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The present experimental in vitro study suggests that a primitive streak (PS) in avian blastoderms is induced by diffusion of morphogenetic substances emanating from Rauber's sickle. Indeed, even without direct contact between a quail Rauber's sickle and the reacting upper layer (by interposition of a vitelline membrane), a PS can be induced in the isolated area centralis or antisickle region of unincubated chicken blastoderms. The so-formed PSs are localized below the vitelline membrane in the immediate neighborhood of the apposed Rauber's sickle material. This seems to indicate that Rauber's sickle organizes the formation of the avian PS according to the basic concept of "positional information." The morphogenetic substances seem to have an effect only on the formation of a PS. Each part of Rauber's sickle seems to have, point by point, the same thickening and PS-inducing effect on each corresponding part of the underlying upper layer (UL). By a mechanism of sliding over the basement membrane and fusion, this finally results in the formation of one single median PS. Our study shows that a PS can be induced in the total absence of hypoblast (sickle endoblast) or caudal marginal zone, by only the presence of Rauber's sickle material. In contrast, the differentiation of mesoblast into blood islands under the influence of Rauber's sickle and neural tissue development are impaired by the interposition of a vitelline membrane. The latter could be due to the absence of a normal interaction of Rauber's sickle-derived sickle endoblast with endophyll and/or upper layer and the absence of cranial migration of the mesoblast. Thus, earlier studies and the present study indicate the existence of a temporospatially bound cascade of gastrulation and neurulation phenomena and blood island formation in the avian blastoderm, starting from Rauber's sickle, the primary major organizer with inducing, inhibiting, and dominating potencies. The latter not only plays a role by secretion of signaling molecules, but also influences development by its cell lineages (junctional endoblast and sickle endoblast)., (Copyright 2003 Wiley-Liss, Inc.)

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

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

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

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

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.