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

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

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

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

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

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

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

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

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

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

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

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

Mosaic versus regulation development in avian blastoderms depends on the spatial distribution of Rauber's sickle material

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

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

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

The marginal zone and its contribution to the hypoblast and primitive streak of the chick embryo

The marginal zone of the chick embryo has been shown to play an important role in the formation of the hypoblast and of the primitive streak. In this study, time-lapse filming, fate mapping, ablation and transplantation experiments were combined to study its contribution to these structures. It was found that the deep (endodermal) portion of the posterior marginal zone contributes to the hypoblast and to the junctional endoblast, while the epiblast portion of the same region contributes to the epiblast of the primitive streak and to the definitive (gut) endoderm derived from it. Within the deep part of the posterior marginal zone, a subpopulation of HNK-1-positive cells contributes to the hypoblast. Removal of the deep part of the marginal zone prevents regeneration of the hypoblast but not the formation of a primitive streak. Removal of both layers of the marginal zone leads to a primitive streak of abnormal morphology but mesendodermal cells nevertheless differentiate. These results show that the two main properties of the posterior marginal zone (contributing to the hypoblast and controlling the site of primitive streak formation) are separable, and reside in different germ layers. This conclusion does not support the idea that the influence of the posterior marginal zone on the development of axial structures is due to it being the source of secondary hypoblast cells.     

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

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

Formation of the avian primitive streak from spatially restricted blastoderm: evidence for polarized cell division in the elongating streak

Gastrulation in the amniote begins with the formation of a primitive streak through which precursors of definitive mesoderm and endoderm ingress and migrate to their embryonic destinations. This organizing center for amniote gastrulation is induced by signal(s) from the posterior margin of the blastodisc. The mode of action of these inductive signal(s) remains unresolved, since various origins and developmental pathways of the primitive streak have been proposed. In the present study, the fate of chicken blastodermal cells was traced for the first time in ovo from prestreak stages XI-XII through HH stage 3, when the primitive streak is initially established and prior to the migration of mesoderm. Using replication-defective retrovirus-mediated gene transfer and vital dye labeling, precursor cells of the stage 3 primitive streak were mapped predominantly to a specific region where the embryonic midline crosses the posterior margin of the epiblast. No significant contribution to the early primitive streak was seen from the anterolateral epiblast. Instead, the precursor cells generated daughter cells that underwent a polarized cell division oriented perpendicular to the anteroposterior embryonic axis. The resulting daughter cell population was arranged in a longitudinal array extending the complete length of the primitive streak. Furthermore, expression of cVg1, a posterior margin-derived signal, at the anterior marginal zone induced adjacent epiblast cells, but not those lateral to or distant from the signal, to form an ectopic primitive streak. The cVg1-induced epiblast cells also exhibited polarized cell divisions during ectopic primitive streak formation. These results suggest that blastoderm cells located immediately anterior to the posterior marginal zone, which secretes an inductive signal, undergo spatially directed cytokineses during early primitive streak formation.     

Cell populations and morphogenetic movements underlying formation of the avian primitive streak and organizer

The cell populations and morphogenetic movements that contribute to the formation of the avian primitive streak and organizer-Hensen's node-are poorly understood. We labeled selected groups of cells with fluorescent dyes and then followed them over time during formation and progression of the primitive streak and formation of Hensen's node. We show that (1) the primitive streak arises from a localized population of epiblast cells spanning the caudal midline of Koller's sickle, with the mid-dorsal cells of the primitive streak arising from the midline of the epiblast overlying Koller's sickle and the deeper and more lateral primitive streak cells arising more laterally within the epiblast overlying the sickle, from an arch subtending about 30 degrees; (2) convergent extension movements of cells in the epiblast overlying Koller's sickle contribute to formation of the initial primitive streak; and (3) Hensen's node is derived from a mixture of cells originating both from the epiblast just rostral to the incipient (stage 2) primitive streak and later from the epiblast just rostral to the elongating (stage 3a/b) primitive streak, as well as from the rostral tip of the progressing streak itself. Collectively, these results provide new information on the formation of the avian primitive streak and organizer, increasing our understanding of these important events of early development of amniotes.     

A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo

In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

Limited left-right cell migration across the midline of the gastrulating avian embryo

During avian development the earliest phase in which the avian embryo expresses axial features of a left-right axis is at the primitive streak stage. Until the stage of definitive primitive streak (streak 4 H&H), the axis seems to possess morphological bilateral symmetry. Morphological asymmetry begins only during the next few hours of incubation, with development of overt morphological and molecular asymmetry within Hensen's node (stage 5 H&H). In this report, we present an experimental study aimed at following the pattern of cell movements during primitive streak formation and gastrulation of specific left-right regions from earlier stages of the avian embryo. To determine the origin of cells contributing to each side of the primitive streak, we applied the dye Lysinated-Rodamine-Dextran (LRD) to one half, either left or right, of the pre-streak blastoderm (stages X–XIII, EG&K). We tried to estimate the relative cell contribution to primitive streak formation, and to the three germ layers evolving during gastrulation in the context of the left-right axis. Moreover, we asked whether the midline serves as a border, that is, as a physiological barrier preventing cell passing during gastrulation. Our results demonstrate that on each side of the axis, either the right or the left, most of the cells originate from the same half of a pre-streak blastoderm, populate the same half of the PS and contribute to tissues largely confined to that particular side. However, along the primitive streak, a few cells were detected on the opposite side of the midline. Moreover, variation in the number of cells crossing the midline at specific regions along the primitive streak was found. Most crossing cells were located near the mid rostrocaudal extent of the primitive streak, from 25–85% of its length. At the posterior end of the primitive streak, fewer crossing cells were detected. At the anterior region of the PS, that is, within Hensen's node, cells do not cross the midline. These results suggest that differences occur in the process of ingression along the rostrocaudal extent of the PS. Dev. Genet. 23:175–184, 1998. © 1998 Wiley-Liss, Inc.     

Mesodermal patterning during avian gastrulation and neurulation: Experimental induction of notochord from non-notochordal precursor cells

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 μm and a mediolateral extent of less than 250 μm, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate. © 1995 Wiley-Liss, Inc.     

Determination of embryonic polarity in a regulative system: evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo

Avian embryos have a remarkable capacity to regulate: when a pre-primitive streak stage embryo is cut into fragments, each fragment can spontaneously initiate formation of a complete embryonic axis. We investigate the signalling pathways that initiate primitive streak formation and the mechanisms that ensure that only a single axis normally forms. As reported previously, an ectopic primitive streak can be induced by misexpression of Vg1 in the marginal zone. We now show that Vg1 induces an inhibitor that travels across the embryo (3 mm distance) in less than 6 hours. We provide evidence that this inhibitor acts early in the cascade of events downstream of Vg1. We also show that FGF signalling is required for primitive streak formation, in cooperation with Nodal and Chordin. We suggest that three sequential inhibitory steps ensure that a single axis develops in the normal embryo: an early inhibitor that spreads throughout the embryo (which can be induced by Vg1), a second inhibition by Cerberus from the underlying hypoblast, and finally a late inhibition from Lefty emitted by the primitive streak itself.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Establishment of anterior-posterior polarity in avian embryos.

In pre-streak chick embryos, the extraembryonic posterior marginal zone is able to induce an embryonic axis at an ectopic site without contributing cells to the induced primitive streak. This region expresses mesoderm-inducing factors that are capable of inducing an ectopic streak. Downstream of these events, chordin and bone morphogenetic protein acting within the central disc may play mutually opposing roles influencing streak formation. Although extraembryonic regions are important in establishing the embryonic axis, there does not appear to be an anterior region with head-inducing activity similar to that of the anterior visceral endoderm of the mammalian embryo.