Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10733-10737, November 1995 Developmental Biology

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast ODED KHANER Department of Cell and Animal Biology, The Hebrew University, Jerusalem, Israel 91904 Communicated by John Gerhart, University of California, Berkeley, CA, July 14, 1995

ABSTRACT In the amniotes, two unique layers of cells, of the hypoblast and that the orientation of the streak and axis the epiblast and the hypoblast, constitute the embryo at the can be predicted from the polarity of the stage XIII epiblast. blastula stage. All the tissues of the adult will derive from the Still it was thought that the polarity of the epiblast is sufficient epiblast, whereas hypoblast cells will form extraembryonic for axial development in experimental situations, although in yolk sac endoderm. During gastrulation, the endoderm and normal development the polarity of the hypoblast is dominant the mesoderm of the embryo arise from the primitive streak, (5, 6). These reports (1-3, 5, 6), taken together, would imply which is an epiblast structure through which cells enter the that cells of the hypoblast not only have the ability to change interior. Previous investigations by others have led to the the fate of competent cells in the epiblast to initiate an ectopic conclusion that the avian hypoblast, when rotated with regard axis, but also have the ability to repress the formation of the to the epiblast, has inductive properties that can change the original axis in committed cells of the epiblast. At the same fate of competent cells in the epiblast to form an ectopic time, the results showed that the epiblast is not wholly depen- embryonic axis. Thus, it has been suggested that the hypoblast dent on the organized hypoblast to establish an axis. normally induces the epiblast to form a primitive streak at a In the present work, an attempt was made to reexamine the specific locus. In the work reported here, an attempt was made experiments of Waddington and of Azar and Eyal-Giladi. to reexamine the issue of induction. In contrast to previous Experiments were designed to examine whether the polarity of reports, it was found that the rotated hypoblast of the chicken the hypoblast is actually superior to the polarity of the epiblast embryo does not initiate formation of an ectopic axis in the in determining the site where the embryonic axis will be epiblast. The embryonic axis always initiates and develops initiated. Moreover, an attempt was made to learn whether according to the basic polarity of the epiblast layer. These there is any influence of the size of the rotated hypoblast or of results provoke a reinterpretation of the issues of mesoderm the direction, left or right, of hypoblast rotation on the induction and primitive streak initiation in the avian embryo. development of the embryonic axis or whether the rotated hypoblast might more effectively initiate an ectopic axis in a Cell interactions during the early stages of avian development blastoderm from which the resident posterior marginal zone are crucial for the process of axis determination. Two unique had been removed (7). layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage of the chicken embryo. The MATERIALS AND METHODS interactions between these layers of cells during the stages of axis formation were studied by Waddington (1, 2), who sep- Chicken eggs (Leghorn x Leghorn) were incubated for the arated the hypoblast from the epiblast, rotated it through 900, acquire stage XIII (EG&K) blastoderms. The operation of the and then replaced it so that the anterior-posterior axes of the blastoderm was done with care according to published proce- two layers were at right angles to one another. As a result, the dures (8, 9). The prospective anterior-posterior polarity was primitive streak curved so that its anterior end pointed toward determined on the basis of Koller's sickle ridge, which marks the original anterior region of the hypoblast. Since these the posterior side of the blastoderm. experiments seemed to demonstrate an effect of the hypo- The hypoblast (in experimental series A and C) and/or the blast's orientation on the direction of elongation of the prim- blastoderm (in experimental series Bi, B2, and C) were itive streak, Waddington suggested that the hypoblast deter- dissected as a disc of one of three sizes: (i) Small disc: The mines the direction of tissue movements in the epiblast, by circular cut was made central to Koller's sickle and the mar- which the primitive streak is formed. ginal zone. (ii) Medium disc: The circular cut was made central Azar and Eyal-Giladi (3), making use of a new normal table to the marginal zone, on the edge of Koller's sickle. (iii) Large of chicken development (4), reexamined this issue in an effort disc: The circular cut was made within the marginal zone, near to define the interactions between the epiblast and the hypo- the posterior junction of the marginal zone and the area opaca. blast during the blastula stage [stage XIII, Eyal-Giladi and This disc contains Koller's sickle and marginal zone cells. Kochav (EG&K)]. The hypoblast at this stage was separated All the experiments reported here were done with blasto- from the epiblast and rotated 900. It was reported that the derms in which morphological features were clearly observ- subsequent primitive streak developed according to the pre- able, as defined in Fig. 1. In experimental series Bi, B2, and sumptive anterior-posterior polarity of the hypoblast. The C, dissected blastoderm regions peripheral to the discs were main interpretation from this research was that, at stage XIII, the experimental conditions of rotation produce a polarity Epiblast An-terior conflict between two gradient fields- one the inductiveness of and Hvpoblast \ the hypoblast and the other the competence of the epiblast- Marghinal zone and that the inductiveness of the hypoblast always dominates. Koller's sickle In subsequent research, it was found that the epiblast can Area Opaca develop a primitive streak and embryonic axis in the absence Posterior FIG. 1. Diagram of the main morphological characteristics of the The publication costs of this article were defrayed in part by page charge chicken blastula (stage XIII, EG&K). Blastoderm is shown in ventral payment. This article must therefore be hereby marked "advertisement" in view-that is, the hypoblast is toward the viewer and the epiblast is accordance with 18 U.S.C. §1734 solely to indicate this fact. away from the viewer. The yolk has been removed. 10733 Downloaded by guest on September 30, 2021 10734 Developmental Biology: Khaner Proc. Natl. Acad. Sci. USA 92 (1995)

discarded while the remaining disc regions were incubated. Table 1. Rotation of the hypoblast 900 relative to the epiblast During the time of incubation, in a humid environment for does not alter orientation in which embryonic axis develops 36-48 hr at 37.5°C the embryos were observed, photographed, (series A and controls) and analyzed with reference to stages of the normal table of Deviation of axis chicken development (10). Operation N R-L 0° 5°-60° Bent Series A: Hypoblast RESULTS rotated From the reports of Waddington and Azar and Eyal-Giladi it Small 9 6-3 6 2 1 is unclear whether the whole hypoblast or only a circular Medium 14 6-8 10 3 1 subregion of it was dissected and rotated. Since previous work Large 28 12-16 18 8 2 (8) had shown that rotated blastoderm discs of different sizes Controls have different effects on the development of the primitive Unoperated 20 - 14 4 2 streak, it was considered important in the present analysis to Operated 10 - 8 1 1 examine the effects of rotated hypoblasts of several sizes. Hypoblast dissected Rotation of the Hypoblast Relative to an Epiblast That (large disc), replaced, Remains in Planar Contact with the Marginal Zone. In this not rotated type of experiment, only the hypoblast was dissected as a disc The hypoblast was rotated to the right or left (R-L). Deviation of the of small, medium, or large size. It was removed and rotated 900 embryonic axis from the orientation predicted from the anterior- in relation to the presumptive posterior side of the blastoderm, posterior polarity of the stage XIII epiblast is shown. Controls included to either the right or the left, and replaced on the epiblast (Fig. unoperated or operated but unrotated blastoderms. N, number of 2, series A). In 60-70% of the operated blastoderms, the blastoderms scored. Deviation of embryonic axis: 00, within 5° of that range displacements from the predicted orientation; axis developed in an orientation strictly in accord predicted; 5°-60°, embryonic bent, cases in which the primitive streak was bent 5°-60° from the with the presumptive anterior-posterior polarity of the epi- predicted orientation. blast. The rotated hypoblast did not influence the primitive streak to deflect toward the anterior end of the hypoblast, in orientation predicted from the presumptive anterior-posterior contrast to Waddington's report (1), or to reorient the prim- polarity of the epiblast, while in -10% the primitive streak itive streak 900 toward its own posterior side (3). The size of initially developed in a straight line oriented with the pre- the rotated disc did not make a significant difference in the sumptive epiblast axis, but later the embryonic axis came to results, nor was there an effect of rotating to the right or left bend 5°-60° from this polarity. However, in about one-half of (Table 1; Fig. 3A-E). In -25% of the cases, the embryonic axis these cases of the 25% and 10% groups, the embryonic axis developed in a straight line displaced 5°-60° from the axis developed from the side toward which the hypoblast was rotated, but in the other half the axis developed from the Series A: Rotation of the Hypoblast 900 opposite side-that is, the deviation was random with respect to the direction of hypoblast rotation. Small Medium Large Two control experiments were important to do. In the first, it was determined how precisely the orientation of the ante- P-% p Vp rior-posterior axis of the embryo could be predicted from the HypoHyc Hypo polarity of the blastoderm (its presumptive anterior-posterior axis) at the stage of operation. A high level of predictability is Kolleir's sickler' crucial for analyzing the results. It was found that in only 70% PT sickle sickle P Epiblast Epiblast Epiblast of the cases the orientation of the anterior-posterior axis of the embryo could be predicted (within 50) from the polarity of the stage XIII blastoderm. In the other 30%, the embryonic axis Series B1: (epiblast + hypoblast) or Series B2: (epiblast alone) deviated 5°-60° (20%) or was secondarily bent by 50-60° (10%). This range of deviations of axes closely resembles that Small Medium Large of the operated rotated blastoderms of series A. -N / ,-se, Marginal/"' The influence of the hypoblast operation on the development \ zone` ) I of the embryonic axis was also assessed. The hypoblast was \- / Koller's \/ dissected (a large-sized disc) from the blastoderm but then put Pt "--sickle Epiblast back in its original orientation. The results demonstrated that in Ep t 80% of the cases the embryonic axis developed according to the k.piblast presumptive anterior-posterior axis, while in 20% of the cases Series C: deviations of5°-600 were observed (Table 1). Thus, the operation Rotation of the Hypoblast 900 itself did not affect the predictability of the orientation of the on an epiblast from which the marginal zone region has been removed embryonic axis, and predictions are accurate in 70-80% of the cases. When these control values are compared with the exper- imental series of rotated hypoblasts, it is evident that rotation of p Small Medium Large p p the hypoblast did not significantly affect the orientation of the Hypoblast - Hypoblast , Hypoblast embryonic axis formed by the epiblast. zone Rotation of the Hypoblast on an Epiblast from Which the \'-. " Koller's Marginal Zone Has Been Removed. The marginal zone of the Pt * sickle chicken embryo is known to play a crucial role in determining Epiblast PE p Epiblast the site at which the primitive streak will be initiated (7, 11). In Epiblast the experiments described above, the marginal zone was left and so was that inductive FIG. 2. Diagram of the experimental operations on the stage XIII connected to the epiblast, it possible avian blastoderm. The hypoblast and/or the blastoderm were dis- influences by the hypoblast were present but were overwhelmed sected as small, medium, or large discs as indicated. The four exper- by region-specific influences from the posterior marginal zone. imental series are diagrammed. Therefore, an attempt was made to assess whether a rotated Downloaded by guest on September 30, 2021 Developmental Biology: Khaner Proc. Natl. Acad. Sci. USA 92 (1995) 10735

FIG. 3. Photomicrographs of operated blastoderms from series A, Bi, and C. All blastoderms were viewed from the ventral side. (A-C) Stage XIII blastoderms at the end of the particular series A operation. (A) Small disc: the hypoblast has been rotated to the right side of the blastoderm. (B) Medium disc: the hypoblast has been rotated to the left side. (C) Large disc: the hypoblast has been rotated to the right. (D) The blastoderm of C was incubated 36 hr. A mature straight embryonic axis has developed; its orientation is that predicted from the anterior-posterior polarity of the preincubation epiblast. (E) The blastoderm of B has been incubated 30 hr. A mature embryonic axis has developed; its orientation deviates 150 from the presumptive posterior-anterior polarity of the preincubation epiblast. (F) Series Bi small disc containing epiblast and hypoblast was cultured alone. (G) A blastoderm as in F incubated 24 hr. A small primitive streak has developed from the presumptive posterior side of the blastoderm. Thereafter, the streak regressed and development did not continue (categorized as RPS in Table 2). (H) Series Bi large disc containing epiblast, hypoblast, and marginal zone was cultured alone. (I) Blastoderm of H was incubated 32 hr. An embryonic axis of smaller than normal size has developed from the presumptive posterior side of the blastoderm (categorized as EA in Table 2). (J) Series C medium disc. Hypoblast was removed and rotated 900 to the left in this example. The disc containing epiblast and hypoblast was cultured alone. (K) Blastoderm of Jwas incubated 16 hr. A small primitive streak developed from the presumptive posterior side of the blastoderm and then regressed after 20 hr of incubation (categorized as RPS in Table 2). C, carbon particles; NC, notochord; S, somite; B, brain; PS, primitive streak; KS, Koller's sickle; E, epiblast; H, hypoblast. (x36.) hypoblast can initiate an ectopic axis in the epiblast when the series of experiments (series B2), the entire hypoblast was first marginal zone has been removed. However, removal of the dissected and discarded from the blastoderm, and then small, marginal zone may in itself affect development, and therefore it medium, or large discs of epiblast were cut. was first necessary to evaluate the development of stage XIII The epiblast discs developed more or less completely and blastoderms from which various regions had been removed. with larger or smaller embryonic axes, depending on the size In this type of experiment (Fig. 2, series B1), the blastoderm of the disc and the presence or absence of marginal zone, was dissected as a small, medium, or large disc. In a related hypoblast, and area opaca (Table 2). Embryonic axes fre- Downloaded by guest on September 30, 2021 10736 Developmental Biology: Khaner Proc. Natl. Acad. Sci. USA 92 (1995) Table 2. The orientation at which the embryonic axis develops in the epiblast is not altered when the hypoblast is rotated 900, even when the marginal zone has been removed (series Bi, B2, and C) Deviation of Extent of development axis Operation N R-L UMT RPS PS HP EA 00 50-600 Series Bi Epiblast and unrotated hypoblast Small 9 8 1 0 0 0 Medium 16 0 10 4 0 2 Large 20 - 0 2 3 0 15 Series B2 Epiblast alone Small 5 - 5 0 0 0 0 Medium 8 3 5 0 0 0 Large 12 0 4 0 1 7 Series C Epiblast and rotated hypoblast Small 4 2-2 3 1 0 0 0 1 Medium 6 2-4 0 4 1 0 1 5 1 Large 10 6-4 0 2 0 2 6 8 2 Series Bi discs of epiblast and hypoblast of small, medium, or large size were cut from the blastoderm, and the peripheral regions were discarded. Series B2 discs of epiblast alone were cut from the blastoderm and the hypoblast and peripheral regions were discarded. Series C, the operation was done as in Bi but the hypoblast was rotated 900 to the left or right relative to the epiblast (R-L). Operated blastoderms were allowed to develop 24-36 hr before scoring the extent of development. Scoring the deviation of the embryonic axis from the orientation predicted from the anterior-posterior polarity of the stage XIII epiblast. N, number of blastoderms scored; R-L, number rotated to the right or left. Deviation of axis: 0°, within 50 of that predicted; 5-600, range displacements from the predicted orientation. Extent of development includes unorganized mesodermal tissue (UMT), regressed primitive streak after it initially forms (RPS), full primitive streak (PS), head process formed (HP), and embryonic axis forms including notochord, somites, and brain (EA). quently developed to mature stages in large blastoderm discs, In 60-70% of the operated blastoderms, the embryonic axis where the marginal zone was left connected to the epiblast. develops in the orientation predicted from the presumptive Large discs could frequently develop small embryonic axes in anterior-posterior polarity of the epiblast. The results did not 75% of the cases when the hypoblast was present (series B1) differ significantly with the three different sizes of rotated and in 60% of the cases when it was absent (series B2). When hypoblast, nor was there an influence of rotation to the right medium discs were prepared, o60% of these could still develop or left side. Control experiments demonstrate that the orien- small primitive streaks even when the hypoblast was absent, but tation of the embryonic axis can be predicted in only 70-80% these streaks tended to regress (Table 2, series B2). The small of the cases in a stage XIII blastoderm. The conclusion from discs, whether or not hypoblast was present, usually formed these results is that there is no evidence for the rotated hypoblast's unorganized mesodermal tissue, which did not contain an iden- inducing an ectopic axis in the epiblast at stage XIII. Either the tifiable primitive streak (Table 2; Fig. 3 F-I). Thus, the best kind hypoblast exerts no inductive influence on the epiblast at this of disc in which to test the effects of hypoblast rotation in the stage in the experimental situation, or, if it does, the epiblast absence of the marginal zone was the medium-sized disc. dominates the effect and does so even when the marginal zone is The results of series C demonstrated that large discs are not removed. These data provide no basis for the inference that the very informative because they still contain the marginal zone, hypoblast exerts such an inductive effect on the epiblast under although they do lack the area opaca (Fig. 2, series C). The unoperated conditions in normal development. medium discs are most informative for they lack the marginal What then led to the previous conclusions of the inductive zone and area opaca, and yet they develop well enough to score role of the hypoblast? Careful examination of Waddington's the position of the primitive streak and occasional embryonic results (1, 2) shows that only 11 operated chicken blastoderms axis. In this set, five of six (83%) developed the streak in an were analyzed. In just 4 of these was the axis bent or slightly orientation predicted from the epiblast polarity; the remaining reoriented in accord with the posterior side of the rotated case was slightly deviated. Finally, small discs are not very hypoblast. In the other 7, the axis did not shift; it developed informative because their development is too incomplete to straight and in an orientation predicted from the original score the orientation of streaks or axes (Table 2; Fig. 3 J and anterior-posterior polarity of the epiblast. The published K). Thus, in the case of the medium-sized discs, the rotated visual records of the bent embryos show clearly that the hypoblast did not influence the primitive streak to shift 900 posterior half of the embryo developed in accord with the toward its own posterior side even though the marginal zone presumptive posterior-anterior polarity of the epiblast, while region had been removed. the anterior part of the axis was bent 15°-45° toward the posterior side of the rotated hypoblast. Waddington worked with incubated blastoderms that include not only the blastula DISCUSSION stage XIII but also slightly older developmental stages [stage In this research, in contrast to previous reports, no evidence XIV, EG&K; stage 2, Hamburger and Hamilton (H&H)] in was found that the rotated hypoblast of the chicken embryo which the primitive streak had started to develop even before initiates the formation of an ectopic axis in the epiblast, even the operation. At these more advanced stages of development, when the marginal zone has been removed from the epiblast. the hypoblast is firmly connected to the streak, and it is Downloaded by guest on September 30, 2021 Developmental Biology: Khaner Proc. Natl. Acad. Sci. USA 92 (1995) 10737 impossible to separate the hypoblast as an intact layer of cells. the epiblast gain its anterior-posterior polarity? It remains Mechanical forces needed to separate the hypoblast might possible that posterior marginal zone and Koller's sickle cells distort the blastoderm and cause the embryonic axis to bend. do give polarity to the epiblast, but they do this in a way that A more recent reexamination of the hypoblast's role was does not involve the hypoblast. In the chicken embryo, the more precise than Waddington's original study since it was gene goosecoid (gsc), which in Amphibia is expressed in the based on a new normal table of chicken development (3, 4) and prospective head mesoderm portion of Spemann's organizer, made use of modern techniques of surgery and incubation, is expressed in the region of the posterior marginal zone and basically the same conditions as used in the present report. Koller's sickle well before and during stage XIII (17). These According to the 1981 report, the embryonic axis was shifted cells may have organizer properties that determine the 900 toward the position of 'the posterior side of the rotated initiation site of gastrulation in the chicken embryo and that hypoblast. However, it is not apparent why a shift of 900 was the marginal zone and Koller's sickle cells do not contribute concluded when the visual records show no shift in the example to the hypoblast but instead represent a very of the primitive streak and only -45° of displacement for the early population example of an embryonic axis. Also, in other illustrated of the "middle layer" cells, some of which actually form the embryos the axis developed in a straight line with a deviation primitive streak and node, and may induce other epiblast of 15°-45° from the predicted anterior-posterior axis but not cells to form the streak as well. This interpretation preserves of 900, as was reported. Nevertheless, the authors concluded a role for posterior marginal zone cells in axis formation, but that at stage XIII, the rotation of the hypoblast by 900 in it makes that role independent of the hypoblast. Another relation to the epiblast does alter the position at which the report proposes that the role of the hypoblast at stage XIII epiblast forms a primitive streak and axis. might be only to induce uncommitted cells of the epiblast to In both investigations (1, 3), there were no control experi- form blood cells by a basic fibroblast growth factor-type ments to test the accuracy of predicting the anterior-posterior inductive signal rather than to induce embryonic axial polarity of the epiblast at the time ofoperation and none to test structures (18). This report supports the notion that the the influence of the operation or the effects of cultivation on hypoblast not only cannot induce the formation of an ectopic the development of control embryos in comparison to the embryonic axis upon rotation but also cannot induce the experimental ones. As reported here, deviations or bends of formation of the original embryonic axis. the embryonic axis by 5-60° were found in nearly 20-30% of the cases as a consequence of incorrect identification of the 1. Waddington, C. H. (1932) Philos. Trans. R. Soc. London B 211, presumptive anterior-posterior axis and/or as a result of the 179-230. operation, even without hypoblast rotation. These control 2. Waddington, C. H. (1933) Wilheim Roux Arch. Entwicklungs- values account for the minority of deviated streaks and axes mech. Org. 128, 502-521. accompanying hypoblast rotations in the experimental series. 3. Azar, Y. & Eyal-Giladi, H. (1981) J. Embryol. Exp. Morphol. 61, A clarification of the role of the is crucial 133-144. hypoblast for 4. Eyal-Giladi, H. & Kochav, S. (1976) Dev. Biol. 49, 321-337. understanding issues of mesoderm induction and axis forma- 5. Mitrani, E. & Eyal-Giladi, H. (1981) Nature (London) 298, tion in the avian embryo. The hypoblast is a complex tissue: 800-802. cells that contribute to it are known to derive from at least two 6. Mitrani, E. & Eyal-Giladi, H. (1984a) Differentiation (Cambridge, sources. Some cells move central from the posterior marginal UK) 26, 107-111. zone and Koller's sickle, while the others ingress from the 7. Azar, Y. & Eyal-Giladi, H. (1979) J. Embryol. Exp. Morphol. 52, epiblast (12). It has been suggested that the cells moving from 79-88. the marginal zone gradually adhere with clustered ingressing 8. Khaner, 0. & Eyal-Giladi, H. (1986) Dev. Biol. 115, 275-281. cells from the epiblast to form a complete lower layer, the full 9. New, D. A. T. (1955) J. Embryol. Exp. Morphol. 3, 326-331. hypoblast. Several reports support the notion that only hypo- 10. Hamburger, V. & Hamilton, H. L. (1951) J. Morphol. 88, 49-92. blast cells from the posterior marginal zone are able to induce 11. Khaner, 0. (1993) in Current Topics in DevelopmentalBiology, ed. the primitive streak in the epiblast (7, 12-14). Other reports Pedersen, R. A. (Academic, New York), Vol. 28, pp. 155-180. claim that those cells do not induce the primitive streak but 12. Eyal-Giladi, H. (1991) Crit. Rev. Poultry Biol. 3, 143-166. only mark the site at which primitive streak cells will arise in 13. Eyal-Giladi, H., Debby, A. & Harel, N. (1992) Development the epiblast (15-17). If cells derived from the posterior mar- (Cambridge, UK) 116, 818-830. zone constitute a dominant 14. Eyal-Giladi, H., Lotan, T., Levin, T., Avner, 0. & Hochman, J. ginal really inductive cell popula- (1994) Development (Cambridge, UK) 120, 2501-2509. tion in the stage XIII hypoblast, why do they not induce an 15. Stern, C. D. & Canning, D. R. (1990) Nature (London) 343, ectopic axis and repress the formation of the original one? In 273-275. light of the negative results presented here, it is uncertain 16. Stern, C. D. (1990) Development (Cambridge, UK) 109,667-682. whether the hypoblast has a role in the process of mesoderm 17. Izpisu'a-Belmonte, J. C., De Robertis, E. M., Storey, K. G. & induction and primitive streak initiation. Stern, C. D. (1993) Cell 74, 645-659. If the hypoblast, which is the precursor of extraembryonic 18. Gordon-Thomson, C. & Fabian, B. C. (1994) Development (Cam- yolk sac endoderm, does not induce the epiblast, how does bridge, UK) 120, 3571-3579. Downloaded by guest on September 30, 2021