Hypoblast Induction of Multiple Area Vasculosae, and Stabilization of the Area Opaca Vasculosa in Young Chick Blastoderm

/. Embryo!, exp. Morph. Vol. 68, pp. 115-126, 1982 1 J 5 Printed in Great Britain © Company of Biologists Limited 1982

Hypoblast induction of multiple area vasculosae, and stabilization of the area opaca vasculosa in young chick blastoderm

By NIKOLAS ZAGRIS1 From the Tissue Culture Laboratory, Department of Biological Sciences, University of Patras

SUMMARY Multiple vascular areas are formed in unincubated and prestreak chick blastoderms under the influence of multiple transplanted hypoblasts. Though the transplanted hypoblasts merge into one continuous layer, they do not participate collectively in forming one streak and embryonic axis with one area vasculosa at its postero-lateral end, but each hypoblast seems to form an embryonic centre or fraction of a centre. It may be that the merged hypoblasts do not lose their individuality or that they have induced their prospective embryonic centre before merging. This is an indication that the hypoblast is able to initiate certain events to which the epiblast responds. Composite blastoderms with stage-2 host epiblasts and with two or more transplanted hypoblasts form the embryonic axis at its prospective plane and two area vasculosae, the area opaca vasculosa (a.o.v.) as in the control blastoderm, and an induced area vasculosa 180° anteriorly. However, composite blastoderms with stage-3 epiblasts and with two or more transplanted hypoblasts behave as the control blastoderms forming the embryonic axis and the a.o.v. at their prospective sites. This indicates that the typical a.o.v. in the chick blastoderm is stabilized to blood island formation at stage 3. The stage dependence which involves progressive restriction of the areas in which blood islands will develop, suggests the existence of a centre which creates organization by integrating short-lived fields. It seems that there are no particular cell groups of the unincubated blastoderm determined to form erythrocytes but that the organizing capacities of the area are necessary to induce the first early commitments of prospective erythroblasts along this course.

INTRODUCTION Blood islands are condensations of splanchnopleuric mesoderm which, in close association with the underlying endoderm, form blood cells (Miura & Wilt, 1969, 1970). Premesoderm. cells invaginate from the epiblast through the primitive streak (PS) to establish the mesodermal layer (Rudnick, 1955; Rosenquist, 1966). However, it is known that in the absence of a normal PS and of the resulting axial mesoderm there is always present some mesenchyme probably formed by a process of diffuse polyinvagination from the epiblast 1 Author's address: Tissue Culture Laboratory, Department of Biological Sciences, University of Patras, Patras, Greece. 116 N. ZAGRIS (Eyal-Giladi & Wolk, 1970; Azar & Eyal-Giladi, 1979; Zagris & Eyal-Giladi, 1982). By about 18 h of incubation, mesodermal cells from the area pellucida in a horseshoe-shaped region posterior and posterolateral to the PS invade the proximal portion of the area opaca adjacent to the area pellucida, and by 24 h of incubation they aggregate into cell clusters which are known as the blood islands (Settle, 1954). This zone into which mesoderm has grown is called the area opaca vasculosa (a.o.v.), because it is from this region that the blood cells and yolk-sac blood vessels arise (reviewed by Wilt, 1967, and Bellairs, 1971). Work of several investigators has contributed significantly to our knowledge of the endodermal layer formation in the avian embryo (Vakaet, 1962, 1970; Modak, 1965, 1966; Nicolet, 1970; Rosenquist, 1972; Fontaine & Le Douarin, 1977; Sanders, Bellairs & Portch, 1978). Though there is little doubt that blood islands are mesodermal in origin, the presence of the endoderm is necessary for the attainment of full haemoglobin (Hb)-forming capacity, possibly because it transmits essential materials from yolk to mesoderm and/or perhaps because it forms the endothelium of the blood islands (Wilt, 1965, 1967). Zagris (1979, 1980) has shown that unincubated blastoderms in which the PS and embryonic axis are inhibited mechanically are capable of forming primitive and definitive erythrocytes and embryonic and adult Hbs. This is evidence that the interacting components for erythroid cell formation need not invaginate through a PS and do not require the continued presence of the embryonic axis. Dantschakoff (1907), Sabin (1917), and Murray (1932) have given classical morphological accounts of the development of the blood islands. Haemoglobin in these islands is first detectable at the 6- to 7-somite stage (Wilt, 1967), and regulatory events occurring prior to the appearance of Hb have been described by several investigations (reviewed by Wilt, 1967, and Bruns & Ingram, 1973). Murray (1932) mapped the haemopoietic regions in chick blastoderms with mature PS, and Rudnick (1938) reported the appearance of erythroblasts in cultures taken from various stages of development of the PS. Settle (1954) mapped the areas of the chick blastoderm capable of forming blood islands in vitro from pre-streak to blastoderms of 4-6 somites and proposed that certain areas of the chick blastoderm became committed to erythropoiesis before the 6-7 h of incubation. Despite the extensive literature on the location of the a.o.v. and erythroid cell development in the chick embryo (Fraser, 1963; Wilt, 1967; Schalekamp, Schalekamp, Van Goor & Slingerland, 1972; Bruns & Ingram, 1973; Brown & Ingram, 1974; Wainwright & Wainwright, 1974; Cirotto, Scotto Di Telia & Geraci, 1975; Tobin, Selvig & Lasky, 1978; Zagris & Melton, 1978; Chapman & Tobin, 1979; Martin, Beaupain & Dieterlen- Lievre, 1980; Zagris, 1980), there is little information about the stabilization of the a.o.v. The present work was undertaken to determine the time this area, and not any other in the blastoderm, is committed and stabilized to blood Stabilization of area opaca vasculosa in chick blastoderm 117 island formation. Various aspects of the hypoblast behaviour, such as the organized influence it exerts on the epiblast, which emerge in this study are discussed in concert with the a.o.v. formation.

MATERIALS AND METHODS Culture Freshly laid fertilized eggs (stage X-roman numeral indicates stage of development according to Eyal-Giladi & Kochav, 1976) of the White Leghorn breed were used. Unincubated blastoderms, and blastoderms incubated up to the definitive streak stage (stage 4 - arabic numeral indicates stage of development according to Hamburger & Hamilton, 1951) were removed from the egg, washed free of the vitelline membrane and any adhering yolk, and carded with a wide-mouthed pipette in a drop of Ringer solution on to a vitelline membrane raft. The blastoderm was flattened, epiblast side against the surface of the vitelline membrane which was stretched over a glass ring as described by New (1955). Hypoblasts from stage XIII blastoderms were loosened from the epiblast and eventually peeled off from it with the use of sharpened fine dissecting needles. Composite blastoderms were constructed by placing a series of two, three or four hypoblasts on to host unincubated blastoderms (stage X)> or on to denuded epiblasts from older blastoderms (up to stage 4). Hypoblasts were manoeuvred so that they were stretched in a polar, triangular or quadrangular pattern. The prospective anteroposterior axis was determined by means of the cell population density which characterizes most unincubated blastoderms (Spratt & Haas, 1960), and the plane of the axis was marked by a line of non-toxic, non-diffusible carbon or carmine powder with a fine needle directly on the hypoblast. The plane of the axis of the host epiblast was similarly marked. In addition, control external marks were made close to the stretched blastoderm on the vitelline membrane raft to serve as double checks of the blastoderm orientation. Blastoderms were cultured in plain Dulbecco's modified Eagle medium (MEM), 1-5 ml/blastoderm in a small Petri dish (internal diameter 5 cm) resting on a moist cotton ring inside a larger support Petri dish (9 cm). The cultures were incubated at 38 °C as described elsewhere (Zagris, 1979). Blasto- derms, culture media, and glassware were handled with sterile precautions.

Staining and determination of haemoglobin The control and experimental blastoderms were cuHured for at least 3 days before their staining with benzidine - peroxide solution (Zagris, 1980) to show presence of Hb for photography. At the end of the culture period, the MEM was removed and the blastoderms were flooded with benzidine - peroxide 118 N. ZAGRIS solution. Colour developed within the first few minutes after application of the staining solution which was removed after about 5 min, and the blastoderms were photographed. For Hb determination, blastoderms, which were in culture usually for 5 days, were lifted from the vitelline membrane carefully, and were homogenized in l-9mlH2O. The homogenate was centrifuged for 10 min at 700rev./min in a clinical centrifuge, and the supernatant was used for Hb estimation by the O-dianisidine-H2O2 procedure (Hell, 1964). The results and conclusions are based on the study of more than 20 blastoderms per group.

RESULTS Two, three or four hypoblasts transplanted on to unincubated blastoderms and on to denuded epiblasts of older blastoderms in a polar, triangular, and quadrangular pattern merge to form a continuous layer about 8 h after their transplantation. Four hypoblasts transplanted onto an unincubated blastoderm in a quadrangular pattern have merged into a continuous layer as shown after 20 h in culture (Fig. 1 A). Multiple vascular areas which form synchronously one at the site of each transplanted hypoblast are orientated each around a prominent tissue aggregation which is usually without distinct morphology as shown after 6 d in culture (Fig. 1B). These vascular areas which are localized usually in a U-shaped configuration resemble the a.o.v. and are referred to as

Abbreviations: «, aborted axis; aov, area opaca vasculosa; ax, embryonic axis; chl, composite hypoblastic layer; h, head region; iav, induced area vasculosa. Fig. 1. Stage-X blastoderm with four hypoblasts placed in a quadrangular pattern shows merging of the hypoblasts into a continuous hypoblastic layer after 20 h (A) in MEM culture. Shown after 6 days (B) in culture, it displays i.a.v. (dark areas), one at the site of each transplanted hypoblast, stained with benzidine-peroxide solution. Tracings (B) mark individual i.a.v. Scale bar = 500 fim. Fig. 2. Stage-X blastoderm with four hypoblasts placed randomly forms four i.a.v. as shown after 6 days in culture. Tracings mark individual i.a.v. Scale bar = 500 /tm. Fig. 3. Stage-2 blastoderm with two additional hypoblasts forms the embryonic axis and the a.o.v. at their prospective sites, and one i.a.v. 180° anteriorly as shown after 6 days in MEM culture. Scale bar = 500 ftm. Fig. 4. Stage-3 blastoderm with two additional hypoblasts forms the embryonic axis and a.o.v. at their prospective sites shown after 5-5 days in MEM culture. Scale bar = 500 fim. Fig. 5. Stage-3 blastoderm with two additional hypoblasts forms the a.o.v. at its prospective site, and the embryonic axis turned 180° as shown after 6 days in MEM culture. Scale bar = 500 fim. Fig. 6. Stage-3 control blastoderm forms the embryonic axis and the a.o.v. at their prospective sites as shown after 6 days in MEM culture. Scale bar = 500 /im. Fig. 7. Stage-10 to -11 blastoderm developed in ovo, shows normal formation of the embryonic axis and a.o.v. at their prospective sites. Blastoderm stained with benzidine-peroxide solution. Scale bar = 1000 ftm. Stabilization of area opaca vasculosa in chick blastoderm 119 1A

hiV

. 2 A7V

aov

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»{ >V i i 120 N. ZAGRIS induced area vasculosae (i.a.v.). However, presence of the prominent tissue aggregation is not necessary and blood formation occurs in its absence. When the hypoblasts are placed randomly and in close proximity, the neighbouring i.a.v. which form interconnect to an intricate, massive plexus but they do not lose their U-shaped orientation around the prominent tissue aggregation. Each of these areas gives the impression of an embryonal organization centre as is shown after 6 days of culture of a host unincubated blastoderm onto which four hypoblasts were transplanted (Fig. 2). The i.a.v. arise synchronously at the site of the transplanted hypoblasts of which anteroposterior orientation on the host epiblast is not important, and blood islands can form at the new posterior end of the transplanted hypoblast which is oriented towards the edge of the composite blastoderm. Host epiblasts from pre-streak blastoderms display the same behaviour as the unincubated blastoderm in that they also support formation of multiple i.a.v. depending on the number of the hypoblasts transplanted. Composite blastoderms with host epiblasts from initial streak (stage-2) blastoderms show formation of only two vascular areas one posteriorly as in the control, the other 180° anteriorly. In these epiblasts the early streak is already imprinted, and, in all cases, it disperses after transplantation of the hypoblasts, and a new streak forms at the original axis plate. Transplantation of two hypoblasts onto the anterior end of a stage-2 blastoderm opposite the host streak and with the anterior end of the transplanted and host hypoblasts almost touching one another results in formation of a definite, although morphologically abnormal, embryonic axis according to its original anteroposterior orientation, and to the formation of two area vasculosas, the a.o.v. as in the control blastoderm, and an i.a.v. 180° anteriorly (Fig. 3). Host epiblasts from intermediate streak (stage-3) and older blastoderms on to which two or more hypoblasts are transplanted form a definite embryonic axis at the original axis plate, and only one vascular area, the a.o.v. at its expected topographical location on the host epiblast. Such a host epiblast on to which two hypoblasts were transplanted forms the a.o.v. at the expected normal location as is shown after 5-5 days of culture (Fig. 4). A similar composite blastoderm shows a posteroanterior turning of its embryonic axis, the head occupying the original tail region in the host blastoderm, thus giving the impression of a 180° shift of the a.o.v. (Fig. 5). This reversal of the anteroposterior polarity, which occurs rarely, apparently involves extensive reorgan- ization of the embryonic axis, but the fact that the a.o.v. was formed at its prospective site provides strong evidence of the stability of the a.o.v. by the intermediate streak stage. A control blastoderm explanted at stage 3 is shown after 6 days in culture (Fig. 6). This specimen was chosen because it shows the typical embryonic axis and also size and location of the a.o.v. as displayed by most blastoderms in the plain MEM culture. It is of interest to note that the a.o.v. of the control Stabilization of area opaca vasculosa in chick blastoderm 121

Table 1. Formation of area vasculosae in epiblasts from chick blastoderms at various stages of development on to which two, three, or four hypoblasts were transplanted

Composite Hypoblastsf Area vasculosae blastodermst Stage* (no. transplanted) (no. formed) (no. examined) 2 2 25 3 3 25 4 4 10 2 2 25 3 3 25 4 4 15 2-3 2 2 25 3 2 25 4 2 10 3-4 2 1 25 3 1 25 4 1 10 * Stage, marks the developmental time blastoderms were explanted and cultured as composite or control blastoderms in MEM for at least 3 days. t Composite blastoderms, were constructed by placing two, three, or four hypoblasts on to unincubated blastoderms or on to denuded epiblasts of the stages indicated. blastoderms is less prominent than the total surface area occupied by the area vasculosae of the composite blastoderms. A stage 10-11 blastoderm which developed in ovo is presented to serve as reference point for the location of the a.o.v. relative to the embryonic axis (Fig. 7). The blastoderm was stained with benzidine-peroxide solution. The number of area vasculosae formed in composite blastoderms at various stages of development can be summarized as shown in Table 1. Each transplanted hypoblast induces formation of one area vasculosa, that is four i.a.v. are formed under the influence of four hypoblasts, in blastoderms at stages X and 1, two area vasculosas, the a.o.v. and one i.a.v., are formed in stage-2 blastoderms, while only the a.o.v. is formed in stage-3 blastoderms. The results in each group were consistent. Some cultures were lost because of technical error, such as puncturing or overstretching the vitelline membrane of the raft which resulted in disfiguring of the blastoderm, but these were discarded. Measurements of the total amount of Hb were made on blastoderms which were explanted at the stages X, 1, 2-3 and 3-4. Control, and composite blastoderms constructed by transplantation of two, and three hypoblasts on denuded epiblasts were cultured in MEM for 5 days. There was about a 2-fold, and a 5- to 6-fold increase of Hb content in composite blastoderms with the two and three transplanted hypoblasts, respectively, in the stage-X and stage-1 groups as compared to their control. Composite blastoderms of the stage-2 to 122 N. ZAGRIS Table 2. Measurement of Hb in control and composite blastoderms (Values expressed in /tg per blastoderm ± standard deviation. The results are the average of three experiments.)

Composite blastodermsf Control , * » Stage* blastoderms Two hypoblasts Three hypoblasts X 018 ±002 0-27 ±001 0-6 ±003 1 0-22 ±003 0-55 ±.006 1-3 ±002 2-3 0-90 ±013 0-82 ±009 0-88 ±008 3-4 0-91 ±003 115 ±009 110±004 * Stage, marks the developmental time blastoderms were explanted and cultured as composite or control blastoderms in MEM for 5 days. t Composite blastoderms, as defined in Table 1.

-3 and stage-3 to -4 groups contain about the same amount of Hb as their control blastoderms. More Hb is present in control blastoderms explanted at older stages, compared to those at younger stages, at the end of the same culture period (Table 2). Blastoderms cultured in MEM form the embryonic axis with a distinctive anteroposterior orientation at its prospective site. The embryonic axis shows a pronounced brain region and neural tube, a lateral mesoderm which rarely becomes segmented, and, in many cases, cardiac tissue which pulsates rhyth- mically (62 pulses/min). However, the U-shaped a.o.v. forms at its prospective site characteristic of normal in ovo development, and normal growth of the blastoderm is not affected. The MEM supports substantial Hb formation as compared to culture in plain albumin in which, although there is blood island formation, Hb presence is rarely detected under the stereoscope even after staining with benzidine (Zagris & Eyal-Giladi, 1981). In addition to its enhancing Hb formation, the MEM provides favourable conditions for prolonged blastoderm survival for at least 10 days, in contrast to culture in plain albumin in which it shows the typical distinct signs of degeneration 2-3 days after the beginning of culture. Enhancement of Hb formation and prolonged embryonic survival in culture are distinct advantages which make the MEM a useful culture medium for the study of erythropoiesis in the early chick blastoderm. In all the experiments described above, the first blood islands appear as numerous cell thickenings in the almost transparent blastoderm at the beginning of the second day in culture. The cell thickenings become slightly tinged with yellow at the end of the second day in culture, and a few hours later appear as dense bright red clusters due to the presence of Hb apparent even on casual observation. Blood islands become more intensely red the following days in culture (Zagris, 1979). The presence of Hb was confirmed by a sharply positive benzidine stain, and was not found, after staining, in any place in which it had not been detected in the unstained state. Stabilization of area opaca vasculosa in chick blastoderm 123

DISCUSSION The unincubated blastoderm, a relatively unstructured tissue with no apparent plane of symmetry, can form multiple embryonal centres or fractions of centres as evidenced by cellular aggregations each associated with a vascular area under the influence of multiple transplanted hypoblasts (Figs. 2, 3). It is well known that the endoderm plays an important role in organizing the mesoderm into blood islands (Miura & Wilt, 1970). In this connexion, it should be noticed that the hypoblast affects both the PS and the a.o.v. Thus, an induced streak is accompanied by an accumulation of blood islands opposite its posterior end. In our results, vascular rings are orientated around tissue aggregations which we interpret as aborted attempts to form embryonic axes. It is of interest to note that, though the transplanted hypoblasts merge to form a continuous thick layer, they do not participate collectively in forming one streak and embryo body with one area vasculosa at the posterior end, It may be that the merged hypoblasts do not lose their individuality, or, more likely, that they have induced their prospective embryonal centre before their merging. It seems that the unincubated blastoderm is a developmental mosaic field out of which one or more embryonic fields may arise. The properties of mosaic fields are discussed in a theoretical paper by Chandebois (1976). Another point of interest which emerges from our results is that the hypoblast does not seem to have an anteroposterior orientation but this is determined by the epiblast. Thus, blood islands always form at the hypoblast posterior oriented towards the periphery of the epiblast. Prestreak blastoderms exhibit similar behaviour as that discussed with the unincubated blastoderm and are not capable of complete embryonic integration. However, embryonic segregation, especially that concerned with embryonic axis formation, occurs in stage-2 and older blastoderms which display remarkable regulative features. Blastoderm with stage-2 host epiblasts and with two or more transplanted hypoblasts form one well centered embryonic axis and two blood island areas one posteriorly as in the control, the other 180° anteriorly. More Hb is present in composite as compared to control blastoderms at stage X and stage 1, the amount of Hb increasing as the number of transplanted hypoblasts increases. This, in concert with the observation that the vascular area(s) of the composite look more prominent morphologically than that of the control blastoderms, may reveal that there is not a fixed pool of prospective blood cells which is shared. It would seem either that there js a non-fixed pool from which prospective blood cells are recruited or that each transplanted hypoblast induces blood cell formation in situ. Waddington (1933) has shown that the hypoblast plays an important tole in directing tissue movements which build the streak in the epiblast but it was not clear whether the hypoblast starts these movements. In our results, the formation of multiple embryonal centres or fractions of centres at the site 124 N. ZAGRIS of the transplanted hypoblast on unincubated blastoderm or pre-streak epiblast shows that the hypoblast is able to initiate certain events to which the epiblast responds. The experiment in which the embryonic axis formed at an 180° angle (Fig. 7), in addition to providing strong evidence demonstrating the stability of the definitive a.o.v. by stage 3, also indicates that the hypoblast can induce a new set of movements to form a PS. This new set of movements seems to coalesce with the original set and may be powerful enough to annul the original thus reversing the posteroanterior orientation of the axis. The fact that an already imprinted streak on the host epiblast disappears in all cases demonstrates that its cells are not stabilized, and may show that to accomplish integration, it is important to re-establish a simple low-layer morphological pattern. According to Spratt & Haas (1960), complete integration depends upon the dominance of one embryo-initiating centre over any others which may be present in a composite system. Then, it is likely that movements might merge into one another to give rise to a single PS in the prospective axial plane of the blastoderm. The stage dependence involving a progressive restriction of the areas in which blood islands will develop, suggests the existence of a centre which creates organization by integrating short-lived fields. It seems that there are no particular cell groups of the unincubated blastoderm determined to form erythrocytes, but that the organizing capacities of the area are necessary to induce the first early commitments of prospective erythroblasts along this course. It is at the stage-3 blastoderm that the distinctive horseshoe-shaped a.o.v. surrounding the posterior and posterolateral parts of the area pellucida is stabilized.

Some of the results of this work were presented at the XlVth International Embryological Conference, 11-17 Sept. 1980, Patras, Greece.

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{Received 6 July 1981, revised 9 October 1981)