/. Embryol. exp. Morph., Vol. 75, 1, pp. 79-90, August 1967
Printed in Great Britain
79
Regeneration and growth control in Nereis
II. An axial gradient in growth potentiality
By D. W. GOLDING 1
From the Department of Zoology, University of Bristol
Caudal regeneration in Nereis diversicolor is that rapid proliferation of segments which occurs after loss of part of the posterior of the body and the reformation of the pygidium. The rate of segment proliferation is highest during the
production of the first new segments. Thereafter, the rate declines rapidly with
each passing week, though if the regenerated segments are amputated, rapid
proliferation is again resumed (Golding, 1967). Two explanations of these
results are possible. First, the declining rate of segment proliferation during
regeneration may be the result of a declining concentration of hormone in the
body fluids. There may be a feed-back from the regenerating tail to the ganglion.
As segments are proliferated, the secretory activity of the ganglion is suppressed.
Secondly, the level of hormone in the body fluids may remain constant, the
proliferating region of the pygidium becoming progressively more insensitive
as regeneration proceeds.
It has also been shown that the rate of proliferation is correlated, not only
with the stage reached in the regenerative process, but also with the number of
segments amputated. The greater the number of segments lost, the faster the
rate of segment proliferation which ensues. This is so with animals with ganglia
in situ, and with those whose ganglia have been replaced by ganglia removed
from intact donors. The same two explanations proposed for the initially high
but rapidly declining rate of segment proliferation during regeneration are
possible with respect to the correlation between the number of segments lost
and the amount of ensuing regeneration. The first alternative is that removal
of a large number of segments results in a high concentration of hormone in the
body fluids, whilst the second is that the proliferating region so formed may be
more sensitive to a given level of hormone than that formed after the loss of
fewer segments.
As a possible way of obtaining experimental evidence to support one or other
of the two alternatives described above, a technique for grafting decerebrate
fragments of worms into host animals was developed, and a number of grafting
experiments performed.
1
Author's address: Department of Zoology, University of California, Berkeley, California, U.S.A.
80
D. W. GOLDING
MATERIALS AND METHODS
Despite the refinement of the technique developed by Durchon (1960), it was
decided that it was not practical for the large number of grafting experiments
that it was wished to carry out.
After progressive improvements in technique, grafting carried out in the
following way was found to be satisfactory.
Preparation of the graft
The first four segments were removed. Four longitudinal cuts were made in
the body wall. One was made just dorsal, and one just ventral, to the parapodia
on each side of the body. Each extended for eight segments from the cut surface
of the decapitated worm. The lateral strips of body wall between the cuts and
bearing the parapodia were removed, but the dorsal and ventral strips were left
attached to the fragment. The length of the intestine thus exposed was excised.
After amputation of the requisite number of posterior segments, the fragment,
together with the dorsal and ventral strips of body wall with which it was to be
stitched into the host, was ready for grafting.
Preparation of the host
Seven transverse cuts were made right through the body wall in the midsegmental position, to the right of the dorsal blood-vessel. The most anterior
cut was made in about the tenth segment and the others in the six segments
immediately posterior to it. The fourth of these cuts was made the width of the
graft, the others were smaller. The graft was then stitched into the host by means
of drawing the strips of body wall through the cuts in the body wall of the host.
Both strips were inserted into the fourth and largest cut, the dorsal strip being
turned anteriorly with respect to the host and passed through the anterior cuts,
the ventral strip being turned posteriorly and treated similarly. In this way, the
anterior cut surface of the graft was closely applied to the cut in the host's body
wall prepared for it, so that the confluence of the coeloms of the host and graft
was ensured. The jaws of the host were cut off where they entered the flesh of
the proboscis, since it was found that, even if the grafting operation was a
success, the graft would often be attacked and eaten by the host.
Upon recovery from the anaesthetic, the grafted animals were introduced into
glass tubes of an appropriate size. The latter was found to be a critical factor
in their survival. If the size was too large, efficient irrigation of the tube by the
animal was impossible, and the death of host and graft ensued sooner or later,
whilst if the size was too small, a host could not easily reverse in the tube and the
graft was pulled out when it attempted to do so. Tubes of a diameter twice that
into which the hosts could crawl by themselves were found to be ideal. The ends
of each tube were covered with bolting silk secured with thick rubber bands.
Material of a gauge which would not allow the animal to crawl through it, but
Regeneration in Nereis. II
81
which, with this qualification, offered the minimum resistance to water flow,
was chosen for this purpose. Each tube was submerged in 50 % sea water, and
any air trapped inside was sucked out using a rubber tube of a suitable internal
diameter. The water was aerated vigorously and continuously. After a grafted
animal had been in a given tube for 7 days, the silk was removed and the animal
induced to crawl into a clean tube. The ends of this were sealed with clean silk
before returning it to clean 50 % sea water. This procedure was necessitated by
the accumulation of mucus in the tube and by the tendency on the part of the
silk to become choked with mucus and algal growth. Mucus strands and
ligatures were removed from the animals at the same time.
This description applies in detail only to the later, more extensive experiments.
In preliminary experiments the technique had not been developed to this
extent, and some regenerating tails were probably lost due to predation by the
host.
The absence of co-ordinated movement on the part of the host and graft,
together with the tendency on the part of the host to prey on the graft, indicate
that the host and graft failed to attain nervous continuity. However, they did
achieve coelomic, cuticular and vascular continuity. In some cases, the gut of the
graft grew forward to join that of the host. The strips of graft body wall, used to
stitch the graft into the host, became incorporated into the body wall of the latter.
The complete compatibility of the tissues of two individuals was convincingly
demonstrated by the grafting of a fragment of one animal onto the posterior
surface of another from which the posterior half had been amputated. The
sections of intestine healed together in such a way as to leave no visible trace
of their separate origin. The cut edges of the body wall also healed together and
there was coelomic confluence. Nervous continuity was not apparently achieved.
RESULTS
(i) Preliminary grafting experiments
In the first preliminary experiment (Exp. A), twenty animals originally 65-75
segments long were used as grafts, and twenty, 80-90 segments long, were
employed as hosts. (This experiment was carried out in late June 1964, and the
hosts used were comparatively immature, despite their size.) The grafts were
prepared in the way already described, and stitched into the large intact hosts.
Posterior segments were amputated, leaving grafts of only 20 intact segments.
After 21 days, the number of segments regenerated by the grafts was determined.
These results are given in Table 1. They show that the majority of posteriorly
amputated grafts were induced to regenerate by hormone having its source in
the intact hosts.
The second preliminary experiment involved ten animals, each 65-75 segments long, which were prepared for use as grafts. Each graft consisted of 20
intact segments, as in the previous experiment. Twenty larger intact worms were
6
JEEM l8
82
D. W. GOLDING
also used. They were divided into two groups, the members of one being used
as hosts, whilst the other acted as a control group. Each animal of both host
and control groups had the right parapodium of the sixth segment from the
posterior end of the body removed—only segments bearing parapodia with
acicula being counted. This operation was carried out so that the number of
segments grown during the experiment could be determined. Members of the
control group were treated identically to those of the experimental group,
except that the grafting operation was not performed upon them..Three weeks
after grafting, the number of segments regenerated by the grafts and the number
grown by the host and control animals were determined. The results are given
Table 1. Preliminary grafting experiments (1)
Exp. A
Exp. B
r
irafts
No.
No.
No.
No.
of animals
surviving 21 days
showing no proliferation
proliferating 1 segment
2 segments
3 segments
4 segments
5 segments
6 segments
Mean no.
S.E.
20
14
3
1
2
3
1
4
0
2-7
0-5
Hosts
(intact)
Grafts
(regenerating)
10
7
7
0
0
0
0
0
0
0
10
7
2
1
0
1
0
1
2
30
10
Exp. B. Control group (10 intact animals without grafts): one animal grew 1 segment only
in Table 1. Virtually no segments were grown by the large animals of either
group, despite the comparatively prolific production of segments by the
regenerating grafts.
In the third experiment, thirty-five host and graft animals, each 80-90 and
65-75 segments long, respectively, were used. Each graft consisted of 20 intact
segments, i.e. many segments were amputated from each, whereas only 3
posterior segments were amputated from each host. After 21 days the numbers
of segments regenerated by hosts and grafts were determined, and are given in
Table 2. Survivors whose grafts had lost segments through entanglement in
mucus or as the result of predation by the host were discarded. The results show
that despite the subjection of the pygidia of both hosts and grafts to the same
hormonal environment the grafts regenerated significantly more segments than
the hosts.
The fourth experiment can be regarded as a mirror image of the third. Exactly
comparable animals were used as hosts and grafts, and the operative procedure
Regeneration in Nereis. II
83
followed was the same as before. However, 30 posterior segments were removed
from each host but only 3 from each graft. The results are given in Table 2 and
show that, in this experiment as in the previous one, the number of segments
regenerated was correlated with the number amputated from that particular tail.
The hosts, from which many segments had been removed, regenerated far more
segments than the grafts, which had lost few segments.
Table 2. Preliminary grafting experiments (2)
Expt. C
No.
No.
No.
No.
No.
of segments amputated
of animals
surviving 21 days
showing no regeneration
regenerating 1 segment
2 segments
3 segments
4 segments
5 segments
6 segments
Mean no.
S.E.
Expt. D
Hosts
Grafts
Hosts
Grafts
3
35
19
15
4
0
0
0
0
0
0-2
—
30
35
19
0
0
3
8
7
0
1
3-4
0-2
30
35
13
2
0
3
35
13
9
4
0
0
0
0
0
0-3
—
1
5
3
2
0
30
04
(ii) Major grafting experiments
For the first major grafting experiment, sixty animals 80-85 segments long,
and sixty animals, 65-75 segments long (to act as hosts and grafts respectively)
were collected on the same day. Each size group was randomly divided into three
groups of equal size. All grafts were prepared in the same way, each consisting
of 20 intact segments. Individuals of one group were grafted into animals of one
of the groups of hosts, according to the method already described. In addition,
10 segments were amputated from the posterior of each host. The second group
of hosts received grafts similarly, but had 25 posterior segments removed.
Forty segments were amputated from the third group of hosts. The grafted
animals were kept for 21 days after which time the numbers of segments
regenerated by hosts and grafts were determined. The results are given in Table 3
and Fig. 1. These results were subjected to statistical analysis. The three groups
of grafts regenerated to a similar extent, as the differences between the mean
numbers of segments are not statistically significant. However, the groups of
hosts regenerated by different amounts. There is a clear correlation between the
number of segments regenerated by the host and the number amputated. The
differences between the means are significant (P < 0-01).
In the second major grafting experiment, 100 animals, each 65-75 segments
long, were collected on the same day. They were randomly divided into five
6-2
84
D. W. GOLDING
groups, each containing 20 specimens. The part of the body posterior to the
thirty-second segment was amputated from each specimen of one group. The
anterior segments of these animals, and the intact worms of the other groups,
were kept individually and fed regularly. At intervals of 7 days this procedure
Table 3. Grafting experiment—differential segment loss
No. of segments amputated
No. of animals
No. surviving 21 days
No. showing no regeneration
No. regenerating 1 segment
2 segments
3 segments
4 segments
5 segments
6 segments
7 segments
8 segments
9 segments
10 segments
11 segments
12 segments
Mean no.
S.E.
H
G
H
G
H
10
20
17
2
2
40*
40*
40
40*
20
16
0
0
20
16
1
0
20
16
0
0
3
0
0
1
0
7
2
0
1
25
20
16
2
0
1
0
2
2
1
0
1
3
0
1
3
6
1
0
2
0
0
0
0
0
0
2
2
2
2
3
1
1
7-8
0-6
5
0
0
0
0
0
0
4-4
0-5
3
3
1
2
1
2
0
70
0-6
4
1
4
3
0
1
0
6-7
0-7
2
2
1
3
1
1
1
7-1
0-7
0
2-5
0-3
20
17
0
0
* Approximately.
H = hosts; G = grafts.
20
40
No. of segments amputated from hosts
Fig. 1. Regeneration in grafts and hosts. The grafts were deprived of approximately
the same number of posterior segments; different numbers of segments were amputated from the hosts.
Regeneration in Nereis. II
85
was carried out on one after another of the groups of intact worms. Twentyeight days after the beginning of the experiment, worms of one group were
intact, whilst those of other groups had been regenerating for 1, 2, 3 and 4 weeks
respectively. The number of segments possessed by each worm was recorded.
One hundred animals, 80-85 segments long, were collected on the same
occasion for use as hosts. They were divided into five groups. Worms of each
Table 4. Grafting experiment—grafts at different stages of regeneration
H
G
H
G
H
G
H
G
H
G
0
2
0
0
1
3
0 Int.
0
4
20
20
20 20
20
20 20
20 20 20
12
12
13
17
9
17
15
13
15
9
1
1
0
0
0
0
1
0
0
0
2
0
0
2
0
0
0
8
2
0
1
1
2
0
0
0
0
0
6
4
1
1
2
1
0
3
4
0
3
2
1
2
1
1
3
4
3
4
2
4
1
2
1
1
1
2
3
2
4
0
1
2
0
1
5
0
0
3
3
3
1
1
1
0
1
0
0
0
1
1
1
0
0
0
0
0
1
0
0
1
0
1
4
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
3
0
0
0
0
1
0
0
0
0
0
0
1
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
1
0
0
0
0
0
0
0
4-9 8-7 3-8 4-9 4-6 2-7 5-2 2-7 5-4 1-6
S.E.
0-5 0-8 0-7 0-5 0-5 0-4 0-5 0-5 0-6 0-2
Int. Grafts with intact tails, equivalent t o t hose at a very late stag*j of regeneration.
Age* of regenerates (weeks)
No. of animals
No. surviving 21 days
No. showing no regenerationf
No. regenerating! 1 segment
2 segments
3 segments
4 segments
5 segments
6 segments
7 segments
8 segments
9 segments
10 segments
11 segments
12 segments
13 segments
14 segments
15 segments
16 segments
Mean no.
* Time since loss of segments.
f No. growing segments with reference to grafts with intact tails.
group had the members of one group of grafts stitched into them. The procedure was the same as before except that further posterior segments were not
removed from the grafts. With the exception of the group of grafts with intact
tails, each graft consisted of 20 intact segments plus those regenerated, since
of the 32,12 segments were either removed or used to provide the strips of body
wall used to secure the grafts. Thirty segments were amputated from each host
upon implantation of the graft.
In this way, groups of host animals at the same stage of regeneration, received
groups of grafts at different stages. The grafted animals were kept for 21 days,
after which the numbers of segments regenerated by hosts and grafts subsequent
to the grafting operation were determined. The number proliferated was the
number grown during the 21 days by the grafts with intact tails, and the number
86
D. W. GOLDING
regenerated by all other grafts and by the hosts. The results are given in Table 4
and Fig. 2.
The results show that the groups of hosts (each group bearing grafts at a
different stage of regeneration) regenerated approximately the same number of
segments, as the differences between the means are not statistically significant.
In contrast, the groups of grafts (which had been regenerating for different
lengths of time before being implanted into the hosts) regenerated by widely
differing amounts. The first group had been regenerating for only 1 week before
the grafting operation, and was therefore regenerating under the hormonal
influence of the host for the second, third and fourth weeks of the regenerative
process. Members of this group regenerated an average of over 8 segments each.
The second group had been regenerating for 2 weeks before grafting. They were
1
2
3
4
weeks
Animals with intact tails.
Duration of regeneration of grafts before implantation
Fig. 2. Regeneration in grafts and hosts. The hosts were at the same early stage in
regeneration; the grafts had been regenerating for different periods of time before
the grafting operation.
influenced by the host during the third, fourth and fifth weeks of regeneration,
and produced significantly fewer segments (approximately 5 each). The third
group was grafted into the hosts from the fourth to the sixth week and regenerated an average of less than 3 segments, as did the fourth group, whose
members were influenced by the hormone from the host from the fifth to
the seventh week of regeneration. The grafts with intact tails were regarded as
being equivalent to animals which had been regenerating for a very long period
of time. The mean number of segments proliferated by this group was less
than 2.
The mean numbers of segments proliferated by the first and second groups
differ significantly from each other, and from those of the third, fourth and fifth
groups (P < 0-01).
The correlation seen in this experiment between the rate of segment proli-
Regeneration
in Nereis. II
87
feration and the stage reached in the regenerative process appears exactly similar
to that observed in normal regeneration, that is, that seen in animals with ganglia
in situ.
DISCUSSION
It has been assumed that the increase in the rate of segment proliferation
which follows segment loss is due to a change in the hormonal influence, whether
the change involves secretion of a hormone not previously present, or secretion
of an increased concentration of a hormone present at all times. This is implied
by Hauenschild & Fischer's statement (1962): 'The number and size of the
segments regenerated... can be used an indication of the secretory activity of the
ganglion.' Similarly, Durchon & Marcel (1962) wrote '...simples sections de
fibres nerveuses...sont suffisantes pour declencher, dans le cerveau, une secretion
d'hormone de regeneration.' A radical change in the hormone level is essential
to the concept of activation, developed by Clark and his collaborators (Clark
& Bonney, 1960; Clark & Evans, 1961; Clark & Ruston, 1963).
The observations reported above, however, point to a different conclusion.
The results obtained from the first two preliminary experiments show that
neither loss of posterior segments nor section of the ventral nerve cord is
necessary for the production of regeneration hormone by the supraoesophageal
ganglion. This is in accord with the conclusion reached by Durchon & Marcel
(1962) as a result of similar experiments. They indicate that hormone may be
in the body fluids at all times, or, if it is released in response to damage inflicted
upon the peripheral nervous system or to the influence of a newly formed
pygidium in vascular continuity with the ganglion, that it does not accelerate
the rate of normal growth.
The other preliminary experiments show that when two pygidia, formed as
the result of the loss of different numbers of segments, are subjected to the
influence of the same hormonal environment, the one from which many segments were amputated will regenerate to a greater extent. This indicates that
pygidia formed after loss of different numbers of segments differ in their
inherent growth potentialities.
This conclusion is strongly supported by results of the first major grafting
experiment. If hosts from which more segments have been amputated regenerate
to a greater extent because of a higher concentration of hormone in the body
fluids, then the standard, comparable grafts under the hormonal influence of
such hosts would be expected to regenerate more segments also. They do not do
so. The extent of regeneration in the host appears to be quite independent of
that in the graft.
Two alternative explanations of the correlation between the number of
segments lost and the number regenerating were suggested above. It was postulated that either the removal of a large number of segments results in a high
level of hormone in the body fluids, or that the proliferating region so formed
88
D. W. GOLDING
has a higher intrinsic growth potentiality than that formed after loss of few
segments. We may conclude that the latter is the one consistent with the
experimental facts.
The second major and critical experiment involved the grafting of decerebrate
fragments of animals at different stages of regeneration into standard comparable hosts, the different groups having been subjected to the amputation of
the same number of segments and being at exactly the same stage of regeneration.
The rate of the proliferation of segments in normal regeneration is initially high,
but declines rapidly. In an experiment such as the one described above the same
phenomenon was observed in the different groups of grafts: the later the stage
reached in regeneration the slower the rate of proliferation. This was so despite
the fact that the hormonal environment of the hosts, under the influence of
which the grafts regenerate, induces about the same amount of regeneration in
the different groups of hosts. It is difficult to reconcile these results with the
generally assumed idea that regeneration is induced by a hormone that is produced in response to segment loss, the concentration of which is initially high
but declines rapidly, since if the initially high rate of segment proliferation is
due to a greater concentration of hormone, hosts having rapidly regenerating
grafts would be expected to regenerate more rapidly themselves than those having
slowly regenerating grafts. This is not so. The rates of segment proliferation in
host and graft are apparently completely independent despite the subjection
of both to the same hormonal environment. In other words, grafts at different
stages of regeneration will proliferate segments at radically different rates if
subjected to the same hormonal influence, the inherent growth potentiality
declining sharply as regeneration proceeds.
The influence exerted by the hormone on segment growth seems to be an
extremely simple one. As far as an immature animal at a given stage of development is concerned, the ganglion cannot be said to control segment growth. It
appears to maintain a steady level of hormone in the body fluids and though this
^hormone is indispensable for segment proliferation, the control of the rate of
growth, which is correlated with the number of segments lost, is a local mechanism. No experimental findings have been advanced in support of the view that
the concentration of the hormone changes during regeneration, and there is no
reason to postulate such changes to account for this phenomenon, which can
be explained satisfactorily in terms of changing growth potentialities.
We may conclude that loss of part of the posterior of the body of an immature
Nereis results in the formation of proliferating region that has a high inherent
growth potentiality, the magnitude of the potentiality depending on the number
of segments lost. This potentiality declines progressively as regeneration proceeds. There appears to be an axial gradient in growth potentiality, declining
posteriorly. The rate of regenerative growth in an immature animal depends
upon the level of the body at which the growth zone is formed.
No conclusions with respect to the basis of the axial gradient can be drawn
Regeneration in Nereis. II
89
at this time. However, the size of the nerve cord transected at different levels
may be a relevant factor (Abeloos & Thouveny, 1960). Other axial gradients
may also be involved, for example, the metabolic gradients of Child (1946) and
the gradients in levels of differentiation postulated by Rose (1957).
SUMMARY
Grafting experiments showed that a graft from which posterior segments have
been removed regenerates segments even if implanted into an intact host. Rapid
proliferation by a graft implanted into an intact host is not associated with any
increase in the rate of proliferation by the latter. Removal of a few segments
from a host, but many from its graft, results in the regeneration of few segments by the host, but many by the graft; and vice versa. The regeneration of
many segments by animals from which many segments have been removed is
not attributable to a high level of hormone in the body fluids. The proliferation
region formed after extensive segment loss has a higher inherent growth potentiality than that formed after loss of few segments. The initially high but declining rate of proliferation which characterizes the regenerative process is not
attributable to an initially high but declining level of hormone. The proliferating
region formed after segment loss has a high growth potentiality but this declines
as regeneration proceeds. There appears to be an axial gradient in growth
potentiality, declining posteriorly. Regenerative growth is dependent on the
presence of the hormone, but the rate of growth depends upon the level of the
body at which segment proliferation is occurring.
RESUME
Regeneration et controle de la croissance chez Nereis
II. Existence d'un gradient axial de potentialites de croissance
Des experiences de greffe ont montre qu'un greffon dont les segments posterieurs ont ete amputes regenere, meme s'il est implante dans un note intact.
La proliferation rapide d'un greffon implante dans un hote intact n'est pas
accompagnee d'un accroissement de la vitesse de proliferation de celui-ci.
L'ablation d'un petit nombre de segments chez l'hote, et de nombreux segments chez le greffon est suivie de la regeneration de peu de segments chez
l'hote, mais de beaucoup de segments chez le greffon; et vice versa. La regeneration de beaucoup de segments chez des animaux dont beaucoup de segments ont
ete amputes n'est pas imputable a une concentration elevee d'hormone dans
les humeurs. La region qui prolifere apres une perte considerable de segments a
une potentialite de croissance plus elevee que celle qui se forme apres la perte de
quelques segments. La vitesse de proliferation, initialement elevee, puis decroissante, qui caracterise le processus de regeneration, ne peut etre attribute a un
taux d'hormone, d'abord eleve, puis decroissant. La region qui prolifere apres
90
D. W. GOLDING
la perte de segments a une potentialite elevee, mais celle-ci diminue en cours de
regeneration. On peut admettre qu'il existe un gradient axial de potentiel de
croissance, son intensite decroit vers la region posterieure. La croissance regeneratrice depend de la presence de l'hormone, mais la vitesse de croissance depend
du niveau du corps ou se produit la proliferation segmentaire.
This work was carried out during 3 years of tenure of an award of the Science Research
Council. The author is grateful to Professor R. B. Clark for his encouragement and constructive criticism.
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M. & THOUVENY, Y. (1960). Conditions histologiques de la regeneration chez
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CHILD, C. M. (1946). Organizers in development and the organizer concept. Physiol. Zob'l.
19, 89-147.
CLARK, R. B. & BONNEY, D. G. (1960). Influence of the supraoesophageal ganglion on
posterior regeneration in Nereis diversicolor. J. Embryol. exp. Morph. 8, 112-18.
CLARK, R. B. & EVANS, S. M. (1961). The effect of delayed brain extirpation and replacement on caudal regeneration in Nereis diversicolor. J. Embryol. exp. Morph. 9, 97-105.
CLARK, R. B. & RUSTON, R. (1963). Time of release and action of a hormone influencing
regeneration in the polychaete Nereis diversicolor. Gen. comp. Endocrinol. 3, 542-53.
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(Manuscript received 14 February 1967)