- Email: [email protected]
Twin larvae from halves of the same egg in ascidians
DEVELOPMENrAL
Twin
larvae
BIOLOGY,
from
5, 8‘6100
Halves
(1962)
of the Same
Egg in Ascidians
G. REVERBERI AND G. ORTOLANI Zoological
Institute, Accepted
University February
of Palermo,
Italy
13, 196.2
INTRODUCTION
The fragmentation of the egg into two or more pieces has been considered an appropriate method for studying its potentialities. If the fragments segment with the “typical” pattern and give rise to complete and well-proportioned, though undersized, organisms, the egg is considered to be “totipotent,” which means that its potentialities are not yet fixed. If, on the other hand, the fragments segment partially and give rise to defective organisms, the egg is considered to be a “mosaic” egg, or an egg with potentialities already fixed. The study of the potentialities of the ascidian egg by fragmentation was begun by Reverberi ( 1931) , who obtained a complete and normal (undersized) tadpole from the bigger piece of the unsegmented, already fertilized egg. This result was considered as proof that the fertilized ascidian egg is “totipotent.” Similar results were obtained by Dalcq (1932) on the unfertilized ascidian egg; both fragments of the egg, after fertilization, develop and give rise to tadpoles. The results obtained, however, varied according to the direction and the level of section. When the cut was latitudinal and above the equator, the bigger, haploid fragment developed into a normal tadpole, whereas the smaller, diploid fragment, developed into an embryo with structures depending on the level of section; in the simplest, but rather exceptional case, the embryo was represented by an ampulla of ectoblast containing a mass of entoblast without any other differentiation. When the cut was below the equator a normal tadpole was obtained from the animal fragment, whereas only entoblast, muscle, mesenchyme, and a small piece of ectoblast developed from the vegetal fragment. When the cut was at the equator, in the most favorable cases two normal tadpoles resulted. 84
TWINS
FROM
ASCIDIAN
EGGS
85
Very peculiar results were obtained by Dalcq (1932) when the sections were made along a meridional plane. In that case he obtained: ( 1) hemiembryos, though infrequently; (2) one fairly normal tadpole, and the other with a very stumpy tail; (3) two similar tadpoles normal in appearance though bent backward, or with distorted tails. These three different categories of results were explained by Dalcq (1935a,b, 1938, 1941) by assuming that the unfertilized egg possesses a predetermined structure; organ-forming substances would be distributed along and around the animal-vegetal axis. According to Dalcq the egg would possess at least two distinct morphogenetic plasms disposed as two opposite crescents around the animal-vegetal axis. These plasms would represent the neural and the chordal systems. Such an organization, however, would not he rigid; the egg, consequently, must not be considered a “true” mosaic egg. In 1958, Ortolani showed that every fragment of the unfertilized or fertilized egg of Ascidia malaca and Phallush, if sufficiently large, will give rise to perfectly normal tadpoles, irrespective of the plane of section. Ortolani also examined the results of sectioning the unfertilized egg along the animal-vegetal axis: the two resulting equal fragments gave rise to normal tadpoles. Against the theoretical deductions drawn by Ortolani from these results (absence of any organization in the unfertilized egg) Dalcq (1960) objected: “Q uant au probEme d’une organisation symmktrique bilat&ale de I’oeuf vierge, il ne peut B mon sens &re rksolu que de deux manihes: ou bien par la dkcouverte d’un test cytochimique qui ritv&le cette organisation . . . ou bien par l’ittude attentive de paires d’embryons obtenus par division m6ridienne de l’oeuf vierge. Sous ce rapport, le travail d’ortolani se borne B p&enter, saris 6tude sur coupes, trois paires de t&ards obtenus chez Ascidia malaca. J’h6site beaucoup Z?L les considdrer comme une objection d6cisive aux conclusions qui j’ai cru pouvoir tirer d’un mat&ial plus &endu et minutieusement examink. Disons done que cette grande question reste enti&rement d’actualitk.” The justifiable critique of Dalcq, to whom we are grateful, suggested the present research. This took into account only equal halves of the unfertilized egg which developed to tadpoles. The halves were obtained by equatorial or meridional or oblique sections. As a result we got twins which, independently of the plane of section, looked always complete and nearly normal; they differed from controls in only a few
86
G. REVEHHERI
AND
G. ORTOLANI
small details, such as distorted tails, and absence or numerical tion in palps and pigmented spots. MATERIALS
AND
reduc-
METHODS
We used the unfertilized eggs of Ascidia malaca. The eggs, deprived of their membranes, were cut freehand into two equal pieces with a glass needle, on a film of agar. They were sectioned: (1) meridionally; (2) equatorially; (3) obliquely, as summarized in Table 1. The TABLE HALVES
FROM
THE
Sketch of the operation
Ham
EGG OBTAINED
1 BY DIFFERENTLY
ORIEXT~D
SECTIONS
Nuclear colrdition of the fragments
Results
19
Haploid Diploid
Tadpoles Tadpoles
38
Haploid Diploid
Tadpoles Tadpoles
16
Haploid Diploid
Tadpoles Tadpoles
Number of operated eggs
c7
0 -c3 v-
orientation of the egg was made with respect to the location of the metaphase spindle which is indicated by a clear area at the animal pole. Since the eggs were cut before fertilization, only one of the fragments possessed the egg nucleus. Consequently, after fertilization, one fragment developed as a haploid, the other as a diploid. The presence of the egg nucleus in a fragment was deduced from the emission of the polocytes (polar bodies). The tadpoles which developed from fragments were studied only
TWINS
FHOM
ASCIDIAN
in viva as all details of their organization, are evident in the living state.
EGGS
87
owing to their transparency,
RESULTS
General Results As often noted (e.g., Conklin, 1905), the ascidian egg immediately after the penetration of the sperm exhibits brief but characteristic changes of form which can be considered as an expression of fertilization dynamism. Its spherical shape changes, becoming irregular, then pyriform, then again spherical. These modifications happen only at fertilization and are essentially distinct from similar modifications that occur before the emission of the polocytes. After fertilization, both fragments were found to exhibit these form changes; in this relation it must be recalled that one fragment does not possess the egg nucleus; the other, however, does. The modifications were synchronous in both fragments, and synchronous with those of the entire egg (Fig. 1). When the normal egg prepares to emit the first polocyte it becomes flat; after the emission of the polocyte it reassumes the spherical shape; when it emits the second polocyte, it flattens again. The same modifications occurred also in the fragments (Figs. 1,2). The segmentations were seen to be synchronous in both fragments, and are also synchronous with those of the entire egg. The time table of development to the formation of the tadpole was also the same in both fragments as in the entire egg. Both fragments segmented in the typical pattern (Fig. 3). The blastomeres resulting from cleavage were symmetrically situated with respect to the plane of symmetry, and their relative proportions were also normal. Their size, however, was smaller than that of the blastomeres of the entire egg. As segmentation continued, the blastomeres became smaller; possibly they stop dividing when the cells have acquired their definitive size limit. If so, the number of cells in an organ of a tadpole resulting from a half egg must be lower than that in a normal larva. Actually this was found to be so for the chordal cells. In some twins (Figs. 47), each partner possessed about 20 chordal cells, or half the number found in a normal tadpole (38-40). This result was also obtained by Morgan (1896) in the eggs of the sea urchin. Both fragments of the same egg gastrulated and neurulated normally; finally, in spite of the difference in their chromosome number at the
88
G. REVERBERI
,0-.. u
i d
C
0
0
0
0
0
0
0
0
0
V
a
0
0
0
0
FIGS.
G. ORTOLANI
0 0
20
fragment;
AND
1, 2. Modifications after fertilization. (A) normal (C) haploid fragment. To the left: time in minutes.
egg;
(B)
diploid
TWINS
FROM
ASCIDIAN
EGGS
C -____
0
/1
41 / m
46
0
0
0
0
0
90
G. HEVERBERI
AND
G. ORTOLANI
32 cell
stage
Di P\oid pymemt
Hap\oid pl.ymmt
FIG. 3. A normal egg, a diploid 16-, and 32-cell stages.
fragment,
and a haploid
fragment
at the 8-,
beginning of development, they gave rise to two tadpoles of equal size, usually of equal shape and complete in their organization. It is possible that the haploid state was maintained throughout development; however the chromosome number in the larvae was not checked.
Detailed Results Equatorial sections. Operations of this type were performed by Dalcq ( 1932), who obtained larvae of “entirely normal constitution” (Dalcq, 1938). It was noticed, however, that the ectoblast was not able to cover the bulge of the inner materials; consequently the hind part of the body was abnormal. “In the best cases both embryos were normal except for a shortening of the tail” (Dalcq, 1938, Fig. 42, v). Equatorial sections were carried out also by Ortolani (1958). As a
TWINS
FROM
ASCIDIAN
EGGS
91
result, Ortolani obtained twins which, although most had twisted tails and some lacked sensory organs and palps, could nevertheless be considered as normal. The results of the present investigation are based on 38 operated eggs and are recorded in Figs. 4 and 5. In all the twins, the organs were present, and in normal position; however small abnormalities or deficiencies were also present. The tail was sometimes short, thick, and distorted, and the palps were fused or reduced in number; also in some the sensory spots were reduced in number or situated on the surface of the body. In a few cases, one twin was better developed than the other, looking like a perfect miniature tadpole. Probably this result was due to a small displacement in the plane of section. Twins with straight tails similar to those of the controls were never obtained. We are of the opinion that half of the egg is quantitatively a critical mass for obtaining a normal embryo. Meridional sections. As indicated above, these operations were performed also by Dalcq (1932), who got three different categories of results. Our results are reported in Fig, 6. The twins were either identical or one was better formed than the other; in any case, except for some minor details they were very similar to the controls. The better formation of one partner in some twin pairs is probably due to a displacement of the cut from the exact meridional plane. The general result shows that there cannot be any relation between the plane of operation and (if it exists) a plane of bilateral symmetry. In any case the small abnormalities observed in the tadpoles must not obscure the substantial fact, on which we strongly insist, that the tadpoles were complete in all their organs, and that these were normally disposed and normally differentiated. If one compares twins obtained by meridional sections with those obtained by equatorial sections (Figs. 4 and 5), one can observe that the former look better than the latter; we think, however, that this fact is coincidental, as will be discussed later. ObEique sections. The fragments of this series were obtained from cuts that were made at any angle with the animal-vegetal axis. Both fragments of the egg developed normally and finally gave rise to tadpoles (Fig. 7) which did not seem different from those derived from equatorial or meridional cuts (cf. Figs. 4-6). As before, the twins
92
G. REVERBERI
AND
G. ORTOLANI
,-. :’ ) aa 89 l* )
e“
FIG. 4. Twin tadpoles produced by an equatorial section. Each pair represents the twin tadpoles developed from the fragments of the same egg, cut through the equatorial plane (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment). For some tadpoles the number of the chordal cells, as counted in uiuo, is indicated.
TWINs
mcnf
ASCIDIAN
EGGS
FIG. 5. Twin tadpoles produced by an equatorial section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment).
94
(:. HE\‘ERBE:HI
20
20
.&NJ) C. OHTOLANJ
&J
. P. @B t 24
20
20
/ :t3 0 8 f
t
20
20
20
r: < B b, l.
i
22
i FIG. 6. Twin tadpoles produced by a meridional section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment).
TWINS
FROM
ASCIDIAN
22
\i ‘? PQ 22
-
95
ECCS
20
-
;‘
c ‘i
24
// jl I 22
L
FIG. 7. Twin tadpoles produced by an oblique section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment),
96
(:. HEVEHBERI
AND
6. OHTOLANI
were not perfectly formed although they were complete, The better conformation of one partner in some pairs can be explained by a deviation in the cutting plane, giving rise to unequal fragments. Fragments larger than one-half egg. The previous results indicate that there is no significant difference in the development of fragments obtained by equatorial, meridional, or oblique sections; they also suggest that a quantity of cytoplasm equal to more than half an egg is necessary in order to obtain a straight tadpole. In order to determine the effect of cytoplasmic mass on development, eggs were sectioned into unequal fragments by supra- and subequatorial sections, as shown in Figs, 8 and 9: (1) Supraequatorial sections. Nineteen operations
28
25
28
27
FIG. 8. Haploid tadpoles developed from a fragment larger than half an egg and obtained by sectioning horizontally. For each tadpole the number of the chordal cells, as counted in uiuo, is indicated.
TWINS
FROM
ASCIDIAN
97
EGGS
were performed. After fertilization only the larger, haploid fragments developed; their segmentation was completely normal; the tadpoles were also normal, and their tails straight; 25-28 chordal cells were counted (Fig. 8). The chromosome number in these tadpoles was not checked; probably the number was haploid. (2) Subequatorial sections. Thirteen eggs were operated. After fertilization only the larger, &plod fragments developed to tadpoles. The development was normal in every detail; the resulting tadpoles were not different from the controls; their tails were usually straight; 22-27 chordal cells were counted (Fig 9). Comparing these results with those obtained by supraequa-
.e e
,
24
24
B :-
FIG. 9. Diploid tadpoles developed from a fragment larger than half and obtained by sectioning horizontally. For each tadpole the number chordal cells, as counted in uiuo, is indicated.
24 an egg of the
98
G. REVERBERI
AND
G. ORTOLANI
torial sections we did not discover any difference between them; the haploid or diploid state of the fragments did not influence the final result. A duplication of the chromo8somes during the development of the haploid fragments is possible, but has not been verified. DISCUSSION
First, we want to emphasize that the results presented here agree in every detail with those presented by Dalcq; the subject of disagreement, if any, concerns only their interpretation. Dalcq interprets his results by supposing the existence of a predetermined architecture in the egg. We, on the other hand, suggest that the more probable hypothesis is that in the unsegmented egg there are no preformations at all. According to our experience, any fragment of an unfertilized egg, provided it is larger than half the egg, always gives rise to a normal tadpole, irrespective of the plane of section. The experiments presented in this paper show that both the equal fragments obtained from the same egg, by equatorial or meridional or oblique sections, segment and gastrulate in “typical” way, ultimately giving rise to complete tadpoles. This statement cannot be affected by the fac$ that tadpoles o’riginating from meridional, or equatorial, or oblique sections showed, nevertheless, some abnormalities. As stated earlier, these abnormalities concern the number of the palps, the number and position (on the surface of the body) of the pigmented spots, and some distortions or reduction in the tails. There is no evidence that these abnormalities are the consequence of an incomplete distribution of some primary factors that exist in the unfertilized egg; they seem rather to be the consequence of some disturbances in the morphogenetic movements of gastrulation and/or neurulation, which are, possibly, more frequent in fragments whose capacity to develop normally is critically determined, as shown above, by their size. It must be also remembered that the developing fragments are not protected by egg membranes; consequently some of their cells can stick to the agar substrate. The abnormalities observed in the tadpoles deriving from fragments are not infrequent in tadpoles from entire eggs which previously were deprived of their membranes. It was also noticed that the eggs of different animals possess a more or less sticky cortex; consequently they form tadpoles with more or less abnormalities. This is the reason why we do not attribute any significance tomthe differences in the tadpoles
TWINS
FROM
ASCIDIAN
EGGS
99
deriving from equatorial and meridional sections. As the tadpoles developed from diferent hatches of eggs, the results are not comparable. In conclusion, the results obtained in these experiments support the statement that the unfertilized ascidian egg is equipotent. This statement represents the final conclusion based on a long series of experiments, beginning with the paper, already mentioned, of Reverberi (1931). The latest evidence comes from centrifugation experiments by La Spina ( 1958, 1961) and Reverberi and La Spina ( 1959) ; and from the experiments by Abbate and Ortolani ( 1961) , who removed portions of cytoplasm or cortex from the egg. SUMMARY
Unfertilized eggs of Ascidia nmlnca were fragmented into two equal pieces by equatorial, meridional, or oblique sections. The fragments were then fertilized. Both fragments developed to the larval stage. The resulting twins were complete and normally proportioned but smaller than the controls. Their shape was also deformed, particularly the tail. No significant differences were noticed in the twins deriving from equatorial, meridional, or oblique sections. These results, and the fact that both twins deriving from the same egg are complete, support the view that in the unfertilized ascidian egg, no preconstituted architecture is present. The unfertilized egg is an “isotropic” system, or, in Driesch’s terminology, “harmonic equipotential system.” The distortions or reductions in the tail of the twins are probably due to the fact that half an egg is the critical cytoplasmic quantity necessary for the building of a normal tadpole. Below that critical size, a normal larval body cannot be formed. Tadpoles derived from all sorts of fragments larger than half an egg differ from the controls only in respect to size. REFERENCES ABBATE, C., and ORTOLANI, G. (1961). The d ev el 0 p merit of Ciona eggs after partial removal of cortex or ooplasm. Actu Embryol. Morphul. Exptl. 4, 56-61. CONKLIN, E. G. (1905). The organization and cell-lineage of the Ascidian egg. J. Acad. Nat. Sci. Phila. 13, 1. DALCQ, A. (1932). Etude des localisations germinales dans l’oeuf vierge d’Ascidie par des expkriences de mkrogonie. Arch. anat. microscop. 28, 223-333. DALCQ, A. (1935a). La rkgulation dans le germe et son interprktation. Compt. rend. sot. biol. 119, 1421-1467.
100
G. REVERBEHI
AND
G. ORTOLAKI
DALCQ, A. ( 1935b). “L’organisation de l’oeuf chez lrs Chord& Etude d’embryologie causale.” Gauthier-Villars, Paris. DALCQ, A. ( 1938). “Form and Causality in Early Development.” Cambridge Univ. Press, London and New York. DALCQ, A. ( 1941). “L’oeuf et son dynamisme organisateur.” Albin Michel, Paris. DALCQ, A. ( 1960). Nouveaux constituents de l’oeuf d’iiscidie r&&s par les colorants vitaux m$tachromatiques. Arch. biol. ( Lidge) 71, 93-139. LA SPINA, R. (1958). Lo spostamento dei plasmi mediante centrifugazione nell’uovo vergine di Ascidia e il conseguente sviluppo. Actu Embryol. Morphol. Exptl. 2, 66-78. LA SPINA, R. ( 1961). Development of Ascidia malaca egg fragments produced by centrifugation. A& Embryol. Morphol. Exptl. 4, 320326. MORGAN, T. H. (1896). Studies of the partial larvae of Sphaerechinus. Arch. Entwicklungsmech. Organ. 2, 81. ORTOLANI, G. ( 1958). Cleavage and development of egg fragments in Ascidians. Acta Embryol. Morphol. Exptl. 1, 247-272. REVERBERI, G. ( 1931). Studi sperimentali sull’uovo di Ascidic. Pubbl. .staz. zool. Napoli 11, 168-193. REVERBERI, G., and LA SPINA, R. (1959). N ormnl larvae obtained from dark fragments of centrifuged Ciona eggs. Experientia 15, 122-125.
Twin
larvae
BIOLOGY,
from
5, 8‘6100
Halves
(1962)
of the Same
Egg in Ascidians
G. REVERBERI AND G. ORTOLANI Zoological
Institute, Accepted
University February
of Palermo,
Italy
13, 196.2
INTRODUCTION
The fragmentation of the egg into two or more pieces has been considered an appropriate method for studying its potentialities. If the fragments segment with the “typical” pattern and give rise to complete and well-proportioned, though undersized, organisms, the egg is considered to be “totipotent,” which means that its potentialities are not yet fixed. If, on the other hand, the fragments segment partially and give rise to defective organisms, the egg is considered to be a “mosaic” egg, or an egg with potentialities already fixed. The study of the potentialities of the ascidian egg by fragmentation was begun by Reverberi ( 1931) , who obtained a complete and normal (undersized) tadpole from the bigger piece of the unsegmented, already fertilized egg. This result was considered as proof that the fertilized ascidian egg is “totipotent.” Similar results were obtained by Dalcq (1932) on the unfertilized ascidian egg; both fragments of the egg, after fertilization, develop and give rise to tadpoles. The results obtained, however, varied according to the direction and the level of section. When the cut was latitudinal and above the equator, the bigger, haploid fragment developed into a normal tadpole, whereas the smaller, diploid fragment, developed into an embryo with structures depending on the level of section; in the simplest, but rather exceptional case, the embryo was represented by an ampulla of ectoblast containing a mass of entoblast without any other differentiation. When the cut was below the equator a normal tadpole was obtained from the animal fragment, whereas only entoblast, muscle, mesenchyme, and a small piece of ectoblast developed from the vegetal fragment. When the cut was at the equator, in the most favorable cases two normal tadpoles resulted. 84
TWINS
FROM
ASCIDIAN
EGGS
85
Very peculiar results were obtained by Dalcq (1932) when the sections were made along a meridional plane. In that case he obtained: ( 1) hemiembryos, though infrequently; (2) one fairly normal tadpole, and the other with a very stumpy tail; (3) two similar tadpoles normal in appearance though bent backward, or with distorted tails. These three different categories of results were explained by Dalcq (1935a,b, 1938, 1941) by assuming that the unfertilized egg possesses a predetermined structure; organ-forming substances would be distributed along and around the animal-vegetal axis. According to Dalcq the egg would possess at least two distinct morphogenetic plasms disposed as two opposite crescents around the animal-vegetal axis. These plasms would represent the neural and the chordal systems. Such an organization, however, would not he rigid; the egg, consequently, must not be considered a “true” mosaic egg. In 1958, Ortolani showed that every fragment of the unfertilized or fertilized egg of Ascidia malaca and Phallush, if sufficiently large, will give rise to perfectly normal tadpoles, irrespective of the plane of section. Ortolani also examined the results of sectioning the unfertilized egg along the animal-vegetal axis: the two resulting equal fragments gave rise to normal tadpoles. Against the theoretical deductions drawn by Ortolani from these results (absence of any organization in the unfertilized egg) Dalcq (1960) objected: “Q uant au probEme d’une organisation symmktrique bilat&ale de I’oeuf vierge, il ne peut B mon sens &re rksolu que de deux manihes: ou bien par la dkcouverte d’un test cytochimique qui ritv&le cette organisation . . . ou bien par l’ittude attentive de paires d’embryons obtenus par division m6ridienne de l’oeuf vierge. Sous ce rapport, le travail d’ortolani se borne B p&enter, saris 6tude sur coupes, trois paires de t&ards obtenus chez Ascidia malaca. J’h6site beaucoup Z?L les considdrer comme une objection d6cisive aux conclusions qui j’ai cru pouvoir tirer d’un mat&ial plus &endu et minutieusement examink. Disons done que cette grande question reste enti&rement d’actualitk.” The justifiable critique of Dalcq, to whom we are grateful, suggested the present research. This took into account only equal halves of the unfertilized egg which developed to tadpoles. The halves were obtained by equatorial or meridional or oblique sections. As a result we got twins which, independently of the plane of section, looked always complete and nearly normal; they differed from controls in only a few
86
G. REVEHHERI
AND
G. ORTOLANI
small details, such as distorted tails, and absence or numerical tion in palps and pigmented spots. MATERIALS
AND
reduc-
METHODS
We used the unfertilized eggs of Ascidia malaca. The eggs, deprived of their membranes, were cut freehand into two equal pieces with a glass needle, on a film of agar. They were sectioned: (1) meridionally; (2) equatorially; (3) obliquely, as summarized in Table 1. The TABLE HALVES
FROM
THE
Sketch of the operation
Ham
EGG OBTAINED
1 BY DIFFERENTLY
ORIEXT~D
SECTIONS
Nuclear colrdition of the fragments
Results
19
Haploid Diploid
Tadpoles Tadpoles
38
Haploid Diploid
Tadpoles Tadpoles
16
Haploid Diploid
Tadpoles Tadpoles
Number of operated eggs
c7
0 -c3 v-
orientation of the egg was made with respect to the location of the metaphase spindle which is indicated by a clear area at the animal pole. Since the eggs were cut before fertilization, only one of the fragments possessed the egg nucleus. Consequently, after fertilization, one fragment developed as a haploid, the other as a diploid. The presence of the egg nucleus in a fragment was deduced from the emission of the polocytes (polar bodies). The tadpoles which developed from fragments were studied only
TWINS
FHOM
ASCIDIAN
in viva as all details of their organization, are evident in the living state.
EGGS
87
owing to their transparency,
RESULTS
General Results As often noted (e.g., Conklin, 1905), the ascidian egg immediately after the penetration of the sperm exhibits brief but characteristic changes of form which can be considered as an expression of fertilization dynamism. Its spherical shape changes, becoming irregular, then pyriform, then again spherical. These modifications happen only at fertilization and are essentially distinct from similar modifications that occur before the emission of the polocytes. After fertilization, both fragments were found to exhibit these form changes; in this relation it must be recalled that one fragment does not possess the egg nucleus; the other, however, does. The modifications were synchronous in both fragments, and synchronous with those of the entire egg (Fig. 1). When the normal egg prepares to emit the first polocyte it becomes flat; after the emission of the polocyte it reassumes the spherical shape; when it emits the second polocyte, it flattens again. The same modifications occurred also in the fragments (Figs. 1,2). The segmentations were seen to be synchronous in both fragments, and are also synchronous with those of the entire egg. The time table of development to the formation of the tadpole was also the same in both fragments as in the entire egg. Both fragments segmented in the typical pattern (Fig. 3). The blastomeres resulting from cleavage were symmetrically situated with respect to the plane of symmetry, and their relative proportions were also normal. Their size, however, was smaller than that of the blastomeres of the entire egg. As segmentation continued, the blastomeres became smaller; possibly they stop dividing when the cells have acquired their definitive size limit. If so, the number of cells in an organ of a tadpole resulting from a half egg must be lower than that in a normal larva. Actually this was found to be so for the chordal cells. In some twins (Figs. 47), each partner possessed about 20 chordal cells, or half the number found in a normal tadpole (38-40). This result was also obtained by Morgan (1896) in the eggs of the sea urchin. Both fragments of the same egg gastrulated and neurulated normally; finally, in spite of the difference in their chromosome number at the
88
G. REVERBERI
,0-.. u
i d
C
0
0
0
0
0
0
0
0
0
V
a
0
0
0
0
FIGS.
G. ORTOLANI
0 0
20
fragment;
AND
1, 2. Modifications after fertilization. (A) normal (C) haploid fragment. To the left: time in minutes.
egg;
(B)
diploid
TWINS
FROM
ASCIDIAN
EGGS
C -____
0
/1
41 / m
46
0
0
0
0
0
90
G. HEVERBERI
AND
G. ORTOLANI
32 cell
stage
Di P\oid pymemt
Hap\oid pl.ymmt
FIG. 3. A normal egg, a diploid 16-, and 32-cell stages.
fragment,
and a haploid
fragment
at the 8-,
beginning of development, they gave rise to two tadpoles of equal size, usually of equal shape and complete in their organization. It is possible that the haploid state was maintained throughout development; however the chromosome number in the larvae was not checked.
Detailed Results Equatorial sections. Operations of this type were performed by Dalcq ( 1932), who obtained larvae of “entirely normal constitution” (Dalcq, 1938). It was noticed, however, that the ectoblast was not able to cover the bulge of the inner materials; consequently the hind part of the body was abnormal. “In the best cases both embryos were normal except for a shortening of the tail” (Dalcq, 1938, Fig. 42, v). Equatorial sections were carried out also by Ortolani (1958). As a
TWINS
FROM
ASCIDIAN
EGGS
91
result, Ortolani obtained twins which, although most had twisted tails and some lacked sensory organs and palps, could nevertheless be considered as normal. The results of the present investigation are based on 38 operated eggs and are recorded in Figs. 4 and 5. In all the twins, the organs were present, and in normal position; however small abnormalities or deficiencies were also present. The tail was sometimes short, thick, and distorted, and the palps were fused or reduced in number; also in some the sensory spots were reduced in number or situated on the surface of the body. In a few cases, one twin was better developed than the other, looking like a perfect miniature tadpole. Probably this result was due to a small displacement in the plane of section. Twins with straight tails similar to those of the controls were never obtained. We are of the opinion that half of the egg is quantitatively a critical mass for obtaining a normal embryo. Meridional sections. As indicated above, these operations were performed also by Dalcq (1932), who got three different categories of results. Our results are reported in Fig, 6. The twins were either identical or one was better formed than the other; in any case, except for some minor details they were very similar to the controls. The better formation of one partner in some twin pairs is probably due to a displacement of the cut from the exact meridional plane. The general result shows that there cannot be any relation between the plane of operation and (if it exists) a plane of bilateral symmetry. In any case the small abnormalities observed in the tadpoles must not obscure the substantial fact, on which we strongly insist, that the tadpoles were complete in all their organs, and that these were normally disposed and normally differentiated. If one compares twins obtained by meridional sections with those obtained by equatorial sections (Figs. 4 and 5), one can observe that the former look better than the latter; we think, however, that this fact is coincidental, as will be discussed later. ObEique sections. The fragments of this series were obtained from cuts that were made at any angle with the animal-vegetal axis. Both fragments of the egg developed normally and finally gave rise to tadpoles (Fig. 7) which did not seem different from those derived from equatorial or meridional cuts (cf. Figs. 4-6). As before, the twins
92
G. REVERBERI
AND
G. ORTOLANI
,-. :’ ) aa 89 l* )
e“
FIG. 4. Twin tadpoles produced by an equatorial section. Each pair represents the twin tadpoles developed from the fragments of the same egg, cut through the equatorial plane (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment). For some tadpoles the number of the chordal cells, as counted in uiuo, is indicated.
TWINs
mcnf
ASCIDIAN
EGGS
FIG. 5. Twin tadpoles produced by an equatorial section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment).
94
(:. HE\‘ERBE:HI
20
20
.&NJ) C. OHTOLANJ
&J
. P. @B t 24
20
20
/ :t3 0 8 f
t
20
20
20
r: < B b, l.
i
22
i FIG. 6. Twin tadpoles produced by a meridional section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment).
TWINS
FROM
ASCIDIAN
22
\i ‘? PQ 22
-
95
ECCS
20
-
;‘
c ‘i
24
// jl I 22
L
FIG. 7. Twin tadpoles produced by an oblique section (see Fig. 4 legend) (on the left, the tadpole which developed from the diploid fragment; on the right, the tadpole from the haploid fragment),
96
(:. HEVEHBERI
AND
6. OHTOLANI
were not perfectly formed although they were complete, The better conformation of one partner in some pairs can be explained by a deviation in the cutting plane, giving rise to unequal fragments. Fragments larger than one-half egg. The previous results indicate that there is no significant difference in the development of fragments obtained by equatorial, meridional, or oblique sections; they also suggest that a quantity of cytoplasm equal to more than half an egg is necessary in order to obtain a straight tadpole. In order to determine the effect of cytoplasmic mass on development, eggs were sectioned into unequal fragments by supra- and subequatorial sections, as shown in Figs, 8 and 9: (1) Supraequatorial sections. Nineteen operations
28
25
28
27
FIG. 8. Haploid tadpoles developed from a fragment larger than half an egg and obtained by sectioning horizontally. For each tadpole the number of the chordal cells, as counted in uiuo, is indicated.
TWINS
FROM
ASCIDIAN
97
EGGS
were performed. After fertilization only the larger, haploid fragments developed; their segmentation was completely normal; the tadpoles were also normal, and their tails straight; 25-28 chordal cells were counted (Fig. 8). The chromosome number in these tadpoles was not checked; probably the number was haploid. (2) Subequatorial sections. Thirteen eggs were operated. After fertilization only the larger, &plod fragments developed to tadpoles. The development was normal in every detail; the resulting tadpoles were not different from the controls; their tails were usually straight; 22-27 chordal cells were counted (Fig 9). Comparing these results with those obtained by supraequa-
.e e
,
24
24
B :-
FIG. 9. Diploid tadpoles developed from a fragment larger than half and obtained by sectioning horizontally. For each tadpole the number chordal cells, as counted in uiuo, is indicated.
24 an egg of the
98
G. REVERBERI
AND
G. ORTOLANI
torial sections we did not discover any difference between them; the haploid or diploid state of the fragments did not influence the final result. A duplication of the chromo8somes during the development of the haploid fragments is possible, but has not been verified. DISCUSSION
First, we want to emphasize that the results presented here agree in every detail with those presented by Dalcq; the subject of disagreement, if any, concerns only their interpretation. Dalcq interprets his results by supposing the existence of a predetermined architecture in the egg. We, on the other hand, suggest that the more probable hypothesis is that in the unsegmented egg there are no preformations at all. According to our experience, any fragment of an unfertilized egg, provided it is larger than half the egg, always gives rise to a normal tadpole, irrespective of the plane of section. The experiments presented in this paper show that both the equal fragments obtained from the same egg, by equatorial or meridional or oblique sections, segment and gastrulate in “typical” way, ultimately giving rise to complete tadpoles. This statement cannot be affected by the fac$ that tadpoles o’riginating from meridional, or equatorial, or oblique sections showed, nevertheless, some abnormalities. As stated earlier, these abnormalities concern the number of the palps, the number and position (on the surface of the body) of the pigmented spots, and some distortions or reduction in the tails. There is no evidence that these abnormalities are the consequence of an incomplete distribution of some primary factors that exist in the unfertilized egg; they seem rather to be the consequence of some disturbances in the morphogenetic movements of gastrulation and/or neurulation, which are, possibly, more frequent in fragments whose capacity to develop normally is critically determined, as shown above, by their size. It must be also remembered that the developing fragments are not protected by egg membranes; consequently some of their cells can stick to the agar substrate. The abnormalities observed in the tadpoles deriving from fragments are not infrequent in tadpoles from entire eggs which previously were deprived of their membranes. It was also noticed that the eggs of different animals possess a more or less sticky cortex; consequently they form tadpoles with more or less abnormalities. This is the reason why we do not attribute any significance tomthe differences in the tadpoles
TWINS
FROM
ASCIDIAN
EGGS
99
deriving from equatorial and meridional sections. As the tadpoles developed from diferent hatches of eggs, the results are not comparable. In conclusion, the results obtained in these experiments support the statement that the unfertilized ascidian egg is equipotent. This statement represents the final conclusion based on a long series of experiments, beginning with the paper, already mentioned, of Reverberi (1931). The latest evidence comes from centrifugation experiments by La Spina ( 1958, 1961) and Reverberi and La Spina ( 1959) ; and from the experiments by Abbate and Ortolani ( 1961) , who removed portions of cytoplasm or cortex from the egg. SUMMARY
Unfertilized eggs of Ascidia nmlnca were fragmented into two equal pieces by equatorial, meridional, or oblique sections. The fragments were then fertilized. Both fragments developed to the larval stage. The resulting twins were complete and normally proportioned but smaller than the controls. Their shape was also deformed, particularly the tail. No significant differences were noticed in the twins deriving from equatorial, meridional, or oblique sections. These results, and the fact that both twins deriving from the same egg are complete, support the view that in the unfertilized ascidian egg, no preconstituted architecture is present. The unfertilized egg is an “isotropic” system, or, in Driesch’s terminology, “harmonic equipotential system.” The distortions or reductions in the tail of the twins are probably due to the fact that half an egg is the critical cytoplasmic quantity necessary for the building of a normal tadpole. Below that critical size, a normal larval body cannot be formed. Tadpoles derived from all sorts of fragments larger than half an egg differ from the controls only in respect to size. REFERENCES ABBATE, C., and ORTOLANI, G. (1961). The d ev el 0 p merit of Ciona eggs after partial removal of cortex or ooplasm. Actu Embryol. Morphul. Exptl. 4, 56-61. CONKLIN, E. G. (1905). The organization and cell-lineage of the Ascidian egg. J. Acad. Nat. Sci. Phila. 13, 1. DALCQ, A. (1932). Etude des localisations germinales dans l’oeuf vierge d’Ascidie par des expkriences de mkrogonie. Arch. anat. microscop. 28, 223-333. DALCQ, A. (1935a). La rkgulation dans le germe et son interprktation. Compt. rend. sot. biol. 119, 1421-1467.
100
G. REVERBEHI
AND
G. ORTOLAKI
DALCQ, A. ( 1935b). “L’organisation de l’oeuf chez lrs Chord& Etude d’embryologie causale.” Gauthier-Villars, Paris. DALCQ, A. ( 1938). “Form and Causality in Early Development.” Cambridge Univ. Press, London and New York. DALCQ, A. ( 1941). “L’oeuf et son dynamisme organisateur.” Albin Michel, Paris. DALCQ, A. ( 1960). Nouveaux constituents de l’oeuf d’iiscidie r&&s par les colorants vitaux m$tachromatiques. Arch. biol. ( Lidge) 71, 93-139. LA SPINA, R. (1958). Lo spostamento dei plasmi mediante centrifugazione nell’uovo vergine di Ascidia e il conseguente sviluppo. Actu Embryol. Morphol. Exptl. 2, 66-78. LA SPINA, R. ( 1961). Development of Ascidia malaca egg fragments produced by centrifugation. A& Embryol. Morphol. Exptl. 4, 320326. MORGAN, T. H. (1896). Studies of the partial larvae of Sphaerechinus. Arch. Entwicklungsmech. Organ. 2, 81. ORTOLANI, G. ( 1958). Cleavage and development of egg fragments in Ascidians. Acta Embryol. Morphol. Exptl. 1, 247-272. REVERBERI, G. ( 1931). Studi sperimentali sull’uovo di Ascidic. Pubbl. .staz. zool. Napoli 11, 168-193. REVERBERI, G., and LA SPINA, R. (1959). N ormnl larvae obtained from dark fragments of centrifuged Ciona eggs. Experientia 15, 122-125.