Unincubated Blastoderm Research Articles

A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development.

The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA “in cubo”, followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the “in cubo” electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector, RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication- Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.

Society for International Development, the North–South Roundtable and the Power of Ideas

Unincubated (stage X) chick blastoderms cultured in plain Dulbecco's modified Eagle medium in the absence of serum, synthesize haemoglobin and melanin 2 . 5--3 . 0 and 6 . 0--7 . 0 days, respectively, after the beginning of culture. The capacity of unincubated blastoderm to form haemoglobin and melanin is a response to intrinsic genotypic instructions rather than to the active intervention of substances continuously supplied by the yolk. 5-Bromodeoxyridine, applied during the first and second days of unincubated blastoderm in culture, is incorporated into DNA and perturbs the formation of haemoglobin and melanin, perhaps, by modifying gene expression.

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).

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.

Origin, fate, and function of the components of the avian germ disc region and early blastoderm: Role of ooplasmic determinants

In the avian oocytal germ disc region, at the end of oogenesis, we discerned four ooplasms (alpha, beta, gamma, delta) 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 alpha ooplasm plays a fundamental role during cleavage. The beta 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 gamma and delta ooplasms occurs so that gamma and delta 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 delta 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 delta ooplasm. The other structure (gamma 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 gamma 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 delta 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 beta ooplasm, endophyll containing delta ooplasm, and Rauber's sickle containing gamma ooplasm). These elementary tissues form before the three classic germ layers have developed.

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.

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).

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

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

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

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

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

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

Induction of (Pre) Gastrulation and/or (Pre) Neurulation by Subgerminal Ooplasm and Rauber’s Sickle in Cultured Anti-Sickle Regions of Avian Unincubated Blastoderms

Induction of (Pre) Gastrulation and/or (Pre) Neurulation by Subgerminal Ooplasm and Rauber’s Sickle in Cultured Anti-Sickle Regions of Avian Unincubated Blastoderms

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

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

Avian Gastrulation and Neurulation Are Not Impaired by the Removal of the Marginal Zone at the Unincubated Blastoderm Stage

Avian Gastrulation and Neurulation Are Not Impaired by the Removal of the Marginal Zone at the Unincubated Blastoderm Stage

Developmentally regulated expression of the preproinsulin gene in the chicken embryo during gastrulation and neurulation.

Despite the absence of a pancreas, which develops between embryonic day 3 (E3) to E4, previous studies showed that insulin receptors are widely expressed in chicken embryos from the blastoderm stage (unincubated embryo, E0) through gastrulation (E0.5-E1), neurulation (E1.5-E2), and organogenesis. We now characterize prepancreatic preproinsulin gene expression and its regulation, using a highly sensitive modification of the polymerase chain reaction. We found preproinsulin messenger RNA (mRNA) expression at all stages, from the unincubated chicken blastoderm through early organogenesis, with the highest expression in embryos undergoing gastrulation. In situ hybridization analysis of E1-E1.5 embryos in toto showed widespread distribution of preproinsulin mRNA in a pattern similar to that of insulin receptor mRNA. In contrast, insulin-like growth factor-I mRNA expression appeared later than preproinsulin mRNA in the embryo; it was first demonstrable in the head portion of E3 and was found in head, trunk, and caudal regions by E4. With a novel culture system for chicken embryos during neurulation, we examined whether glucose regulated prepancreatic preproinsulin mRNA expression. Embryos cultured in glucose-free medium had increased preproinsulin mRNA with respect to the value in ovo, but the addition of 17 mM glucose had no stimulatory effect. In marked contrast, in organ cultures of E13 pancreas, insulin mRNA expression decreased in glucose-free medium by 50% relative to that in ovo. The addition of glucose restored the levels to a concentration similar to that found in ovo. Exogenous insulin added to cultured E1.5 embryos increased protein and DNA synthesis. We conclude that the preproinsulin gene is widely expressed in chicken embryo structures throughout gastrulation and neurulation. This prepancreatic preproinsulin mRNA is differentially regulated compared to the pancreatic mRNA. Preproinsulin gene products may have a role in cell proliferation, differentiation, or survival in very early avian embryos at a time when insulin-like growth factor-I expression is absent or undetectable.

Emergence of Myogenic and Endothelial Cell Lineages in Avian Embryos

The roles of cell cycles and of cell-cell interactions in the emergence of myogenic and endothelial cell lineages were studied in avian embryos using the quail-chicken marker system. Quail embryos were treated with drugs preventing either DNA replication or the movement of cells. Portions of drug-treated or untreated quail blastoderms were grafted into chicken wing buds. After an incubation for an additional 4 to 10 days, the embryos were analyzed for the presence of quail muscle or quail endothelial cells by the Feulgen reaction and by immunostaining. Both cell lineages differ in the time of their commitment as well as in the conditions necessary for their emergence. Muscle cells did not differentiate from unincubated blastoderms nor did they develop from drug-treated blastoderms. These results corroborate that the commitment of myogenic cells occurs during gastrulation and indicate that this commitment requires both DNA replication and cellular movements allowing cell-cell and/or cell-matrix interactions. Endothelial cells, on the contrary, developed both from drug-treated and from unincubated blastoderms, indicating that their commitment occurs before and independent of gastrulation and does not require DNA replication during gastrulation.

A Novel Chicken Homeobox-Containing Gene Expressed in Neurulating Embryos

We have isolated a cDNA clone from chicken embryo that contains a homeobox sequence (CHox-cad2). Analysis at the nucleotide and amino acid levels revealed closest similarity to the Xenopus Xcad1 and to other homeoboxes related to Drosophila caudal. RNA blot analysis showed hybridization of CHox-cad2 to two transcripts of 2.6 and 1.5 kb, present at day 1 of embryogenesis (E1). Using the highly sensitive polymerase chain reaction (PCR) to amplify cDNAs from embryonic RNAs from E0 (unincubated blastoderm) to E4, we confirmed the restricted expression of this homeobox sequence to the period of neurulation (E1).

On the origin of cells determined to form skeletal muscle in avian embryos.

Pieces of quail embryos from various developmental stages ranging from unincubated blastoderms (before the appearance of a primitive streak) to embryos having formed somites were grafted to the wing buds or into the coelomic cavity of chicken embryos. The grafts, which can be identified on a cellular level by virtue of the prominent nucleolus-associated chromatin, present in the quail and absent in the chicken, were screened after suitable periods of reincubation for the presence or absence of skeletal myotubes containing quail nuclei. Grafts having contributed to such skeletal myotubes were considered as having contained determined myogenic cells at the time of the grafting procedure. Determined myogenic cells appeared first in the primitive streak and in the mesodermal cells formed by the invagination (gastrulation) of epiblastic cells through the primitive streak. This is true for both the head process and the paraxial mesoderm. Epiblastic cells never gave rise to skeletal myotubes. Therefore it can be said, that the onset of myogenic determination coincides with gastrulation. It remains, however, to be established, whether these two events are causally related to one another.

Determination of Erythroid Cells in Unincubated Chick Blastoderm: stem cells/erythrocytes/unincubated chick blastoderm/critical mass).

Portions the size of 1/6 to 1/32 part of unincubated blastoderm cultured in an albumen-salineagar solid medium form erythroid cells under conditions which suppress normal co-ordinated movements for mesoderm induction. Anterior and posterior regions of unincubated blastoderm have the same potentiality to form erythroid cells. The marginal zone seems to be the contributor of prospective erythroid cells. Portion 1/16 part of unincubated blastoderm forms morphologically distinct erythroid cells of the primitive and definitive lines as in normal development in ovo. It seems progenitor cell(s) is pre-programmed to a particular pattern of differentiation and/or includes differentiation to various erythroid cell types as an obligatory step. This system provides novel experimental possibilities in the study of erythroid cell determination and differentiation.