J. Embryo/, exp. Morph. Vol. 21, 3, pp. 445-66, June 1969
Printed in Great Britain
445
Analysis of follicle cell patterns in dextral
and sinistral Limnaea peregra
By GEERTJE A. UBBELS1, J. J. BEZEM 2 & CHR. P. RAVEN2
From the Zoological Laboratory, University of Utrecht
Several experiments done in our laboratory make it likely that in the egg of
Limnaea stagnalis there is a cortical morphogenetic field (Raven, 1949, 1952,
1966). One of us (Raven, 1963,1964,1967) has studied the origin of this morphogenetic field. In the newly laid egg cell there is a vegetative pole plasm, occupying
a sector of about .110 degrees with its apex near the centre of the egg. It is
situated somewhat obliquely with respect to the longitudinal axis of the first
maturation spindle. Moreover, a circle of six lenticular subcortical patches of
cytoplasm are found in the equatorial region of the egg. These 'subcortical
accumulations' (SCA) are arranged according to a regular pattern. Four of
them are situated close together on one side, occupying about 180 degrees of the
egg circumference; two somewhat larger SCA lie on the opposite side. The
SCA, together with the obliquity of the vegetative pole plasm, define a pattern
which is polar, dorsoventral and nearly symmetric, though a slight asymmetry
seems to be present.
The following facts make it very likely that the pattern of cytoplasmic
differentiations of the recently laid egg is important for cell determination at
later development: (1) the plane of symmetry of this pattern coincides with the
median plane of the future embryo (Raven, 1967); (2) the egg substances
contained in the vegetative pole plasm and in the SCA are distributed among
the cleavage cells according to a definite programme (Raven, 1946, 1967); (3) a
disturbance of this distribution by centrifugation during early cleavage leads to
abnormal development (Raven, 1964).
This cytoplasmic pattern arises by ooplasmic segregation during the passage
of the egg cell through the female genital tract of the parent. Centrifugation
experiments have shown that the normal course of ooplasmic segregation is
apparently controlled by factors residing in the egg cortex (Raven, 1964, 1967;
Raven & van der Wai, 1964). Therefore, it must be assumed that the cytoplasmic
differentiations of the uncleaved egg arise under the influence of a pre-existent
mosaic pattern of the molecular structure of the cortex. This cortical pattern is
probably formed during oogenesis, through interactions between the oocyte
1
Author's address: Hubrecht Laboratory, Uppsalalaan 1, Universiteitscentrum 'De
Uithof', Utrecht, Netherlands.
2
Authors' address: Zoological Laboratory, Janskerkhof 3, Utrecht, Netherlands.
446
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
and the surrounding structures of the gonad. This was concluded from the fact
that the arrangement of the SCA of the oviposited egg agrees with the configuration of the follicle cells surrounding the oocyte in the gonad, while the position
of the vegetative pole plasm corresponds to the part of the egg surface formerly
applied to the gonad wall (Raven, 1963, 1967). The hypothesis was put forward
that the 'blue-print information' (Raven, 1958) in the egg cortex is transmitted
from the parent to the offspring by way of the follicle, whose structure is, so to
speak, 'imprinted' upon the egg during oogenesis.
Since most of these relationships have been studied in the single species
L. stagnalis, the question arises whether these conclusions have a more general
bearing. It seems appropriate, therefore, to test them by the study of the eggs
of other species.
The cortical morphogenetic field does not only direct the orderly displacement of the egg substances during ooplasmic segregation, but also controls the
positions and directions of nuclei and spindles, and thereby the place and
direction of cleavage furrows (Raven, 1964). In this way the two processes of
ooplasmic segregation and cell division are bound together into a unified
pattern, ensuring the orderly distribution of the egg components among the
cleavage cells.
In this connexion the question becomes important whether the asymmetry
of cleavage and of further development in gastropods is due to an asymmetry of
the cortical field. It has long been known that the asymmetry of coiling of the
shell is correlated with the asymmetry of spiral cleavage, the cleavage of sinistral
species of snails being the mirror image of that of dextral snails. That the same
regularity holds within a species has been shown by one of us (Ubbels, 1966).
In L. peregra two races exist, one showing dextral coiling and the other
sinistral coiling of the shell. The cleavage pattern of eggs from which dextrals
arise is the mirror image of that shown by sinistrals. Thus, as in other gastropods, asymmetry of coiling of the shell is correlated with the asymmetry of
spiral cleavage. According to Sturtevant's (1923) hypothesis, which is based on
the work of Boycott & Diver (1923; cf. Boycott, Diver, Garstang & Turner
1931) the direction of coiling of the shell in L. peregra is determined by one pair
of Mendelian factors, the allele for dextral coiling being dominant. Moreover,
the direction of coiling does not depend on the animal's own genotype, but on
the genotype of the female parent. Therefore the asymmetry of the future embryo
must be laid down in the egg during oogenesis (Sturtevant, 1923).
If the asymmetry of the cortex plays a part in the determination of the
cleavage pattern it may be expected that the cortical patterns of eggs derived
from genetically dextral and sinistral L. peregra will mirror each other. If the
cortical patterns in their turn are related to the topography of the elements
surrounding the growing oocyte, the follicle cell patterns of the two races may
be expected to be different, and to mirror each other too. Therefore the arrangement of follicle cells in genetically dextral and sinistral L. peregra was studied.
Follicle cell patterns in dextral and sinistral Limnaea peregra 447
MATERIALS AND METHODS
Ovotestis material derived from genetically dextral (D) and sinistral (L)
individuals was fixed in Zenker's or Bouin's fluid, embedded in paraffin, sectioned at 4 (i and stained with azan, or according to the 'pikro-blauschwarzkernecht rot' procedure. The follicle cell patterns in the two races were studied
with the aid of wax reconstructions. The final magnification was about x 1100.
The nuclei and the cell bodies of the follicle cells, and the area of attachment of
the oocyte cell membrane to the basal membrane of the acinus were indicated
in each model.
250
•200
> <u
„ -5 150
o
o
8i
50
No. of
follicle cells-
1
3
8
Fig. 1. Relationship between size of the oocyte and number of follicle cells. 4- =
oocytes from genotypically sinistral snails; O = oocytes from genotypically dextral
snails.
Fig. 1 shows the relationship between oocyte size (expressed as the weight of
the corresponding wax reconstruction) and the number of follicle cells. It appears
that during the first phase of slow growth the oocyte is gradually surrounded
by an increasing number of follicle cells. They are presumably recruited from the
cells of the germinal epithelium. Cell divisions were never observed in follicle
cells of Limnaea, while cells which could be considered as transitional in their
morphologic appearance between cells of the germinal epithelium and follicle
cells were often encountered in the neighbourhood of the oocytes (Ubbels,
1968). When rapid growth begins, the inner layer of follicle cells immediately
adjoining the oocyte is completed. It appears that this consists in L. peregra of
29
J EEM 21
448
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
seven or eight cells as a rule; six cells are found in a number of smaller follicles,
whereas occasional follicles with nine elements do occur.
The fact that the number of follicle cells varies in this species thwarts the
analysis of follicle cell pattern, as different follicle classes have to be treated
separately. It is further complicated by the fact that the distribution of 'dextral'
and 'sinistral' follicles among the various classes is rather uneven. Of the fullgrown follicles studied in detail (not considering the 6-cell follicles, which are
generally smaller), among those from genetical dextrals there are nine follicles
with seven cells, two with eight and two with nine cells; among the sinistrals
two with seven cells, nine with eight cells and one with nine cells (cf. Table 1).
Owing to variations in the shape of the oocyte and the follicle cells a direct
spatial comparison of the various follicle cell patterns proved to be difficult.
Therefore in each model an imaginary plane was brought through as many
Table 1. The arrangement offollicle cell nuclei around the oocyte
N o . of
follicle
cells
Type and
no. of the
oocyte
6
D20
D44
7
D45
L 9
L 23
L 38
D 13
D 16
D19
D21
D27
D29
D30
D31
D46
L 11
L 51
8
D26
D47
L
L
L
L
L
L
L
L
L
9
7
8
10
22
33
34
36
37
40
D18
D28
L 24
Positions of the centre;5 of the successive
follicle cell nuclei
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26 137 171 214
21 114 175 247
28 123 190 247
44 131 186 240
40 93 173 266
46 184 230 267
61 176 222 245
16 102 149 157
26 114 193 246
29 79 122 202
36 83 165 212
34 94 146 197
20 59 85 170
31 170 196 229
19 117 136 204
41 183 195 236
20 39 137 176
31 99 174 198
20 155 175 214
27 90 126 180
30 68 113 173
36 81 126 180
25 82 115 172
48 112 168 176
45 135 165 195
29 125 191 228
21 56 141 162
38 90 150 210
29 66 118 154
41 95 115 190
47 94 149 164
317
299
313
305
320
313
283
258
272
281
260
249
262
268
282
254
215
236
262
207
203
234
213
232
240
279
197
233
213
210
219
329
321
325
338
348
334
334
327
330
319
350
267
301
279
270
288
278
280
278
301
289
263
279
278
266
329
330
333
330
315
335
320
315
331
331
338
301 338
292 340
274 313
Follicle cell patterns in dextral and sinistral Limnaea peregra 449
follicle cell nuclei as possible. The nuclei which were not included in this plane
were projected upon it. The distances between the centres of the successive
nuclei or of their projections were measured in a clockwise direction as seen
from the apical side. All distances were converted into arcs of a circle with a
circumference equal to the sum of the distances.
In Table 1 the positions of the centres of the follicle cell nuclei are expressed
in degrees of a circle, arbitrarily taking the position of one of the nuclei as zero.
Tf the follicle cells within a certain class are arranged according to a pattern,
it must be possible to rotate the models in such a way that their patterns coincide.
Since there is no fixed point of reference, several possible combinations have to
be tried out. This has been done by means of a computer by one of us (J. J. B.).
The following considerations determined the method of approach of the
problem.
Estimation of patterns
It has been mentioned above that the available material can be classified
according to the race of the animal (dextral or sinistral) and to the number of
follicle cells (varying from 6 to 9), yielding eight classes in all. Let us consider
the problem of determining the existence of a pattern common to the members
of a single class. If such a pattern exists, the follicle cells in different oocytes of
the same class must occupy corresponding positions, although, of course, a
certain amount of individual variability is admissible. We will, therefore, treat
the actual positions of corresponding follicle cells as random variables, and
define the common pattern as the set of their population means. The problem
is how to obtain an estimate of this pattern from the data in Table 1. The
difficulty lies in the fact that the oocyte possesses no fixed point of reference
that would enable us to establish with certainty which follicle cells are in correspondence. This question has to be decided by means of the data themselves,
and they are by no means unambiguous in this respect. Therefore, a criterion is
needed leading to a decision that is optimal in some sense. Minimum variance
seems to be a reasonable choice. In the following we will explain how this
criterion is applied.
Suppose a class contains n oocytes with p follicle cells each. If the way in
which the cells correspond is somehow fixed, they can be numbered (corresponding ceils receiving the same number), and the np observations can be arranged
in a table with n rows and p columns. The following notation will be used:
ati = observation in row / and column j (i.e. position of follicle celly
in oocyte /), (/ = 1 ... n, j = 1 ... p);
Mi = Ti^u/p = mean of row i;
j
Qj = Jlanln = mean of column j ;
%
m = YiQijlnp = mean of all observations.
ij
29-2
450
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
In each oocyte the positions are measured from an arbitrary origin. One of
the follicle cells was chosen as the origin and was given the number 1, so that
all values in the first column of Table 1 are 0. The set of column means <z-; is an
estimate of the pattern. It is independent of the choice of origins in the individual
oocytes, in the sense that shifting one of these changes all column means by the
same amount and so results only in a shift of the origin from which the means
are measured. The column variances, on the other hand, depend on the choice
of origins and, according to the criterion mentioned above, we have to choose
them in such a way that the sum of these variances is minimized. Choosing a
new origin for a given oocyte means changing all values in the corresponding
row by the same amount, say <xi for row /. The quantities ai5 with mean value
<* = Ttocjn, must be such that the total sum of squares 2 Yi{aij + cci-ai-ay
is
i
j
i
minimized. This condition does not define the quantities ce>i uniquely. We can
still impose a second condition, for which we choose a = 0, which is equivalent
to requiring that the column means remain unchanged. The values o^ satisfying
both conditions, are given by c^ = m - miri and the minimum value q of the
sum of squares by
q = S 2 ( % - f l j - w « + /w)2.
3
i
To obtain the best estimate of the pattern in the sense defined above, we
would have to compute the value of q for every possible correspondence of
follicle cells, and finally choose the one yielding the lowest value. Unfortunately,
a complete survey of all pn~x possibilities is impracticable. For instance, in the
case of L-oocytes with eight follicle cells each, where p = 8 and n = 9, this
would require 88 computations of q, which even with the use of a computer
would take too much time. It is possible, however, to make a partial survey
which is readily carried out by a computer and yet gives a reasonable guarantee
that the best estimate is indeed obtained. Starting with a given oocyte in a fixed
position, a second one is added and the best correspondence is found by the
method described above. Then both oocytes remain fixed and the process is
repeated with a third one, and so on until all members of the class have been
added. A complete cycle now requires only p(n-1) computations, while the
calculation is further simplified by the fact that at each stage k of the process the
sum of squares qk can be derived from that of the preceding stage by means of
the formula
\
k — If
Vic =flfe-i+ - y | 2 (aki - a,)2 -p(mk - m) 2 |.
The result obtained in this way depends on the sequence in which the oocytes
are added. Though each sequence tends to produce an arrangement with low
variance, it does not automatically lead to the lowest value. It is, therefore,
necessary to perform many cycles with widely differing sequences until a
sufficient number of them produce identical results. Since the computing time
for one cycle is very small, this is easily accomplished.
Follicle cell patterns in dextral and sinistral Limnaea peregra 451
Comparison O/D- and L-patterns
The same minimum variance criterion can be used to decide between hypotheses regarding the relations between patterns in different classes. To this end
the classes are combined to a single unit and minimum variance arrangements
are determined, corresponding to the various hypotheses to be compared. Once
again the lowest value of the variance indicates which of the hypotheses is to be
preferred.
In particular, it can be investigated in this way whether the difference in
D- and L-asymmetry is reflected in the follicle cell patterns. If the comparison is
restricted to oocytes with the same number of follicle cells, the hypotheses to be
compared are simple alternatives: either D- and L-patterns are identical, or one
is the mirror image of the other. To choose between these alternatives, the
minimum variance arrangement is determined for D- and L-oocytes combined,
first with the order of the follicle cells unchanged and then with this order
reversed in one of the two types. The first or the second alternative is accepted
according to whether the lowest value of the variance is found in the first or in
the second case.
The use of this mathematical criterion to decide between two biological
possibilities emphasizes the necessity of checking its reliability in some way. The
changes in value of the variance caused either by renumbering or by reversing
the order of the follicle cells are generally small compared with this value itself
(cf. Tables 2 and 3 in the next paragraph). Consequently, it is impossible to
verify the conclusions statistically, since in most cases the differences would
not be significant. It is, however, possible to check the reliability of the criterion
by applying it to cases where the result it should produce is known beforehand.
To this end the method described in the previous paragraph for a single class,
consisting of oocytes of the same type and with the same number of follicle cells,
is extended slightly. Each time a new oocyte is added, it is tried not only with the
follicle cells in their original order but also with this order reversed and, if
necessary, the order is fixed in the latter state. The criterion can be considered as
satisfactory if in the final arrangement the majority of oocytes are in agreement
with regard to the order of their follicle cells.
RESULTS
The computations sketched in the foregoing paragraphs were carried out on
an Electrologica X8-computer provided with an ALGOL-processor. The
programs, written in ALGOL, will not be discussed here but they are available
on request.
(a) Reliability of the method
The results obtained by checking the method in the way described above are
presented in Table 2. For each class, with the exception of the class 9L which
452
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
contains only one oocyte, two values of the minimum variance are recorded.
The first value holds if the order of the follicle cells is fixed, the second value if
this order can be either maintained or reversed. In the columns headed by
a + or — sign are shown the numbers of oocytes in which the original order of
the follicle cells in the final arrangement is maintained or reversed, respectively.
Table 2 shows that the agreement between oocytes in the minimum variance
arrangement with variable order of follicle cells is satisfactory. In the larger
classes 7D and 8L the majority of oocytes (7 or 8 out of 9) are in agreement.
With the smaller classes, containing only two or three oocytes each, there is
agreement in two cases (6D and 7L), disagreement in two cases (6L and 8D),
while in one case (9D) the question remains undecided because the difference
in variance with respect to the fixed order arrangement is very small. All things
considered, these results seem to justify the use of the criterion for purposes of
comparison.
Table 2. Minimum variances in single classes with fixed or variable
order offollicle cells
+ = original order maintained; — = order reversed.
Fixed order
Class
6D
6L
7D
7L
8D
8L
9D
Variable order
+
-
Variance
+
-
Variance
3
3
9
2
2
9
2
0
0
0
0
0
0
0
101
157
194
86
204
99
3
1
7
2
1
8
1
0
2
2
101
143
173
86
169
85
102
108
0
1
1
1
(b) Comparison ofD- and L-patterns
The results obtained by combining D- and L-oocytes with the same number
of follicle cells are presented in Table 3. For each number of follicle cells three
values of the minimum variance are recorded. The first value holds if for D- and
L-oocytes both the original order of the follicle cells is maintained, the second
value if this order is reversed in one of the two types, and the third value if this
order can be maintained or reversed in each individual oocyte of both types.
The numbers of oocytes concerned are shown in the same way as in the previous
table.
It is difficult to draw any definite conclusions from Table 3 because the results
do not all point in the same direction. The best way will be to consider the four
classes separately.
In the class of oocytes with six follicle cells a lower value of the variance
results if the two types are taken in opposite order. The difference, however, is
Follicle cell patterns in dextral and sinistral Limnaea peregra 453
small: 159 against 167. Moreover, there is no complete agreement between the
L-oocytes since the variance is further reduced to the value 149 if only two of
them are reversed while the third one is taken in the same order as the D-oocytes.
The only appreciable difference is found in the class of oocytes with seven
follicle cells. Here the arrangement with both types in the same order is clearly
preferred. Reversing the order in the L-oocytes increases the variance from 205
to 270. Leaving both possibilities open for each individual oocyte results in a
still smaller value 187, but even then the two L-oocytes are in agreement with
the majority of the D-oocytes.
Table 3. Minimum variances in combined classes
+ = original order maintained; — = order reversed.
Fixed ordei
Fixed ordei
Variable order
A
Class
+
Variance
-
6D
3
0
L
7D
3
9
0
0
L
8D
2
2
0
0
L
9D
9
2
0
0
L
1
0
1 167
J
1 205
J
I 119
J
1 92
J
+
Variance
3
0
0
9
3
0
0
0
2
2
9
2
0
0
0
1
I 159
J
1 270
J
I 114
J
1 87
J
+
Variance
3
0
1
7
2
2
2
1
0
1
8
2
0
0
1
1
1 149
J
1 187
J
1 100
J
1 87
J
The class of oocytes with eight follicle cells shows the same picture as that
with six cells. There is a very small difference (114 against 119) in favour of the
arrangement of the two types in opposite order, but here too the variance
assumes a lower value (100) if only one of the two D-oocytes is reversed while
the other one is taken in the same order as the L-oocytes. In the class with nine
follicle cells the number of oocytes as well as the difference between the values
of the variance is too small to give an indication in either direction.
Taking into account all facts listed above, it may be concluded that, although
the data are by no means unanimous, there is more evidence in favour of the
hypothesis that D- and L-patterns are identical. The estimates given below are
based on this assumption.
(c) Estimates of patterns and optimal arrangements
In view of the last conclusion, oocytes of both types with the same number of
follicle cells were taken together with all cells in the same order. Optimal
arrangements and estimates of patterns were computed for each number of
454
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
follicle cells. They are presented in Tables 4 and 5. To give an idea of
the accuracy of the estimates, standard deviations of the means are likewise
recorded in Table 5, where they are indicated by the symbol S.
Table 4. Optimal arrangement offollicle cells for individual oocytes
Oocyte
no.
Position of follicle cells
D20
D44
D45
L 9
L 23
L 38
D 13
D 16
D 19
D21
D27
D29
D30
D31
D46
L 11
L 51
D26
D47
L 7
L 8
L 10
L 22
L 33
L 34
L 36
L 37
L 40
D18
D28
L 24
11 37 148 182 225 328
12 33 126 187 259 311
5 33 128 195 252 318
4 48 135 190 244 309
346 39 119 212 266 306
342 28 166 212 249 295
5
10
0
3
340
345
20
2
358
8
348
4
5
358
5
355
0
342
356
15
3
356
43 89 120
26 112 159
53 88 114
32 82 125
27 109 156
45 97 148
40 79 105
41 100 133
17 115 134
26 91 132
7 105 144
42 73 135
53 92 121
25 88 124
35 73 118
40 76 121
25 82 115
38 86 126
41 79 116
66 88 118
24 59 144
26 101 123
355 24
348 29
18 26
181
167
202
205
204
200
190
164
202
173
342
331
334
178
178
166
172
166
161
147
165
161
205 277 331
296
268
281
284
292
285
282
303
280
315
183 318
166 197
341
304
311
354
329
328
327
328
265 340
151 171 306 326
208
220
213
214
206
176
200
213
61 113 149 208
83 103 178 198
65 112 159 206
275
274
278
278
296
272
292
273
274
266
261
335
328
335
334
326
338
334
333
296 333
280 328
276 331
Table 5. Estimates of patterns
No. of
follicle
cells
Mean of position
0
0
0
0
36 137 196 249 311
33 97 134 188 291
38 82 124 165 202
26 69 109 162 204
—
330
281
267
—
—
333
284
—
—
—
330
5-3
4-3
3-3
5-5
Follicle cell patterns in dextral and sinistral Limnaea peregra 455
Description of the follicle cell pattern
The average positions of follicle cells, given in Table 5, determine the patterns
of 6-, 7-, 8- and 9-celled follicles respectively. The question arises whether there
is a relationship between these patterns. If, as suggested by Fig. 1, the number of
follicle cells gradually increases by the recruitment of cells from the germinal
epithelium, one may expect that the additional follicle cells will be intercalated
between the cells already present. The latter will in general keep their positions,
270°-
Fig. 2. Average follicle cell positions in follicles of different cell number. The
average positions of follicle cells (1-6) in 6-cell follicles, as seen from the animal
pole, are indicated in the inner circle. The cells of 7-, 8- and 9-cell follicles, respectively, have been put in the outward following circles in matching positions.
Table 6. Combination of the patterns
No. of
follicle
cells
Mean of position of cell no.
1
9
6
5
7
4
3
0
8
24
31
—
—
—
94
101
111
103
111
160
150
155
157
—
180
182
187
213
213
220
213
275
277
264
256
2
8
324 —
314 —
306 347
296 349
456
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
perhaps with the exception of the immediately adjacent cells moving up a short
distance to make room for the newcomer. To establish the relationship between
follicles with different cell numbers, the four patterns given in Table 5 must be
rotated in such a way that the average positions of the cells show the best
correspondence to these expectations. This results in the positions given in
Table 6 and the corresponding Fig. 2.
In a previous publication (Raven, 1963) the follicle cells of L. stagnalis have
been numbered in a counter-clockwise direction. To preserve a certain uniformity of treatment, we have done the same in the present case; this does not
X9
Fig. 3. Individual positions of follicle cells in genetical sinistrals. The plan of the
figure and the notation of the cells correspond to Fig. 2.
imply, to be sure, that cells with the same number in the two species are considered homologous. The cells of the six-celled follicle of L. peregra are numbered from 1 to 6, starting with the cell at the left end of the largest gap in the
series and proceeding counter-clockwise (Fig. 2). With the increase in follicle
cell number, cell 7 is inserted between 4 and 5, cell 8 between 1 and 2, and cell 9
between 1 and 6, but nearer to the latter. These numbers have also been placed
above the columns of Table 6. It is evident both from the figure and the table
that identical cells in general have constant positions within about 10 degrees
Follicle cell patterns in dextral and sinistral Limnaea peregra 457
(cf. columns 6, 5, 7 and 4 of Table 6), with the exception of cells 1 and 2, which
move apart to make room for cell 8, and cell 3, taking part in this movement to a
lesser extent. This is in agreement with the above-mentioned expectations.
While Fig. 2 gives the average positions of the cells, in Figs. 3 and 4 the
individual values of Table 4, but readjusted so as to correspond with the
average positions of Table 6 and Fig. 2, have been inserted for the follicles of
genetical sinistrals and dextrals, respectively. It appears from these figures that,
Fig. 4. Individual positions of follicle cells in genetical dextrals.
although the positions of corresponding cells in different follicles exhibit a
certain amount of scatter, for most cells they are distinctly clustered around the
average values. The areas occupied by adjacent cells can in most cases clearly
be delimited against each other, and hardly overlap. This argues for the accuracy
of the pattern. Moreover, in comparing Figs. 3 and 4, it is evident that L- and
D-follicles do not differ from each other. This confirms the conclusion reached
above that L- and D-patterns are identical rather than mirrored.
Subcortical accumulations in the eggs ofL. peregra
In a previous paper (Raven, 1963) the conclusion was reached that the
arrangement of the subcortical accumulations (SCA) of the oviposited egg of
458
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
L. stagnalis reflects the positions of the follicle cells surrounding the growing
oocyte in the gonad. In this connexion it appeared important to investigate
whether SCA can also be distinguished in the uncleaved eggs of L. peregra, and
if their arrangement can be related to the pattern of follicle cells in this species
too.
Fig. 5 A. Egg P 52-2. Extrusion of first polar body. Egg with six SCA, of which three
are visible (c, d, e). Magnification x 560. B. Egg P52-4. Telophase of first maturation division. Egg with six SCA, of which five are visible (a-e; e only marginally
sectioned). Magnification x 560. C. Egg P52-4. SCA c-e at higher magnification
(x 875). D. Egg 74-7. Anaphase of second maturation division. Egg with seven
SCA. SCA e a n d / a t higher magnification; e marginally sectioned. Magnification
x875.
Follicle cell patterns in dextral and sinistral Limnaea peregra 459
To this end 106 eggs of L. peregra, ranging from anaphase of the first maturation division to anaphase of the second maturation division, have been studied.
These eggs had been preserved in 1963, to serve for another investigation, and
were probably from genetical sinistrals. They had been fixed in Bouin's fluid,
sectioned at 5 /i, and stained with iron haematoxylin and erythrosin.
A study of the sections shows that in these eggs distinct SCA can be recognized. They have a similar appearance and localization as in L. stagnalis,
occupying more or less lens-shaped areas immediately beneath the plasma
membrane in the equatorial and vegetative regions (Fig. 5 A, B). Their boundary
against the internal cytoplasm is rather irregular, and indented by the vacuoles
surrounding y-granules. It is often rather difficult to establish their outline, as
they gradually thin out beneath the plasma membrane. In such cases the
separation between neighbouring SCA may also be rather vague.
The SCA consist of a 'dense' cytoplasmic matrix, staining brownish in iron
haematoxylin-erythrosin preparations. Dense masses of delicate basophil
granules are embedded in this matrix; they stain blue-black with iron haematoxylin. The mitochondria, occurring in the animal pole plasm, around the
maturation spindle and asters, and in the cytoplasmic meshes between the
vacuoles, exhibit the same staining, but they are coarser and probably rodshaped, whereas the SCA-granules seem to be mainly spherical. The latter are
therefore probably not mitochondria. These granules are accumulated for the
greater part immediately beneath the plasma membrane in masses often several
layers thick, but they may partly also extend inwards in the triangular partitions
between adjacent vacuoles (Fig. 5C, D). The /?- and y-granules of the protein
yolk are lacking in the SCA, at least in uncleaved eggs. It is evident that the
composition of the SCA agrees with that found in L. stagnalis (Raven, 1967).
In order to study the positions of the SCA, graphical reconstructions were
made of all eggs. The method employed was the same as that used for the eggs
of L. stagnalis (Raven, 1967). As in that investigation, the positions of the SCA
established in the original drawings were transposed by projection on to an
idealized equatorial plane, on which the parts of the SCA extending into the
vegetal hemisphere were drawn as they would be seen by looking at the egg from
the direction of the vegetative pole. By the method outlined in the abovementioned paper, these diagrams were brought into corresponding positions
by establishing a system of common meridians, and the co-ordinates of the
SCA of each egg in a group were measured. From the median values of these
measurements a 'median projection' of the group was constructed.
It soon appeared that the 106 eggs studied fell into two groups: in 72 eggs
six SCA could be distinguished, whereas the remaining 34 eggs had seven SCA.
Originally the eggs were subdivided into age classes (anaphase of first maturation division, etc.), and median projections of all eggs with the same number of
SCA within a single class were constructed. It appeared, however, that no
significant changes in the positions of the SCA with age occurred during the
460
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
period considered (anaphase of first to anaphase of second maturation division).
Therefore, the eggs of all age classes with the same number of SCA could be
taken together, and a common median projection of them constructed.
Fig. 6. Limnaea peregra. Median projection of 72 eggs with six SCA. Diagram of
vegetal hemisphere; vegetative pole in centre, a-f: the six SCA. Arrows 1-6: corresponding positions of cells 1-6 of 6-cell follicles. Interrupted line: approximate
plane of symmetry.
Fig. 6 shows the median projection of all (72) eggs with six SCA. The SCA
are arranged in a circle beneath the equator of the egg. They are not evenly
spaced, however, but on one side there is a wider gap between two adjacent
SCA, while on the opposite side at least the centres of two SCA are somewhat
wider apart than elsewhere. If we draw a line bisecting these two angles, this
defines a meridian plane, with respect to which the centres of the individual
SCA are approximately symmetrically arranged. The only apparent asymmetry
is in the size of the SCA denoted by c and d in the drawing, d being much more
extended in a latitudinal direction.
Follicle cell patterns in dextral and sinistral Limnaea peregra 461
The median projection of the (34) eggs with seven SCA is shown in Fig. 7.
Again the SCA are arranged in a subequatorial circle, but their distribution is
now clearly asymmetric. If we again draw the meridian plane bisecting the
Fig. 7. Limnaea peregra. Median projection of 34 eggs with seven SCA. Arrows 1-7:
positions of cells 1-7 of 7-cell follicles. Interrupted line: meridian plane bisecting
widest gap between SCA.
widest gap in the series, three SCA are situated on one side of this plane and
four on the other side, though slightly overlapping into the other half. Between
the two groups there is a somewhat larger gap than between adjacent SCA of
the same group, which may even partly overlap in their latitudinal extension.
DISCUSSION
1. Within the limitations of the method used and of the restricted material
to which it has been applied, the results described above seem to lead to the
conclusion that the follicle cells in egg follicles of L. peregra are not arranged
462
G. A . U B B E L S , J. J. B E Z E M & C. P . R A V E N
haphazardly around the oocyte, but according to a definite pattern. The following arguments for this conclusion can be adduced:
(a) In the execution of the computations, for every class of follicles (classified
according to the race of the animal and the number of follicle cells) many cycles
with widely differing sequences of the individual follicles were performed in
order to find the arrangement with the lowest variance. It appeared that an
identical configuration was found for a majority of the cycles.
(b) If the arrangement of the follicle cells is haphazard, one can expect that a
substantial reduction of the variance can be attained by permitting the reversal
of the order of the cells in part of the follicles. It appears, however, that this
expectation is not fulfilled; in general, the original order of the follicle cells
was maintained, and the reversal of some follicles gave only a slight reduction
of the variance (cf. Tables 2 and 3).
(c) The patterns resulting from the computations in the various classes show
a great resemblance. N o t only does it seem probable that the patterns of L- and
D-follicles with the same number of cells are identical rather than reversed, but
the patterns of follicles with different cell numbers can be arranged in such a way
that they conform to reasonable assumptions concerning their mutual relationship (Fig. 2).
{d) The most convincing evidence on the reality of the pattern results from the
inspection of Figs. 3 and 4, in which the positions of the cells in the individual
follicles are summarized. It appears from these figures that: (1) corresponding
cells in different follicles of all size classes are situated in circumscribed areas,
which hardly overlap; (2) the single values within an area show a certain amount
of scatter, but most of them are distinctly clustered in a certain part of the area;
(3) the patterns of L- and D-follicles obtained in this way are superimposable;
(4) in both patterns there is a corresponding region of 40-50° of the circumference which is entirely free of follicle cells.
If one takes into account that the recognition of the species-specific pattern
of the follicle is bound to be obscured, not only by the normal variability
inherent in all biological structures, but also by a summation of distortions
caused and errors committed in all preceding phases of the investigation (fixation
and sectioning, drawing and making of reconstructions, measurement of cell
distances), it is surprising that the main characteristics of the pattern are clearly
expressed in these figures. The possibility that such a result could have been
produced by the mere process of ordering of follicles with random arrangement
of the cells, by the criterion of minimum variance as employed in this investigation, seems rather far-fetched. In this connexion it is important to note that the
afore-mentioned errors could not have been biased by anticipation of the
expected result, since the latter appeared only afterwards as outcome of the
computations. Therefore, it is believed that the resulting pattern conforms to
reality.
2. The pattern of follicle cells in L. peregra is not identical with that found in
Follicle cell patterns in dextral and sinistral Limnaea peregra 463
L. stagnalis (Raven, 1963), even as regards the 6-cell follicles of the former
species. However, a certain general resemblance between the two patterns
cannot be denied. In both, the arrangement of the cells is not radially symetrical. On one side of the follicle, the cells are more narrowly spaced than on the
other; in L. peregra there is even a wide gap between the cells on one side.
Therefore, one can distinguish, in addition to the polar axis connecting the
centre of the basal surface with the free pole of the oocyte, a second axis at
right angles to the first. In L. stagnalis, it has been proved that this 'dorsoventrality' of the egg follicle coincides with that of the later embryo (Raven, 1967).
It remains to be determined whether this also holds for L. peregra.
3. In L. stagnalis, the pattern of follicle cells is nearly symmetrical with respect
to the plane containing the two axes, though a slight asymmetry seems to exist.
In L. peregra, in 6- and 8-cell follicles the arrangement of the cells is also
roughly symmetrical; in 7- and 9-cell follicles, on the other hand, there is a
distinct asymmetry. This asymmetry is the same in L - and D-follicles. Our
expectation that the follicle cell patterns of the two races of L. peregra mirror
each other has therefore not been fulfilled. Apparently, the determination of the
asymmetry of the future embryo (of which we know that it probably occurs
during oogenesis) does not take place by way of the asymmetry of the follicle
cell pattern, but according to some other mechanism.
In a recent book Waddington (1966) states, referring to our work, ' . . .it has
been found that the pattern of asymmetry... is reversed for left-handed and
right-handed forms'. Though we have alluded to the present work in some
previous publications, we have never made any definite statement to this effect.
It now appears that this view can no longer be upheld.
4. In L. stagnalis, in uncleaved eggs six subcortical accumulations (SCA) have
been found, the arrangement of which duplicates the pattern of follicle cells
previously surrounding the oocyte in the gonad (Raven, 1963, 1967). Since the
SCA arise only after ovulation by the accumulation of certain components of
the ooplasm beneath particular regions of the plasma membrane, and because
their pattern is restored after the displacement of these components by centrifugal force, it was concluded that it reflects a pre-existent mosaic pattern of the
egg cortex, which in its turn is formed during oogenesis in correspondence with
the structures surrounding the oocyte.
It has now been found that similar relationships occur in L. peregra.
Uncleaved eggs of this species have either six or seven SCA. They are arranged
according to a definite pattern, which is nearly symmetric in the eggs with six
SCA (Fig. 6), but clearly asymmetric in those with seven (Fig. 7). In both cases
there is a wider gap between the SCA on one side.
This description already points to a general similarity between the pattern of
SCA and the pattern of follicle cells discussed above. A closer inspection shows
that the two patterns are nearly superimposable. In Fig. 6 the average positions
of the centres of the follicle cells in 6-cell follicles have been indicated by
30
J E E M
21
464
G. A. U B B E L S , J. J. B E Z E M & C. P . R A V E N
arrows. (It should be noted that in Fig. 2 the follicle cell patterns have been
represented as seen from the animal pole, whereas in the reconstruction of the
SCA in Fig. 6 the egg is viewed from the vegetal side; therefore, the follicle cells
from 1 to 6 here follow each other in a clockwise direction). It is evident that
there is a striking similarity between the two patterns, each of the SCA strictly
corresponding to the position of a single follicle cell. In Fig. 7, where eggs with
seven SCA are compared with 7-cell follicles, the correspondence between the
two is not as good, but in view of the fact that the opposition of four follicle
cells on one side against three on the other is matched by the arrangement of the
SCA, it seems satisfactory. It should be stressed that the patterns of follicle cells
on the one hand, and SCA, on the other, have been established by two independent lines of research, using different methods, and that their great similarity
only revealed itself in the final elaboration of the results. Therefore, the two
mutually confirm each other, and strongly argue for the reality both of the
pattern of follicle cells and of SCA.
5. A difficulty might seem to arise from the fact that only eggs with six or
with seven SCA have been found, whereas many egg follicles in L. peregra have
eight or even nine cells. If the pattern of SCA is supposed to be a reflection of
the follicle cell pattern, one should therefore expect also to find egg cells with
eight or nine SCA.
For the moment the most plausible explanation of this discrepancy is provided by the observation that, while originally the follicle cells are tightly
apposed to the oocyte surface, during later growth stages a follicle cavity
appears between the two. This has been observed both in L. stagnalis (Ubbels,
1968) and in L. peregra. The formation of the follicle cavity coincides with the
beginning of the final stage of rapid growth; in L. peregra at that time about
six follicle cells have been formed (Fig. 1). At this stage also the formation of a
zona radiata becomes evident, pointing to a change in the superficial regions of
the oocyte. One may assume that the influence of the follicle cells, 'imprinting'
their pattern on the egg surface, is greatly reduced or entirely abolished when
they withdraw from the oocyte. In that case, the mosaic pattern of the egg
cortex will not represent the final configuration of the follicle cells, but rather
their arrangement at the moment of formation of the follicle cavity. Future
research must show whether this supposition is right.
6. We may conclude that the results of this investigation lend support to the
hypothesis that part of the developmental information is transmitted from the
parent to the offspring by way of the egg follicle, whose structure is 'imprinted'
upon the egg during oogenesis. It seems hardly probable that the correspondence
between the follicle cell pattern, on the one hand, and the arrangement of the
SCA of the oviposited egg, on the other, in both L. stagnalis and L. peregra is
merely accidental. It has been shown in L. stagnalis that the polarity and dorsoventrality of the later embryo arise in conformity to this pattern (Raven, 1967).
It is therefore likely that the structure of the egg follicle determines the polarity
Follicle cell patterns in dextral and sinistral Limnaea peregra 465
and dorsoventrality of the egg cell. However, the present investigation seems to
show that the asymmetry of the embryo is not determined in a similar way.
SUMMARY
1. The structure of the egg follicles in dextral and sinistral Limnaea peregra
has been studied.
2. The number of follicle cells surrounding large oocytes varies between 6
and 9.
3. The follicle cells are not arranged in an arbitrary way, but according to a
definite pattern. This pattern is polar and dorsoventral, and either nearly
symmetric or asymmetric depending on the cell number.
4. The patterns of follicles with different cell numbers are connected in a
simple way.
5. The patterns of follicles in genetically dextral and sinistral snails do not
mirror each other, but are identical.
6. In uncleaved eggs of L. peregra, a system of six or seven subcortical
accumulations (SCA) is found. In eggs with six SCA their arrangement is
identical with that of the cells in 6-celled follicles; in eggs with seven SCA there
is a great resemblance to the cell pattern in 7-celled follicles.
7. It is concluded that the pattern of SCA reflects the arrangement of the
follicle cells at the moment when the follicular cavity begins to form.
8. The results lend support to the hypothesis that the polarity and dorsoventrality of the later embryo are determined by the structure of the egg follicle.
The determination of the asymmetry of the embryo takes place in another way.
RESUME
1. La structure des follicules germinatifs des Limnaea peregra dextres et
sinistres a ete etudiee.
2. Le nombre des cellules folliculaires entourant les grandes ovocytes varie
entre 6 et 9.
3. Les cellules folliculaires ne sont pas arrangees de facon arbitraire, mais
selon un patron distinct. Celui-ci est polaire et dorsoventral, et symetrique ou
asymetrique dependant du nombre des cellules.
4. Les patrons des follicules dont le nombre des cellules est different sont
lies d'une maniere simple.
5. Les patrons des follicules des animaux dextres et sinistres ne sont pas
inverses mais superposables.
6. Dans les oeufs insegmentes de L. peregra il y a un systeme de 6 ou 7
accumulations souscorticales (SCA). Dans les oeufs a 6 SCA, leur arrangement
est identique a celui des cellules dans les follicules a 6 cellules; dans les oeufs a
7 SCA leur arrangement se ressemble au patron cellulaire des follicules a 7
cellules.
30-2
466
G. A. UBBELS, J. J. BEZEM & C. P. RAVEN
7. On conclut que Farrangement des SCA reflechit le patron des cellules
folliculaires au moment ou la cavite folliculaire commence a se former.
8. Les resultats soutiennent rhypothese que la polarite et la dorsoventralite
de l'embryon sont determinees par la structure du follicule germinatif. La
determination de l'asymetrie de l'embryon se fait d'une autre maniere.
We are indebted to Professor C. H. Waddington for his kindness in providing us with
dextral and sinistral L. peregra from a stock kept in the Institute of Animal Genetics in
Edinburgh.
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{Manuscript received 3 October 1968)