The mechanisms involved in the generation of axial structures in the chick are well documented, yet, little is known about the actual factors that generate such a complex pattern. The recent demonstrations that all-trans-retinoic acid (RA) acts as a morphogen during limb development (Thaller and Eichele, 1987) lead us to examine whether during axis formation in the developing chick, RA could be one of the factors involved. We now show that retinoic acid can block a very unusual property of normal early chick embryonic cells, mainly their capacity to grow in semisolid medium. We also present experiments that suggest that RA may play a direct role during axis formation in the developing chick.

Considerable progress has been attained in understanding the role the various parts of the early chick blastula play in generating embryonic axial structures (see Eyal-Giladi, 1984). In spite of this, little is known about the actual factors that generate such a complex pattern. The recent demonstrations that RA acts as a morphogen during limb development (Thaller and Eichele, 1987) lead us to test whether RA could also be involved in the establishment of the chick embryonic axis.

Between stage X and stage XIII, the one-layer-thick chick blastoderm containing the peripheral area opaca, the marginal zone and the central epiblastic area, gradually forms a second hypoblastic layer underlying the central epiblastic region (see Table 1). Normally, through the inductive action of the hypoblast, the embryonic axis starts to develop from the posterior end of the epiblast (Azar and Eyal-Giladi, 1979; Mitrani and Eyal-Giladi, 1981). If the hypoblast is removed, the marginal zone has the capacity to regenerate a new inductive hypoblast (Azar and Eyal-Giladi, 1979). Rotation of the hypoblast by 90° is followed by a similar rotation of the embryonic axis (Waddington, 1932; Azar and Eyal-Giladi, 1981). If the polarity of the hypoblast is destroyed, the orientation of the primitive streak can be determined by the epiblast (Mitrani and Eyal-Giladi, 1981).

Table 1.

Pattern of axis formation following grafts of DEAE paper soaked in 1 mg ml ȡ1 RA, to the lateral marginal zone of chick blastoderms

Pattern of axis formation following grafts of DEAE paper soaked in 1 mg ml ȡ1 RA, to the lateral marginal zone of chick blastoderms
Pattern of axis formation following grafts of DEAE paper soaked in 1 mg ml ȡ1 RA, to the lateral marginal zone of chick blastoderms

RA is essential in the control of epithelial differentiation (see Roberts and Sporn, 1984). It induces differentiation of F9 mouse teratocarcinoma stem cells (Strickland and Mahdavi, 1978) and inhibits the growth of some malignant cells (Roberts and Sporn, 1984; Haussier et al. 1983). RA induces digit pattern duplication when locally applied to the anterior aspect of the developing chick wing bud (Tickle et al. 1982, 1985). It has recently been shown that chick limb buds contain endogenous RA distributed as a concentration gradient across the limb anlage with a high-point at the posterior domain of the limb bud (Thaller and Eichele, 1987). Recently, a broader understanding as to how retinoids may act was obtained with the identification of the RA receptor (RAR) and the discovery that RAR bears homology to the steroid and thyroid hormone receptors (Petkovich et al. 1987; Giguere et al. VT. Benbrook et al. 1988). RARs contain discrete DNA-binding and ligand-binding domains and thus it is likely that the hormone-receptor complex induces a cascade of regulatory events that results from the activation of specific set of genes (Benbrook et al. 1988). In the chick limb bud, a spatial distribution of cellular protein binding to RA (CRABP) has been observed (Maden et al. 1988). In the amphibian limb bud, mRNA coding for RAR has been found to be localized specifically in the regenerating cells that control limb pattern (Giguere et al. 1989).

In the present work we examine the possible interaction between RA and early chick embryonic cells in two steps. First we explore a simpler system to test for response to RA. We take advantage of an unusual property of early embryonic cells, mainly their capacity to grow in agarose (Mitrani, 1984). We show that when RA is applied to early chick cells grown in agarose, the cells lose the capacity to form colonies. Second we test for the capacity of RA to interfere with axis formation by applying it, in a localized manner, into a manipulated blastoderm grown in vitro. The results of these experiments suggest that RA may be involved in the control of axis formation in the developing chick.

Culture of early chick embryo cells

Stage X-XII blastoderms (Eyal-Giladi and Kochav, 1976) were incubated in trypsin-EDTA for 10 min, transferred to culture medium and dissociated by gently forcing them through increasingly finer Pasteur pipettes. Cells were plated on standard culture Petri dishes at 105 cells ml-1 (Mitrani and Eyal-Giladi, 1982). The culture medium used was RPMI1640 containing 10% fetal calf serum and penicillin and streptomycin at a concentration of 1 %. The cells were incubated at 37 °C in 5 % CO2 and air at 100% humidity. Each experiment was performed in duplicate and the same number of cells was plated on each dish. A stock solution of all trans-retinoic acid (Sigma type XX) was prepared fresh each time in dimethyl sulphate (DMSO, Fluka) at a concentration of 1 mgml-1 and diluted to the required concentrations directly in the culture media. At the end of the culture period, a relative estimation of cell number was obtained by comparing the total nucleic acid present in each monolayer culture. Nucleic acids were extracted as described previously (Mitrani et al. 1987).

Anchorage-independent cell growth

Single cells were derived from whole stage X-XII blastoderms (Eyal-Giladi and Kochav, 1976) and grown in agarose as described previously (Mitrani, 1984). Briefly, 0·8 ml of 0·33% agarose (Sea Plaque, Marine Colloids, USA) in culture medium was prepared as an underlayer in 35 mm Petri dishes. Stage X-XII blastoderms were incubated in trypsin-EDTA for 10 min, transferred to culture medium and dissociated by gently forcing them through increasingly finer Pasteur pipettes. The dissociated cells were suspended in the same medium with a final agarose concentration of 0·3 % and layered onto the previously agarose-coated Petri dish. After gelling, the dishes were incubated at 37 °C in an atmosphere of 5% CO2. Cells were plated at 3×104 cells per dish. The culture medium used was RPMI 1640 containing 10 % fetal calf serum and penicillin and streptomycin at a concentration of 1 %. RA solutions were applied as described in the previous section. Each experiment was performed in duplicate and the same number of cells was plated on each dish.

Growth of blastoderms in vitro

Blastoderms were removed from the egg at the appropriate stage and grown in vitro using the New technique (New, 1955). Blastoderms were operated in Ringer’s solution. The hypoblast and a small fragment (0·6×0·6mm) derived from the posterior aspect of the marginal zone (PMZ) were removed from the blastoderm. At the same time, a small strip of Whatman diethylaminoethyl cellulose (DEAE) paper soaked in a RA solution in DMSO, was implanted in the lateral marginal zone region, 90° counterclockwise to the posterior end (see Table 1). The operated blastoderms were then placed lower side up on a vitelline membrane stretched on a glass ring and cultured directly on egg albumin at 37°C for 24–48 h.

Growth in agarose

Untreated cells and cells treated with 0·05 % DMSO showed the first signs of colony formation already after 36 h and continued to form colonies of about 20–100 cells during the next 24–48h (see Fig. 1). Treated cells at RA concentrations as low as 5×10−8M could not form colonies in agarose. Only when the dose was lower (5×10−9M) the first signs of colony formation could be observed. As a control for toxicity, cells derived from stage X-XII blastoderms were grown in standard monolayer cultures (Mitrani and Eyal-Giladi, 1982). In the monolayer cultures, cells grown in the presence of 10−8M-RA, reached confluency at about the same time as untreated cells and were morphologically indistinguishable from them. RA-treated cells grown in monolayers remained viable for the same period of time as the untreated cells even when the RA concentration was 5 × 10−6 M, which is 100-fold higher than the concentration necessary to inhibit growth in agarose (Fig. 2).

Fig. 1.

Colony growth in the’ presence of RA. Cells from whole stage XI-XII blastoderms grown in agarose in a 0·05% DMSO solution, after 54 h in culture. (A) In the absence of RA, large number of colonies can be seen.(B) 5 ×10“10 M-RA, the number of colonies is not significantly different from untreated cells. (C) 5×10−9M-RA, the number and size of the colonies starts to decrease. (D) 5×10−8M-RA, no colonies were observed at this concentration of RA, only single cells can be seen in the culture. (×80).

Fig. 1.

Colony growth in the’ presence of RA. Cells from whole stage XI-XII blastoderms grown in agarose in a 0·05% DMSO solution, after 54 h in culture. (A) In the absence of RA, large number of colonies can be seen.(B) 5 ×10“10 M-RA, the number of colonies is not significantly different from untreated cells. (C) 5×10−9M-RA, the number and size of the colonies starts to decrease. (D) 5×10−8M-RA, no colonies were observed at this concentration of RA, only single cells can be seen in the culture. (×80).

Fig. 2.

Selective inhibition of the anchorage-independent growth of early chick cells by retinoic acid. Left axis indicates number of colonies, each containing more than twenty cells, as a percentage of the number of colonies obtained in the absence of RA (closed circles). 3×104 cells were plated per 35 mm Petri dish. Each experiment was performed in duplicate. Retinoic acid was dissolved in DMSO and the amount of solution applied was never more than 0·05 % of the total culture volume. In the absence of RA, DMSO was applied as 0·05 % of the culture volume. Right axis indicates cell number after 24 h in monolayer cultures expressed as a percentage of cell number obtained from an identical monolayer culture in the absence of RA (open circles).

Fig. 2.

Selective inhibition of the anchorage-independent growth of early chick cells by retinoic acid. Left axis indicates number of colonies, each containing more than twenty cells, as a percentage of the number of colonies obtained in the absence of RA (closed circles). 3×104 cells were plated per 35 mm Petri dish. Each experiment was performed in duplicate. Retinoic acid was dissolved in DMSO and the amount of solution applied was never more than 0·05 % of the total culture volume. In the absence of RA, DMSO was applied as 0·05 % of the culture volume. Right axis indicates cell number after 24 h in monolayer cultures expressed as a percentage of cell number obtained from an identical monolayer culture in the absence of RA (open circles).

Blastoderm cultures

Table 1 summarizes the manipulations performed and the results obtained in this experimental series. More than 150 blastoderms were treated with grafts soaked in RA concentrations ranging from 10 ng ml-1 to 10 mg ml-1. The more reproducible results were obtained with RA doses of Imgml-1 and only those results have been included in the table. At stage XI, all RA-treated blastoderms developed normally, with axes emerging from the original posterior end (Fig. 3A). At stage XII, 87·5% of RA-treated blastoderms developed normally, with axes emerging from the original posterior end. At stage XIII, in the absence of RA, normal axes developed from the posterior end in all cases (Fig. 3B). However, in the presence of RA, the formation of axes from the posterior end was inhibited in 90·1 % of the cases and, instead, an agglomeration of cells along an axis perpendicular to the posteroanterior axis of the blastoderm was observed (Fig. 3C,D). With time these structures deteriorated and the blastoderms developed no further.

Fig. 3.

(A) Stage XI blastoderm deprived of the PMZ and of the emerging hypoblast and grown in the presence of a DEAE graft soaked in a 1 mg ml-1 solution of RA in DMSO (gR). A normal axis develops from the posterior end (p) (×20). (B) Stage XIII blastoderm deprived of the PMZ and of the hypoblast and grown in the presence of a DEAE graft soaked in DMSO only (g). A normal axis develops from the posterior end (p) (×20). (C) Stage XIII blastoderm deprived of the PMZ and of the hypoblast and grown in the presence of a DEAE graft soaked in a 1 mg ml-1 solution of RA (gR). An agglomeration of cells occurs at the site of the graft. No axis develops from the posterior end (p) (×20). (D) Transverse section of a stage XIII blastoderm grown in the same conditions as the one described in C, parallel to the anteroposterior axis of the blastoderm, through the area of cell agglomeration. A concentration of unorganized ‘mesenchymal’ cells can be seen at the central area of the section (×80).

Fig. 3.

(A) Stage XI blastoderm deprived of the PMZ and of the emerging hypoblast and grown in the presence of a DEAE graft soaked in a 1 mg ml-1 solution of RA in DMSO (gR). A normal axis develops from the posterior end (p) (×20). (B) Stage XIII blastoderm deprived of the PMZ and of the hypoblast and grown in the presence of a DEAE graft soaked in DMSO only (g). A normal axis develops from the posterior end (p) (×20). (C) Stage XIII blastoderm deprived of the PMZ and of the hypoblast and grown in the presence of a DEAE graft soaked in a 1 mg ml-1 solution of RA (gR). An agglomeration of cells occurs at the site of the graft. No axis develops from the posterior end (p) (×20). (D) Transverse section of a stage XIII blastoderm grown in the same conditions as the one described in C, parallel to the anteroposterior axis of the blastoderm, through the area of cell agglomeration. A concentration of unorganized ‘mesenchymal’ cells can be seen at the central area of the section (×80).

The first set of experiments shows that early chick embryonic cells lose the capacity to grow in semisolid medium when cultured in the presence of low doses of RA. The mechanisms that lead the primary chick cells to grow in agarose in the absence of exogenous factors are far from clear. A large number of growth factors and/or oncogenes can confer anchorage-independence growth properties to untransformed cell lines (see Roberts et al. 1985; De Larco and Todaro, 1978; Sasada et al. 1988). Roberts et al. (1985) have shown that induction of anchorage-independent growth by various growth factors involves different cellular pathways, which can be distinguished by their sensitivity to RA. The clonal cell system described above may help to elucidate similar growth factor interactions in normal primary early embryonic cells, first in relation to their capacity to grow in agar and second in the broader and complex aspect of axis formation.

The fact that cells from stage XI-XI1 chick blastoderms respond to RA by losing the capacity to grow in agarose suggests that already at those early stages they possess receptors for RA and that these receptors may have a functional role in the normal developing embryo. Such reasoning led us to perform experiments with blastoderm expiants. The results of these experiments indicate that whilst at stage XI, local application of RA at 90° from the posterior aspect will not hamper the development of a normal axis from the posterior end, at stage XIII such treatment will inhibit the formation of the normal axis at the posterior side and will cause instead an agglomeration of cells along an axis perpendicular to the posteroanterior axis of the blastoderm.

Transplantation experiments have shown that there is a gradient of inductivity within the hypoblast (Waddington, 1932; Azar and Eyal-Giladi, 1979; Mitrani and Eyal-Giladi, 1981; Mitrani et al. 1983) and within the marginal zone of preprimitive streak blastoderms (Kahner and Eyal-Giladi, 1986). The posterior aspect of the hypoblast has the highest inductive activity (Mitrani et al. 1983). Similarly, the marginal zone behaves as a ring-like gradient field, the maximal value of which is at the PMZ end (Kahner and Eyal-Giladi, 1986, 1989). When the PMZ of a normal stage XI blastoderm is removed a normal axis will still appear from the posterior end. However, if the PMZ is removed and transplanted to a position 90° from the posterior side, a new axis develops according to the new position specified by the PMZ implant. The normal axis, which would otherwise develop from the posterior end, is inhibited (Kahner and Eyal-Giladi, 1986). The gradient of inductivity also has a temporal component so that at stage X, inductivity of the PMZ is maximal and decreases as development progresses. By stage XIII, the highest inductivity is concentrated on the hypoblast and is weaker at the MZ (see Kahner and Eyal-Giladi, 1989).

Clearly, axis formation is the result of a delicate balance of forces. The dominating field determines the site of axis formation and inhibits, in most cases, the creation of secondary ectopic axes. In the present experiments, we removed both the PMZ and the hypoblastic layer to minimize the natural inductive forces. At stage XI, when RA is applied at 90° from the posterior aspect, the dominant force is still the remaining marginal zone. At stage XIII, in the absence of RA, the remaining marginal zone, although much weaker than at stage XI (see Kahner and Eyal-Giladi, 1989), is still capable of generating a new inductive field. Yet when RA is applied at this later stage, it partially overcomes the weaker inductive action of the remaining marginal zone, so that the formation of an axis from the posterior aspect of the blastoderm can no longer materialize and an agglomeration of cells is created instead along the gradient of diffusion of RA.

There is evidence suggesting that during limb development a set of cartilage elements can form in the absence of RA, and that the function of RA could be to convert an exactly periodic pattern into a non-equivalent one (Slack, 1987). Axis formation may constitute a more complex problem in which different types of cells are being induced to take alternative differentiation pathways in a very precise manner. Perhaps, as is becoming clear in Xenopus development, more than one factor is at play during axis formation (see Slack, 1987; Godsave et al. 1988; Symes et al. 1989). The present results suggest that, in the chick at least, RA may be one of them.

This work was supported by the American-Israel Binational Science Foundation, Jerusalem, Israel number 85-00296 to E.M.

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