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.     

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

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

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

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

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

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

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).     

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.     

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.     

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.     

Early steps in neural development

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

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

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

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

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

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

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

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

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

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

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.     

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo.

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived.Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [³H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.     

Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells

The origin of prospective M cells, which are median neuroepithelial cells that become wedge-shaped during bending of the neural plate and eventually form the midline floor of the neural tube, was determined by constructing quail/chick chimeras and using the quail nucleolar marker to identify quail donor cells in chick host blastoderms. Two possible sites of prospective M-cell origin in the epiblast were examined: a single, midline rudiment located just rostral to Hensen's node and paired rudiments flanking the cranial part of the primitive streak. Our results suggest that M cells arise exclusively from the midline, prenodal rudiment. From this rudiment, M cells extend caudally throughout the entire length of the neuroepithelium. This new information on the origin of prospective M cells will aid in the analysis of their role in neurulation.     

Timing and cell interactions underlying neural induction in the chick embryo

Previous studies on neural induction have identified regionally localized inducing activities, signaling molecules, potential competence factors and various other features of this important, early differentiation event. In this paper, we have developed an improved model system for analyzing neural induction and patterning using transverse blastoderm isolates obtained from gastrulating chick embryos. We use this model to establish the timing of neural specification and the spatial distribution of perinodal cells having organizer activity. We show that a tissue that acts either as an organizer or as an inducer of an organizer is spatially co-localized with the prospective neuroectoderm immediately rostral to the primitive streak in the early gastrula. As the primitive streak elongates, this tissue with organizing activity and the prospective neuroectoderm rostral to the streak separate. Furthermore, we show that up to and through the mid-primitive streak stage (i.e., stage 3c/3+), the prospective neuroectoderm cannot self-differentiate (i.e. , express neural markers and acquire neural plate morphology) in isolation from tissue with organizer activity. Signals from the organizer and from other more caudal regions of the primitive streak act on the rostral prospective neuroectoderm and the latter gains potency (i.e., is specified) by the fully elongated primitive streak stage (i.e., stage 3d). Transverse blastoderm isolates containing non-specified, prospective neuroectoderm provide an improved model system for analyzing early signaling events involved in neuraxis initiation and patterning.     

The Initiation of Gastrular Ingression in the Chick Blastoderm

Normal gastrular ingression in the chick blastoderm occurs intwo steps. The first consists in de-epithelialization of thecells in the middle of the young primitive streak. The cellsthat will ingress converge as a sheet towards the primitivestreak; this convergence builds up the elongating primitivestreak. These cells come from a large posterior area of thearea pellucida. In this area they show many blebs at their ventralside. These blebs are not visible in the more lateral regionsof the upper layer at this stage. During the second step ofingression, de-epithelialization goes on in the middle of theprimitive streak, but convergence within the upper layer hascome to an end, while migration of the ingressed middle layercells starts, away from the primitive streak. To observe thefirst stages of ingression, we studied secondary primitive streaks,induced by grafting a nodus posterior into the entophyllic crescentof a host blastoderm. We fixed blastoderms in which, thougha secondary primitive streak was not yet visible, spreadingof the graft had taken place so as to make evocation of a streakmost probable. From this study we conclude that the initiationof de-epithelialization in experimental and probably in normalchick gastrulation is not preceded by an overall lysis of thebasal lamina at the future site of ingression. Ingression startsand goes on as a de-epithelialization of individual cells.     

Distribution and functional role of laminin during induction of the embryonic axis in the chick embryo

Laminin is a major glycoprotein of basement membranes and has been shown to promote cell adhesion, and movement of various nonepithelial cells and tumour cells. Using antibodies to laminin in paraffin sections and cultured embryos, we have studied the distribution of laminin and its involvement in the first morphogenetic events, beginning with the first extensive cellular migrations and interactions that result in the induction of the primitive streak (PS) and of the neural plate in the early chick embryo. Laminin immunogold labeling was not detected in the blastoderm at stage X. At stage XIII, laminin immunoreactivity was detected at the ventral surface of the epiblast and in the entire hypoblast. The intense labeling of the hypoblast indicated that these cells are active in laminin synthesis. Extracellular matrix (ECM) started accumulating as the first embryonic spaces were forming, before the morphogenetic movements of gastrulation were initiated. Immunogold labeling revealed a punctate pattern of laminin distribution in the ECM in the blastocoele, and in the space below the neural plate. Laminin, which is a multidomain molecule known to interact with other molecules of the ECM and with the cell surface, could serve as the scaffold for highly specific contact points of migrating cells and for the folding of epithelial sheets during this time in the developing embryo. We incubated blastoderms at stages X and XIII with laminin antibodies (1:30 dilution) for 4 h, then cultured the blastoderms further in plain egg albumin. The laminin antibodies did not interfere with triggering of PS cell movements, but perturbed the normal migration pattern of these cells. A normal PS did not form and, as a consequence, the embryonic axis was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)