/ . Embryol. exp. Morph. Vol. 60, pp. 373-387, 1980
Printed in Great Britain © Company of Biologists Limited 1980
373
Commitment of stem cells to
nerve cells and migration of nerve cell
precursors in preparatory bud
development in Hydra
By STEFAN BERKING 1
From the Zoologisches Institute Universitiit Heidelberg
SUMMARY
Budding in Hydra starts as an evagination of the double-layered tissue in the parent
animal's gastric region. Five hours later the density of nerve cells in the bud's tissue doubles,
representing the first detectable difference from the cellular composition of the surrounding
tissue. These new nerve cells derive from multipotent stem cells which are in S-phase one
day before evagination starts. Some of the bud's new nerve cells derive from stem cells
which have migrated into the future bud's tissue after their commitment, apparently
attracted by the bud anlage. The bud anlage recruits precursors of nerve cells even during
starvation, during which nerve cell production ceases in other parts of the body. Furthermore,
the bud anlage controls the duration of the development from commitment to final
differentiation of the resulting nerve cells.
Experiments with an inhibitor purified from hydra tissue indicate a tight correlation
between stages of preparatory bud development and stages of recruitment of nerve cells
for the bud. Whether or not precursors of nerve cells are involved in the control of bud
formation in normal hydra, as compared to epithelial hydra which still bud though
consisting of epithelial cells only, will be discussed.
INTRODUCTION
Budding in Hydra may serve as a model system for processes basic to
morphogenesis. A new bud is formed at a certain distance from head and foot
in tissue of rather uniform composition (Bode et ah 1973). A small area becomes
specified to develop into the tip of a new bud. This tip is subsequently able to
organize the surrounding tissue (Li & Yao, 1945; Berking, 1979a). The bud is
formed by evagination of the double-layered tissue of the parent animal. This
process may be comparable to processes involved in tissue invagination in
gastrulation.
A useful tool for studying the processes which precede visible bud development was a low-molecular-weight inhibitor purified from hydra tissue (Berking,
1977). This substance prevents bud development reversibly in very low con1
Author's address: Zoologisches Institut, Universitat Heidelberg, Im Neuenheimer
Feld 230, D-6900 Heidelberg 1, Federal Republic of Germany.
374
STEFAN BERKING
"Surrounding tissucv
Bud
Planes of sectioning
Fig. 1. Excision of the bud and the 'surrounding tissue'.
centrations. Upon release from inhibition almost all animals start immediately
with the same preparatory developmental step. About half a day later
evagination begins.
The position of the bud was found to be specified only after the end of the
treatment with inhibitor (Berking & Gierer, 1977). Thus it is possible to
investigate preparatory developmental steps involved in budding from the very
beginning of this process.
The present paper deals with the formation of new nerve cells in the course
of budding because this is one of the earliest events in bud development. The
first visible change in the cellular composition of the young bud, which starts
with the same cellular composition as its surroundings, is an increase in the
density of nerve cells (Bode et ah 1973). The method was to label animals at
various times before the bud evaginates by injection of [3H]thymidine (David
& Campbell, 1972). The label is incorporated in cells which are in the S-phase
at the time of injection. These include stem cells which can give rise to nerve
cells and nematocytes. Thus labelling the animals at certain times before or
after treatment with inhibitor results in the labelling of the cells of bud anlagen
of defined developmental stages.
There was a further reason to look at the relation between nerve cell development and budding. In whole animals the inhibitor was found to prevent the
determination of stem cells to nerve cells (Berking, 1979£). Thus it would be
interesting to know whether or not the influence of the inhibitor on preparatory
bud development can be correlated with its influence on nerve cell formation
during budding.
MATERIALS AND METHODS
Hydra attenuata were used for all experiments. All experiments were done
at 20 °C. For details see Bode et al. (1973). To determine the cellular composition of the bud and its surroundings, the tissues were cut off as shown in
Fig. 1 and disintegrated in acetic acid, glycerol and water (1:1:13). The cells
Nerve cell formation in preparatory bud development in Hydra 375
were classified according to David (1973). The category 'nerve cells' includes
all types of nerve cells bearing processes which can be detected with the light
microscope. 'Interstitial cells' (/-cells) denote single i-cells and nests of two
(in Table 1 nests of four are included); about half of this population appears to
consist of multipotent stem cells (Sproull & David, 1979).
Hydra cells were labelled with [Me-3H]thymidine (20 Ci/mM) by injecting
0-2 /d containing 0002 fiC\ of the isotope directly into the gastric cavity (David
& Campbell, 1972). A single injection result in a pulse of about 1 h duration.
Labelled cells were classified in cell macerations by autoradiography using
Kodak AR10 stripping film.
The experimental error was calculated either by use of the binomial distribution or by ^-analysis. For all experiments purified inhibitor was used which
was enriched at least 500-fold from crude extract (Berking, 1977). Crude extract
was used as standard for comparison with purified extract. An optical density
(280 nm) of 0-033 per ml crude extract was arbitrarily defined as one biological
unit (I BU). Retardation of budding was observed to occur by treatment with
0-5-1 BU (Berking & Gierer, 1977). Treatment with inhibitor was found to
not hinder the incorporation of the label into the stem cells, the precursors
of the nerve cells (Berking, 19796).
For the transplantation experiments animals were stained by feeding them
for some days with Artemia nauplii cultured in a solution of Evans Blue
(Merck, Darmstadt, FRG) according to Wilby & Webster (1970).
Standard procedure. Animals were fed daily with Anemia nauplii. One day
after the last feeding animals bearing one bud of age up to 6 h were collected
(except first experiment, Table 1) and were fed.
Visible onset of budding. The transition from a smooth surface of the budding
region to the protrusion of a tiny tip in this region which is stable upon contraction and elongation of the animal is termed visible onset of budding. This
process is completed within 1 h.
RESULTS
In the tissue of the young bud the density of the nerve cells is twice as high as in
the surrounding tissue
The visible beginning of bud formation is an evagination of the doublelayered tissue of the parent animal's gastric region. The very young bud has
the same cellular composition as the tissue surrounding the bud. Later an
increase in the density of the nerve cells was observed to occur (Bode et ah 1973).
About 170 additional nerve cells become detectable between 4 and 8 h after
evagination has started. This corresponds to a doubling of the nerve cell
number (Table 1).
376
STEFAN BERKING
Table 1. In buds 8 h old the frequency of nerve cells has increased (x2', P < 0-001)
caused by 170 ±29 additional nerve cells in the bud's tissue (9900 cells per bud,
i.e. about 10 % of the animals tissue; mean of 23 buds)
Age of bud . . .
No. of tissue pieces
No. of counted cells
Epithelial cells (%)
Big /-cells (%)
Little /-cells (%)
Nematoblasts (%)
Nerve cells (%)
Gland cells (%)
3 h 4 5 min ± 1 h
Bud
Surrounding
tissue
10
2058
270
24-6
23-2
20-6
1-55
3-1
10
2135
25-9
24-5
22-6
22-5
1-41
31
7 h 30 min
Bud
13
2015
27-8
25-6
19-4
19-9
3-13
4-3
8 h 15 min ± 1 h 20 min
Bud
23
1924
26-6
25-7
21-1
19-2
3-26
3-9
Surrounding
tissue
23
1969
26-6
26-2
22-2
210
1-4
2-9
The new nerve cells appear in 5 h old buds 1 day after their precursors were in
S-phase
Animals bearing one bud were fed (zero time), labelled at 5 h, treated from
6 to 8 h with inhibitor and then allowed to form buds (Fig. 2). The buds were
excised at various times after evagination had started and the frequency of
labelled nerve cells per bud was determined.
The bud's new nerve cells are formed at a certain developmental stage of the
bud (Fig. 2C) and not a fixed period of time after labelling (Fig. 2D). They
appear at 5 h after the bud visibly begins to evaginate.
Buds of this age change their morphology (Fig. 2B). The labelling index
of 50 % indicates that almost all new nerve cells of the young bud (termed
'primary nerve cells') derive from precursors which are in S-phase at the time
of labelling.
Effect of feeding and inhibitor treatment on the production of the bud's primary
nerve cells
Feeding allows stem cells which are just in the middle of their S-phase at
the time of feeding to develop into nerve cells (the S-phase has a length of about
12 h (David & Gierer, 1974)). Stem cells which are exposed to inhibitor while
in the first half of their S-phase (inhibitor sensitive phase, ISP) are prevented
from developing into nerve cells after the next mitosis. Those in the second
half of their S-phase at the time of treatment are not affected (Berking, 1919 b).
The development of the primary nerve cells appears to follow the same rules.
Labelled primary nerve cells are found if the label is injected less than 7 h after
the beginning of the treatment (Fig. 3). Thus stem cells which end their S-phase
at 7 h or later do not develop into nerve cells. Those which end their S-phase
Nerve cell formation in preparatory bud development in Hydra 377
A Feeding Labelling Treatment with
\ ^/ |
^ inhibitor
Allowed to form
new buds
4
6
Time (h)
8
Bud stage 3
10
Bud stage 4
A
100"*
50
en
80 2 " 40
60 -S c 30
40 « 1 20
- 20 | ^ 1 0
2
3
4
5
Age of the bud (h)
^
28
29
30
31 32
Time after feeding (h)
Fig. 2. New nerve cells appear in buds 5 h old. Starting conditions as usual
(see Materials and Methods). (A) Selected animals were fed (zero time) labelled
5-6 h) and treated with inhibitor (6-8 h, hatched area, 12 BU). The next day the
animals began to form new buds which were excised at different ages and prepared
for autoradiographic analysis (C, D); (O—O), percentage labelled nerve cells in
the bud; (A—A) percentage labelled nerve cells in the respective surrounding tissue.
Each point in the graph is obtained from counts of 45-75 nerve cells of preparations
of 5-9 buds. (C) The percentage of labelled nerve cells in buds of different ages and
respective surrounding tissues plotted against the age of the buds; (D) the same
frequencies plotted against the time interval between feeding (zero time as in most
of the following representations) and preparation of the animals. The horizontal
bars indicate the range of the age of the used buds. For clarity the standard
deviations are omitted. (B) Bud stages were classified according to Otto and
Campbell (1976).
earlier, for instance at 5 h after the beginning of a treatment, are not prevented
from developing into nerve cells because they are exposed to inhibitor only
during the second half of their S-phase. Thus in this experiment (Fig. 3) the
primary nerve cells derive mainly from those cells which are just in the middle
of their S-phase at the time of feeding. Furthermore, feeding on the day of
experiment (before the first treatment) greatly enhances bud formation (Berking
& Gierer, 1977). Thus it is suggested that both effects of feeding, allowance of
budding and allowance of nerve cell formation, are coupled to one another.
The development into the primary nerve cells of the bud is controlled by the
bud anlage
The result of the following experiment confirms the notion that the developmental period of the bud's first nerve cells is of variable length.
Animals were treated with inhibitor (12 BU) for 2 and 8 h respectively. In
both cases the treatment was terminated just before the label was injected at
378
STEFAN BERKING
i
60
A
50
40
-
•
D
D
•
•
i
B
-
30
20
10
o
°A
Feeding
" I
\
#
Time (h)
-
•
M "
i
i
i
i
i
t
i
10
12-
28
30
32
34
36
Time after feeding (h)
Fig. 3. Localization in time of the S-phase of those stem cells which give rise to
the primary nerve cells. Standard conditions. (A) Animals were fed (zero time) and
treated with inhibitor (12 BU) from 20 min to 8 h (hatched area). The percentage of
labelled nerve cells (O, A, # , A) and /-cells ( • ) found the next day in buds 5 h old
is plotted (A) against the time after feeding at which the label was injected into the
animals and (B) against the length of the time interval between feeding and preparation. Each point is the result of countings of at least 100 /-cells and 35-60 nerve
cells respectively.
8 h 20 min after feeding. Labelling following 8 h of treatment causes only a few
(9 ± 3 %) primary nerve cells to be labelled, as expected from the above
experiment, whereas labelling following 2 h of treatment (after 6 h 20 min
inhibitor-free development) results in the labelling of almost all primary nerve
cells (40 ± 4-5 %) in buds 5-6 h old. Thus commitment of stem cells to nerve cells
takes place over a certain period of time following feeding, and the beginning
of an inhibitor treatment delimits the period in which commitment can take
place. However, the finally formed nerve cells are found to be formed not after
a fixed period of time after beginning of the treatment but rather when the bud
becomes 5 h old (Fig. 2C, D).
In contrast, throughout growth, excluding morphogenetic processes like
budding, nerve cell development appears to be accomplished within a rather
fixed interval after commitment (Berking, 19796). Thus the duration of the
development from stem cells to the primary nerve cells appears to be coupled
to the process which causes evagination.
The precursors of the primary nerve cells can migrate
Animals were treated and labelled so as to label the precursors of the primary
nerve cells (Fig. 4). Then the animals were sectioned in the budding region and
transplanted to unlabelled animals which had been treated and sectioned in
the same way. The half-animals were transplanted together so that budless
animals of normal morphology were obtained. Thus the budding region contains
labelled and unlabelled stem cells which have become committed to develop
Nerve cellformation in preparatory bud development in Hydra 379
Bll'F ANIMALS
-• ll.-ccdinB1""'™'"
| inhibitor
t/ I
y/\
\
i
Bucllcss animals
obtained
VMn
"*
Interstitial cells
Type of transplants
from which buds were analysed
L-lpithdial cells
I
/
No of counted
cells
\
'.I labelled
\
\
',; labelled
No. of counted
cells
C7 labelled
cells
No. of counted
cells
86
194
53
219
16
143
240
56
289
20
85
249
50
222
21
122
216
46
259
20
Cl
95
200
500
0
C2
79
225
302
0-7
C3
179
501
601
0-2
Fig. 4. The precursors of the primary nerve cells appear to migrate. Animals with
one just visible bud, normally red coloured ones and blue, vitally stained ones were
fed (zero time) and treated with inhibitor (12 BU). The blue ones were labelled;
other conditions as usual. The animals were sectioned and transplanted between
4 h 40 min and 8 h, however, in each case immediately after the end of treatment.
The table shows the frequency of labelled cells of various types as found either in
excised buds (4-10 per given cell count) of totally labelled animals or of buds
formed at transplants. In the buds of transplants formed of unlabelled tissue the
frequency of labelled nerve cells is higher than the frequency of labelled /-cells
(x"; P < 0001) whereas in totally labelled animals the reverse was found (x2;
002 < P < 005).
into nerve cells, including those which develop into the primary nerve cells of
a bud. The next day buds are formed. Those formed from labelled and unlabelled tissue respectively were excised and analysed separately. Buds formed
from unlabelled epithelial cells were found to contain about 5 % labelled
primary nerve cells but only very few (about 0-5 %) labelled /-cells (about one
half of the /-cells appear to be stem cells (Sproull & David, 1979)). The
Experiments were designed so that stem cells which are not committed at the
time of transplantation to become nerve cells should not give rise to the
primary nerve cells of the future bud. But if one argues that the primary nerve
cells develop only from stem cells which become committed in the future bud's
tissue, one has to explain how a population of stem cells of which 0-4 % are
labelled could give rise to nerve cells of which 10 % are labelled (in the bud
5 % of the nerve cells were found to be labelled, half of the bud's nerve cells
are new ones). This indicates that mainly commited stem cells immigrate into
the future bud's tissue, and not uncommitted stem cells.
380
STEFAN BERKING
Treatment with
inhibitor
Labelling
Type
Allowed to form
new buds
10
l
60 -
B
50
T
40
1
1
1
1
1
r -"~l
1
1
1
-
T
o
l
O
Ti
30
T
20
o
10
~T
1
- C
_L
~
1
Time, h
T
O
o
o
-
|
8
|
1
1
1
1
10
12
Age of the bud (h)
I
i
14
24
i
i
i
i
1
1
1
26
28
30
Time after treatment (h)
1
30
Fig. 5. Stem cells which happen to start their S-phase close to the end of the
treatment with inhibitor give rise to the secondary nerve cells. Starting conditions
as usual. (A) animals were treated (12 BU), from 1 h to 9 h 50 min and labelled
either between 7 h 55 min and 8 h 25 min or between 9 h 50 min and 10 h 25 min.
Presentation of the result comparable to Fig. 2.
Secondary nerve cells derive from stem cells which start their S-phase at the end
of the treatment with inhibitor
In buds 8-15 h old the nerve cell density doubles a further time; these nerve
cells will be termed 'secondary nerve cells'. However, the stepwise increase in
nerve cell density is caused by the treatment which starts shortly after feeding
(first treatment).
Stem cells which start their S-phase close to the end of the treatment with
inhibitor give rise to nerve cells (I.e.). Secondary nerve cells were formed in the
same way: animals were either labelled immediately before or immediately
after the end of a treatment (Fig. 5). In buds of early labelled animals fewer
nerve cells were found to be labelled than in buds of late labelled animals.
The development of stem cells to secondary nerve cells can be prevented by means
of a second treatment with inhibitor
Animals were treated twice with inhibitor, as shown in Fig. 6, the first time
as usual and the second time at 4 h after the first treatment for a period of
2 h 40 min. The animals were labelled either before or after the second treatment. Then the animals were allowed to form buds which were excised for
autoradiographic analysis.
The result (Fig. 6B) indicates that the secondary nerve cells derive mainly
Nerve cell formation in preparatory bud development in Hydra 381
Excision of the new buds
for autoradiographic analysis
Treatment with inhibitor
_ ..
first
second
Feeding i
,
;— Labelling
l I IT'
0
3
Time, h
15 18
21 24 27 30 33 36 39 42 45 48
Time necessary to Appearencc
Stem cells
develop processes of labelled
Mitosis 1
,—nerve cells
S-Phase
Ga-Phase
= = Q l m m m
12
Development into
labelled nerve cells
=0=
1
B
1
1
i
i
i
i
i
i
i
T "
•
1 -
50 —
rve cells
C
•I
T
•
i
40 —
T
•
1
1
i
30 —
•o
% labellc
1
—
T
O
1
T
O
20 -
1
T
o
1
T
O
10
1
1
i
14
i
I
16
i
i
i
i
18
20
Age of the bud (h)
I
1
T
O
1 _
i
22
Fig. 6. Formation of secondary nerve cells after two treatments with inhibitor.
Starting conditions as usual. The further procedure is shown in the upper half of the
scheme (A). The animals were treated with inhibitor (20 BU) and labelled either at
the first (O) or at the second time ( # ) shown in the scheme. The lower half of the
scheme shows the stem cell populations which give rise to labelled secondary nerve
cells in the case the second treatment prevents stem cell determination. The result of
the experiment is shown in (B). The 10-20 % labelled secondary nerve cells found in
the buds of the early labelled animals (O—O) may partly derive from stem cells
which have become labelled at the end of their S-phase and have become determined ( t ) within their next S-phase.
from stem cells which start their S-phase after the end of the second treatment.
Most of the stem cells which would give rise to nerve cells after the first treatment
(those just starting their S-phase) are prevented from becoming nerve cells by
the second treatment.
25
EMB 60
382
STEFAN BERKING
First treatment
with inhibitor
Second
treatment
Labelling
I
6
Time (h)
10
Type
1-2BU
Allowed
A to form
3-2 BU
D
12
% labelled nerve cells in tissue of
Type
16
18
20
22
24
Bud
Bud surrounding
55-6 ± 5 0
54-0 ±4-4
12-8 ± 3 0
12-8 ± 2 1
44-0 + 3-4
59-5 ±4-7
9 1 ±2-1
110 ±2-8
5 0 0 ±4-0
4 9 1 ±4-6
4-4 ± 1-7
5-3 ± 1-7
26
Time after first treatment (h)
Fig. 7. Effect of a second treatment with low concentrations of inhibitor on the
formation of nerve cells. Starting conditions as usual. The further procedure is shown
in the scheme (A). (B) shows the inhibitory effects of the treatments on bud
formation. The table shows the frequency of labelled nerve cells found in the newly
formed buds (7-10 per given frequency) 8-13 after evagination has started.
Further, the frequency of labelled nerve cells in the tissue surrounding the
respective buds is shown. With increasing concentrations of the inhibitor the
frequency of labelled nerve cells is only slightly lower in the tissue of the bud
(X2; 0-3 < P < 05) whereas in the tissue surrounding the bud less than half of the
normal frequency is observed (x2; i* < 0001).
Low concentrations of inhibitor prevent mainly the formation of new nerve cells
in the tissue surrounding the bud
The animals were treated twice with inhibitor, the first time as usual and
the second time with low concentrations for a period of 2 h. Between both
treatment the animals were labelled (Fig. 7). The buds which formed the next
day were excised and analysed and compared to the surrounding tissue. The
data indicate that the secondary nerve cells were formed at almost the same
frequency whether the animals were treated or not. However, in the tissue
surrounding the bud fewer nerve cells were formed as a result of the treatment.
This indicates that either the sensitivity of stem cells to externally supplied
inhibitor is lower in the tissue of the future bud or that committed stem cells
within the bud's surrounding tissue do migrate preferentially into the tissue
of the future bud. Thus, in terms of sensitivity to inhibition of nerve cell
recruitment, the bud anlage appears to start acquiring a typical property of
a head.
Nerve cellformation in preparatory bud development in Hydra 383
Table 2. Formation of nerve cells in head, bud and tissue surrounding the bud
upon starvation and treatment with inhibitor
Labelled nerve cells (%) in tissue of
Time labelled
10 h 30min
12 h
Head
Bud
Bud surrounding
3-6±l- 4
1-4±1- 0
44±6 •1
38 ±5 •4
1-1 ± 1 0
0 <10
Starting conditions as usual. The animals were fed and subsequently treated
with inhibitor (18 BU) from 30 min to 22 h. At 7 h 30 min after the end of the treatment the animals were treated a second time and labelled at the times given. The
procedure allows stem cells which start their S-phase within a short period after
the end of the first treatment to give rise to labelled nerve cells; buds 8-14 h old
were analysed.
One day after treatment and starvation stem cells become determined to develop
into secondary nerve cells in the bud but only rarely into nerve cells in other parts
of the body
Animals were treated with inhibitor for 22 h after feeding and were then
allowed to restart bud development. At 10 or 12 h after the end of the treatment
the animals were labelled. The next day the animals were dissected and prepared
for autoradiographic analysis. The result (Table 2) indicates that only a few
nerve cells develop from labelled stem cells in the head and gastric regions, just
as in untreated animals starved for the same period of time (I.e.). However,
secondary nerve cells were found to be formed in normal frequency. Thus it is
argued that the bud anlage generates a signal which determines stem cells to
nerve cells and/or attracts committed stem cells.
DISCUSSION
The first nerve cells of the bud (primary and secondary) are recruited by two
mechanisms: (1) by local determination from multipotent stem cells; after bud
induction this process proceeds even during starvation, at a time when nerve
cell determination ceases in other parts of the animal; (2) by immigration of
stem cells committed to become nerve cells from outside the future bud's
tissue. Uncommitted stem cells appear to enter the area of the future bud only
rarely, as a result of random movement. These findings raise the question as to
what extent the increase of the nerve cell density in head and foot regenerating
tissue and the normal replacement of nerve cells during steady state growth of
these structures is also caused by immigration of committed stem cells.
The duration of the developmental period of the primary nerve cells from
end of S-phase onwards varies among animals. They become detectable,
synchronously, at 5 h after the bud's evagination begins. Thus the differentiation of the primary nerve cells is under strong control of the bud anlage
25-2
384
STEFAN BERKING
(whatever that may be). Nerve cells start to protrude processes at about 6 h
after mitosis (David & Gierer, 1974). Thus it is argued that the precursors of
the primary nerve cells undergo mitosis synchronously around the beginning
of evagination.
Budding in normal and epithelial hydra
In normal hydra the position of the bud was shown to be specified in two
steps. First, a belt-like area is specified at a certain distance from head and
foot; second, a small area within the circumference of this belt is specified
(Berking & Gierer, 1977). The kinetics of bud development were studied by
applying inhibitor pulses to the animal at various times. Four qualitatively
different developmental phases can be distinguished between a stage where
the final position of the future bud is not yet specified and the beginning of
evagination (Berking & Gierer, 1977). The experiments in this paper indicate
a tight correlation between these phases and the development of stem cells to
the bud's nerve cells. Both the start of bud development and the commitment
of the future bud's new nerve cells are initiated by feeding; both processes are
not restricted to the presumptive bud's tissue but extend into a rather large area.
In the following time, pulses of inhibitor applied within certain periods are
able to cancel both preparatory bud development and the development of stem
cells to the nerve cells of the bud. It is certainly possible, although unlikely,
that the inhibitor affects both processes independently at the same time. It is
more plausible that one process has a strong influence on the other.
The occurrence of buds in epithelial hydra (Campbell, 1976) which are
depleted of interstitial cells and their derivatives, the nerve cells and the
nematocytes, has shown that epithelial cells alone can produce all the requirements for bud formation.
Thus nerve cell commitment may be argued to depend on signals generated
by epithelial cells. However, the kinetics of nerve cell commitment as determined
by means of inhibitor treatment was found to be the same during preparatory
bud development and during growth throughout the whole animal. It thus
appears difficult to understand how the inhibitor should affect the bud's
pre-pattern which in turn affects the commitment of stem cells to nerve cells
when nerve cell commitment alone shows the same kinetics. In addition, the
effect of the inhibitor on budding and on nerve cell formation cannot be
explained by a single mechanism.
On the other hand, a simple possible explanation would be that in normal
hydra not only epithelial cells but other cell types also have an influence on
budding. The experiments with epithelial hydra do not exclude that in normal
hydra cell types which are not present in epithelial hydra effectively control
budding. These cells may just be faster or more efficient than epithelial cells
in generating the appropriate signals themselves or in stimulating epithelial
cells to generate these signals earlier than epithelial cells would on their own.
Nerve cellformation in preparatory bud development in Hydra 385
A possible role of nerve cell precursors in the control of bud formation
One possible explanation for the experimental data is that the development
of the future nerve cells of the bud is the velocity-determining step in preparatory
bud development. According to this hypothesis evagination is triggered if
a certain density of nerve cell precursors which have reached G2-phase is attained
in the presumptive bud's tip. These precursors will differentiate as primary and
secondary nerve cells. Furthermore, within a short period at the very beginning
of the inhibitor sensitive phase (ISP) stem cells are most sensitive to inhibitor.
Based on these assumptions the influence of the inhibitor on preparatory bud
development consists of only one effect, to prevent stem cells from becoming
nerve cells:
The first phase of bud development starts in all animals immediately after
the end of the first treatment. This phase was found to be the most sensitive
one. Phase 1 has a variable length between 1 and 10 h. Evagination starts at
12 h after end of phase 1. The explanation would be that the first phase
represents the period in which precursors of nerve cells which later contribute
to the threshold density when they reached their G2-phase are still in the very
early, i.e. the most inhibitor sensitive, part of their ISP.
At the end of phase 1 the last of these 'necessary' precursors leaves the most
inhibitor sensitive part of its ISP and continues to travel through S-phase,
which takes about 12 h. When it enters G2-phase the threshold density is
reached, therewith triggering evagination. The variable length of phase 1 may
reflect differences between the animals in the ability to recruit the appropriate
number of stem cells for the development into nerve cells in the future bud's
tissue.
The second phase represents the period in which necessary precursors are
still in their ISP but not at its very beginning.
A second treatment within this period was found to prevent stem cells from
becoming committed to develop into the secondary nerve cells. After the
second treatment a fresh start into secondary nerve cells takes place. This can
explain why a second treatment within the first 6 h after the first treatment
influences the bud anlagen of all animals. They are all within their first or
second phase of bud development. The treatment cancels the result of the
bud's preparatory developmental processes from the end of the first treatment
onwards. After the end of the treatment the development starts again with
phase 1.
After the 7th hour some bud anlagen have become insensitive to inhibitor
(3rd phase). The first buds which will be formed after the second treatment
start evagination at the same time as the first of a control group treated only
once. However, the number of newly formed buds per time unit is lower in
the group treated twice and a plateau, a maximal frequency of formed buds, is
reached. The slope of the curve which describes this increase per unit time
386
STEFAN BERKING
depends on the beginning of the second treatment (the later the steeper), not
on its end. Further, the slope of the curve does not depend on the concentration
of the inhibitor applied if it is above a certain threshold. The explanation would
be that a treatment which starts 7 h after the first treatment is no longer able
to prevent the commitment of all stem cells (the ISP has a length of less than 7 h).
The result is that the density of committed stem cells in animals treated twice
is lower than normal, also in the tissue of the future bud and its surrounding.
The later the treatment starts, the more stem cells have been committed and
left their ISP. Thus the number of stem cells which have been committed is
determined by the beginning of the second treatment, not by its duration and
not, above a certain threshold, by the applied concentration. Consequently
the threshold density in the future bud can be attained only if committed stem
cells migrate into the future bud's tissue from an abnormally large area, and
this would take more time. Thus the treatment causes some bud anlagen to
not attain the threshold density at all, and some only after an unusually long
period.
The fourth phase starts immediately before evagination. The effect of an
inhibitor treatment is almost immediately reversible. Its explanation may
require a further assumption.
It should be emphasized that this discussion of the role of nerve cell precursors
in control of budding - as one possible explanation of the data - does not
exclude the involvement of epithelial cells. However, it may indicate that if cell
types other than epithelial cells are present in animals, some of them can
effectively control budding just by being faster or more efficient than epithelial
cells to generate the signals which finally push epithelial cells to start evagination.
In normal hydra, as in epithelial hydra, epithelial cells may initiate bud
development by generating the pre-pattern of the bud which causes, if possible,
a local accumulation of nerve cell precursors in the future bud's tissue and
later on evagination. In normal hydra, however, it cannot be excluded that
even the very beginning of budding is controlled by precursors of nerve cells.
It is possible that they control their own accumulation out of the presumptive
bud's surroundings, like slime moulds in fruiting body formation control their
own accumulation.
I would like to thank Dr W. Miiller for support and providing laboratory space; Dr
A. Gierer, Dr H. Meinhardt and L. Graf for critical reading of the manuscript, R. Heisner
for excellent technical assistance and the Deutsche Forschungsgemeinschaft for support.
REFERENCES
S. (1977). Bud formation in Hydra: Inhibition by an endogenous morphogen.
Wilhelm Roux' Arch, devl Biol. 181, 215-225.
BERKING, S. (1979a). Analysis of head and foot formation in Hydra by means of an
endogeneous inhibitor. Wilhelm' Roux Arch, devl Biol. 186, 189-210.
BERKING,
Nerve cellformation in preparatory bud development in Hydra 387
S. (19796). Control of nerve cell formation from multipotent stem cells in Hydra.
J. Cell Sci. 40, 193-205.
BERKTNG, S. & GIERER, A. (1977). Analysis of early stages of budding in Hydra by means
of an endogeneous inhibitor. Wilhehn Roux1 Arch, devl Biol. 182, 177-129.
BERKING.
BODE. H. R., BERKING, S., DAVID, C. N., GIERER, A., SCHALLER, H. C. & TRENKNER, E.
(1973). Quantitative analysis of cell types during growth and morphogenesis in Hydra.
Wilhehn Roux' Arch. EntwMech. Org. Ill, 269-285.
CAMPBELL, R. D. (1976). Elimination of Hydra interstitial and nerve cells by means of
colchicine. /. Cell Sci. 21, 1-13.
DAVID, C. N. (1973). A quantitative method for maceration of Hydra tissue. Wilhehn Roux'
Arch. EntwMech. Org. Ill, 259-268.
DAVID, C. N. & CAMPBELL, R. D. (1972). Cell cycle kinetics and development of Hydra
attenuata. 1. Epithelial cells. / . Cell Sci. 11, 557-568.
DAVID, C. N. & GIERER, A. (1974). Cell cycle kinetics and development of Hydra attenuata.
III. Nerve and nematocyte differentiation. /. Cell. Sci. 16, 359-375.
Li, H. P. & YAO, T. (1945). Studies of the organizer problem in Peimatohydra oligactis.
III. Bud induction by developing hypostome. /. exp. Biol. 21, 155-160.
OTTO, J. J. & CAMPBELL, R. D. (1976). Budding in Hydra attenuata bud stages and fate map.
/ . exp. Zool. 200, 417-428.
SPROULL, F. & DAVID, C. N. (1979). Stem cell growth and differentiation in Hydra attenuata.
I. Regulation of self-renewal probability in multiclone aggregates. /. Cell Sci. 38 155-169.
WILBY, O. K. & WEBSTER, G. (1970). Studies on the transmission of hypostone inhibition
in Hydra. J. Embryol. exp. Morph. 24, 583-593.
{Received 27 March 1980, revised 4 June 1980)