amphibian embryos. They observed that, after transplantation of a piece of dorsal blastopore lip of the early gastrula to the ventral side of another embryo, a secondary embryonic axis developed on this side. So the existence of induction between different associated parts of embryonic tissues was decisively shown in favor of epigenetic development. Spemann's organizer influences the fate of a part of the host cells and induces them to form axial structures, more particularly a neural plate, the rudiment of the central nervous system.

In 1969, Nieuwkoop discovered that, during early blastulation in amphibia, signals are required from a region that is localized vegetal to the prospective dorsal blastopore lip to initiate development of the mesodermal (or Spemann's organizer). This vegetal region is now designated as Nieuwkoop's center. In amphibians, Wnts seem to be the primary axis (formation of a head and trunk–tail regions) -inducing factors, as microinjection of several Wnts into the ventral cells of early embryos leads to a complete duplication of the body axis (Cadigan and Nusse, 1997; Deardorff et al., 1998). This duplication is believed to arise from the formation of a second Nieuwkoop's center (dorsal vegetal cells) of the early blastula, which then induces overlying tissue to become Spemann's organizer (homologous to the avian Hensen's node; Waddington, 1932). Indeed in birds and mammals the properties of Spemann's organizer are performed by the node that is the rostral end of the elongated primitive streak (PS). Besides induction events occurring between neighboring structures of the avian blastoderm, our experimental studies in birds also indicates that induction events occurred from neighboring extrablastodermic ooplasms, such as the nucleus of Pander (1817; Callebaut et al., 2004b) or from incorporated neighboring γ ooplasm such as Rauber's sickle, forming junctional endoblast (Callebaut and Van Nueten, 1994), indicating the existence of ooplasmic determinants in the avian embryo. For a better understanding of the phenomena that take place during early embryonic development, we must consider first the substrate in which the blastoderm will develop: the avian oocyte at the end of oogenesis. By radioactive or trypan blue-induced fluorescent labeling of the oocytal layers we followed the fate of the four ooplasms (α, β, γ, δ) in the avian blastoderm and neighboring ooplasm. For the visualization of the ooplasms, we used preferentially the quail rather than the chick because the former is much smaller, grows faster, and the application of radioactive products is much less expensive. After a description of events in the extrablastodermal subgerminal ooplasm and subgerminal space at the moment of laying, we studied the inductive influences of tissues, with nonembryonic fate, i.e., Rauber's sickle and the deep elements of the area centralis (sickle canal, endophyll, and sickle endoblast) on the upper layer, from which the whole embryo proper will form, resulting in the formation of a PS and Hensen's node.

The deep central origin of the primordial germ cell material from the nucleus of Pander and its relationship with the endophyll was discussed. Finally, we followed the evolution of marginal blastoderm structures, localized between area centralis and area opaca, i.e., the caudal marginal zone (caudally) and the anti-sickle region (cranially).

DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

At the end of its final growth period (postlambrush stage: Callebaut, 1973), the ooplasm of the avian germ disc exhibits both histochemically and autoradiographically a concentric, radially symmetric distribution. DNA- and RNA-containing material is bound to mitochondria forming the so-called ticos (tritiated thymidine-incorporating cytoplasmic organelles; Callebaut, 1972, 1983a; D'Herde et al., 1995; Fig. 1). This radially symmetric distribution is similar to the mitochondria-bound radial and circular crown-shaped symmetry, which has been described in Xenopus oocytes (Tourte et al., 1984; Mignotte et al., 1987).

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Figure 1. Schematic drawing of the radial symmetry in the premature quail (Coturnix coturnix japonica) oocyte, visible by the presence of RNA-rich subcortical cytoplasmic organelles (ticos, aggregates of mitochondria represented by small circles, one tico is indicated by arrow); the large circular line represents the periphery of the germinal vesicle with spherical postlampbrush chromosomes (represented by dots, indicated by arrowhead) in its center (Callebaut, 1972; D'Herde et al., 1995). In this and all subsequent figures, the orientation is indicated by A (anterior) and B (posterior).

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At the end of the avian oogenesis, we described the existence of four morphologically and functionally different types of ooplasms: α, β, γ, δ. These are localized, respectively, from peripheral and superficial to central and deep (Callebaut, 1987; Fig. 2A). Each of these ooplasms will play a distinct role in the early development of the avian germ both in space and time. The most peripheral and superficial α ooplasm becomes very mobile immediately after fertilization. It contains numerous osmiophilic granules and penetrates along with the cleavage furrows into the more central and deeper ooplasms (Fig. 2B–D). Specifically, the α ooplasm penetrates into the superficial part of the δ ooplasm-containing nucleus of Pander (with typical primordial yolk granules; Fig. 2C,D) and so delimits the blastomeres of the early blastodisc from the deep part of the nucleus of Pander. This area is the region where the primordial germ cell yolk is localized (Callebaut, 1983b, 1984). The α ooplasm disappears from the blastodermeres after the cleavage. The β ooplasm, originally localized in the peripheral region of the blastodisc, becomes mainly localized in the PS and forms somatic structures.

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Figure 2. A: Schematic drawing of a midsagittal section through a quail oocyte just before maturation, representing the four ooplasmic regions with an onion-peel organization: the α ooplasm (red), most superficial and peripheral; the deep β ooplasm (blue); the γ ooplasm (green) surrounding the nucleus of Pander; and the deepest central δ ooplasm (yellow) in the nucleus of Pander. The oblique split represents the spatially unequal formation of the subgerminal space, resulting in the spatial oblique uptake in the deeper part of the blastoderm of peripheral γ ooplasm forming the Rauber's sickle (RS), and the uptake of the δ ooplasm, forming endophyll (E) in the future caudal region of the blastoderm; GV, germinal vesicle. B: Surface view of an early avian cleavage stage covered with superficial α ooplasm (in red). C: Schematic representation of the localization of the α (red), β (blue), and δ (yellow) ooplasms in the first large “closing” central blastomeres as represented in B. The forming subgerminal space is lined by a narrow α layer. F, cleavage furrow; NP, nucleus of Pander containing δ ooplasm (Callebaut, 1987) containing primordial yolk globules, indicated by dots. D: Section through an Epon embedded quail germ (early cleavage stage) after a lipid-conserving fixation. Here also the superficial α ooplasm (containing numerous osmiophilic granules) is seen to penetrate into the depth, along with the cleavage furrow (F) and lines also its deepest distended part (d), forming the anlage of the subgerminal space. Scale bar = 100 μm in D.

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The γ ooplasm surrounds the nucleus of Pander (Figs. 2A, 3A). It is in this γ ooplasm-containing region that Rauber's sickle (Rauber, 1876) develops at the moment of bilateral symmetrization (Figs. 3B,C, 4A,B). This bilateral symmetrization, characterized by the appearance of a Rauber's sickle (Callebaut, 1993a), takes place approximately 6 hr before laying (Clavert, 1960) and is determined by gravity (Kochav and Eyal-Giladi, 1971). This change occurs by the eccentric displacement and tilting of the nucleus of Pander with surrounding γ ooplasm below the overlying more superficial blastoderm part, close to the upper germ wall, under the influence of the rotation and oblique position of the egg in ovo (Callebaut, 1993a–c; Fig. 4B). Indeed by this distortion, the deep cellularized part of the germ disc comes into intimate contact with the underlying ooplasm. In the diametrically opposed anti-sickle region, close to the lower germ wall, on the contrary, the contact with the underlying ooplasm is disrupted (Figs. 3C, 4B), and the quantity of incorporated ooplasm in this region of the blastoderm remains low or absent. This finding is particularly clearly seen on autoradiographs of the fifth laid egg after an injection of [3H]tyrosine into the mother quail. In a midsagittal section of such a blastoderm (Fig. 5), we see that, in the Rauber's sickle region, the labeled ooplasm (surrounding the δ ooplasm of the nucleus of Pander) adheres vertically to the upper layer. This adhesion seems to be at the origin of the formation of the “Sichel Rinne” (Koller, 1882), that is, a superficial sulcus in the upper layer above Rauber's sickle probably formed by the inward traction on the upper layer (Fig. 5). By contrast, in the diametrically opposed anti-sickle region, all the homologous ooplasm has lost contact with the upper layer as the result of disruption from this ooplasm. This process is already a first indication that ooplasm taken up by the blastoderm, or not, plays a fundamental role during the further development of the involved part. By oblique positioning of the avian germ disc in ovo (Callebaut, 1993a; Fig. 4), the formation of a Rauber's sickle can be evoked in any part of the γ ooplasm region. Thus, originally the ooplasm that helps to form Rauber's sickle is circularly distributed. From a comparative literature study, Eyal-Giladi (1997) concludes that the establishment of the axis in chordates (axialization with bilateral symmetrization) depends on the translocation of oocytal (maternal) determinants from the vegetal pole toward the future dorsocaudal side of the embryo. On arrival at their destination, the activated determinants form in all chordates an induction center homologous to the amphibian “Nieuwkoop center” (or avian Rauber's sickle), which later will induce, respectively, the formation of the “Spemann's organizer” or avian Hensen's node. In the case of birds, these determinants seem to be originally present in the deep peripheral circular γ ooplasm, surrounding the δ ooplasm-containing nucleus of Pander. The underlying massive yolk mass presses, by unilateral mechanical pressure during the in utero rotation, part of this γ ooplasm against the deep peripheral cells of the blastoderm rim, so forming Rauber's sickle. In the unincubated egg, these Rauber's sickle cells and later junctional endoblast cells still continue to take up large volumes of ooplasm by active encircling movements (Callebaut, 1993b).

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Figure 3. A: Schematic representation of the circular localization (seen in vertical projection) of the γ ooplasm (in green) in the avian germ disc region before bilateral symmetrization. The γ ooplasm surrounds the more central δ ooplasm (yellow in the figure) of the nucleus of Pander. B: Schematic representation of the ovoid localization of the γ ooplasm (in green, seen in vertical projection) in the bilaterally symmetrized blastoderm. The condensed Rauber's sickle (RS) and the expanded anti-sickle region (AS) have eccentrically formed in the γ ooplasm-containing region, which surrounds the δ ooplasm (yellow in the figure). C: Schematic representation (simplified after Callebaut, 1993b) of the localization of two intraoocytally radioactively labeled layers (respectively, 4 or 6 days after a maternal radioactive injection) in the γ and δ ooplasms on a midsagittal section through an unincubated avian blastoderm (bilaterally symmetrized). Note the permanent boot-shaped deformation of the ooplasmic layers around the nucleus of Pander (NP) composed of δ ooplasm (yellow) and in the surrounding γ ooplasm (green). The toe-shaped part of both γ and δ layers are expanded and horizontally flattened. They remain in the underlying ooplasm below the anti-sickle region (AS), and they have lost contact with the blastoderm. Caudally, in the heel-shaped part, the γ ooplasm is more condensed and adheres vertically to the upper layer, forming Rauber's sickle (RS); the heel and middle part of the layers containing δ ooplasm of the nucleus of Pander (NP) are taken up in the caudal part of the area centralis of the blastoderm and later segregate progressively as endophyll (E), which also contains δ ooplasm (Callebaut, 1987, 1993a); in blue is upper layer mainly composed of β ooplasm.

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Figure 4. A: Schematic representation (not to scale) of the air-bubble (AB) method to stabilize quail germs in ovo in a vertical or oblique orientation. The external eccentric aspect of a quail blastoderm during early bilateral symmetrization is also represented. AP, area pellucida; AS, anti-sickle region; LGW, lower germ wall (future cranial region); RS, anlage of Rauber's sickle; UGW, upper germ wall (future caudal region). B: Stereomicroscopic surface view of a living quail blastoderm after 12-hr in ovo incubation in a vertical orientation in a extracted white shelled egg, as represented in A. Labels are as in A (after Callebaut, 1993a). Scale line = 2.5 mm.

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Figure 5. Autoradiograph of a midsagittal section through a spontaneously developed unincubated quail blastoderm growing in the fifth labeled egg yolk ball (after a maternal injection of [3H]tyrosine). The labeled γ ooplasm-containing layer is boot-shaped, surrounding a caudal heel-shaped (H) and a cranial toe-shaped (T) unlabeled ooplasm. The disruption of the cranial part of the blastoderm from the underlying toe-shaped–labeled γ ooplasm (forming a hook) is clearly seen, whereas in the caudal part a vertical contact remains at the level of Rauber's sickle; arrow indicates the “Sichel Rinne,” i.e., a superficial sulcus in the upper layer, probably due to adhesion with the underlying Rauber's sickle. Scale bar = 300 μm (after Callebaut, 1994).

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The relationship between the spatial orientation of the early vertebrate blastoderm on its egg yolk and the ensuing development of its caudocephalic axis has been described first by Wintrebert (1922) in the egg of Selachians. In the Selachian egg, also any part of the periphery of the blastoderm can give rise to the embryonic caudal edge: the only condition is that it should correspond temporally to its highest point. There is much homology, thus, with the situation in avian blastoderms. Our study indicates that also in amniote embryos ooplasmic determinants play a fundamental role.

Apart from the initiation of gastrulation by the uptake of γ ooplasmic determinants, also the initiation of neurulation can be explained by the uptake in the blastoderm of δ ooplasmic determinants. I have described (Callebaut, 1987, 1993b) the eccentric formation of the avian endophyll (mainly localized caudally and centrally) by the oblique formation of the subgerminal space through the nucleus of Pander (Fig. 3C, line 6, with central part and heel taken up in the blastoderm). Both endophyll and nucleus of Pander contain δ ooplasm and induce an early neural plate (with neural groove and folds) in the neighboring upper layer (Callebaut et al., 2004b). This also suggests a role for ooplasmic determinants present in the δ ooplasm.

We think that the formation of the central nervous system occurs in two periods: an early neural plate stage with pronounced thickening of the neighboring upper layer and characterized by a more or less pronounced neural groove with median basal plate anlage and voluminous lateral folds (Fig. 6). Later, floor plate tissue, derived from Hensen's node (if present), bisects the basal plate anlage and the definitive spinal cord is formed (Catala et al., 1995, 1996; and our unpublished results).

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Figure 6. Transverse section through isolated cranial quadrant of quail blastoderm cultured in vitro for 8 hr. E, endophyll; UL, thickened upper layer presenting an early neural plate aspect with neural groove and walls. Both endophyll and upper layer keep distance even at their rims (arrowheads). Scale bar = 200 μm.

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Since the in ovo blastoderm fragmentation experiments of Lutz (1949), it was assumed that the avian unincubated blastoderm, oriented approximately according to the rule of Von Baer (1828) still has a potential radial symmetry (forming a so-called regulative system). Also Spratt and Haas (1960, 1965) describe the radial pattern of differential growth in cultured isolated quarters of unincubated chicken blastoderms, however, often without an exact original orientation, because the localization of the Rauber's sickle was not used as a definitive landmark. According to the radially symmetry hypothesis any isolated blastoderm quadrant should give rise to a complete miniature embryo. After placing isolated lateral and caudal quadrants (oriented with reference to the localization of Rauber's sickle and each containing a part of Rauber's sickle) in culture, we observed that these quadrants effectively always form a complete miniature embryo with a neural plate and a PS, induced by Rauber's sickle material (Callebaut and Van Nueten, 1995). By contrast, in cultured isolated cranial quadrants, we observed two kinds of development (Callebaut et al., 2000c). In approximately 50–60% of the cases only a broad early neural plate formed. Sections through the latter show a pronounced thickening of the upper layer, with neural groove and neural walls, separated from the endophyll by a broad space (Fig. 6). No signs of ingression or PS formation were observed. In the other cases, both a PS and a neural plate were observed, forming usually a centrally directed embryo. The reason for the different developmental behavior was either the absence of Rauber's sickle horn tips in the cranial quadrants, in the first case, or their presence in the latter case. Endophyll was present in both cases and was responsible for the inductive action in the upper layer with the formation of a neural plate in all the cranial quadrants. This finding again demonstrates that endophyll (primary hypoblast) exists as a separate entity, different from Rauber's sickle material and also different from sickle endoblast (secondary hypoblast), which is derived from Rauber's sickle (Callebaut and Van Nueten, 1994). Thus, in the unincubated avian blastoderm, three fundamentally different, elementary tissues can be discerned: the upper layer mainly composed of β ooplasm, the endophyll containing δ ooplasm, and Rauber's sickle material containing γ ooplasm forming nearly three quarters of a circle.

The different components of the unincubated avian blastoderm and subgerminal ooplasm are represented in Figure 7A,B. We used mainly the terminology of Vakaet (1970) during the description of the tissues present in the early avian embryo. The term upper layer (UL), which covers the whole blastodermal area, is used because, in the unincubated avian blastoderm, this layer is still multipotent and gives rise to not only the ectoderm proper but also to the mesoderm, definitive endoderm, and to neurectoderm. The deep part of the unincubated blastoderm is composed successively from caudally to rostrally, of the deep part of the caudal germ wall, the deep part of the caudal marginal zone, Rauber's sickle, the incomplete endophyll corresponding to the primary hypoblast (Eyal-Giladi and Wolk, 1970). Eyal-Giladi and Kochav (1976) describe the endophyll in their Stage X (freshly laid chicken blastoderm removed from its yolk ball) as a meshy layer just cranial to Rauber's sickle. They regarded this stage as the first sign of primary hypoblast formation. The latter endophyll does not reach into the most cranial part of the unincubated blastoderm as was suggested and depicted by Vakaet (1962b, 1970). Indeed in this cranial part, there is usually an empty space (lacune) where no deep layer tissue is found (Harrisson et al., 1991) except in the most cranial part (anti-sickle region) where some loose disrupted yolk masses or cell aggregates are found forming sometimes a crescent-shaped anti-sickle. The latter can in some blastoderms be seen from the surface (Fig. 8) and can even be confused with the real Rauber's sickle. Therefore, we think that Vakaet (1970) in his description of the unincubated blastoderm was misled and concluded that endophyll was found in the whole deep part of the area centralis.

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Figure 7. A: Schematic representation of the components of an unincubated quail blastoderm seen from below, after removal of the subgerminal ooplasm, ready for in vitro culture. AC, area centralis enclosed by Rauber's sickle (RS) and the sickle horns; AS, anti-sickle region; CA GW, caudal germ wall; CMZ, caudal marginal zone, more or less transparent; CR GW, cranial germ wall; EN, incomplete endophyll sheet (yellow); L, lacuna in the deep layer, the deep side of the UL is visible (blue); SH, fragmentary far-cranially extending sickle horns. B: Schematic representation of a midsagittal section (indicated in A by a vertical line) through an unincubated quail blastoderm with surrounding ooplasms (β ooplasm, blue; γ ooplasm, green; δ ooplasm, yellow) after fixation in situ on the egg yolk ball. CHR, chromosome clusters; CSO, central subgerminal ooplasm in which the central nucleus of Pander (NP; Pander, 1817) is seen; E, edge of the blastoderm;EN, incomplete endophyll layer (in yellow); LN, bended latebra neck; OG, early overgrowth zone (Callebaut and Meeussen, 1988); PAO, paragerminal ooplasm forming a tubulin-rich ring (TUB) at distance from the edge of the blastoderm (Callebaut et al., 1996b); PEO, perigerminal ooplasm; RS, Rauber's sickle (in green); SGS, subgerminal space forming a caudal pocket A (axilla shaped) and a cranial recess (R) in which free yolk masses or sometimes cells are found forming the anti-sickle (AS); t, toe-shaped and h, heel-shaped part of the nucleus of Pander; T, toe-shaped and H, heel-shaped part of the surrounding yolk layers (γ ooplasm) as result of the rotation in utero (the large arrow indicates the direction of rotation and compression of the yolk mass under the combined influence of gravity and egg rotation; UL, upper layer (in blue) with “Sichel Rinne” superficial sulcus in the upper layer above Rauber's sickle (arrowhead); UL CMZ, upper layer from the caudal marginal zone (the caudal marginal zone being a more or less transparent part adherent to the caudal peripheral subgerminal ooplasm [PSO] by means of a deeper part [DL CMZ]); YE, early development of the yolk endoblast, growing into the peripheral subgerminal ooplasm (PSO); YM, the voluminous yolk mass of the egg yolk ball in which the eccentricity of the successive yolk layers (after daily injections of [3H]tyrosine) parallel with the eccentricity in the blastoderm is represented. Callebaut, 1983, 1993a). Note that, by contrast to the caudal region, the cranial region is disrupted from the underlying peripheral subgerminal ooplasm (Callebaut, 1993a–c).

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Figure 8. Surface view of a living unincubated quail germ in situ on top of its egg yolk ball. AO, area opaca with clear-cut edge (E); AS, anti-sickle region containing numerous disrupted ooplasmic masses and cells; NP, remaining deeper part of the nucleus of Pander, visible through the transparent area pellucida (AP); PZ, perigerminal clear zone limited by a white halo (WH), forming the paragerminal β tubulin-containing ooplasm; SR, sickle of Rauber. Scale bars = 3 mm (after Callebaut and Meeussen, 1988).

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To have a clear view on the relationship between the blastoderm proper and the underlying ooplasms and yolk mass, it is often necessary to fix the blastoderm on its egg yolk ball in toto (Callebaut, 1994). We will now successively focus on the localization and function of each of these components.

SUBGERMINAL SPACE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

The subgerminal space is already partially formed during the cleavage stage. Our study (Callebaut, 1987) indicated that the α ooplasm, which penetrates during the cleavage stage into the underlying γ and δ ooplasm along with the cleavage furrows, lines also the subgerminal space at depth. Just in front of Rauber's sickle, the subgerminal space ends abruptly by the adhesion of deep and superficial layers and forms there a wide arm-pit–like groove (axilla) in which usually no free material is seen (Fig. 7B). By contrast, the cranial part of the subgerminal space (recess) forms a narrow split under the anti-sickle region and the cranial germ wall. This recess is not closed but opens into the perigerminal space. It contains numerous flattened isolated yolk masses, some loose cells, and amorphous material forming the anti-sickle. This recess is formed by the disruption of the local blastoderm part from the underlying ooplasm during the period of bilateral symmetrization. The cranial part of the blastoderm contains no deep layer (no γ ooplasm) and, thus, no Rauber's sickle activity, because the ooplasm identical to Rauber's sickle is not taken up and remains in the subgerminal ooplasm beneath (Figs. 3C, 5). From their study, Kochav et al. (1980) concluded, only on a morphological basis, that at the moment of the area pellucida formation, there occurs a massive cell-shedding process. They presumed that all deeper cells of the germ fall into the subgerminal cavity and finally assemble in its lowest (future cranial) part under influence of gravity. We could not confirm this hypothesis, because after trypan blue–induced fluorescent labeling of the yolk in quail eggs, the labeled cells or yolk masses both in the caudal and cranial part of the subgerminal space always remained localized in the prolongation of the labeled subgerminal yolk layers (Callebaut, 1987). That study confirms the presence of free material under the lowest part of the area pellucida and under the lower (cranial) germ wall (Fig. 4) but suggests that this phenomenon is due to an early local tearing process between germ and underlying expanded subgerminal ooplasm (Fig. 5). This suggestion can probably explain the oblique formation of the subgerminal space (Fig. 2A) and why the sickle of Rauber is the most stable part of the unincubated blastoderm where most of the γ ooplasm remains in the blastoderm (Callebaut, 1987) probably by prolonged contact with/and local late colonization of the subgerminal ooplasm, forming the junctional endoblast.

SUBGERMINAL OOPLASM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Nucleus of Pander

In the center of the subgerminal ooplasm the nucleus of Pander (Pander, 1817; containing δ ooplasm: Callebaut, 1987) can be seen from the surface in the living state (Fig. 8). This nucleus of Pander is usually considered as a kind of “lost” yolk mass, having no influence on blastoderm development. However, we have shown that when a prelaid nucleus of Pander (from a quail egg extracted before laying) is placed on the deep side of the uncommitted upper layer of an isolated anti-sickle region of an unincubated chicken blastoderm, it induces an early neural plate (Callebaut et al., 2004b). This finding probably also occurs temporally in situ in the unaltered germ disc region during early development in combination with the also temporal effect of endophyll (both containing δ ooplasm). Indeed, in situ in the egg yolk ball, at the end of the intrauterine period and also shortly after laying, the nucleus of Pander and endophyll are in very close contact with the upper layer. In the nucleus of Pander, I have observed the presence of some round Feulgen-positive spherules (Callebaut, 1994). After [3H]thymidine, [3H]tyrosine, [3H]uridine, or [3H]tyrosine administration in ovo, before laying, an intense incorporation of these precursors was observed in and around the nucleus of Pander, which indicates DNA, RNA, and/or protein synthesis, perhaps in relation with the inductive influence emanating from the nucleus of Pander. The aspect and evolution of the nucleus of Pander during early incubation was studied by magnetic resonance imaging (Falen et al., 1991), however, without histological data.

Peripheral Subgerminal Ooplasm

The peripheral subgerminal ooplasm (localized mainly in the β ooplasm; Fig. 7B) is found in a broad circular area below the periphery of the blastoderm (Callebaut, 1994). After in toto Feulgen staining, large numbers (250 per mm2) of 40-μm diameter clusters of grouped mitotic figures are seen. After in ovo application of [3H]thymidine, [3H]uridine, or [3H]tyrosine, also an intense labeling of these sub-blastodermic chromosome groups and surrounding intervitelline material was seen. The function of these elements localized in a giant annular syncytium is not known.

Peri- and Paragerminal Ooplasm

On the surface of the germ disc region of the unincubated egg just peripherally to the usually sharply defined edge of the unincubated living blastoderm, a broad clear circular perigerminal ooplasmic zone is seen that surrounds the opaque area opaca of the blastoderm (Fig. 8). More peripherally at a distance of approximately 400 μm from the edge of the blastoderm, a white halo (paragerminal ooplasm) is observed (Fig. 8). We found a high concentration of β-tubulin in this paragerminal ooplasmic ring (Callebaut et al., 1996b). The distribution of β-tubulin in the peri- and paragerminal ooplasm has much homology with the tubulin distribution seen in meroblastic teleost eggs (Solnia-Krezel and Driever, 1994). All the described avian extrablastodermic structures can perhaps be considered as belonging to one giant syncytial yolk cell described in the also meroblastic Danio eggs. In the latter, it has been shown that mesoderm-inducing signals are derived from the yolk cell and pass through the syncytial layer to the blastoderm (Mizuno et al., 1996). Also, during the early cleavage stages in teleost development, most ooplasmic determinants are segregated into the yolk cell, rather than in the blastoderm cells (Devillers, 1961). The present review indicates that, in birds, the polarized uptake of part of the extrablastodermic ooplasmic structures by the blastoderm, plays a fundamental role in the further early embryonic development (gastrulation and neurulation).

RAUBER'S SICKLE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

In the past, the very existence in unincubated avian blastoderms of a Rauber's sickle (1876) has been debated vigorously, for decades. The term Rauber's sickle is the exact synonym of Koller's sickle. The reason for the doubt about the actual existence of a Rauber's sickle in every unincubated avian blastoderm seems to be that it was only visible from the surface of the freshly laid quail egg in approximately 30% of cases (Fig. 8; Lutz, 1964; Fargeix, 1964).

At the level of Rauber's sickle, the upper layer does not appear as a uniform cell layer but rather as a syncytium with poorly delineated cell borders (Harrisson et al., 1991). The basal surface of the upper layer is irregular, but blebbing is not observed. Because during several decades Rauber's sickle was not considered as a deep layer component distinctly separable from the caudal marginal zone, its function remained unknown until Callebaut and Van Nueten (1994) first systematically isolated and transplanted (without surrounding tissues) quail Rauber's sickles (which are usually much larger than chicken Rauber's sickles). They demonstrated the strong inductive activity of Rauber's sickle for the formation of mesoblast and definitive endoderm. Spratt and Haas (1965) made no clear distinction in the unincubated chicken blastoderm between Rauber's sickle and the caudal marginal zone. Therefore, they were not able to recognize the anti-sickle region (Figs. 7, 8) in which no Rauber's sickle material can be detected morphologically or functionally (Callebaut and Van Nueten, 1995).

Rauber's Sickle and Not the Caudal Marginal Zone Initiates Primitive Streak Formation in the Area Centralis

Although it is now clear that Rauber's sickle has strong PS-inducing effect (Callebaut et al., 2003a), earlier studies suggested that the inductive signal is provided by the caudal marginal zone (Eyal-Giladi and Khaner, 1989; Stein, 1990; Eyal-Giladi et al., 1992; Khaner, 1998). However, it was not always clear whether a part or all of Rauber's sickle was taken together with the caudal marginal zone in their transplantation experiments. Furthermore, no distinction (or an arbitrary one) was made between the area centralis (encircled by Rauber's sickle, which was not always seen) and the larger area pellucida, which also includes the caudal marginal zone. According to Eyal-Giladi et al. (1992, 1994), cells from the caudal marginal zone contribute to both the forming hypoblast and the forming PS by centripetal migration by means of Rauber's sickle. By contrast Bachvarova et al. (1998, Bachvarova et al. 1999) claimed that the caudal marginal zone is able to induce an ectopic PS without contributing to the streak. Thus, according to the latter authors, the caudal marginal zone should have Nieuwkoop center (Nieuwkoop, 1969, 1973) activity (inducing endomesoblast). In their study, a quail caudal marginal zone was associated in vitro with a chick cranial half blastoderm. In such associations, a centrally directed PS was often observed starting from the cranial part of this cranial half. However, we have shown that this finding also often occurs spontaneously in the absence of caudal marginal zone material (Callebaut and Van Nueten, 1993) by the presence of far cranially extending, unrecognized sickle horn material in the cranial half.

By the use of the quail–chicken chimera technique (Callebaut et al., 1996a), we observed that, during normal blastoderm development, the upper layer of the caudal marginal zone (at the convexity of Rauber's sickle) is not undergoing gastrulation movements, in contrast to the upper layer of the area centralis (in the concavity of Rauber's sickle). This was also confirmed by Lawson and Schoenwolf (2001). They concluded from labeling experiments that PS precursor cells originate from upper layer cells overlying the rostral border of Rauber's sickle and, thus, confirm our observations obtained by the use of quail–chick chimeras that Rauber's sickle is the early gastrulation organizer (Callebaut and Van Nueten, 1994; Callebaut et al., 2003a). So Rauber's sickle during early incubation remains the natural boundary between these two upper layer regions with a different fate.

In all our experimental studies, we have always observed a strong PS-inducing effect by Rauber's sickle, even in the total absence of the caudal marginal zone (Callebaut et al., 1997). After transplantation of quail caudal marginal zones or upper layer fragments taken from above Rauber's sickle on the cranial quadrants or anti-sickles of whole unincubated chicken blastoderms, we could not observe induction phenomena (Callebaut et al., 1998). However, we recently found that the presence of the endogenous Rauber's sickle can have a dominating and suppressive effect on the inducing capacities of other deep layer components of the same cell lineage, i.e., on sickle endoblast placed on the area centralis (Callebaut et al., 2003b). Therefore, we have tested the eventual PS-inducing effect of the caudal marginal zone placed on an isolated anti-sickle region (Rauber's sickle free) or on cranial blastoderm parts, which contain no Rauber's sickle material (as represented in Fig. 9). We could demonstrate, thus, that the avian caudal marginal zone by itself has no PS-inducing effect (Callebaut et al., 2002b).

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Figure 9. A: Schematic representation of a method to obtain a Rauber's sickle-free blastoderm fragment by excision and removal of Rauber's sickle and surrounding tissues (caudal and lateral germ walls, caudal and lateral marginal zones, and neighboring parallel area centralis region) from an unincubated chicken blastoderm. AS, anti-sickle region; CAGW, caudal germ wall; CMZ, caudal marginal zone; CRGW, cranial germ wall; END CH, chicken endophyll; RS, Rauber's sickle and its often far cranially extending horns (SH). B: The excised cranial and central part of the unincubated chicken (Gallus domesticus) blastoderm is covered with a quail vitelline membrane (VM) to avoid sliding and is placed in culture.

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Function of Rauber's Sickle in Avian Blastoderms Is Homologous to the Function of Nieuwkoop's Center in Amphibian Blastulas

This finding has been confirmed recently by the study of goosecoid genes in avian blastoderms. Indeed Lemaire et al. (1997) found strong expression of one of the goosecoid genes (Gsx) in the upper layer above Rauber's sickle, suggesting induction by the latter. So goosecoid expression was not found in Rauber's sickle, but above it. Later, this upper layer ingresses and also forms the Gsx-expressing PS and Hensen's node. The latter is close to the amphibian organizer in Spemann's Mangold's definition (1924). Arendt and Nübler-Jung (1999) made a detailed comparative morphology study of gastrulation in embryos of amphibians, reptiles, and birds. They also conclude that structures such as the avian hypoblast (sickle endoblast) and Rauber's sickle may have homologues with the organizer region (Nieuwkoop center) of amphibian embryos.

Although material derived from Rauber's sickle (sickle endoblast and junctional endoblast) and endophyll (Callebaut et al., 1998; Fig. 13) have an indispensable inductive function for the development of the definitive embryonic tissues (which are all derived from the upper layer) during gastrulation and neurulation, they never give rise to any definitive structure (Callebaut et al., 2003a). Thus, they belong to the so-called extraembryonic part of the blastoderm (as is also the case for the Nieuwkoop center; Guger and Gumbiner, 1995).

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Figure 13. Schematic drawing of the anchor-shaped spreading (seen from the dorsal side after removal of the upper layer and primitive streak region at stages 3–4 of Hamburger and Hamilton (1951) of the two cell lineages (sickle endoblast, SE; junctional endoblast, JE), derived from Rauber's sickle (colored in green); the small circles represent cellular connections between the sickle endoblast or junctional endoblast and the removed superficial layer. LC, left part of the sickle canal; RC, right part of the sickle canal; TE, transitional endoblast; DEF END (in blue), place where the definitive endoderm extends radially; ENDOPHYLL CR, localization of the endophilic crescent and wall (in yellow; simplified after Callebaut and Van Nueten, 1994).

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From Radial Symmetry by Means of Sickle-Shaped Bilateral Symmetry to Primitive Streak Formation

Once the formation of the Rauber's sickle is provoked by unidirectionally disturbing the mechanical equilibrium between the components of the blastoderm and the subgerminal ooplasm, the blastoderm becomes permanently fixed in an equilibrium in which the radial symmetry is definitively broken and replaced by bilateral symmetry characterized by the sickle shape of Rauber's sickle and also the sickle shape of the predisposed anlage fields found in the area centralis of the unincubated blastoderm (Callebaut et al., 1996a; Fig. 10). There exists also a parallelism between the sickle shape of the predisposed anlage fields and the also sickle-shaped layers in the underlying ooplasm (Callebaut et al., 2000c), which seems to indicate that, just as is the case for Rauber's sickle and the nucleus of Pander (Callebaut et al., 2004b), inductive processes emanate vertically from the underlying subgerminal ooplasm.

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Figure 10. Fate map of the early avian blastoderm: schematic representation of the main localization of the predisposed (not definitively committed) anlage fields (appropriately arranged but partial overlapping of neighboring fields not indicated) in the upper layer of a chicken (Gallus domesticus) unincubated blastoderm (slightly simplified after Callebaut et al., 1996a). Note the general eccentric sickle-shaped aspect of the anlage fields in the area centralis. Rauber's sickle (green); definitive endoderm, blue; lateral plates, somites, and notochord (chorda), red; central nervous system and intraembryonic ectoderm (I.E.ECT.), not colored. There is an obvious parallelism between the sickle shape of the anlage fields in the upper layer (UL) and the ovoid central subgerminal ooplasmic layers (Callebaut et al., 2000c). The curved arrows on the anlage fields indicate the directions of converging movements of the upper layer cells during formation of the primitive streak (Wetzel, 1929) and neurulation (Bortier and Vakaet, 1992).

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This sickle shape of the anlage fields also suggest that before the eccentric tilting of the subgerminal ooplasm occurred, there existed a perfect circular configuration probably in relation with the histological demonstrable radial symmetry before. The fate of these sickle-shaped anlage fields in the unincubated blastoderm is at that moment, however, not yet definitively determined. This determination will occur only after 5 to 6 hr of incubation, when the deep layer elements have already influenced the upper layer (UL) as we could demonstrate for the neural anlage fields (Callebaut et al., 1996a). These observations suggest the existence of a kind of early ooplasmic and embryonic predisposition. It is, thus, by unidirectional changing of the established equilibrium (principle of unidirectional chaos: Prigogine and Lefever, 1968) in the germ disc region that inductive signals appear, leading to the epigenetic phenomena. So during the ensuing gastrulation, the upper layer material of the sickle-shaped predisposed anlage fields will converge and ingress by means of the PS, localized over the permantly functional radius below which a maximum of ooplasm is concentrated (Figs. 10, 11). Therefore, all the material from each sickle-shaped anlage field concentrates on this single radius, which forms the final body axis. Here there is a strong similarity with the gastrulation and neurulation phenomena described by Vandebroek (1969) in selachian germs (also developing on very large eggs). Indeed, in the latter vertebrate group there also exist analogous sickle-shaped anlage fields both for gastrulation and neurulation and localized in the upper layer in the same succession order as in birds.

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Figure 11. Combined schematic drawing representing (1) the hypothetic diffusion of morphogens or signalling molecules (indicated by small dots) emanating from Rauber's sickle (RS) and its sickle horns (SH, green) into the neighboring tissues of the avian blastoderm, i.e., into the area centralis (AREA CENTR in blue), into the caudal marginal zone (CMZ), and into the caudal germ wall (CGW), where they can influence (induce or inhibit) ectopically placed structures (endophyll, other Rauber's sickle fragments, or junctional endoblast) to form or not to form a second streak; (2) the broad movements (indicated by curved arrows) of cell groups in the upper layer of the area centralis, in the direction of the median primitive streak (partially after Wetzel, 1929). The curved legs of the U-shaped lines indicate moving fronts of cell groups (corresponding to local primitive streak anlagen) that will ingress after convergence into the final median primitive streak (Callebaut et al., 2003c).

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Not only the fate but also gene expression patterns indicate a role of Rauber's sickle as an early avian organizer (Boettger et al., 2001). Accordingly, cells near Rauber's sickle express a whole battery of genes related to genes associated with the amphibian organizer, such as Gsc, Gsx, cNot1, cNot2, Hnf3 beta, otx2, and chordin (Bally-Cuif et al., 1995; Ruiz I Altaba et al., 1995; Stein and Kessel, 1995; Lemaire et al., 1997; Stein et al., 1998; Streit et al., 2000). All these expression domains are initially sickle-shaped, i.e., spread more or less broadly and transversely to the future longitudinal axis. These expression domains are localized in the concavity of Rauber's sickle, where we have localized the similarly sickle-shaped anlage fields in the upper layer of the unincubated chicken blastoderm (Callebaut et al., 1996a).

How can we explain that, finally, only one PS is formed in the middle region of the thickened upper layer? A recent experimental in vitro study (Callebaut et al., 2003c) suggests that a PS in avian blastoderms is induced by diffusion of morphogenetic substances emanating from Rauber's sickle (Fig. 11). 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 anti-sickle region of unincubated chicken blastoderms. The so-formed PS is localized below the vitelline membrane in the immediate neighborhood of the apposed Rauber's sickle material. This finding seems to indicate that Rauber's sickle organizes the formation of the avian PS according to the basic concept of “positional information” (Wolpert, 1969, 1998). 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 medial sliding over the basement membrane and fusion, this finally results in the formation of one single median PS (Bortier et al., 2001). Our study shows that a PS can be induced in the total absence of sickle endoblast or caudal marginal zone, by the only presence of Rauber's sickle material. We have shown by radioactive labeling that the upper layer cells slide “en bloc” over the basement membrane (Bortier et al., 2001), which greatly improves the migration of these cell groups and can explain the rapid fusion of neighboring PSs as represented by the movement of the curved lines moving in the direction of the final median PS (Fig. 11). The represented gross morphological movements correspond exactly to the observations of Wetzel (1929) after in ovo vital staining of the upper layer of chicken blastoderms. As was the case with an ectopically placed junctional endoblast (Callebaut et al., 2000b), our studies also seem to indicate that the permanent presence of Rauber's sickle (with a continuous release of signaling molecules), forming a local maximum, establishes a diffusion gradient throughout the blastoderm (schematically represented in Fig. 11). This gradient can dominate and inhibit the development of a PS at a distance. Our studies suggest 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.

By colonization and incorporating the underlying and neighboring γ ooplasm, Rauber's sickle progressively transforms into the voluminous junctional endoblast (Fig. 12) and extends a sheet of sickle endoblast in a cranial and centripetal direction under the upper layer (Fig. 13). The more voluminous junctional endoblast has a stronger PS-inducing effect than the less-developed Rauber's sickle from which it is derived (Callebaut et al., 2000b). This finding suggests that the quantity of the incorporated subgerminal extrablastodermic γ ooplasm plays a dominating role in inducing a PS.

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Figure 12. An early primitive streak chick blastoderm placed in culture, with deep side directed upward, to show the localization of the junctional endoblast (indicated by three arrowheads on the left side); just as Rauber's sickle from which it is originally derived, the junctional endoblast extends far cranially close to the cranial blastoderm quadrant and far beyond the level of the rostral end of the primitive streak. Scale bar = 1 mm.

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Sickle Canal

We have observed the existence in avian blastoderms of a voluminous blind ending (approximately 2–4 mm long; Fig. 14), previously unrecognized, sickle-shaped canal (termed sickle canal or pararchenteric canal; Callebaut et al., 2000a). It bulges into the subgerminal space medially from the junctional endoblast (derived from Rauber's sickle). In its bottom, we find a sickle endoblast which separates it from the subgerminal space (Figs. 15, 16). The sickle canal is found both in the chicken and quail blastoderm. The origin and evolution of the sickle canal have been followed (using quail–chicken chimeras), by apposing quail Rauber's sickle fragments on Rauber's sickle-free fragments of unincubated chicken blastoderms. Very obvious, on sections through these chimeras, is the intimate contact between the V- or U-shaped quail junctional endoblast and the first-formed blood islands, developing from mesoblast that migrates peripherally over the sickle canal and parallel junctional endoblast. Hence, the junctional endoblast induces blood islands.

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Figure 14. Stereomicrograph of a living primitive streak blastoderm (quail) incubated for 20 hr and removed from its egg yolk ball, seen from its deep side. The V-shaped thin walled blind ending transparent sickle canal (arrowheads) is very obvious; arrows indicate junctional endoblast on each side. n, Hensen's node; PS, primitive streak region; GC, germinal crescent formed by cranially displaced endophyll (Callebaut et al., 2000a). Scale bar = 1 mm.

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Figure 15. Section through the caudal part of a quail embryo after 23 hr incubation, fixed in situ on its egg yolk ball. PS, caudal part of the primitive streak region; SC, the lumen of one side of the sickle canal is very wide, whereas the lumen of the other side (indicated by an arrow) is narrow; both lumina are separated by an oblique incomplete septum (S). BI, onset of formation of blood islands below the epiblast; the subgerminal space contains a coagulate that is tightly fixed to the sickle endoblast (two arrowheads) or junctional endoblast (one arrowhead). Iron hematoxylin and eosin staining. Scale bar = 300 μm (Callebaut et al., 2000a).

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Figure 16. Enlarged view of the lateral part of Figure 15. BI, blood island; the arrows indicate mesoblast migrating laterally over the junctional endoblast (JE), forming a small blood island (BI); the arrowhead indicates a charcoal particle that was placed in vivo on the transparent V-shaped zone visible alive from the surface over the sickle canal region; C, coagulate in the subgerminal space, tightly adhering to the sickle endoblast or junctional endoblast; L, lateral; M, medial; SC, lumen of sickle canal. Scale bar = 100 μm.

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Effect of Rauber's Sickle-Derived Junctional Endoblast on the Development of the Coelom and Associated Vascular System (at the Convexity of Junctional Endoblast)

Shortly after the first blood islands have appeared, (Fig. 17A), parallel coelomic vesicles develop in a more superficial mesoblast sheet (coelomic–mesoblast; Callebaut et al., 2002a; Fig. 17B). Both the coelom and the vascular system are intimately associated in coelomates (Dollander and Fenart, 1973). This is also clearly seen in the avian embryo. A three-dimensional relation between the junctional endoblast and peripherally migrating mesoblast, forming blood islands and coelomic vesicles, is shown in Figure 18. Our junctional endoblast ablation experiments seem to suggest that the pericardium and primary heart tube form progressively (in time and space) under the inductive influence of the cranial horns of Rauber's sickle and junctional endoblast (Callebaut et al., 2004a). However, an associated defect of the endoderm also could have an influence. The far-extending lateral localization of the sickle horns could be the reason for the original double anlage of the endocardium of the heart and pericardium. Lawson and Schoenwolf (2001) report that upper layer cells induced either by median or lateral parts (sickle horns) of Rauber's sickle follow a different way during their ingression by means of the PS. Before the development of the heart, a homeobox protein (nkx 2.5) appears, which may cooperate with its known relatives in defining an anteroventral field, including the developing heart and pharyngeal endoderm (Brand et al., 1997; Lopez-Sanchez et al., 2002). Very recently, two spatially distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos have been observed (Walmsley et al., 2002). The first population gives rise to embryonic blood and vitelline veins and to the endocardium of the heart in the anterior ventral blood island. The second population resides in the dorsal lateral plate mesoderm and contains precursor adult blood stem cells and the major vessels. In Xenopus laevis, many models depict induction from the Spemann organizer (the descendants of which are notochord and head mesoderm) as a gradient of dorsalizing factors that diffuse across the marginal zone. The distance of a marginal zone cell from the Spemann organizer at gastrulation would then determine its dorsoventral identity. Thus, the central blood islands, which were proposed to arise from tissue furthest away from the Spemann organizer, were thought to be specified by the absence of organizer signaling. In the chicken blastoderm, the blood islands also develop at maximal distance from the notochord and head mesoderm (localized in the cranial part of the area centralis). According to Huber et al. (1998), blood formation during vertebrate embryogenesis may require at least three distinct functions, mesoderm induction (by activin or fibroblast growth factor), ventral mesoderm patterning (by bone morphogenetic proteins [BMPs]), and hematopoietic-specific cell specifications (by GATA factors). Some of these steps seem to correspond to those we saw during avian blood island formation. In any case, it is remarkable that, under inductive influence of Rauber's sickle material alone, blood vessels and associated coelomic cavities can develop from the originally multipotent upper layer of isolated anti-sickle regions (Callebaut et al., 2002b).

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Figure 17. A: Schematic representation of the transformation of the peripheral part of the mesodermal mantle (M) into blood island (BI) through induction by junctional endoblast (JE, green) as it slides between the epiblast (E) and this junctional endoblast to become settled above the more lateral yolk endoblast (YE); LAT, lateral; MED, medial; SC, sickle canal; SE, sickle endoblast (green). B: Schematic representation of the formation of coelomic vesicles (C) in a more superficial sheet of mesoblast (M) above each blood island (BI), formed some what earlier. E, epiblast; LAT, lateral; MED, medial; JE, junctional endoblast (green); SE, sickle endoblast (green).

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Figure 18. Schematic three-dimensional representation of the formation of blood islands (B, red) and associated coelomic vesicles (C) in the avian blastoderm under the inductive influence of Rauber's sickle-derived junctional endoblast (JE in green); the paths followed by the mesoblast cells destined to form finally the blood islands are colored in red; after ingression (indicated by medially directed arrows) of upper layer cells from the area pellucida by means of the primitive streak (PS); they form mesoblast cells and then migrate laterally (indicated by lateral arrows) under the upper layer (partially removed) and over the junctional endoblast into the caudal marginal zone and area opaca (AO) and so form there the area vasculosa.

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ENDOPHYLL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

The endophyll is also known as the primary hypoblast (Eyal-Giladi and Kochav, 1976). It contains δ ooplasm (primordial yolk spheres; Callebaut, 1987). In the unincubated blastoderm, it forms a thin, often incomplete sickle-shaped sheet localized in the caudal part of the area centralis in the concavity of the Rauber's sickle (Fig. 7A). When placed in vitro on a isolated anti-sickle from an unincubated chicken blastoderm or on a caudal marginal zone, it induces an early neural plate (with neural groove and walls) (Callebaut et al., 1999; Fig. 19A,B). Thus, under the inductory influence of endophyll alone, an early neural plate is formed in the upper layer. Also after culture, only an early neural plate forms in an unincubated chicken blastoderm from which the Rauber's sickle, its horns, and surrounding tissues are excised (Fig. 9). This blastoderm fragment is only composed of the anti-sickle region and the neighboring central part. It contains only upper layer and some endophyll material localized in the caudal part. No Rauber's sickle or sickle endoblast are present. The early neural plate forms in the immediate neighborhood of the endophyll under its inductive influence (Callebaut et al., 2003b). A PS never forms, because no Rauber's sickle material is present. That the observed thickening is an early neural plate and not the result of a specific induction can be demonstrated by apposing the upper layer of the isolated center (node anlage) of an early quail blastoderm (incubated for 8–9 hr). This resulted in the formation of a quail notochord fragment and a neural groove with quail floor plate, intercalated between both chicken lateral neural folds (unpublished results). By contrast, if sickle endoblast is placed on this blastoderm fragment then a PS also forms (Callebaut et al., 2002c). During early incubation, the endophyll is displaced cranially by the cranially growing sickle endoblast (derived from Rauber's sickle) and finally forms the endophyll wall (germ cell crescent; Fig. 13). It is an unusual hollow structure that contains endophyll and primordial germ cells in its deeper part that has been displaced into the former anti-sickle region. This endophyll wall can be used for grafting pieces of embryonic tissue (Vakaet, 1964; Gallera, 1964, 1969, Gallera and Nicolet, 1969).

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Figure 19. A: Induction area (C) provoked after 20 hr of culture by a quail endophyll fragment placed on the caudal marginal zone of an unincubated chicken blastoderm. In the induction area, a narrow axis is seen, directed caudally. I, primary chicken embryo. B: Section through the axis seen in the clear zone (C) visible on A reveals the existence of early neural plate with large groove (indicated by the arrowhead) directly above the grafted quail endophyll (E), separated by a large space (S) in which no intervening middle layer is present.

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In cultured isolated prenodal rostral blastoderm parts (containing the endophyll wall) typically prosencephalon-like structures develop (Vakaet, 1962a). Vakaet considered this as a “spontaneous neurulation,” which takes place in the upper layer of the cranial part of the blastoderm. We think that this so-called “spontaneous neurulation” is in reality due to the induction by the endophyll included in this region. Indeed in younger isolated and cultured anti-sickle regions (where only upper layer is present), a “spontaneous neurulation” never takes place, because only ectoderm develops. Schoenwolf et al. (1989), using transsection assays of prenodal rostral blastoderm isolates, also sometimes found neuroid structures in these isolates after culture in vitro. Other possibilities are that, before the excision and isolation of the germ cell crescent region (prenodal fragment of the blastoderm), not only endophyll is present but also prechordal plate. The latter is known to induce prosencephalon-like structures (Pera and Kessel, 1997). By interaction with sickle endoblast arising from Rauber's sickle (the early gastrulation organizer: Callebaut and Van Nueten, 1994; Callebaut et al., 1997) or from Hensen's node (a later avian organizer: Waddington, 1932), endophyll orients or re-orients the head region and the caudocranial direction of an induced miniature embryo (Callebaut et al., 1999). Chapman et al. (2003) suggest that successive inductive interactions between primary hypoblast, anterior definitive endoderm and epiblast act to promote an anterior character. As endophyll and sickle endoblast are displaced rostrally by anterior definitive endoderm, signals from the latter stabilize and maintain this rostral identity in the overlying neurectoderm. Rostrally located deep layer (stages XII–XIV) expresses Lim 1, Hnf 3b, otx2, Gsc, Cerberus, Hex, and Crescent (Chapman et al., 2002). Ganf is the earliest marker detected in the rostral epiblast in response to anteriorizing signals from the lower layer and neural specification by the head organizer. This rostral epiblast seems to correspond later with the early induced neural plate seen in our experiments. Head organizer cells leave Hensen's node, as ingressing axial mesoderm, permitting the remaining population to perform the role of trunk/tail organizer.

SICKLE ENDOBLAST (SECONDARY HYPOBLAST)

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Sickle endoblast forms a unicellular far-extending layer proliferating from the medial rim of the Rauber's sickle or junctional endoblast in a centripetal and cranial direction during early incubation (Callebaut and Van Nueten, 1994; Callebaut et al., 1999; Fig. 13). Principally, it contains γ ooplasm.

Sickle Endoblast Belongs to the Same Cell Lineage as Rauber's Sickle and Has a Similar Behavior but Is Dominated by Rauber's Sickle

If a quail sickle endoblast fragment is placed on the anti-sickle region of an unincubated chicken blastoderm from which Rauber's sickle has been selectively scraped away, then a whole embryo with a PS, definitive endoderm, Hensen's node, and a neural plate develops in a diametrically opposed direction, starting from the anti-sickle region (Callebaut et al., 2003a, b). The same occurs when a fragment of quail sickle endoblast is placed on the isolated central part of an unincubated chicken blastoderm (Callebaut et al., 2002c). Central subgerminal ooplasm artificially placed in contact with Rauber's sickle material or sickle endoblast in culture, can function as a substrate for cellular proliferation with again inducing and/or regenerating capacities in the neighboring upper layer (Callebaut et al., 2000c). Even activation of embryo formation can occur by unfertilized quail blastodiscs (Callebaut et al., 2000d).

When quail sickle endoblast is placed on the isolated anti-sickle region of an unincubated chicken blastoderm in culture, an early neural plate develops. By contrast, when a piece of quail sickle endoblast is placed on the anti-sickle region of a whole unincubated chicken in culture, it has no inducing effect. This finding indicates that Rauber's sickle dominates or inhibits ectopically placed sickle endoblast, which is derived from the same cell lineage. This sickle endoblast, if withdrawn from the influence of Rauber's sickle, has gastrulation- and/or neurulation-inducing potencies on the upper layer of the unincubated blastoderm, but it has no influence on blood island formation. The homeobox gene cHex is expressed in Rauber's sickle and sickle endoblast (Yatskievych et al., 1997). cHex transcripts were also detected within blood islands beginning at stage 4 (Hamburger and Hamilton, 1951) and in extraembryonic and intraembryonic vascular endothelial cells. Because we have shown that Rauber's sickle and junctional endoblast have an inducing effect on blood island formation, we can postulate an unknown relationship with the cHex gene.

Influence of Sickle Endoblast on Neurulation and Gastrulation

The molecular basis of neural induction has been extensively studied in Xenopus laevis, and it was found to be tightly coupled to the establishment of the dorsoventral axis (De Robertis and Sasai, 1996; Hemmati-Brivanlou and Thomsen, 1995; Hemmati-Brivanlou and Melton, 1997). In frogs, the prospective ectoderm is induced by BMPs. In contrast, a neural development requires the inactivation of BMPs and is achieved by direct complex formation between BMPs and neural-inducing factors such as chordin, noggin, or follistatin (Piccolo et al., 1996; Zimmermann et al., 1996). In the chick blastoderm at early stages, the prospective epidermis is characterized by the expression of the homeobox gene DLX5, which remains an epidermal marker during gastrulation and neurulation and enables it to be distinguished from the more central neural plate (Pera et al., 1999). That vertical signals from the lower layer are necessary for the establishment of the neural plate has been shown by the latter authors by repeated extirpations of the underlying endoblast. In the absence of the lower germ layers, the epidermis expanded into the region that normally forms the neural plate.

Knoetgen et al. (1999a, b) analyzed the GANF (Gallus anterior neural fold) -inducing potential of various tissues at different stages during chick development by transplantation to the outer margin of the area pellucida, where the epiblast cells are fated to become epidermis (Spratt, 1952; Rosenquist, 1966; Schoenwolf and Sheard, 1990; Bortier and Vakaet, 1992; Garcia-Martinez et al., 1993). Transplants of Hensen's node (HH3+/HH4) on whole blastoderms led to the induction of a neuroectodermal structure with a strong expression of GANF in its cranial margin. Grafting of the young head process (HH4) to the lateral cranial area pellucida caused a thickening of the epiblast and an induction of GANF expression in juxtaposed cells.

A secreted molecule named “Cerberus,” which is expressed in anterior endoderm, has the property to induce ectopic head structures when microinjected into ventral regions of Xenopus embryos (Bouwmeester et al., 1996; Bouwmeester, 1997). The patterning of the chick forebrain anlage by the prechordal plate has been described by Pera and Kessel (1997). According to these authors also, the avian neural plate is evident before the first mesendodermal or axial mesodermal cells ingress, excluding the prechordal plate and the notochord as primary sources for neural induction. During early gastrulation, cells invaginate through the tip of the growing streak and spread radially to form the definitive (gut) endoderm (Vakaet, 1970). During this radial expansion, the latter definitive endoderm pushes the sickle endoblast also radially (Callebaut and Van Nueten, 1994; Fig. 13). The cranial hemicircular sickle endoblast slides under upper layer cells that transform into a hemicircular neural plate anlage (Bortier and Vakaet, 1992). The latter cells are localized close to the former anti-sickle region, exactly in the concavity of the cranially displaced endophilic crescent. The remaining more caudal sickle endoblast is localized under the upper layer, which will give rise to the PS-forming area centralis region. This different evolution in the cranial (anti-sickle) region vs. the central (area centralis) region probably can be explained by the different reactivity in these two upper layer regions (Callebaut et al., 2002c).

The absence of neural induction after the grafting experiments with the deep layer on whole blastoderms by Gallera and Nicolet (1969) and by Knoetgen et al. (1999a, b) can probably be explained by the full presence of Rauber's sickle material. This finding indicates also that the earlier conclusions from grafting experiments on whole unincubated blastoderms (containing Rauber's sickle, the primary major organizer, or on PS blastoderms, containing Hensen's node, a secondary major organizer) must be reconsidered. Therefore, we cannot agree with either of the conclusions of Knoetgen et al. (1999b) that the endoblast on its own elicits any detectable change in the adjacent host ectoblast after transplantation or that the avian organizer is confined to Hensen's node only. Foley et al. (2000) studied the eventual role of the early deep layer (endophyll and/or sickle endoblast) on the expression of the molecular markers Sox3 (Uwanogho et al., 1995) and Otx2 (Bally-Cuif et al., 1995) in the upper layer. From Hamburger and Hamilton stage 6–7 on, Sox3 is specifically expressed in the entire chicken neural plate and Otx2 is expressed throughout the forebrain and midbrain. Foley et al. (2000) found that the early deep layer regulates an early transient phase of Otx2 and Sox3 expression in the adjacent upper layer. Therefore, they concluded that the early deep layer does not induce neural tissue or forebrain definitively. However, their transplantation experiments were not performed on Rauber's sickle—or junctional endoblast-free blastoderm fragments but on whole blastoderms. Recently, Knezevic and Mackem (2001) found evidence that two genes, later associated with the gastrula organizer (Gnot-1 and Gnot-2), are induced by the deep layer signals in prestreak embryos. According to the latter authors, these genes could perhaps regulate axis formation in the early embryo, which could also explain the induction of a streak in the isolated part of the area centralis by sickle endoblast (Callebaut et al., 2003b).

HENSEN'S NODE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Hensen's node develops at the cranial end of the fully elongated (Fig. 14) PS. Sickle endoblast seems to be the link between Rauber's sickle (the primary major organizer of the avian blastoderm) and the node (the secondary major organizer). Indeed in the absence of Rauber's sickle, but in the presence of sickle endoblast, a whole embryo with node is induced in the UL after culture (Callebaut et al., 2003a). It is not exactly known how the node develops (Le Douarin, 2001). However, Catala et al. (1995) demonstrated by the quail–chicken chimera technique that the avian node gives rise to the so-called midline structures: the floor plate (intercalated between the lateral folds of the neural plate), the underlying notochord, and the dorsal part of the endoderm. We found that the anlage fields of these three structures in the unincubated blastoderm are located in close apposition to the midline through Rauber's sickle (Callebaut et al., 1996a).

PRIMORDIAL GERM CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Nussbaum (1880, 1901) was the first to propose the preformation thesis in birds, i.e., that the primordial germ cells have inherited their yolk from the precursor oocyte from which their egg yolk ball and blastoderm are derived. The primordial germ cells (PGCs) are only first unequivocally distinguishable in the one- to eight-somite chicken blastoderm between epiblast and endophyll of the germ cell crescent (Swift, 1914). Because PGCs are found close to the endophyll and seem to emerge from it, Vakaet (1962a) supposed that they were derived from the endophyll. Dubois (1967, 1969) also concluded that the endophyll is at the origin of the formation of PGCs in birds. The precursors of the PGCs, which are often initially morphologically indistinguishable from the surrounding somatic cells in earlier stages, are called presumptive primordial germ cells (pPGCs). These cells divide mitotically to produce one PGC and one somatic cell. In a hypothetical map of the anlage fields in the unincubated blastodisc, Vakaet (1962a) localized the pPGCs in the peripheral deep rim of the area pellucida. Thus, in general, it was accepted that chicken germ cells originate from the deep layer. By contrast, Eyal-Giladi et al. (1981) concluded by using chick–quail chimeras, made before PS formation, (i.e., stage XIII: 10–12 hr of incubation) that avian PGCs were from epiblastic origin. Avian PGCs where then thought to arise through a gradual epigenetic process. However, in these older blastoderms, the deep layer is no longer composed of endophyll but mainly formed by sickle endoblast, derived from Rauber's sickle. Indeed, the endophyll and associated PGCs are then already displaced cranially and adhere to the deep cranial part of the epiblast and to the hemicircular fibrous bands there (England, 1983). They will form part of the endophilic crescent in older stages. The experiments of Cuminge and Dubois (1992) seemed to confirm the thesis of Eyal-Giladi, but they also investigated similar blastoderm stages in which the deep layer greatly differs from the deep layer in unincubated blastoderms. By using trypan blue–induced fluorescent labeling of the yolk layers of quail oocytes during their final postlampbrush stage, I could demonstrate that primordial germ cells together with the endophyll (Fig. 20) contain yolk from the deep central region of the germ disc, i.e., δ ooplasm from the superficial part of the nucleus of Pander (Callebaut, 1983b, 1984). So, nearly 95% of PGCs can be labeled 6–7 days after one single injection of trypan blue in the mother quail. Oocytal yolk labeling, 1–4 days after an injection, gives no labeling of the primordial germ cell yolk but gives labeling of more superficial somatic cells that contain more superficial ooplasms (β or γ). The observed trypan blue–induced fluorescent yolk labeling in the caudally localized endophyll in the unincubated quail blastoderm (Callebaut, 1987) is in agreement with the observed localization of the pPGC (also containing δ yolk) after transsection experiments (Fargeix, 1967; Rogulska, 1968; Dubois and Croisille, 1970), i.e., mainly in the caudal region of the unincubated blastoderm. The original deep and central localization of pPGC material has been confirmed recently by the use of a chicken vasa homologue (Tsunekawa et al., 2000). Chicken vasa protein forms part of the mitochondrial cloud in younger chick oocytes and localizes to the central cleavage furrows (which extend into the δ ooplasm of the nucleus of Pander) until stage IV (Eyal-Giladi and Kochav, 1976). At that moment, six to eight cells of the approximately 300 blastomeres containing germ, contain vasa protein and are probably pPGCs (Tsunekawa et al., 2000). The data of Callebaut (1984) and Tsunekawa et al. (2000), thus, indicate that a kind of preformation may be the mechanism for germ cell specification in birds. As in Xenopus, in the quail there are two known populations of oocytal mitochondria, which become finally localized in the early embryo: one population becomes localized in the vegetal pole where it forms a component of the germ plasm in Xenopus (Tourte et al., 1984; Mignotte et al., 1987) and a component of the nucleus of Pander (δ ooplasm) in the quail (Callebaut, 1984; D'Herde et al., 1995). The other population of mitochondria is localized much more superficially and forms the obvious radially and concentrically disposed group around the germinal vesicle both in Xenopus (Mignotte et al., 1987) as in the quail (Ticos: Callebaut, 1972, 1983a; D'Herde et al., 1995). These mitochondria will populate the somatic tissues of the offspring (in Xenopus: Dawid and Blackler, 1972; in quail: Watanabe et al., 1985). Our conclusion is in agreement with Wolpert (1998) and Extavour and Akam (2003) that epigenetic germ cell development is an exception and that most animals use localized ooplasmic determinants to specify the germ line.

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Figure 20. Fluorescence microscope photograph of a transverse section through a one-somite quail embryo, developed in the sixth laid egg (formed 9 days after a maternal injection with trypan blue). E, unlabeled ectoderm. The primordial germ cell (indicated by the arrow) rests on the unlabeled deep layer and contains yolk spheres containing trypan blue–induced fluorescent yolk granules. The fluorescent marking by trypan blue is also very obvious in the yolk of the endophyll wall (EW), indicating a common origin of primordial germ cell ooplasm and endophyll from the nucleus of Pander. Scale bar = 100 μm (after Callebaut and Vakaet, 1981).

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CAUDAL MARGINAL ZONE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

The caudal marginal zone was defined by Eyal-Giladi and Kochav (1976) as the more or less transparent belt that separates the Rauber's sickle from the area opaca (Fig. 7). It, thus, forms a part of the area pellucida peripheral to Rauber's sickle. The caudal marginal zone and Rauber's sickle are neighboring structures, but the way in which they form is distinctly different. By the use of the trypan blue–induced yolk layer labeling, we have shown that Rauber's sickle is the only part of the blastoderm that remains in situ in close apposition with the subgerminal ooplasm (mainly γ ooplasm) and, thus, forms the most vegetal part of the blastoderm (Callebaut, 1987; Fig. 7). By contrast, with the same method, we have shown that the caudal marginal zone and neighboring area opaca developed by centrifugal expansion of cells from the blastoderm over the peripheral ooplasm at the end of the intrauterine period (Callebaut and Meeussen, 1988; Fig. 7). The first blood islands appear normally in the caudal marginal zone parallel with and under the inductive influence of the V-shaped junctional endoblast (Callebaut et al., 2002a). This formation is not a specific feature of the caudal marginal zone, because by placing a fragment of Rauber's sickle on the isolated anti-sickle, blood vessels and associated coelomic vessels are also induced (Callebaut et al., 2002b).

If a quail sickle endoblast is placed on the caudal marginal zone of a whole unincubated blastoderm, then a secondary miniature chicken blastoderm will develop in this caudal marginal zone. The PS and accompanying neural plate of this secondary embryo are directed peripherally into the caudal germ wall away from Rauber's sickle (Callebaut et al., 2002b). Thus, the resulting “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 by the presence of sickle endoblast. In normal development, a streak only forms in the area centralis in the concavity of Rauber's sickle. Rauber's sickle, thus, not only forms a morphological limit around the area centralis but constitutes also a functional boundary.

ANTI-SICKLE REGION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

The anti-sickle region forms the most rostral segment of the unincubated avian blastoderm. It contains no Rauber's sickle material and no endophyll, because it is localized rostrally from the tips of the sickle horns. The anti-sickle region is formed at the end of the intrauterine period at the moment of bilateral symmetrization of the blastoderm (with formation of Rauber's sickle; Fig. 4). By the end of this critical period the embryo has slid rostrally relative to the γ ooplasm, with a shear zone just under the blastoderm (Fig. 3C; Callebaut, 1993c, 1994). This movement is reminiscent of cortical rotation in the animal cap of the frog embryo (Bachvarova, 1999) that determines the embryonic axis shortly after fertilization. In both cases at the moment of bilateralization, the superficial layer moves relative to the deeper layer, with accompanying redistribution of ooplasm. BMP4 RNA is present in the amphibian animal cap at the start of gastrulation (Fainsod et al., 1994; Hemmati-Brivanlou and Thomsen, 1995). This secreted growth factor (BMP4) plays a pivotal role wherein BMP signalling induces epidermal differentiation. The absence of BMP signalling, accomplished by BMP antagonists, including noggin, follistatin, and chordin, leads to the formation of neural tissue (Hemmati-Brivanlou and Melton, 1997). Thus, neural differentiation, not epidermal differentiation, should be the default state of embryonic ectoderm.

Glossary

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

Chronological Events

  • The germ disc of the avian oocyte presents radially placed groups of mitochondria (ticos) (Fig. 1).

  • Before fertilization, four ooplasms (α, β, γ, δ) with onion peel-like disposition can be distinguised with the α ooplasm most superficially and in the central core, the δ ooplasm forming the nucleus of Pander (Fig. 2A).

  • After fertilization during the cleavage, the superficial mobile α ooplasm penetrates along with the cleavage furrows into the deeper ooplasms (Fig. 2C).

    Uptake of ooplasmic determi nants before laying:

    • 1
      Part of the γ ooplasm is taken up in the caudolateral part of the blastoderm, forming Rauber's sickle (RS) (Fig. 3B), which, after incubation by further ingrowth in the γ ooplasm, will transform into junctional endoblast (Fig. 12).
    • 2
      Part of the δ ooplasm is taken up in the centrocaudal region of the blastoderm and forms endophyll (primary hypoblast) (Fig. 3C).
  • At the moment of laying the unincubated blastoderm presents a sickle-shaped bilateral symmetry (Fig. 7A) with 3 elementary tissues:

    • The Rauber's sickle containing γ ooplasm and encircling the area centralis.

    • The caudocentral endophyll with δ ooplasm in the concavity of Rauber's sickle.

    • The upper layer (UL) or epiblast covers the whole blastoderm and contains mainly β ooplasm; in the area centralis of the UL we found prediposed sickle-shaped anlage fields parallel with Rauber's sickle (Fig. 10).

  • During early incubation, a sheet of sickle endoblast (secondary hypoblast) grows from the inner rim of Rauber's sickle and pushes the endophyll (primary hypoblast) in a cranial direction to form the endophyllic crescent (Fig. 13); hypoblast or endoblast are not precise terms.

  • Gastrulation: during early incubation above the sickle endoblast a primitive streak (PS) develops in the upper layer on the midline through the middle of RS and junctional endoblast (Fig. 12), under inductive influence of the latter, Hensen's node develops in the cranial end of the full grown PS.

  • Neurulation: 1) An early neural plate with groove and lateral folds develops under inductive influence of the endophyll. 2) Later, cells derived from Hensen's node bisect the middle region of the early neural plate in the depth of the neural groove and form there the floor plate.

  • The vitelline blood circulation and asociated coelom develop under inductive influence of junctional endoblast, a massive V-shaped structure localized between the area centralis and the caudolateral marginal zone.

  • Primordial germ cells: mother cells of the germ cells; they contain originally δ ooplasm derived from the nucleus of Pander.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES

I thank Prof. Dr. Gary Schoenwolf for his kind invitation to write this review. Without the outstanding microsurgical assistance of Mrs. Emmy Van Nueten and her more than 30 years of collaboration, this work would never have been possible. Also much thanks to Mr. F. De Bruyn for excellent artwork, Mr. F. Schallenberg for photography, and to Mrs. V. De Maere for typing the manuscript. The author is very grateful to Prof. Dr. F. Harrisson and to Prof. Dr. H. Bortier for the possibility to continue to work in their laboratory.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENT OF THE AVIAN GERM DISC REGION FROM THE POSTLAMPBRUSH OOCYTE STAGE TO THE FERTILIZED UNINCUBATED BLASTODERM STAGE
  5. SUBGERMINAL SPACE
  6. SUBGERMINAL OOPLASM
  7. RAUBER'S SICKLE
  8. ENDOPHYLL
  9. SICKLE ENDOBLAST (SECONDARY HYPOBLAST)
  10. HENSEN'S NODE
  11. PRIMORDIAL GERM CELLS
  12. CAUDAL MARGINAL ZONE
  13. ANTI-SICKLE REGION
  14. Glossary
  15. Acknowledgements
  16. REFERENCES
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