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Protein synthesis in epiblast versus hypoblast during the critical stages of induction and growth of the primitive streak in the chick embryo
DEVELOPMENTAL
Protein
BIOLOGY
45, 358-365 (19%)
Synthesis
in Epiblast
Versus
Stages of Induction Primitive
Streak
Hypoblast
and Growth
of Zoology,
The Hebrew
the Critical
of the
in the Chick Embryo
HEFZIBAH EYAL-GILADI, ISAAC FARBIASZ, DAVID OSTROVSKY,’ Department
during
University
Accepted
April
of Jerusalem,
AND JACOB Jerusalem,
HOCHMAN
Israel
9, 1975
In the chick the inducing power of the hypoblast for primitive streak was assumed to reach its maximum at the beginning of the primitive streak stage and to last until its completion. It was therefore of interest to trace the protein synthetic activity of the epiblast and hypoblast during five successive developmental stages and to correlate them with the known morphogenetic events. The investigation was done along two lines: 1) A quantitative survey was made of the uptake of tritiated phenylalanine into epiblasts versus hypoblasts and their incorporation into trichloroacetic acid-precipitable protein. 2) Incorporation of label into protein was followed by a comparative investigation of the electropherograms of epiblast versus hypoblast at the different stages. The quantitative survey has shown an almost uniform and rather law incorporation of label into protein in the hypoblast layer with a very short period of doubled activity between full hypoblast and initial primitive streak (p.s.). During this period the inductive capacity of the hypoblast for primitive streak was supposed to reach its maximal value. The qualitative survey indicated different patterns of incorporation in the two layers studied. Of special interest are two peaks (III and IV) which appear in the hypoblast previous to p.s. formation at the time of its augmented synthetic activity which also coincides with the onset of its inductive capacity. At later stages two similar peaks appear in the epiblast. It is suggested that a protein included in the above peaks might represent the inductor of the primitive streak. INTRODUCTION
An investigation of the biochemical changes underlying cell differentiation during early vertebrate development is complicated by both the small size of the embryos and the inherent complexity of the process of differentiation. Usually whole embryos (mainly amphibian and mammalian) are studied and changes in RNA, DNA and protein synthesis traced through a sequence of embryonic stages. These studies are primarily descriptive, confirming that changes occur, but it is difficult to causally relate a particular change to a specific morphogenetic event. The pessimistic conclusion of Deuchar (1973) that “Studies of DNA, RNA and protein of embryonic cells are therefore no longer expected to tell us why the cells ’ Present address: Department of Biology, ville State College, Millersville, PA 17551.
Millers-
differ, but only again that they differ. . . .” can therefore be understood. We felt that a much more precise and accurate analysis should be undertaken of a developmental event that is thought to be the only one going on in the embryo and of which the morphogenetic aspects are well known. Neural induction by the mesoderm of the archenteron was long regarded as the first inductive process in a developing embryo and was therefore called primary induction. Recently however it was shown, by Nieuwkoop (1969) in amphibians and Eyal-Giladi (1970) and Eyal-Giladi and Wolk (1970) in the chick, that neural induction is preceded by the induction of the mesoderm by the entoderm. In the chick this process involves two embryonic layers, the hypoblast as the inducing and the epiblast as the reacting system, resulting in the formation of the primitive streak.
358 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
EYALGILADI
ET AL.
Protein
The specific molecular factors responsible for this induction are unknown. Tiedemann (1968) succeeded in isolating a protein fraction from g-day-old chick embryos which was capable of inducing mesoderm. Although there is no evidence that this particular protein fraction is involved in primitive streak induction, Tiedemann’s results suggest that it would be logical to investigate first the protein synthesis of the chick embryo during the period of primitive streak induction, as the primitive streak is destined to give rise to the embryo’s primary mesoderm. It therefore seemed appropriate to trace the pattern of quantitative and qualitative differences in protein synthesis between epiblast and hypoblast, starting at the full hypoblast-stage XIII (Eyal-Giladi and Koand continuing chav, in preparation) through four stages of primitive streak formation. MATERIALS
AND METHODS
Freshly laid eggs from hybrid New Hampshire 6 x Leghorn p chickens were collected from chicken houses in KiryatAnavim or from our own laboratory. In vitro labeling. Eggs were incubated at 37°C to the desired developmental stage. The blastoderms were removed in cold Ringer’s solution under sterile conditions. Only blastoderms that adhered to the vitelline membrane were used. Each embryo and its surrounding vitelline membrane were cleaned of adhering yolk platelets and placed in a watch glass containing 0.5 ml of Ringer’s solution. A small glass ring (diameter, 1.0 cm) was put on the vitelline membrane so as to secure the embryo, tighten the membrane, and remove folds (New, 1955). The watch glass was placed in a humid petri dish and the embryo incubated for 1 hr at 37°C. Following incubation, the Ringer’s solution was replaced by carefully adding 0.5 ml of fresh Ringer’s solution at 37°C containing 30 PCi of tritiated phenylalanine (specific activity, 5.0
Synthesis
in Chick Primitive
Streak
359
Wmmole). The preparation was then incubated for an additional hour after which the embryo was transferred to 0°C for 10 min and washed three times in cold Ringer’s solution to remove excess label and adhering yolk platelets. Stages used. The following developmental stages were used: A, a blastoderm with a fully developed primary hypoblast, stage XIII of Eyal-Giladi and Kochav (in preparation); B, a blastoderm with an initial primitive streak; C, a blastoderm with ‘/? of a primitive streak; D, a blastoderm with l :i of a primitive streak; and E, a blastoderm with over ‘15of a primitive streak. Stages B-E correspond to stages 2-5 of Vakaet (1962) and to stages l+, 2, 3, and 3+ of Hamburger and Hamilton (1951).
Separation of hypoblast and epiblast. The primary hypoblast was removed using either a hair loop or fine surgical pins. The morphogenetic cell movements during primitive streak formation, including the streaming of cells into the mesoderm and their penetration into the hypoblast to contribute to the definitive entoderm, were taken into consideration. Consequently the part of the hypoblast regarded by us as primary hypoblast was determined for each of the above stages relying on the observations of Rosenquist (1966; 1972) and Nicolet (1970; 1971) on the cell movements in the hypoblast . The primary hypoblast sections as indicated by the striped areas on the drawings of the different stages in Figs. 2 and 3 could be very easily freed at the time of the operation from the epiblast and from the very few adhering mesodermal cells. Next the area opaca of the blastoderm was removed. The remaining area pellucida and its adherent derivatives, namely, mesoderm and entoderm which invaginated from the primitive streak, were regarded as epiblast. The stage of development was determined for each blastoderm at the time of incubation, rechecked at the time of introduction of phenylalanine, and once again
360
DEVELOPMENTALBIOLOGY
at the point of separation of the epiblast and hypoblast. Processing of samples. Samples from either pooled or individual preparations were homogenized in a total volume of 1 ml of Ringer’s solution at 0°C with a Pontes all-glass homogenizer using ten strokes of the pestle. Following homogenization, cold trichloroacetic acid (TCA) was added to a final concentration of 10% and the preparation left overnight at 4°C. The material was then centrifuged at 20,OOOg for 1 hr in a Sijrval Superspeed RC-PB refrigerated centrifuge. The supernatant fluid containing the amino acid pool was saved for counting. The protein pellet was washed three times with cold TCA. The wash was also saved for counting. The protein pellet was dissolved in 0.1-0.4 ml of 0.2 N NaOH containing 2% sodium dodecyl sulphate (SDS) (Maize1 1969). Aliquots of the solubilized protein (5 ml from pooled samples and 25 ml from individual samples) were added to vials containing 10 ml of scintillation fluid (667 ml of tolueve, 337 ml of Triton X, 5.5 g of PPO, 0.5 g of POPOP). Radioactivity was measured in a Packard Tri-Carb Model 3003 liquid scintillation spectrometer at a counting efficiency of 50%. Protein concentration was determined by the method of Lowry et al. (1951). Electrophoresis of embryos. Preparations intended for electrophoresis were dialysed for 24 hr against two changes of Tris buffer, pH 6.7, containing 1% SDS. Samples of 30-100 ~1 of protein were used for SDS-polyacrylamide-gel electrophoresis (Maizel, J. V., unpublished manuscript; Maurer, 1971). Samples were run at a constant current of 1.0 mA/gel in the spacer gel and 1.5-2.0 mA/gel in the resolving gel. At the conclusion of the run, gels were stained with Coomassie brilliant blue G-250.
Gels intended for the determination of counts per minute (cpm) in the protein bands were frozen with Dry Ice at the
VOLUME 45, 1975
completion of the run and sliced into 2.0-mm sections. The spacer gel was also saved for counting. Gel sections were added to scintillation vials and solubilized in 0.5 ml of hydrogen peroxide-SDS solution (24% H,O, and 2% SDS). Ten milliliters of scintillation fluid were added to the solubilized gels. The counting efficiency for the solubilized gel slices was 40%. RESULTS
Amino Acid Uptake and Incorporation Proteins
into
A preliminary experiment demonstrated that a peak level of phenylalanine incorporation into embryo proteins occurred when 0.5 ml of label at a concentration of 60 &i/ml was added to the in vitro embryo (Fig. 1). This concentration was, therefore, used throughout the study. A significant difference in the rate of phenylalanine incorporation into epiblast and hypoblast proteins was seen at stage A (Table 1). The epiblast had nearly five times the specific activity of the hypoblast. The fact that the hypoblast had a greater concentration of unincorporated phenylalanine in the amino acid pool, 350 cpmlmg of protein as opposed to 134 cpm/mg of protein for the epiblast, indicated that the difference in specific activity between the two germ layers reflected a difference in
Labeled
Amino
Acld
concentrotlon(pc/mll
FIG. 1. The effect of labeled amino acid concentration upon uptake. The experiment, was done on single intact stage A embryos.
EYAL-GILADI
Protein
ET AL.
Synthesis
TABLE A REPRESENTATIVE Embryonic layer
Epiblast Hypoblast a The epiblast
Incorporated cpm
28,500 1,800 and hypoblast
in Vitro
in Chick Primitive
1
INCORPORATION
Pool cpm
Amount of protein (mid
4,700 3,500
35 10
EXPERIMENTS
Specific activity of unincorporated phenylalanine (crmhg protein)
Comparative Biosynthetic Activity Between Epiblast and Hypoblast During Successive Developmental Stages Figure 2 shows the specific activities of epiblasts and hypoblasts at five developmental stages. Major differences can be seen between the activities of these germ layers. The epiblast demonstrates two periods of distinctive activity: One at the end of hypoblast formation (stage A) and the other at the mid-primitive-streak formation (stage D). There is a plateau between stages B and C when the activities of the epiblast and the hypoblast are nearly equal. The hypoblast demonstrates a distinct rise just prior to stage B. Figure 3 is based on the averaged data of all the experimental material and demonstrates the fluctuations of the “activity coefficient” (A.C.) throughout the studied period of gastrulation. From stage A (A.C., 4.0) there is a significant fall of the activity coefficient to its lowest value of 1.45 at stage B (highest relative activity of hypoblast). As gastrulation proceeds the activity coefficient rises to a maximum of 6.95 at mid-gastrulation (stage D) and then immediately drops again to 1.65 at late gastrulation (stage E).
Specific activity of incorporated phenylalanine (cpdmg protein)
134 350
of a single stage A embryo were separated
the rate of protein synthesis rather than a difference in precursor availability. This difference in incorporation rate was also expressed in terms of an activity coefficient defined as the ratio of epiblast to hypoblast specific activity.
361
Streak
Activity coefficient (815/180)
815 180
4.5
and processed individually.
Figure 3 suggests that although the activity coefficient varies considerably from stage to stage, its value at each point is characteristic for the stage investigated. Electrophoretic
Patterns
The relative incorporation rates of epiblast and hypoblast protein fractions separated by SDS-acrylamide-gel electrophoresis are shown in Fig. 4. There are distinct differences between the pattern of phenylalanine incorporation into epiblast and hypoblast proteins at all the stages studied. Considering only the most obvious and consistant peaks, the following can be noted. In the epiblast of stage A there are two peaks, I and II, of relatively high incorporation. In the hypoblast of the same stage there is only one prominent peak. It has the same electrophoretic mobility as peak I of the epiblast. From stage B onwards, peak I disappears both in the epiblast and hypoblast, whereas peak II, characteristic of the epiblast alone, can still be discerned at stages B and C. At stage B there is a high rate of incorporation in two hypoblast protein fractions, peaks III and IV. Peak III includes over 25% of the total cpm. These peaks disappear from the hypoblast at later stages but peaks with the same electrophoretic mobility appear in the epiblast of stage D. Also, at this stage, peak II, characteristic of the epiblast at earlier stages, disappears.
362
DEVELOPMENTALBIOLOGY
- - -
VOLUME 45, 1975
@
epiblost hypoblost
/’
-\
------e--
c-A
STAGE FIG. 2. Incorporation of labeled amino acid into TCA-precipitable proteins of separated epiblasts and hypoblasts through successive stages of primitive streak formation. On the schematic drawings of the stages, the primary hypoblast is indicated by a striped area, while the secondary hypoblast from primitive streak origin by a white area. DISCUSSION
Thus far studies on protein synthesis in early vertebrate embryos have been carried out mainly on whole embryos in an attempt to detect changes occurring with progressive development (from one stage to another). As has been postulated by Van Blekrom and Manes (1974) on the grounds of their results concerning preimplantation rabbit embryos: “if differentiation, as defined by differential protein synthesis is occurring in a certain cell population of the blastocyst, it would not be detected in the study of total embryos.” The present study differs from previous investigations on vertebrate embryos by not merely looking for bulk changes in protein synthesis. An attempt has been
made to trace the dynamics of such changes both regionally and temporally and to correlate them to the earliest inductive interaction known thus far in the chick (Eyal-Giladi and Wolk, 1970). Our unpublished observations on the chick agree with the conclusion of Van Blekrom and Manes (1974) for the rabbit, namely, that the pattern of protein synthesis does not change in in vitro studies. This enabled us to perform in vitro labeling, which greatly facilitated our work. The present investigation has been carried out along two comparative lines, quantitative and qualitative. The very interesting phenomenon indicated by the quantitative study, as represented in Fig. 2, concerns the very short period between stages A and B. Here there is on the one
EYAL-GILADI ET AL.
Protein Synthesis in Chick Primitiue Streak
hand a very sharp decline in amino acid incorporation into protein in the epiblast and an increase in the synthetic activity of the hypoblast, bringing the incorporation rate of the two layers to the same level. After the first appearance of the primitive streak this phenomenon is reversed, there is a remarkable decline in the activity of the hypoblast whereas the epiblast resumes accelerated activity reaching a new peak at stage D. The period of the rising synthetic activity of the hypoblast is at the end of hypoblast formation. On the basis of the transfilter induction studies of EyalGiladi and Wolk (1970) this stage was assumed to be the starting point for the inducing activity of the hypoblast on the
363
epiblast, resulting in the gradual formation of the primitive streak. SDS-acrylamide-gel electrophoresis was carried out to correlate these protein changes observed during primitive streak induction with specific protein fractions. Stained gels showed no characteristic differences in the banding patterns between epiblasts and hypoblasts either at the same developmental stage or among the five stages studied. The protein fractions, however, had distinct regional and temporal differences in the incorporation patterns of the label. A similar observation was made by Brahma and van der Saag (1972) for whole amphibian embryos. In stage A there is some similarity in the
STAGE FIG. 3. Pattern of activity coefficient (specific activity of epiblast/specific activity of hypoblast) the studied stages. The number of experiments for each point is enclosed in parentheses.
throughout
DEVELOPMENTALBIOLOGY
% lot
A
VOLUME 45. 1975
HYPOBLAST
EPIBLAST =I
6
% 6 B 2
2.02X103 CPM
HYPOBLAST
E PIBLAST
125x10"
%
F
CPM
1.05X lo3
CPM
?
FIG. 4. Electropherograms of epiblasts and hypoblasts at the five studied stages of development. Below the abcissas, the banding on a polyacrylamide gel of five proteins with known molecular weights is shown for comparison. The proteins were run parallel to the experimental material. a, Ovalbumin dimer, 94 x lo3 MW; b, bovine serum albumin, 65 x log; c, ovalbumin monomer, 47 x 103; d, trypsin, 25 x 1OJ; e, cytochrome c, 13 x 103.
EYALGILADI ETAL.
Rotein
pattern of incorporation of the epiblast and the hypoblast, the electropherograms of both showing one peak (I) with the same electrophoretic mobility. Whereas peak I is the only significant one in the hypoblast of stage A, in the epiblast a much more prominent additional peak II exists. Peak II is unique for the epiblast and persists through the first three developmental stages A, B and C. The initial appearance of the primitive streak at stage B corresponds to the appearance of peaks III and IV in the hypoblast. There is also an increase in the rate of amino acid incorporation into hypoblast protein at this time. Interestingly enough, although peaks III and IV disappear from the hypoblasts of successive stages, two similar peaks with the same electrophoretie mobility appear in the epiblast at stage D, which again was shown quantitatively to be at the maximum of incorporation activity (Fig. 2). From the above facts a working hypothesis may be suggested: After stage A, which is the completion of hypoblast formation, there is a burst of synthetic activity in the hypoblast, mainly of one protein (peak III). As this same period is considered (EyalGiladi and Wolk 1970) to be the beginning of the process of primitive streak induction, this protein may be the inductor. This hypothesis is supported by the fact that a protein or proteins with the same electrophoretic mobility appear later on (after their disappearance from the hypoblast) in the reacting epiblast, which might suggest that an inductor has been transferred to the epiblast or that the synthesis of a similar protein has been induced in the epiblast. Further studies are called for in order to check these hypotheses. REFERENCES BRAHMA, S. K., and VAN DER SAAG, P. T. (1972). Studies on biosynthesis of soluble proteins in early amphibian development by isoelectric focusing in
Sy nthesis in Chick Primitioe
Streak
365
thin layer polyacrylamide gel. Exp. Cell Res. 75, 527-530. DEUCHAR, E. M. (1973). Biochemical aspects of early differentiation in vertebrates. Advanc. Morphog. 10, 175-225. EYAL-GILADI, H. (1970). Differentiation potencies of the young chick blastoderm as revealed by different manipulations. II. Localized damage and hypoblast removal experiments. J. Embryol. Erp. Morphol. 23, 739-749. EYAL-GILALN, H., and KOCHAV, SH. (in preparation). A complementary normal table of the chick’s first stages of development. EYAL-GILADI, H., and WOLK, M. (1970). The inducing capacities of the primary hypoblast as revealed by transfilter induction studies. Wilhelm Roux Arch. Entwicklungsmech. Organismen 165, 226-241. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAURER, H. R. (1971). “Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis.” Walter de Gruyter, Berlin. NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3, 320-331. NICOLET, G. (1970). Analyse autoradiographique de la localization des diffkrentes ebauches pr&omptives dans la ligne primitive de l’embryon de poulet. J. Embryol. Exp. Morphol. 23, 79-108. NICOLET, G. (1971). Avian gastrulation. Aduan. Morphog. 9, 231-260. NIEUWKOOP, P. D. (1969). The formation of mesoderm in Urodelean amphibians. I. Induction by the endoderm. Wilhelm Roux Arch. Entwicklungsmech. Organismen 162, 341-373. ROSENQUIW, G. C. (1966). A radioautographic study of labelled grafts in the chick blastoderm development from primitive-streak stages to stage 12. Co&rib. Embryol. Carnegie Inst. Wash. 38, 71-110. ROSENQUIST, G. C. (1972). Endoderm movements in the chick embryo between the early short streak and head process stages. J. Exp. Zool. 180,95-103. TIEDEMANN, H. (1968). Factors determining embryonic differentiation. J. Cell Physiol. 72, Suppl. 1, 129-144. VAKAET, L. (1962). Some new data concerning the formation of the definitive endoblast in the chick embryo. J. Embryol. Exp. Morphol. 10, 38-57. VAN BLECKROM, J., and MANES, C. (1974). Development of preimplantation rabbit embryos in uiuo and in uitro. II. A comparison of qualitative aspects of protein synthesis. Deuelop. Biol. 40, 40-51.
Protein
BIOLOGY
45, 358-365 (19%)
Synthesis
in Epiblast
Versus
Stages of Induction Primitive
Streak
Hypoblast
and Growth
of Zoology,
The Hebrew
the Critical
of the
in the Chick Embryo
HEFZIBAH EYAL-GILADI, ISAAC FARBIASZ, DAVID OSTROVSKY,’ Department
during
University
Accepted
April
of Jerusalem,
AND JACOB Jerusalem,
HOCHMAN
Israel
9, 1975
In the chick the inducing power of the hypoblast for primitive streak was assumed to reach its maximum at the beginning of the primitive streak stage and to last until its completion. It was therefore of interest to trace the protein synthetic activity of the epiblast and hypoblast during five successive developmental stages and to correlate them with the known morphogenetic events. The investigation was done along two lines: 1) A quantitative survey was made of the uptake of tritiated phenylalanine into epiblasts versus hypoblasts and their incorporation into trichloroacetic acid-precipitable protein. 2) Incorporation of label into protein was followed by a comparative investigation of the electropherograms of epiblast versus hypoblast at the different stages. The quantitative survey has shown an almost uniform and rather law incorporation of label into protein in the hypoblast layer with a very short period of doubled activity between full hypoblast and initial primitive streak (p.s.). During this period the inductive capacity of the hypoblast for primitive streak was supposed to reach its maximal value. The qualitative survey indicated different patterns of incorporation in the two layers studied. Of special interest are two peaks (III and IV) which appear in the hypoblast previous to p.s. formation at the time of its augmented synthetic activity which also coincides with the onset of its inductive capacity. At later stages two similar peaks appear in the epiblast. It is suggested that a protein included in the above peaks might represent the inductor of the primitive streak. INTRODUCTION
An investigation of the biochemical changes underlying cell differentiation during early vertebrate development is complicated by both the small size of the embryos and the inherent complexity of the process of differentiation. Usually whole embryos (mainly amphibian and mammalian) are studied and changes in RNA, DNA and protein synthesis traced through a sequence of embryonic stages. These studies are primarily descriptive, confirming that changes occur, but it is difficult to causally relate a particular change to a specific morphogenetic event. The pessimistic conclusion of Deuchar (1973) that “Studies of DNA, RNA and protein of embryonic cells are therefore no longer expected to tell us why the cells ’ Present address: Department of Biology, ville State College, Millersville, PA 17551.
Millers-
differ, but only again that they differ. . . .” can therefore be understood. We felt that a much more precise and accurate analysis should be undertaken of a developmental event that is thought to be the only one going on in the embryo and of which the morphogenetic aspects are well known. Neural induction by the mesoderm of the archenteron was long regarded as the first inductive process in a developing embryo and was therefore called primary induction. Recently however it was shown, by Nieuwkoop (1969) in amphibians and Eyal-Giladi (1970) and Eyal-Giladi and Wolk (1970) in the chick, that neural induction is preceded by the induction of the mesoderm by the entoderm. In the chick this process involves two embryonic layers, the hypoblast as the inducing and the epiblast as the reacting system, resulting in the formation of the primitive streak.
358 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
EYALGILADI
ET AL.
Protein
The specific molecular factors responsible for this induction are unknown. Tiedemann (1968) succeeded in isolating a protein fraction from g-day-old chick embryos which was capable of inducing mesoderm. Although there is no evidence that this particular protein fraction is involved in primitive streak induction, Tiedemann’s results suggest that it would be logical to investigate first the protein synthesis of the chick embryo during the period of primitive streak induction, as the primitive streak is destined to give rise to the embryo’s primary mesoderm. It therefore seemed appropriate to trace the pattern of quantitative and qualitative differences in protein synthesis between epiblast and hypoblast, starting at the full hypoblast-stage XIII (Eyal-Giladi and Koand continuing chav, in preparation) through four stages of primitive streak formation. MATERIALS
AND METHODS
Freshly laid eggs from hybrid New Hampshire 6 x Leghorn p chickens were collected from chicken houses in KiryatAnavim or from our own laboratory. In vitro labeling. Eggs were incubated at 37°C to the desired developmental stage. The blastoderms were removed in cold Ringer’s solution under sterile conditions. Only blastoderms that adhered to the vitelline membrane were used. Each embryo and its surrounding vitelline membrane were cleaned of adhering yolk platelets and placed in a watch glass containing 0.5 ml of Ringer’s solution. A small glass ring (diameter, 1.0 cm) was put on the vitelline membrane so as to secure the embryo, tighten the membrane, and remove folds (New, 1955). The watch glass was placed in a humid petri dish and the embryo incubated for 1 hr at 37°C. Following incubation, the Ringer’s solution was replaced by carefully adding 0.5 ml of fresh Ringer’s solution at 37°C containing 30 PCi of tritiated phenylalanine (specific activity, 5.0
Synthesis
in Chick Primitive
Streak
359
Wmmole). The preparation was then incubated for an additional hour after which the embryo was transferred to 0°C for 10 min and washed three times in cold Ringer’s solution to remove excess label and adhering yolk platelets. Stages used. The following developmental stages were used: A, a blastoderm with a fully developed primary hypoblast, stage XIII of Eyal-Giladi and Kochav (in preparation); B, a blastoderm with an initial primitive streak; C, a blastoderm with ‘/? of a primitive streak; D, a blastoderm with l :i of a primitive streak; and E, a blastoderm with over ‘15of a primitive streak. Stages B-E correspond to stages 2-5 of Vakaet (1962) and to stages l+, 2, 3, and 3+ of Hamburger and Hamilton (1951).
Separation of hypoblast and epiblast. The primary hypoblast was removed using either a hair loop or fine surgical pins. The morphogenetic cell movements during primitive streak formation, including the streaming of cells into the mesoderm and their penetration into the hypoblast to contribute to the definitive entoderm, were taken into consideration. Consequently the part of the hypoblast regarded by us as primary hypoblast was determined for each of the above stages relying on the observations of Rosenquist (1966; 1972) and Nicolet (1970; 1971) on the cell movements in the hypoblast . The primary hypoblast sections as indicated by the striped areas on the drawings of the different stages in Figs. 2 and 3 could be very easily freed at the time of the operation from the epiblast and from the very few adhering mesodermal cells. Next the area opaca of the blastoderm was removed. The remaining area pellucida and its adherent derivatives, namely, mesoderm and entoderm which invaginated from the primitive streak, were regarded as epiblast. The stage of development was determined for each blastoderm at the time of incubation, rechecked at the time of introduction of phenylalanine, and once again
360
DEVELOPMENTALBIOLOGY
at the point of separation of the epiblast and hypoblast. Processing of samples. Samples from either pooled or individual preparations were homogenized in a total volume of 1 ml of Ringer’s solution at 0°C with a Pontes all-glass homogenizer using ten strokes of the pestle. Following homogenization, cold trichloroacetic acid (TCA) was added to a final concentration of 10% and the preparation left overnight at 4°C. The material was then centrifuged at 20,OOOg for 1 hr in a Sijrval Superspeed RC-PB refrigerated centrifuge. The supernatant fluid containing the amino acid pool was saved for counting. The protein pellet was washed three times with cold TCA. The wash was also saved for counting. The protein pellet was dissolved in 0.1-0.4 ml of 0.2 N NaOH containing 2% sodium dodecyl sulphate (SDS) (Maize1 1969). Aliquots of the solubilized protein (5 ml from pooled samples and 25 ml from individual samples) were added to vials containing 10 ml of scintillation fluid (667 ml of tolueve, 337 ml of Triton X, 5.5 g of PPO, 0.5 g of POPOP). Radioactivity was measured in a Packard Tri-Carb Model 3003 liquid scintillation spectrometer at a counting efficiency of 50%. Protein concentration was determined by the method of Lowry et al. (1951). Electrophoresis of embryos. Preparations intended for electrophoresis were dialysed for 24 hr against two changes of Tris buffer, pH 6.7, containing 1% SDS. Samples of 30-100 ~1 of protein were used for SDS-polyacrylamide-gel electrophoresis (Maizel, J. V., unpublished manuscript; Maurer, 1971). Samples were run at a constant current of 1.0 mA/gel in the spacer gel and 1.5-2.0 mA/gel in the resolving gel. At the conclusion of the run, gels were stained with Coomassie brilliant blue G-250.
Gels intended for the determination of counts per minute (cpm) in the protein bands were frozen with Dry Ice at the
VOLUME 45, 1975
completion of the run and sliced into 2.0-mm sections. The spacer gel was also saved for counting. Gel sections were added to scintillation vials and solubilized in 0.5 ml of hydrogen peroxide-SDS solution (24% H,O, and 2% SDS). Ten milliliters of scintillation fluid were added to the solubilized gels. The counting efficiency for the solubilized gel slices was 40%. RESULTS
Amino Acid Uptake and Incorporation Proteins
into
A preliminary experiment demonstrated that a peak level of phenylalanine incorporation into embryo proteins occurred when 0.5 ml of label at a concentration of 60 &i/ml was added to the in vitro embryo (Fig. 1). This concentration was, therefore, used throughout the study. A significant difference in the rate of phenylalanine incorporation into epiblast and hypoblast proteins was seen at stage A (Table 1). The epiblast had nearly five times the specific activity of the hypoblast. The fact that the hypoblast had a greater concentration of unincorporated phenylalanine in the amino acid pool, 350 cpmlmg of protein as opposed to 134 cpm/mg of protein for the epiblast, indicated that the difference in specific activity between the two germ layers reflected a difference in
Labeled
Amino
Acld
concentrotlon(pc/mll
FIG. 1. The effect of labeled amino acid concentration upon uptake. The experiment, was done on single intact stage A embryos.
EYAL-GILADI
Protein
ET AL.
Synthesis
TABLE A REPRESENTATIVE Embryonic layer
Epiblast Hypoblast a The epiblast
Incorporated cpm
28,500 1,800 and hypoblast
in Vitro
in Chick Primitive
1
INCORPORATION
Pool cpm
Amount of protein (mid
4,700 3,500
35 10
EXPERIMENTS
Specific activity of unincorporated phenylalanine (crmhg protein)
Comparative Biosynthetic Activity Between Epiblast and Hypoblast During Successive Developmental Stages Figure 2 shows the specific activities of epiblasts and hypoblasts at five developmental stages. Major differences can be seen between the activities of these germ layers. The epiblast demonstrates two periods of distinctive activity: One at the end of hypoblast formation (stage A) and the other at the mid-primitive-streak formation (stage D). There is a plateau between stages B and C when the activities of the epiblast and the hypoblast are nearly equal. The hypoblast demonstrates a distinct rise just prior to stage B. Figure 3 is based on the averaged data of all the experimental material and demonstrates the fluctuations of the “activity coefficient” (A.C.) throughout the studied period of gastrulation. From stage A (A.C., 4.0) there is a significant fall of the activity coefficient to its lowest value of 1.45 at stage B (highest relative activity of hypoblast). As gastrulation proceeds the activity coefficient rises to a maximum of 6.95 at mid-gastrulation (stage D) and then immediately drops again to 1.65 at late gastrulation (stage E).
Specific activity of incorporated phenylalanine (cpdmg protein)
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of a single stage A embryo were separated
the rate of protein synthesis rather than a difference in precursor availability. This difference in incorporation rate was also expressed in terms of an activity coefficient defined as the ratio of epiblast to hypoblast specific activity.
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Streak
Activity coefficient (815/180)
815 180
4.5
and processed individually.
Figure 3 suggests that although the activity coefficient varies considerably from stage to stage, its value at each point is characteristic for the stage investigated. Electrophoretic
Patterns
The relative incorporation rates of epiblast and hypoblast protein fractions separated by SDS-acrylamide-gel electrophoresis are shown in Fig. 4. There are distinct differences between the pattern of phenylalanine incorporation into epiblast and hypoblast proteins at all the stages studied. Considering only the most obvious and consistant peaks, the following can be noted. In the epiblast of stage A there are two peaks, I and II, of relatively high incorporation. In the hypoblast of the same stage there is only one prominent peak. It has the same electrophoretic mobility as peak I of the epiblast. From stage B onwards, peak I disappears both in the epiblast and hypoblast, whereas peak II, characteristic of the epiblast alone, can still be discerned at stages B and C. At stage B there is a high rate of incorporation in two hypoblast protein fractions, peaks III and IV. Peak III includes over 25% of the total cpm. These peaks disappear from the hypoblast at later stages but peaks with the same electrophoretic mobility appear in the epiblast of stage D. Also, at this stage, peak II, characteristic of the epiblast at earlier stages, disappears.
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VOLUME 45, 1975
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epiblost hypoblost
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STAGE FIG. 2. Incorporation of labeled amino acid into TCA-precipitable proteins of separated epiblasts and hypoblasts through successive stages of primitive streak formation. On the schematic drawings of the stages, the primary hypoblast is indicated by a striped area, while the secondary hypoblast from primitive streak origin by a white area. DISCUSSION
Thus far studies on protein synthesis in early vertebrate embryos have been carried out mainly on whole embryos in an attempt to detect changes occurring with progressive development (from one stage to another). As has been postulated by Van Blekrom and Manes (1974) on the grounds of their results concerning preimplantation rabbit embryos: “if differentiation, as defined by differential protein synthesis is occurring in a certain cell population of the blastocyst, it would not be detected in the study of total embryos.” The present study differs from previous investigations on vertebrate embryos by not merely looking for bulk changes in protein synthesis. An attempt has been
made to trace the dynamics of such changes both regionally and temporally and to correlate them to the earliest inductive interaction known thus far in the chick (Eyal-Giladi and Wolk, 1970). Our unpublished observations on the chick agree with the conclusion of Van Blekrom and Manes (1974) for the rabbit, namely, that the pattern of protein synthesis does not change in in vitro studies. This enabled us to perform in vitro labeling, which greatly facilitated our work. The present investigation has been carried out along two comparative lines, quantitative and qualitative. The very interesting phenomenon indicated by the quantitative study, as represented in Fig. 2, concerns the very short period between stages A and B. Here there is on the one
EYAL-GILADI ET AL.
Protein Synthesis in Chick Primitiue Streak
hand a very sharp decline in amino acid incorporation into protein in the epiblast and an increase in the synthetic activity of the hypoblast, bringing the incorporation rate of the two layers to the same level. After the first appearance of the primitive streak this phenomenon is reversed, there is a remarkable decline in the activity of the hypoblast whereas the epiblast resumes accelerated activity reaching a new peak at stage D. The period of the rising synthetic activity of the hypoblast is at the end of hypoblast formation. On the basis of the transfilter induction studies of EyalGiladi and Wolk (1970) this stage was assumed to be the starting point for the inducing activity of the hypoblast on the
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epiblast, resulting in the gradual formation of the primitive streak. SDS-acrylamide-gel electrophoresis was carried out to correlate these protein changes observed during primitive streak induction with specific protein fractions. Stained gels showed no characteristic differences in the banding patterns between epiblasts and hypoblasts either at the same developmental stage or among the five stages studied. The protein fractions, however, had distinct regional and temporal differences in the incorporation patterns of the label. A similar observation was made by Brahma and van der Saag (1972) for whole amphibian embryos. In stage A there is some similarity in the
STAGE FIG. 3. Pattern of activity coefficient (specific activity of epiblast/specific activity of hypoblast) the studied stages. The number of experiments for each point is enclosed in parentheses.
throughout
DEVELOPMENTALBIOLOGY
% lot
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HYPOBLAST
EPIBLAST =I
6
% 6 B 2
2.02X103 CPM
HYPOBLAST
E PIBLAST
125x10"
%
F
CPM
1.05X lo3
CPM
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FIG. 4. Electropherograms of epiblasts and hypoblasts at the five studied stages of development. Below the abcissas, the banding on a polyacrylamide gel of five proteins with known molecular weights is shown for comparison. The proteins were run parallel to the experimental material. a, Ovalbumin dimer, 94 x lo3 MW; b, bovine serum albumin, 65 x log; c, ovalbumin monomer, 47 x 103; d, trypsin, 25 x 1OJ; e, cytochrome c, 13 x 103.
EYALGILADI ETAL.
Rotein
pattern of incorporation of the epiblast and the hypoblast, the electropherograms of both showing one peak (I) with the same electrophoretic mobility. Whereas peak I is the only significant one in the hypoblast of stage A, in the epiblast a much more prominent additional peak II exists. Peak II is unique for the epiblast and persists through the first three developmental stages A, B and C. The initial appearance of the primitive streak at stage B corresponds to the appearance of peaks III and IV in the hypoblast. There is also an increase in the rate of amino acid incorporation into hypoblast protein at this time. Interestingly enough, although peaks III and IV disappear from the hypoblasts of successive stages, two similar peaks with the same electrophoretie mobility appear in the epiblast at stage D, which again was shown quantitatively to be at the maximum of incorporation activity (Fig. 2). From the above facts a working hypothesis may be suggested: After stage A, which is the completion of hypoblast formation, there is a burst of synthetic activity in the hypoblast, mainly of one protein (peak III). As this same period is considered (EyalGiladi and Wolk 1970) to be the beginning of the process of primitive streak induction, this protein may be the inductor. This hypothesis is supported by the fact that a protein or proteins with the same electrophoretic mobility appear later on (after their disappearance from the hypoblast) in the reacting epiblast, which might suggest that an inductor has been transferred to the epiblast or that the synthesis of a similar protein has been induced in the epiblast. Further studies are called for in order to check these hypotheses. REFERENCES BRAHMA, S. K., and VAN DER SAAG, P. T. (1972). Studies on biosynthesis of soluble proteins in early amphibian development by isoelectric focusing in
Sy nthesis in Chick Primitioe
Streak
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thin layer polyacrylamide gel. Exp. Cell Res. 75, 527-530. DEUCHAR, E. M. (1973). Biochemical aspects of early differentiation in vertebrates. Advanc. Morphog. 10, 175-225. EYAL-GILADI, H. (1970). Differentiation potencies of the young chick blastoderm as revealed by different manipulations. II. Localized damage and hypoblast removal experiments. J. Embryol. Erp. Morphol. 23, 739-749. EYAL-GILALN, H., and KOCHAV, SH. (in preparation). A complementary normal table of the chick’s first stages of development. EYAL-GILADI, H., and WOLK, M. (1970). The inducing capacities of the primary hypoblast as revealed by transfilter induction studies. Wilhelm Roux Arch. Entwicklungsmech. Organismen 165, 226-241. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAURER, H. R. (1971). “Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis.” Walter de Gruyter, Berlin. NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3, 320-331. NICOLET, G. (1970). Analyse autoradiographique de la localization des diffkrentes ebauches pr&omptives dans la ligne primitive de l’embryon de poulet. J. Embryol. Exp. Morphol. 23, 79-108. NICOLET, G. (1971). Avian gastrulation. Aduan. Morphog. 9, 231-260. NIEUWKOOP, P. D. (1969). The formation of mesoderm in Urodelean amphibians. I. Induction by the endoderm. Wilhelm Roux Arch. Entwicklungsmech. Organismen 162, 341-373. ROSENQUIW, G. C. (1966). A radioautographic study of labelled grafts in the chick blastoderm development from primitive-streak stages to stage 12. Co&rib. Embryol. Carnegie Inst. Wash. 38, 71-110. ROSENQUIST, G. C. (1972). Endoderm movements in the chick embryo between the early short streak and head process stages. J. Exp. Zool. 180,95-103. TIEDEMANN, H. (1968). Factors determining embryonic differentiation. J. Cell Physiol. 72, Suppl. 1, 129-144. VAKAET, L. (1962). Some new data concerning the formation of the definitive endoblast in the chick embryo. J. Embryol. Exp. Morphol. 10, 38-57. VAN BLECKROM, J., and MANES, C. (1974). Development of preimplantation rabbit embryos in uiuo and in uitro. II. A comparison of qualitative aspects of protein synthesis. Deuelop. Biol. 40, 40-51.