/. Embryol. exp. Morph., Vol. 17, 1, pp. 35-81, February 1967
Printed in Great Britain
35
Observations on the discovery of a
dorso-ventral axis in Hydra
By RONALD V. BAIRD 1 & ALLISON L.BURNETT 2
From the Department of Biology, Illinois Institute of Technology,
and Department of Biology, Western Reserve University
I. INTRODUCTION
By following the development of buds of Hydra pseudoligactis following experimental
treatment of the parent animal, it is shown that, once bud initiation has occurred, bud morphogenesis proceeds independently of parental regulation. It is proposed that the factors regulating body form, the tentacle pattern, and the two axes of the bud are produced by the
developing bud. It is also demonstrated that a dorso-ventral axis, apparent during bud
development, persists at the level of the growth and budding regions after bud maturation. The hypothesis is proposed that the growth regulatory effect of the dorso-ventral axis
of polarity is demonstrated by the formation of a bilaterally symmetrical bud tentacle
pattern, by the orientation of the regenerated tentacle pattern and by the determination of the
site of the first bud produced after maturation or starvation of animals. A possible
mechanism of the action of the dorso-ventral axis of polarity in bud initiation, development
and maturation is discussed in terms of a model of bud development.
Form, in the biological sense, exists from the molecular to the organismal
level of organization. That is, as a result of the activity of the molecular components or products of cells, proliferation and differentiation of cells occur.
These processes result in diverse populations of cells. Differential activity of cell
populations, in turn, is manifested as body regions! Finally, the appearance of
body regions results in the gross structural differentiation constituting the
characteristic form of the organism. The interaction and regulation of these
processes at all levels are evident in view of the uniformity of the net process
and the end result. Since levels of organization are conceptual in the sense that
their limits are not always well denned, analysis at one specific level of organization leads into other levels of organization. Answers to questions, for example,
posed at the organismal level present questions to be answered at the tissue and
lower levels of organization. At each level, the structural components possess
form and activity. The goal of the developmental biologist is to describe the
components with respect to their form and the regulation of their activity during
development.
1
Author's address: Department of Biology, Illinois Institute of Technology, Chicago,
Illinois.
2
Author's address: Developmental Biology Center, Department of Biology, Western
Reserve University, Cleveland, Ohio 44106.
3-2
36
R. V. BAIRD & A. L. BURNETT
Conceivably, during the course of development, the morphogenetic activity
and form of the structural components of the developing system progressively
change, thereby producing a progressive change in the form of the organism.
Since form embodies the spatial relationships of the structural components
constituting the pattern of the system, development can be considered as the
process of construction of a series of patterns until the final pattern of the
organism is attained. The question then arises whether development is an interaction between the developing and non-developing components of the system
or whether development, once initiated, is regulated by morphogenetic phenomena restricted to the developing component of the system. There must be an
interaction between the components of a system in order for part of a nondeveloping system to give rise to a developing system or for part of a uniformly
developing system to begin developing in a differential manner. Analysis of a
developmental system in this frame of reference must answer the following
questions: (1) What is the relationship of the developing and non-developing
components of a developing system? (2) What is the relationship of the developing components to the final form of the system?
Hydra is useful in developmental studies because of the relative structural
simplicity of the animal. The tubular body consists of an outer epidermis separated from an inner gastrodermis by a mesolamella. The continuity of the body
is interrupted distally by a ring of hollow tentacles surrounding the oral opening,
the hypostome. Proximally, the body of the animal terminates in the abrupt
convergence of the body walls forming a basal disc with an aboral pore. The
linear body of the animal is differentiated into seven body regions: hypostome,
tentacle region, growth region, gastric region, budding region, peduncle and
basal disc. Hydra is essentially a bipolar system of growth (Tripp, 1928; Brien
& Reniers-Decoen, 1949; Burnett, 1959) in that cells are sloughed from the
distal tips of the tentacles apd the proximal basal disc. These cells are replaced
by the movement of cells, distally and proximally, originating primarily in the
growth region located at the base of the tentacles. Consequently, the cells
comprising the gastric region eventually become the cells of the peduncle before
being sloughed off the base of the animal. Cells in the bases of the tentacles
traverse the length of the tentacles before being sloughed off the tentacle tips.
Thus, hydra do not age in the classical sense of the word. The continuous production of cells, primarily in the growth region, replaces lost cells and results in
an animal that does not stop growing (Brien & Reniers-Decoen, 1949; Burnett,
1959).
Burnett in his early growth model (1961) has shown that the hypostome produces a growth-stimulating factor resulting in a region of active growth proximal
to the hypostome. Burnett hypothesizes that cellular activities in the growth
region, in turn, produce a growth-inhibiting factor. Both the stimulatory and
inhibitory factors diffuse proximally and distally from the growth region, and
during this process the inhibitor becomes less active as it passes down the
Dorso-ventral axis in Hydra
37
animal. This results in a progressive alteration of the stimulator:inhibitor ratio
along the body column. Burnett (1961, 1966) suggests that growth is regulated
by the net activity of the stimulator. At the level of the budding region, the net
activity of the stimulator is sufficient to form additional growth regions which
produce buds and additional inhibitor. L. Davis (personal communication) has
successfully isolated an inhibitory factor from hydra culture water and from the
tissues of hydra. Burnett (1962) and Lesh & Burnett (1964) have isolated a
growth-stimulating factor from the tissues of hydra.
In addition to reproducing sexually by forming gonads in the epidermal layer
of cells, hydra also reproduce asexually by producing buds in a restricted level
of the body column, the budding zone. Buds begin as a localized outpushing
of the body wall of the parent animal (Braem, 1910;Kanajew, 1930;Brien, 1955).
Tentacles are elaborated, a hypostome forms, a peduncle develops, and finally
a basal disc is formed. Basal-disc formation is followed by detachment of the
bud from the parent animal. After a period of growth, the detached animals
begin producing buds. During the period before producing buds, recently
detached animals increase in size and additional tentacles are frequently
formed.
Developing tentacles on buds form a pattern relative to the disto-proximal
axis of the parent animal (Haacke, 1879; Hertwig, 1906; Koelitz, 1910; Hyman,
1930; Rulon & Child, 1937). Rulon & Child (1937) speak of the dorsal side of
the bud (Koelitz, 1910) being more active than the ventral side of the bud
while discussing the developmental sequence of the first six tentacles, and
explain the difference in activity as a reflexion of the origin of the dorsal side of
the bud from a higher level of a tentacle regeneration gradient of the parent
animal (Weimer, 1928; Rulon & Child, 1937). More specifically, there are five
polar characteristics of the bud tentacle pattern of Hydra pseudoligactis (see
also Section III A):
(1) After the synchronous appearance of the first two tentacles, tentacle
formation alternates between the dorsal and ventral aspects of the bud.
(2) Tentacle formation on the dorsal side of the bud precedes tentacle formation on the ventral side of the bud.
(3) There are usually more tentacles produced dorsally on the bud than
ventrally.
(4) The pattern is essentially bilaterally symmetrical.
(5) The dorso-ventral axis of the bud coincides with the disto-proximal axis
of the parent animal.
Although the dorso-ventral polarity is apparent only at the distal end of the
bud, the regularity of the tentacle pattern indicates a significant difference in
the developmental capacity of the two sides of the bud. In view of the normal
growth pattern of hydra, there may be a dorso-ventral difference between the
two sides of the mature animal.
In terms of the budding process of hydra, the two general questions raised
38
R. V. BAIRD & A. L. BURNETT
earlier can be restated: (1) What is the developmental relationship between the
parent animal and bud morphogenesis? (2) Is there a relationship between the
morphogenetic phenomena of bud development and maintenance of the
definitive form of hydra? Answers to these questions would provide insight into
the more general issues of localization of the morphogenetic processes in a
developing system and the possible role of developmental phenomena in the
maintenance of the form of the definitive, i.e. non-developing, system. The
approach used in this analysis will utilize surgical deletion, grafting, and marking
techniques of experimental embryology.
II. MATERIALS AND METHODS
-, „ ,
A. Culture methods
u
1. Mass cultures
The animals used in all experiments were from a clone of Hydra pseudoligactis and were cultured at 20 °C under constant illumination in bicarbonateversene-distilled water to which calcium had been added (modified from
Loomis & Lenhoff, 1956). Two stock cultures were maintained in 8 in. preparation dishes filled with culture water. The cultures were fed Artemia salina
nauplii every other day. After the hydra had been allowed to feed for 1 h, they
were removed from the dish by rubbing the inside surface of the bowl and then
pouring the culture water containing hydra and uningested Artemia into a
strainer. Only Artemia and small detached buds passed through the strainer.
Tap water was run over the animals in the strainer to remove uningested
Artemia and the strainer was placed in a small bowl containing tap water while
the culture dish was cleaned. The animals were then transferred to the clean
culture dish by pouring fresh culture water over the inverted strainer. In this
manner, approximately 800 animals were cultured in a single bowl. Aeration of
the cultures was observed to increase the incidence of budding animals and to
reduce the incidence of sexual animals. Consequently, the cultures were continuously aerated by gently bubbling air through an air stone.
2. Experimental animals
All experimental animals were cultured in 60x15 mm dishes containing
25-30 ml culture water. The lids were left ajar to permit circulation of air. In
order to determine optimum culture conditions, the effect of population density
and of the interval between feeding upon the budding rates of untreated animals
were investigated.
(a) Population density. Preliminary experiments suggested that the budding
rate tends to decrease as the number of animals per dish is increased. The
observed differences, however, were not statistically significant.
(b) Feeding interval Five non-budding animals were placed in each of six
dishes and the interval in days between feeding was varied from zero to five.
The animals were cleaned daily. A 30-day record was kept of the number of
Dorso-ventral axis in Hydra
39
buds detaching per dish. Detached buds were removed from the experimental
dishes. The results (Fig. 1) show that the budding rate for a 30-day period
decreases as the interval between feedings is increased. Although the highest
budding rate was in those animals fed daily (zero days interval), these animals
developed abnormally long budding regions with as many as ten buds attached
at one time.
100 -i
90 80 70 60 50 40 30 20 10 -
0
1 2
3
4
Days between feedings
5
Fig. 1. The effect of increasing the interval between feedings on the
budding rate during a 30-day period of time.
(c) Standard culture conditions. All experimental animals were 24 h starved
animals. A regime of feeding animals every other day was selected for experimental animals and, unless single animal culture was required, five animals
were cultured in each dish. Individually cultured animals were cleaned every
other day after the animals had been fed. On alternate days between feedings,
fresh culture water was added to the dishes. Dishes containing five animals were
cleaned daily. Upon completion of an experiment, both experimental and
control animals were discarded although most of the buds detaching from both
groups of animals during the course of an experiment were returned to the stock
cultures. Used culture dishes were washed in 7 X (Lindbro Chemical Co. Inc.),
rinsed five times in distilled water and air-dried before being re-used.
40
R. V. BAIRD & A. L. BURNETT
B. Staging of animals
The development of a bud is a continuous process and generally takes 6 or 7
days. However, by using the appearance of well-defined anatomical structures
of the bud, the process can arbitrarily be divided into six stages (modified from
Yao, 1945, and Burnett, 1961). Although each stage persists approximately 24 h,
the duration of each stage may be variable.
Stage 0. Animals fed Artemia nauplii accumulate the pigment of the Artemia
in gastrodermal digestive cells of the gastric and budding regions. Twenty-four
hours before the appearance of a bud, there is a well-defined line of demarcation
between the pigmented cells of the gastrodermis of the budding region and the
non-pigmented cells of the peduncle.
Stage I. The first sign of bud formation is the appearance of an outpushing
of the parental body wall in a restricted area of the budding region. Initially the
outpushing is rounded, but later it develops a small pimple-like structure at the
distal tip.
Stage II. This stage is characterized by the formation of the first two tentacles.
Stage III. The characteristic feature of this stage is the formation of the third
and fourth tentacles.
Stage IV. In this and subsequent stages, tentacle formation continues. However, the characteristic event is the development of the peduncle. This is recognized by the proximal appearance of gastrodermal cells lighter in color than the
gastrodermal cells of the distal regions of the bud. This region also becomes
elongated and narrower than the more distal regions of the developing bud.
Stage V. During this stage, the basal disc of the bud is formed. The physical
separation of the bud from the parent animal becomes more apparent as the
development of the bud is completed.
Stage VI. The buds have detached from the parent animals, and, compared with mature (budding) animals, are characterized by their small size.
.,,-,..
C. Experimental techniques
1. Excision
*
*
Excisions were performed under the x 1-5 magnification of a dissecting
microscope. Individual animals were placed in a Petri dish containing culture
water and left undisturbed until they had become extended. The animals were
then severed by a single cut with a curved-blade scalpel. Portions of the animals
adhering to the scalpel were pushed off the scalpel with forceps and the desired
portions of the animals were then transferred with a pipette to a small culture
dish.
2. Grafting
Grafts of entire body regions were performed in paraffin-lined preparation
dishes containing short pieces of vertically embedded human hair and filled with
culture water. Animals to be grafted were transferred to the dishes and allowed
Dorso-ventral axis in Hydra
41
to relax. The animals were then severed with iridectomy scissors and the pieces
were oriented on the hairs with the hair passing through the enteron of each
piece. The culture water level was then lowered to the top of the hairs, thereby
holding the pieces in contact with each other and preventing the animals from
climbing off the hairs. After allowing the animals to remain undisturbed for a
minimum of 2 h, culture water was added to the dish and the grafted animals
were gently pushed off the hairs and transferred to small culture dishes containing culture water. Unsuccessfully grafted animals were discarded. Twentyfour hours after grafting, the animals were fed and subsequently maintained
under standard culture conditions.
-45°
\
45'
^ /
90°
-90°
-135°
/
\
135
180°
Fig. 2. Diagram showing the classes of data involving measurements of the distance
and direction between two lateral points of reference.
Mid-point
0°
45°
90°
135°
Class
- 2 2 - 5 - 22-5°
22-5- 67-5°
67-5-112-5°
112-5-157-5°
Mid-point
180°
-135°
- 90°
- 45°
Class
157-5—157-5
-157-5—112-5
- 1 1 2 - 5 — 67-5
- 6 7 - 5 — 22-5
3. Markers
Three techniques were utilized to mark particular sides or regions of animals.
Vital dye staining was used to mark particular regions of buds, and the position
of specific tentacles and/or buds was used to mark specific sides of animals.
(a) Vital dye. Developing buds were marked by touching the tip of a drawn out
capillary tube containing a 5 % solution of Nile Blue Sulfate (Weimer, 1927) in
5 % agar to the desired region of the bud. If necessary, the animals were remarked on subsequent days to prevent loss of the marker.
(b) Tentacle. Tentacles were marked by excising approximately the distal
three-fourths of a specific tentacle on stage IV buds. Cropping of the tentacle
was done by pinching the tentacle with forceps and then quickly pulling on the
pinched tentacle. Once a particular tentacle had been cropped, the marker was
42
R. V. BAIRD & A. L. BURNETT
maintained by recropping when necessary. Since cropping a tentacle produces an
opening for the liquid contents of the enteron to leak out, cropping of tentacles
was always performed before the animals were fed to prevent fouling of the
water and subsequent depression of the animals. Whenever the identification
of the marker tentacle was not certain, the animal was discarded. Occasionally
the marker was lost by the formation of a new tentacle of equal length or by the
marker tentacle growing to the same length as other tentacles. In some instances,
the animal became physiologically depressed and the marker was obscured by
clubbing or disintegration of tentacles. In all cases, these animals were discarded.
D. Grouping of data
In all experiments involving estimation of the circumferential distance
between two lateral points on a given animal, the perimeter of the animal was
arbitrarily divided into eight 45° arcs as shown in Fig. 2. The position of the
point of reference (0°) was taken as the mid-point of a 45° arc. If the shortest
distance from the first to the second point of reference was in a clockwise
direction, the distance was given a positive sign or, if it was in a counterclockwise direction, a negative sign. Each class of data, representing all points
within one of the eight 45° arcs, is designated by the mid-point of that class for
the sake of clarity.
III. RESULTS
A. Preliminary observations of the tentacle pattern of developing buds
The normal pattern of tentacle formation on developing buds of Hydra
pseudoligactis was determined by following the daily progress of tentacle
development on budding animals. Twenty-five non-budding (stage 0) animals
were cultured individually under standard conditions, and a 14-day record was
kept of the relative positions of the tentacles on each bud produced. Detached
buds were discarded.
During the 14-day period, a total of thirty-nine buds were produced. At the
time of detachment, four buds each had four tentacles, thirty-four buds each
had six tentacles, and one bud possessed seven tentacles. In all of the buds, the
relative positions and chronological sequence of the first four tentacles were
identical (Fig. 3d). As shown in Fig. 3a, the side of the developing bud closer to
the distal region of the parent animal is considered to be dorsal. The first.two
tentacles appear synchronously on the mid-lateral sides of the bud (Fig. 3 b).
The tentacle forming on the right of the bud is designated no. 1 and the tentacle
on the left as no. 2. These numerical designations are used only to distinguish
between two tentacles appearing at the same time and are in no way intended to
indicate a chronological difference. However, none of the remaining tentacles
generally appear synchronously and the numerical designations represent a
chronological sequence. After the appearance of the first two tentacles,
tentacle (T) no. 3 forms dorsally midway between T l and T 2 (Fig. 3 c).
Dorso-ventral axis in Hydra
43
Tentacle no. 4 then develops ventrally midway between T 1 and T 2 (Fig. 3d).
In 26 of the 35 buds, the fifth tentacle formed dorso-laterally between T 1 and
T 3 and the sixth tentacle formed dorso-laterally between T 2 and T 3 (Fig. 3e).
On eight of the thirty-five buds, the order was reversed, with T 5 forming
between T 2 and T 3 and T 6 forming between T 1 and T 3 (Fig. 3/). One bud
Distal parent
I
Dorsal aspect of bud
O
I
I
Ventral aspect of bud
•
Proximal parent
Fig. 3. Diagrammatic representation of the developing bud tentacle pattern of
Hydra pseudoligactis. (a) Distal view of the bud prior to tentacle (T) formation
showing the polar relationships of the bud and parent animal, (b) T 1 and T 2 form
laterally and synchronously, (c) T 3 develops mid-dorsally between T 1 and T 2.
{d) T 4 appears mid-ventrally between T 1 and T 2. (e) T 5 is produced dorsolaterally between T 1 and T 3 or, as shown in (/), between T 2 and T 3. (g) T 6 is
formed dorso-laterally in the interval unoccupied by T 5 between T 2 and T 3 or, as
shown in (h), between T 1 and T 3. (/) T 7 forms ventro-laterally between T 1 and
T 4 or, as shown in (J), between T 2 and T 4. (k) T 8 is produced ventro-laterally in
the unoccupied interval between T 2 and T 4 or between T 1 and T 4.
formed T 5 between T 2 and T 4 and T 6 between T 2 and T 3. The single
seventh tentacle formed appeared between T 1 and T 4 (Fig. 3 i). The observations regarding the positions of T 7 and T 8 were made on detached buds.
These observations show that the relative positions of tentacles on developing
buds are highly ordered. In these animals and several hundred animals cultured
44
R. V. BAIRD & A. L. BURNETT
individually or in small groups and examined daily, no variation has been
observed in the relative positions or chronological sequence of the first quartet
of tentacles. However, variants have been observed in the mass cultures. Buds
have been observed with one distal tentacle or with the third tentacle on the
ventral aspect of the bud. Since these variants have not been observed among the
experimental animals, they are concluded to be the result of culture conditions—
possibly physical injury during the cleaning procedure—or as the result of
cannibalism rather than representing the normal tentacle pattern. Although T 5
usually appears between T 1 and T 3, the relatively frequent position reversal
of T 5 and T 6, often observed on different buds on the same animal, is considered to be a normal variation of the tentacle pattern (Fig. 3g). Tentacle
formation generally ceases once six tentacles have been formed although animals
with seven or eight tentacles are not rare. Again, the position reversal of T 7
and T 8 (Fig. 3 h) occurs frequently and will be considered normal.
B. The developmental relationship between bud morphogenesis
and the parent animal
During the early stages of bud development, the anatomical boundaries
between the bud and the parent are indistinct. It is not until after the peduncle
of the bud forms that the junction between the parent animal and the bud
becomes well defined. Because of the anatomical continuity, the morphogenetic
factors giving rise to bud development may be localized in either the parent or
in the developing bud. Bud development may also be the result of interaction
between the bud and the parent animal. If bud development is the result of interaction between the bud and parent animal, removal of the regulating parts of
the parent would result in altered bud development. The following experiments
were performed to investigate the effect upon bud development of progressive
removal of regions of the parent animal.
1. The effect of excision of the distal regions of the parent on bud development
Ten animals at each of the first six stages of bud development were selected
from the stock cultures. The distal region of five of the ten animals in each
group was excised by making a transverse cut between the gastric and budding
regions. The distal regions were then discarded. The remaining five animals at
each stage of bud development received no surgical treatment. Each group of
five animals was cultured in small culture dishes and all animals were starved
during the 10-day duration of the experiment. Daily observations were made of
bud development and the tentacle pattern of all buds. The number of attached
buds was recorded on the last day of the experiment.
The results from two identical series of experiments (Table 1) show that the
bud tentacle pattern and body form are not influenced by excision of the distal
portion of the parent animal. The tentacle pattern on all developing buds,
regardless of their stage of development at the time of excision, was normal.
Dorso-ventral axis in Hydra
45
A f-test for matched samples was performed on the difference between the total
number of buds produced (detached plus attached buds) combining data from
the two experiments for groups of five animals at each of the six stages of
budding. This analysis showed no significant difference in the number of buds
produced by animals with excised distal regions and the non-excised control
Table 1. The effect of excision of the parental regions distal to
the budding region on bud initiation and development*
Parental
regeneration.
Series:
Abnormal
bud tentacle
patterns.
Series:
Initial stage
Detached
buds.
Series:
Attached
buds.
Series:
development
a
b
a
b
a
b
a
b
0
4
6
8
6
8
6
5
5
6
8
9
31
40
2
4
7
7
8
6
6
7
5
7
5
7
33
38
4
0
0
0
3
0
1
0
0
0
3
0
11
0
3
0
2
0
3
0
2
0
2
0
0
1
12
1
0
—
0
—
0
—
1
—
0
—
0
—
1
—
0
—
1
—
0
—
1
—
1
—
1
—
4
—
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
A
0
I
II
III
IV
V
Total
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
* Observations made; over a 10-day period of time.
animals (f u = 1*1, P > 0-10). The same analysis applied to the difference
between the number of detached buds produced by the control and experimental animals, again combining data from two experiments, shows a significantly greater number of detached buds for the control group (tn = 2-25,
P < 0-025). Bud initiation is apparently unaffected whereas bud development
is retarded by removal of the distal regions of the parent animal. Since neither
experimental nor control animals were fed during the course of the experiment,
bud initiation ceased shortly after five or six buds had been formed by each
group of animals. It was therefore impossible to test the long-term effects of
excision of the distal parent animal. However, the absence of any short term
effects of distal parent excision upon bud development suggests that development of the body form and tentacle pattern of the bud is independent of the
regions of the parent animal distal to the budding region.
46
R. V. BAIRD & A. L. BURNETT
2. The effect of excision of the proximal regions of the parent on bud development
A similar experiment was performed to analyse the relationship between bud
development and the proximal regions of the parent animal. Two groups of
thirty animals were selected from the stock cultures starved for 24 h. Each
group consisted offiveanimals at each of the first six stages of bud development.
The parental peduncles were excised and discarded and the five animals bearing
single buds at the same stage of development were cultured in the same dish.
The five control animals at each stage of bud development were also cultured in
Table 2. The effect of excision of the parental regions proximal to
the budding region on bud initiation and development*
Iniitial stage
rfbud
dev elopment
III
IV
V
Total
a
3
4
6
8
3
12
11
11
7
13
7
10
9
12
12
8
49
66
ON
II
Abnormal
bud tentacle
patterns.
Series:
A
1
b
ON
I
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Parental
regeneration.
Series:
A
A
t
7
9
9
10
12
10
43
47
a
ON VO
0
Attached
buds.
Series:
Detached
buds.
Series:
6
6
4
6
6
8
2
3
7
6
34
35
b
a
b
a
b
3
14
9
12
5
9
11
3
7
6
9
7
44
51
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
0
0
0
0
0
0
0
0
0
0
0
* Observations made over a 10-day period of time.
single dishes. All animals were cultured under standard conditions. Daily
observations were made for 10 days of the development and tentacle pattern of
all buds. Detached buds were discarded. On the tenth day, the number of buds
attached to the parent animals was recorded.
The results of the two experimental series are summarized in Table 2. The
body form and tentacle pattern of all buds were normal and statistical analysis
of the pooled data from two experiments showed no significant difference
between the total number of buds produced by the control and experimental
animals (/ n = -1-1945, P > 0-10) or between the number of detached buds
produced by the control and experimental animals (tn = 1-70, P > 0-10). These
Dorso-ventral axis in Hydra
47
results demonstrate the independence of bud development and tentacle pattern
from the regions of the parent animal proximal to the parental budding region.
On the basis of the results of the two preceding experiments, two conclusions
can be drawn: (1) surgical removal of the regions distal to the budding zone,
while retarding bud development, does not affect bud initiation or the bud
tentacle pattern; (2) surgical excision of the regions proximal to the budding
zone has no effect on the rate of bud initiation, development, or tentacle pattern.
Bud development, including the tentacle pattern, does not appear to be regulated
by the regions of the parent animal proximal and distal to the budding region.
The following experiment was performed to test this possibility.
Table 3. The effect of isolation of the budding region on bud
initiation and development*
c
development
Experimental
Control
I
Experimental
Control
II
Experimental
Control
III
Experimental
Control
IV Experimental
Control
V
Experimental
Control
Total Experimental
Control
0
a
0
1
5
6
7
7
8
5
5
7
5
6
30
32
r
Abnormal
bud tentacle
patterns.
Series:
b
c
a
b
c
a
b
c
a
b
c
1
4
7
7
0
4
6
8
5
8
6
5
5
6
8
9
30
40
0
0
0
0
1
0
0
0
3
0
2
0
3
0
4
0
1
0
2
0
1
0
2
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
7
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
0
0
0
0
0
0
0
0
0
0
0
0
0
ON OO
Initial stage
Parental
regeneration.
Series:
Attached
buds.
Series:
Detached
buds.
Series:
7
7
9
7
11
7
43
38
0
0
0
0
1
0
0
2
1
15
1
* Observations made over a 10-day period of time.
3. The development of buds on isolated budding regions
The budding region was excised from groups of five animals at each of the
first six stages of budding. A daily record was kept of the development of body
form and tentacle pattern of all buds and the number of detached buds for each
group of experimental animals and for identical but non-surgically treated
groups of animals. Each group of five animals at the same initial stage of bud
development were cultured in the same dish and all animals were starved during
the 10 days the experiment was in progress. Detached buds were discarded. At
48
R. V. BAIRD & A. L. BURNETT
the end of the experimental period, the number of attached buds in each group
was recorded.
The results of three experimental series, summarized in Table 3, show that
the body form and tentacle pattern of all buds were normal. Statistical analysis
of the combined data demonstrates that there is no significant difference in the
number of buds produced (t17 = 1-41, P > 0-10) or in the number of detached
buds (/17 = 1-0, P > 1-0) between the control animals and the isolated budding
regions. These results support the hypothesis that bud development is not
directly regulated by the regions of the parent animal distal and proximal to the
budding zone.
4. The dorso-ventral axis of the bud tentacle pattern
Since the dorso-ventral axis of the bud tentacle pattern always coincides with
the disto-proximal axis of the parent animal, the disto-proximal polarity of the
parent animal appears to be determining the dorso-ventral axis of the bud. If this
is the case, then reversal of the parental disto-proximal polarity would result in a
reversal of the dorso-ventral axis of the bud. This was tested by grafting excised
budding regions from stage 0 animals back into the animals after the distoproximal axis of the excised budding region was rotated 180°. Grafted animals
were then cultured individually under standard conditions and daily observations were made of the tentacle pattern of all buds.
The budding regions of fifty animals were excised, reversed 180° and grafted
back into place. Twelve grafts were successful. The remaining animals separated
into two or three pieces before the experiment was terminated. Within 1 week
after grafting, the animals began budding and the tentacle pattern of the first
bud on all twelve animals was normal to the disto-proximal polarity of the
budding region but rotated 180° relative to the disto-proximal polarity of the
parent animal. Following the development of one or two buds with rotated
tentacle patterns, the polarity of all subsequent buds, with one exception, was
normal to the disto-proximal polarity of the parent animal. Three animals, after
producing one bud, ceased producing buds and numerous bumps appeared on
the budding region. The bumps initially resembled stage I buds, but no tentacles
were formed. As the older bumps moved down on to the peduncle of the animals,
they lost their rounded appearance, becoming irregularly shaped masses of white
tissue. The form of the peduncle became irregular and occasionally the peduncle
was observed to separate along its length from the body of the animal. During
the next 6 months, none of the abnormal animals became normal in their
appearance. One animal formed a single bud approximately 2 months after
becoming abnormal and the tentacle pattern of this single bud was rotated 180°
relative to the disto-proximal axis of the parent animal.
These observations, while indicating a causal relationship between the distoproximal polarity of the parent animal and the dorso-ventral axis of the bud
tentacle pattern, also indicate that the parent animal is not always able to
Dorso-ventral axis in Hydra
49
regulate a disto-proximal polarity reversal of the budding region and suggest
that reversal of polarity may persist indefinitely. The difference between these
observations and Davis's (1965) experiments where complete success in polarity
reversal was obtained may be a species difference.
5. The development of isolated buds
The following series of experiments were designed to answer questions concerning the developmental relationship between the budding region and bud
morphogenesis. The first question to be considered is whether bud development
is regulated by the budding region or is the result of morphogenetic phenomena
established within the bud at the time of bud initiation. If bud development is
Table 4. The effect of bud isolation on bud development
Stage
II
III
IV
II
III
IV
Day
Stage of
development
No. of
animals
Experimental
I
III
IV
V
V
II
IV
V
III
IV
V
IV
IV
V
V
4
5
Control
I
III
IV
V
V
V
IV
V
III
IV
V
V
IV
V
5
4
1
5
5
5
5
5
3
2
5
5
5
5
5
2
3
5
5
5
5
5
5
5
5
1
Tentacle
pattern
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
5
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
JEEM
Ij
50
R. V. BAIRD & A. L. BURNETT
regulated by the budding region, the development of buds separated from the
budding region would be arrested or anomalous. If, however, bud development proceeds independently of the budding region after bud initiation,
isolated buds would develop normally. The following experiment was carried
out to resolve this question.
Four groups of ten animals, each bearing a single bud, were selected from the
stock cultures. The four groups represented the first four stages of bud development. Five animals, randomly selected from each group, served as non-surgically
treated control animals. The buds were removed from the remaining five animals
per stage of bud development by making two cuts at the junction of the bud and
parent animal. Care was taken to remove as much of the parent animal from the
buds as possible without damaging the buds. The five control animals and five
isolated buds at each stage were cultured in separate dishes. Neither group was
fed during the course of the experiment. A daily record was kept of the development of body form and tentacle pattern of all buds.
There is no apparent difference in the development of buds attached to the
parent animal and those excised from the parent animal regardless of the initial
stage of bud development (Table 4).
Table 5. The effect of bud isolation on the orientation of the bud tentacle
pattern: position of marker relative to the tentacles
Animal
no.
Presumptive
tentacle
no.
Mature bud
tentacle
no.
Difference
(°)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
5
\-4
3-5
6
1-4
6
4
3
2
5
3
6
2
1^
4
5
4
5
6
1^
6
1-4
6
2
5
5
2
2
2-4
4
0
45
45
0
0
0
45
45
0
0
45
45
0
45
0
6. The dorso-ventral axis of isolated buds
In order to rule out the possibility of an alteration of the dorso-ventral axis
of the bud following bud isolation, fifteen stage I buds were marked laterally
with Nile Blue Sulfate. The position of the mark on each bud was recorded
relative to the site of presumptive tentacles. The buds were then excised and,
Dorso-ventral axis in Hydra
51
at the time tentacle development was complete, the position of the mark relative
to specific tentacles was again recorded.
The difference between the original and final position of the mark (Table 5)
is not greater than 45°. This is slightly more than one-half the distance between
two adjacent tentacles and, as such, does not represent a significant alteration
of the dorso-ventral axis of the bud tentacle pattern by isolation of the bud from
the parent animal.
Table 6. The development of body form and tentacle
pattern on isolated stage I buds
Day
Stage of
development
No. of
animals
Tentacle
pattern
Experimental
0
1
2
3
I
III
III
IV
V
—
20
20
6
14
20
Normal
Normal
Normal
Normal
20
20
8
12
20
Normal
Normal
Normal
Normal
Control
0
1
2
3
I
III
III
IV
V
—
7. Maturation of isolated buds
Although the development of isolated buds is normal, the possibility exists
that isolation of buds may result in altered bud maturation. The following
experiment was performed to investigate this possibility. Forty animals, each
bearing a single stage I bud, were selected from the 24 h starved stock culture.
The buds were excised from twenty of the animals. The remaining twenty
animals received no surgical treatment All control animals and experimental
buds were cultured five in a dish and maintained under standard conditions. As
the buds detached from the control animals, the parent animals were discarded.
The development of both control and experimental buds was recorded daily and
the time taken for the animals in both groups to begin budding was measured.
Again the tentacle pattern and body form of the isolated buds were normal
(Table 6). All of the isolated buds were capable of producing buds although the
time required to produce the first bud was generally longer than that for the
control animals (Fig. 4). These experiments show that, once bud initiation has
occurred, body form and tentacle pattern can develop normally without the
influence of the parental budding region. This implies that the morphogenetic
phenomena regulating body form, the tentacle pattern, and the two axes of the
bud are controlled by factors residing in, or produced by, the developing bud.
4-2
52
R. V. BAIRD & A. L. BURNETT
C. The relationship of bud morphogenesis to maintenance
of the adult form
The experiments in the preceding section indicate that the body form and
tentacle pattern of buds develop independently of the parent animal. Once bud
initiation has occurred, the developmental process of the bud is not altered by
any surgical treatment of the parent animal. In other words, bud development
is the expression of morphogenetic processes restricted to the bud. Since this is
the case, the same morphogenetic processes may be operative in the mature
animal. If the dorso-ventral axis of polarity of the bud persists in the mature
animal, it should be possible to demonstrate its developmental effects.
20 -
at
S
'5
Control
00
.5 10
Experimental
J3
5 -
-I
10
1
15
1
20
1
25
30
r
35
Days
y since isolation
Fig. 4. Graphic representation of the maturation of normal and isolated stage I buds.
1. The dorso-ventral axis of polarity at the level of the growth region
(a) The pattern of regenerated tentacles. Ten non-budding animals were excised
immediately below the tentacle bases and the ring of tentacles including the
hypostome was discarded. The animals were cultured individually under
standard conditions and daily observations were made of the relative positions
of regenerated tentacles. The animals were observed until regeneration had
ceased and budding had begun.
The results show that virtually all observed stages of tentacle regeneration
have counterparts in the stages of bud tentacle development (Fig. 5). There is,
however, no counterpart in bud tentacle development to the distally produced
T 3 of the regenerated tentacle pattern of animal no. 10.
Although the positional relationships of the regenerated tentacle patterns are
comparable to those of the bud tentacle pattern, the chronological sequences
of the two patterns are generally not identical. Exceptional in this respect is
animal no. 5 (Fig. 5), where both the positional and chronological relationships
Dorso-ventral axis in Hydra
53
of the regenerated tentacles parallel the same relationships of the bud tentacle
pattern. However, in many of the animals, various intermediate stages seen in
bud tentacle development were not observed. For example, in animal no. 1 the
apparent simultaneous formation of the first three tentacles was followed by the
apparent synchronous development of the fourth and fifth tentacles. This contrasts to the synchronous formation of the first two tentacles followed by the
Bud tentacle development
o -o -6- -<>-
A>
Adult tentacle regeneration
Depressed' A
Fig. 5. Diagrammatic representation of the patterns of tentacle regeneration on ten
adult animals. The patterns of bud tentacle formation are shown for comparison.
diachronic formation of the remaining tentacles of the bud tentacle pattern.
Since observations were made at 24 h intervals, the possibility exists that the
intermediate stages of regeneration were present but simply were not observed.
However, the resultant pattern is identical with the bud tentacle pattern.
(b) The dorso-ventral axis of the regenerated tentacle pattern. Although the
regenerated tentacle pattern and the bud tentacle pattern are considered to be
identical, in order to state that the regenerated tentacle pattern is regulated by a
persisting dorso-ventral axis of polarity of the bud, coincidence must be
demonstrated between the position of specific tentacles formed on the bud and
the corresponding regenerated tentacles. The following group of experiments
54
R. V. BAIRD & A. L. BURNETT
was designed to detect any correlation between the orientation of the two
patterns. .
A preliminary experiment to be discussed later shows that bud movement
relative to the parent animal is restricted to a disto-proximal progression as the
buds develop. There was no evidence of lateral displacement of developing buds.
This was determined by noting the daily position of buds relative to a marked
tentacle of the parent animal. Consequently, it is possible by combining a
marked tentacle and a developing bud to maintain a reference point on the body
of a regenerating animal. In the first experiment, four groups of five stage 0
animals were cultured under standard conditions. As the buds forming on the
animals developed peduncles, one of the tentacles on the developing bud was
cropped. In two of the dishes T 3 was cropped and in the remaining two dishes
T 4 was cropped. As the animals were examined daily, stage IV buds were
marked and buds previously marked were recropped when necessary to maintain the marker. Detached buds bearing a marked tentacle were isolated and
cultured under standard conditions. As the isolated animals began to bud, the
position of the first bud relative to the marked tentacle was noted and a transverse cut was made through the growth region of the budding animal. In this
way, the dorso-ventral aspects of the animal previously indicated by the marked
tentacle were now indicated by the position of the bud. At 24 h intervals the
positions of regenerated tentacles relative to the marker bud were recorded.
Measurement of the degrees of rotation of the regenerated tentacle pattern
from the bud tentacle pattern (Fig. 6) shows 82 % of the regenerated tentacle
patterns within ±45° of the bud tentacle pattern. The largest single class is
composed of regenerated tentacle patterns coinciding with the bud tentacle
pattern (0° rotation). Apparently the regenerated tentacle pattern coincides
with the bud tentacle pattern. However, the dorsal marker bud on all of the
animals may have been orienting the regenerating tentacle pattern. A second
experiment was done using animals with ventral marker buds. The results,
shown in Fig. 7, are essentially the same as the results using dorsally marked
animals. These results demonstrate the coincidence of the dorso-ventral axes
of the bud and regenerated tentacle patterns.
(c) Persistence of the dorso-ventral axis of the regenerated tentacle pattern.
The persistence of the dorso-ventral axis of the bud tentacle pattern in the adult
animal has been demonstrated. However, this was done using recently detached
animals that had not produced more than two buds. In this respect, they were
young animals. The number of tentacles on buds frequently increases after
bud detachment. Consequently, the observed results may indicate only that
tentacle development was. not complete at the time the experiments were
performed. If this is the case, then coincidence of the regenerated tentacle
pattern and the bud tentacle pattern would decrease as the animals age chronologically. Conversely if the dorso-ventral axis of the bud tentacle pattern persists indefinitely, coincidence of the bud tentacle pattern and the regenerated
Dorso-ventral axis in Hydra
55
tentacle pattern would not decrease. These mutually exclusive alternatives were
tested by following tentacle regeneration on chronologically older animals. Ten
animals with T 3 marker tentacles and ten animals with T 4 marker tentacles
were cultured individually under standard conditions. As the animals began
(c)
15 (b)
(a)
10 -,
10 -i
5 -
5 -
IS
S
ca
10 b
C*-l
(•"1
w
d
5b
-TV
180
90
0 -90
180 90
0 -90
135
45 - 4 5 - 1 3 5
135 45 - 4 5 - 1 3 5
i
180
m 90a
i
135
i
a
i
a
i
0
45
b
b
a
a
i
i
i
-90
-45 -135
Degrees of rotation
Degrees of rotation
Fig. 6. Rotation of the tentacle axis during regeneration on T 3 marked animals
(a) and on T 4 marked animals (b). Composite of (a) and (b) shown in (c). Each animal
possesses a single dorsal bud.
(c)
20 - i
15 -
(a)
10 -,
10 -i
5-
5-
n
180
90
0 -90
180 90
0 -90
135
45 - 4 5 - 1 3 5
135 45 - 4 5 - 1 3 5
Degrees of rotation
10 -
5 -
180 135 90 45
0
-90
-45
-135
Degrees of rotation
Fig. 7. Rotation of the tentacle axis during regeneration on T 3 marked animals
(a) and on T 4 marked animals (6). Composite of (a) and (b) shown in (c). Each
animal possesses a single ventral bud.
budding, the markers were maintained and detached buds were discarded.
After 15 days of budding, the animals were starved for 1 week and then fed every
other day. As the animals resumed budding, the position of the first bud was
noted relative to the marker tentacle and the ring of parental tentacles was
56
R. V. BAIRD & A. L. BURNETT
removed. The pattern of tentacle regeneration relative to the marker bud was
recorded at 24 h intervals.
As shown in Table 7, the maximum rotation of the regenerated tentacle
pattern is 45°. This is slightly more than one-half the distance between adjacent
tentacles and, as such, does not represent a significant rotation of the dorsoventral axis of the regenerated tentacle pattern. The dorso-ventral axis of polarity
established during bud development persists in the growth region of mature
animals.
Table 7. The orientation of the regenerated tentacle pattern
on chronologically older animals
T 3 marked
Position of marker bud
o |-i irviJi 1
A
no.
Initially
Terminally
Rotation (°)
1
6
3
+ 45
0
+ 45
0
-45
-45
+ 45
2
3
4
5
6
7
8-10
3
3
3-6
3-5
5
5
1
1-4
4
2-4
2
6
No regeneration
T 4 marked
animal
no.
1
2
3
4
5
6
7
8
9 and 10
1-4
4
6
3
1
4
3-5
5
2-6
2-6
5
5
5
5
1
1
No regeneration
+ 45
+45
+45
+ 45
0
0
0
0
2. The dorso-ventral axis of polarity at the level of the budding region
Based on the high degree of coincidence between the bud tentacle pattern and
the regenerated tentacle pattern observed in the preceding group of experiments,
the conclusion can be drawn that the dorso-ventral axis of polarity of the bud
tentacle pattern persists following bud detachment. This conclusion suggests a
dorso-ventral polarity of the animal with the dorsal side having a greater
physiological activity than the ventral side. Since the persisting dorso-ventral
polarity was shown to be present in the growth region of the adult animal, one
would expect the physiological activity of the more proximal regions of the
animal also to have a dorso-ventral polarity. One of the most striking morphogenetic events of the animal is initiation and development of buds. A difference
Dorso-ventral axis in Hydra
57
in the physiological activities of the two sides of the animal might well have a
regulative role in these processes. If the dorsal side of the animal has the greater
physiological activity as indicated by the dorso-ventral polarity at the level of
the growth region, the first bud would be expected to appear on the dorsal
surface of a recently detached animal.
(a) The position of the first bud. Observations at 24 h intervals of twenty-five T3
marked and twenty-one T4 marked animals cultured individually under standard
conditions following detachment show (Fig. 8) 23 out of 25 (92%) of the first buds
on T 3 marked animals and 19 out of 21 (90 %) of the first buds on T 4 marked
animals appearing within ± 45° of the mid-dorsal surface of the animals. It was
also noted that the position of specific buds relative to the marker tentacles did
not change before detachment. This demonstrates that buds may be used as
accurate markers and shows the existence of a difference in the physiological
activity of the dorsal and ventral sides of chronologically young animals.
1
Fig. 8. Diagrammatic representation of the position of the first bud produced by
twenty-five recently detached T 3 marked animals (a) and twenty-one recently
detached T 4 marked animals (6). The dorsal aspect of each figure is indicated by
the bracket.
(b) Dorso-ventral alternation of bud initiation. Since the animals have a dorsoventral polarity of physiological activity causing the first bud to appear on the
dorsal side of the animal, one might expect continued regulation of the positions
of consecutive buds. The observed dorso-ventral alternation of tentacle formation may have a counterpart in the site of consecutive bud initiation. The relationship between the site of bud initiation and the dorso-ventral axis of the
parent animal was analysed by recording the position of ten consecutive buds
relative to the dorsal and ventral sides of the budding animal. Twenty-five
animals had T 3 marked tentacles and twenty-one were T 4 marked.
A summary of the results (Figs. 9, 10, 11) shows that the site of bud initiation
tends to alternate between the dorsal and ventral sides of the parent animal.
However, the sequence is interrupted by reversals between the fourth and fifth
buds and the eighth and ninth buds on T 3 marked animals. The number of
58
R. V. BAIRD & A. L.BURNETT
24
*
-,
24
20-
<» 20
.§16-1
1 12 Q
8
-
4
-
O
on
"p
16
w
o
12
«Q 8
0
o
^
!
4
i
04
4
-
!
8
-
•I 8
I12
| i 2 -
| 1 6 -
> 2 0 24
i
1
i
2
i
3
r
4
i
5
i
6
i
7
i
8
i
9
24
i
10
1
2
3
4
5
6
7
8
9
Consecutive bud no.
Consecutive bud no.
Fig. 9
Fig. 10
Fig. 9. Graphic representation of the first ten consecutive buds produced relative
to the dorsal and ventral aspects of twenty-five T 3 marked animals. The number of
mid-lateral buds in each group is indicated by the points between the crossbars
on the line representing the distribution of buds in each class.
Fig. 10. Graphic representation of the position of the first ten consecutive buds
produced relative to the dorsal and ventral aspects of twenty-one T 4 marked
animals. The number of mid-lateral buds in each group is indicated by the points
between the crossbars of the graph.
45 40 35 •8
30-
M
25 -
1
20-
10 -
E
B
a
•s
d
Z
5 0
5 10 15 20 25 30 35 40 45
1
2
3
4
5
6
7
8
9
10
Consecutive bud no.
Fig. 11. Composite of Figs. 9 and 10, showing the position of the first ten consecutive buds produced relative to the dorsal and ventral aspects of twenty-five T 3
marked animals and twenty-one T 4 marked animals. The number of mid-lateral
buds in each group is indicated by the interval between the crossbars in the graph.
10
Dorso-ventral axis in Hydra
59
seventh buds forming dorsally was virtually the same as the number forming
ventrally. The same result was observed for the eighth buds. Similarly, the
sequence on T 4 marked animals was interrupted by a reversal at the time of the
fifth bud. The number of dorsal and ventral buds was nearly equal as the fifth,
eighth, and ninth buds were formed. These results, while indicating a dorsoventral alternation of bud initiation, can also be interpreted as indicating a
unidirectional rotation of bud initiation between opposite sides of the animal.
(c) The pattern of consecutive buds. Because of the difficulty in obtaining and
maintaining large numbers of marked animals, measurements of the distances
between consecutive buds on unmarked animals were made. The distance
between two consecutive buds was estimated as described in an earlier section.
Fifty non-budding animals were selected from the mass cultures and cultured
individually under standard conditions. As each animal began budding, daily
observations were made for 15 days. Detached buds were discarded.
The results, summarized in Fig. 12, show that in each group the majority of
consecutive buds are ^ ± 90° from the preceding bud. Although the groups of
buds are not comparable, pooling the data (Fig. 13) illustrates the proportion
of buds forming ^ + 90° from the preceding bud.
As stated previously, the sequential positioning of initiated buds could be interpreted as a unidirectional alternation of bud initiation between opposite sides
of the parent animal. If this is the case, the direction (or sign) of the smallest
angle between consecutive buds would be constant. This is not the case.
Grouping the data, 111 buds were positive to the preceding bud, 94 were directly
opposite, 115 were negative to the preceding bud, and two were distal. There is
therefore no directional preference for the species, nor is there a preferential
direction indicated by any of the groups of consecutive buds. Of the fifty animals
in the experiment, all of the buds on each of only seven animals were initiated
in the same direction from the preceding bud. Two animals produced all buds
either positive to or directly across from the preceding buds and five animals
initiated all buds either negative to or directly across from the preceding buds
The pattern of bud initiation on these animals would approximate a spiral
around the budding region. The remaining forty-three animals appear to present
a pattern of opposing pairs of buds.
These experiments show that the site of initiation of the first bud coincides
with the dorsal side of the parent animal. As subsequent buds are formed, the
pattern of consecutive bud initiation appears to be independent of the dorsoventral polarity of the parental budding region. The position of consecutive
buds subsequently appears to be relative to the position of the preceding bud.
The mechanism of control may be similar to the hypothesis of local inhibition
described by Wigglesworth (1954) to explain the positional relationships of the
epidermal bristles on the abdomen of larval Rhodnius. Apparently development of a bristle inhibits the formation of additional bristles in the immediate
area.
60
R. V. BAIRD & A. L. BURNETT
(d) Persistence of the dorso-ventral polarity of the budding region. If there is a
dorso-ventral polarity at the level of the budding region the apparent independence of the site of the bud initiation from the dorso-ventral polarity of the
parent animal once the first bud has been initiated suggests a reduction or loss
of the parental dorso-ventral polarity at this level. In view of the normal distoproximal growth pattern of the animal, the dorso-ventral polarity at the level
of the parental growth region would be expected to result in a persisting dorsoventral polarity at the level of the budding region. If the dorso-ventral polarity
of the animals does persist at the level of the budding region, it apparently is
ineffective in regulating the site of consecutive buds. Should it persist, it could
15 -
•3
10
15 -
•3 10
"
CO
B
•a
C3
ed
O
z
5-
5 -
0
45 90 135 180
-90
-135
-45
Degrees from bud no. 1 to bud no. 2
i
i
0
45
i
i
i
i
r
i
90 135180
-90
-135
-45
Bud no. 2 to bud no. 3
15 -
15 -\
-310-
«g 10 -
SSH
0
45
I
I
l
l
90 135 180
-90
-135
I
45 90 135 180
-45
-135
-90
-45
Bud no. 4 to bud no. 5
Bud no. 3 to bud no. 4
Fig. 12. Each graph represents the direction and angle of the shortest distance
between groups of consecutive buds produced by fifty animals during a 15-day
period of time.
Dorso-ventral axis in Hydra
61
10 -i
20 -
5-
15•8
6
Z
I
10-
45 90 135 180
0
i
-90
-135
o
Z
-45
Bud no. 6 to bud no. 7
510-
i
0
i i T T
45 90 135 180
I
r _
-90
-135
-45
Degrees from
Bud no. 5 to bud no. 6
sH
I
E
a
J
0
45 90 135 180
-90
-135
O
Bud no. 7 to bud no. 8
I I I I
I
I I
0 45 90 135 180 -90
z
-135
-45
-45
Bud no. 8 to bud no. 9
^
s -i
5 -
e
•a
i
i
0
45
i
i
T
i
90 135 180
r
.
-90
-135
6 0
Z
-45
I
I
0
45
I
I
90 135 180
-135
-90
-45
Bud no. 10 to bud no. 14
Bud no. 9 to bud no. 10
Fig. 12 (cont.)
possibly be detected by the position of the first bud formed on a budding animal
following a period of starvation during which bud initiation ceases. To test this
possibility, twenty T 3 marked animals and twenty-seven T 4 marked animals were
cultured individually under standard conditions and allowed to bud for 2 weeks.
The animals were then starved for 1 week and the position of the last bud initiated
by each animal was noted. The animals were then re-fed until bud initiation
resumed and the position of the first bud on each animal was recorded.
62
R. V. BAIRD & A. L. BURNETT
The data, summarized in Fig. 14, show that 55 % of the last buds formed by
the T 3 marked animals (Fig. 14a) were on the dorsal surface of the animals and
44-4 % of the buds on T 4 marked animals (Fig. 14 c) were on the dorsal surface.
As budding resumed after the period of starvation, 70 % of the newly
formed buds on T 3 marked animals (Fig. 146) and 81-4 % of the new buds on
T 4 marked animals (Fig. I4d) were on the dorsal aspect of the parent animals.
70%
100 9080-
70<«
6 0
"
.§
50 H
•S
40H
o
30%
20%
44-4%
81-4%
44-4%
18-5%
3020-
10-
0
45
90
135 180
-135
-90
-45
Distance between consecutive
buds (°)
Fig.13
Fig. 14
Fig. 13. Composite of Fig. 12, showing the direction and angle of the shortest
distance between all consecutive buds produced by fifty animals during a 15-day
period of time.
Fig. 14. Graphic representation of the position of the last bud formed as budding
animals were starved and the position of the first bud formed when the animals
were re-fed. The numbers inside of each diagram represent the tentacle pattern of
the parent animal and the number outside of each diagram represent the number
of animals forming buds at the indicated positions, (a) The position of the last bud
formed on T 3 marked animals as budding ceased, (b) The position of the first bud
formed on T 3 marked animals as budding resumed, (c) The position of the last
bud formed on T 4 marked animals as budding ceased, (d) The position of the first
bud formed on T 4 marked animals as budding resumed.
In both groups of animals, there was an increase in the incidence of dorsal buds:
15 % of T 3 marked animals and 37 % for the T 4 marked animals. If the position
of the newly formed buds was regulated by the position of the last bud initiated,
the dorso-ventral distribution of the newly formed buds would be approximately
Dorso-ventral axis in Hydra
63
the reverse of the distribution of the last buds formed. This is not, however, the
case. In both groups the incidence of dorsal buds increased irrespective of the
position of the last bud initiated. These data support the hypothesis of a persisting dorso-ventral polarity of the budding region. Although the position of
10 -i
5-
5-
o
0
45
90 135180
-90
-135
I
45 90 135 180
-45
-90
-135
Degrees from bud
no. 1 to bud no. 2
-45
Bud no. 2 to bud no. 3
10 -
20-i
10 H
o
Z
n
45 90 135 180
-135
45 90 135 180
-90
-45
Bud no. 3 to bud no. 4
-135
-90
-45
Composite
Fig. 15. Graphic representation of the direction and angle of the shortest distance
between groups of consecutive buds produced during a 15-day period of time by
fifty animals from which the regions distal to the budding region had been excised.
consecutive buds is regulated by the position of the preceding bud, the dorsoventral axis of polarity of the parent animal regulates the position of the first
bud formed following bud maturation and also regulates the position of the first
bud formed as bud initiation resumes after a non-budding period.
(e) Regulation of the site of consecutive buds. Since it was demonstrated that
consecutive buds tend to form opposite one another, it appears that regulation
of the site of consecutive buds may be achieved at the level of the budding region
rather than by morphogenetic phenomena distal and/or proximal to the budding
region. To investigate this possibility, the effect of excision of the distal or
proximal regions of the parent animal on the pattern of consecutive buds was
determined.
64
R. V. BAIRD & A. L. BURNETT
3. The effect of excision of the distal regions of the parent animal
Two groups of fifty non-budding animals were selected from the stock cultures
and cultured individually under standard conditions. Day 1 for each animal was
taken as the day the first bud appeared. On day 1, each animal in the experi•a 5-,
I o
13 10
'£
T-*H
0
45
1
I
I " I ' I"
90 135 180
-90
-135
et
-45
Bud no. 2 to bud no. 3
5 -
n
0
45
0
90 135180
-90
-135
-45
Degrees from
bud no. 1 to bud no. 2
45 90 135 180
-90
-135
-45
Bud no. 3 to bud no. 4
10-
B
•a
o
Z
5-
I
I
I
i
i
0
45
90
135
180
r
-90
-135
-45
Composite
Fig. 16. Graphic representation of the direction and angle of the shortest distance
between groups of consecutive buds produced by.fifty animals during a 15-day period
of starvation.
mental group was excised at the junction of the gastric and budding regions and
the distal regions were discarded. The animals in both the experimental and
control groups were starved for the 15-day duration of the experiment. Daily
Dorso-ventral axis in Hydra
65
measurements were made of the relative distance and direction between consecutive buds.
Statistical analysis of the data shown in Fig. 15 and Fig. 16 shows the experimental and control results are comparable. In the experimental animals, 44 out
of 61 (72-1 %) of the buds were > ± 135° from the preceding bud and 58 out
of 61 (95-1 %) were ^ ± 90° from the preceding bud. In the control animals, 34
out of 46 buds (73-9 %) were ^ ± 135° from the preceding bud and 45 out of 46
were > ± 90° from the preceding bud. These data are comparable (x2 = 2-26,
P > 0-5). Excision of the distal region of the parent animal has no effect on the
pattern of consecutive bud initiation.
4. The effect of excision of the proximal regions of the parent animal
The pattern of bud initiation on animals with excised peduncles was observed
to determine the role of the proximal parental regions. Fifty non-budding
animals were cultured individually, and as each animal produced the first bud
the parental peduncle was excised and discarded. The shortest distance and
direction between consecutive buds on each animal was then recorded daily for
15 days. Similar observations were made on fifty identical but non-surgically
treated animals. Both groups were maintained under standard culture conditions.
The results, summarized in Figs. 12,13, and 17,18, are comparable (x2 = 6-35,
P > 0-5). The parental peduncle does not regulate the distance between consecutive buds. However, in the control group there was a 1-4 % incidence of new
buds forming proximally to the older buds compared with an 8 % incidence of
proximal buds on the experimental animals.
The data show that removal of the distal regions of the parent animals does
not alter the pattern of consecutive bud initiation. However, since budding
ceased soon after the first few buds were produced, the evidence is only suggestive. The conclusion can be drawn, however, that the proximal regions of the
parent animal do not regulate the pattern of consecutive bud initiation since the
animals were able to feed and budding continued throughout the experimental
period. There is evidence, however, that, although the peduncle does not regulate
the distance between consecutive buds, it may play a role in regulating the
level of the budding region at which bud initiation occurs. At any rate, it appears
that the pattern of consecutive bud initiation is regulated within the budding
region of the parent animal by the location of the preceding bud.
IV. DISCUSSION
A. Normal bud development
Although bud morphogenesis is a continuous developmental process, it consists of three integrated phases each beginning with a distinctive morphogenetic
event. Phase I begins with bud initiation and continues with the elaboration of
the body regions of the bud. Phase II is characterized by tentacle formation.
5
JEEM 17
66
R. V. BAIRD & A. L. BURNETT
15 -i
10 -
o
Z
10 -i
5 -
5 -
o
Z
0 45 90135180 - 9 0
-135 - 4 5
0
45 90135180
-90
-135
Degrees from
bud no. 1 to bud no. 2
-45
Bud no. 2 to bud no. 3
15 -i
10 H
10-
o
5-
5 -
z
o
Z
• I
0 45 90 135180 - 9 0
-135 - 4 5
B
6 0-
Z
1
I I
I
0 45 90 135
0
3 518(
8 0) _-90
-135 - 4 5
Bud no. 5 to bud no. 6
5i
6
Z
0 45 90135180 - 9 0
-135 - 4 5
Bud no. 7 to bud no. 8
n 5-,
6
Z
n
i
—
rTl-
i
o
i
I—|
i—
•aa
i
Bud no. 4 to bud no. 5
Bud no. 3 to bud no. 4
—
i
0 45 90 135180 - 9 0
-135 - 4 5
rrr
n
0 45 90135180 - 9 0
-135 - 4 5
o
Z
0 45 90135180 - 9 0
-135 - 4 5
Bud no 6 to bud no. 7
•3 5-,
I
n
, 0 45 90 135180
-90
-135
-45
Bud no. 8 to bud no. 9
-3 S - ,
'S
0 45 90 135180 - 9 0
-135 - 4 5
Bud no. 10 to bud no. 11
Bud no. 9 to bud no. 10
Fig. 17. Legend on opposite page.
Dor so-ventral axis in Hydra
67
Shortly after the initial outpushing of the parental body wall, the first two
tentacles form simultaneously and laterally. A line drawn through their midpoints is at right angles to the longitudinal axis of the parent animal and coincides with the junction of the dorsal and ventral aspects of the developing bud.
Although the first two tentacles appear simultaneously, the remaining tentacles
are formed diachronically: tentacle no. 3 appears mid-dorsally, tentacle no. 4
forms mid-ventrally, tentacles 5 and 6 appear in the two dorso-lateral positions,
60 -i
50 -
40 -
S
30
03
I 20
10 -
0
45
90 135 180
-135
-90
-45
Consecutive distance between all buds
Fig. 18. Graphic representation of the direction and angle of the shortest distance
between groups of consecutive buds produced by fifty animals from which the
regions proximal to the budding region had been excised (composite of Fig. 17).
and, if seven or eight tentacles are formed, the additional tentacles develop in the
ventro-lateral positions. The beginning of phase III is marked by the detachment of the bud from the parent animal and ends with the onset of budding by
the recently detached bud. During this phase, the animals generally increase in
size and tentacles may be added.
B. The developmental relationship between bud morphogenesis
and the parent animal
Removal of the part of the parent animal distal to the developing bud, irrespective of the stage of bud development, does not alter the tentacle pattern or
body form of the developing buds or buds formed after incision. Since the
Fig. 17. Graphic representation of the direction and angle of the shortest distance
between pairs of consecutive buds produced during a 15-day period by fifty animals
from which the regions proximal to the budding region had been excised.
5-2
68
R. V. BAIRD & A. L. BURNETT
experimental animals were unable to feed after excision, it was impossible
to determine long-range effects. However, if the form of the animal is produced
primarily in the growth region, there must be a causal relationship between the
distal regions of the parent animal and bud morphogenesis. In view of the
statistical identity of the number of buds produced by the experimental animals
and the intact, starved control animals, the results probably reflect the activity
of the more proximal regions of the parent animal. The difference in the number
of detached buds can be explained in three ways which are not mutually exclusive: (1) the distal regions could be regulating bud development; (2) a wound
response could be involved; (3) inhibitor could be diffusing distally from the
peduncle. Evidence to be submitted later indicates that wound response is the
most likely possibility.
If inhibitor diffuses distally from the peduncle, it is possible that the slowness
of tentacle formation on the ventral side of the bud relative to the dorsal side
of the bud and the reduction in the number of detached buds on animals with
the distal regions excised may be the result of peduncular inhibition. This could
also play a role in regulation of the frequency of bud initiation. However, both
of these possibilities can be excluded. The tentacle pattern and body form of
all buds produced by animals from which the peduncles have been excised are
completely normal. In addition, there is no effect upon the number of buds
initiated or detached. In this case, it was possible to demonstrate that the
peduncle of the parent animal plays no long-range role in bud initiation or
development. The inability of any of the animals to regenerate a peduncle can
be interpreted as a possible requirement of peduncular tissue in order for the
proximal tissues of the budding region to differentiate into tissue characteristic
of the peduncle.
In the preceding experiments, the dorso-ventral axis of the tentacle pattern
of all buds initiated before and after excision of the parent animal was
normal. However, in both experiments, one end of the parent animal remained
on the budding region. While the intact ends have no demonstrable role in bud
morphogenesis, they may have regulated the dorso-ventral axis of the bud by
maintaining a difference in the growth-factor gradients across the budding
region. However, removal of the regions of the parent animal both proximal and
distal to the budding region does not alter the axis of the bud tentacle pattern.
In addition, the pattern and form of all buds produced were normal and there
was no alteration in the number of buds initiated or detached. Previously, removal of the distal regions of the parent animal resulted in fewer buds being
detached and it was suggested that a wound response might be involved or that
inhibitor might be diffusing from the peduncle. An additional possibility was that
the distal regions of the parent animal were regulating bud development but not
bud initiation. The number of detached buds from animals from which both the
distal and proximal regions were excised would be altered if distal regulation
were involved. This is not the case. Removal of the peduncle has no effect on
Dorso-ventral axis in Hydra
69
the number of detached buds; this rules out the possibility of inhibitor diffusing
from the peduncle into the budding region and there inhibiting bud development. The remaining possibility, wound response, seems the more likely possibility, for a differential effect initiated by wounding on one end of the budding
region could be equalized by wounding both ends. At any rate, it appears that
the isolated budding region is sufficient to regulate the dorso-ventral axis of the
bud. Should this be the case, rotation of the budding region 180° in a distoproximal direction would lead to the development of buds with a tentacle
pattern rotated 180° around the dorso-ventral axis of the bud. This is precisely
what occurs even though the polarity reversal is effected before bud initiation.
The growth-factor gradients are apparently rigorously established within the
budding region. Substantiating this conclusion is the observation that some
animals are not able to regulate polarity reversal of the budding region. Budding
ceased and the peduncle of these animals increased in length to a point where it
could no longer physically support the animal. Numerous masses of tissue
developed on the rotated budding regions which initially resembled stage I buds
but became irregular disintegrating masses of tissue as they moved proximally
on the peduncle. It is significant that, after 2 months in this abnormal state, one
animal successfully produced a bud and the tentacle pattern was rotated 180°
relative to the disto-proximal axis of the parent animal. Although the delayed
polarity reversal was observed in only one bud, it seems reasonable to conclude
that the dorso-ventral polarity reversal of the bud reflects a persisting distoproximal polarity reversal of the budding region. A resulting convergent, as
opposed to the normal continuous, growth would effectively increase the length
of the animal.
Having shown that the dorso-ventral axis of the bud is determined by the
disto-proximal axis of the budding region and that bud initiation and development are not regulated by the distal and proximal regions of the parent animal,
the developmental relationship between bud morphogenesis and the budding
region remains to be analysed. By surgically isolating buds from the budding
region of the parent animal, it was demonstrated that, regardless of the stage of
bud development at the time of excision, all isolated buds completed what
appeared to be normal development. Marking the buds with vital dye demonstrated that the dorso-ventral axis of the bud was not altered. In addition, although
the period of maturation was extended, all isolated buds were subsequently able
to produce buds.
In completing this portion of the analysis, it can be concluded that bud
initiation consists of the establishment of a disto-proximally and dorsoventrally polarized area of localized growth. Once bud initiation has occurred,
bud morphogenesis proceeds independently of parental regulation. The morphogenetic phenomena regulating the body form, tentacle pattern, and the two
axes of the bud are restricted to the developing bud.
70
R. V. BAIRD & A. L. BURNETT
C. The relationship of bud morphogenesis to maintenance of the adult form
1. The dorso-ventral axis of the growth region
In the preceding section the results show that, once bud initiation has occurred,
bud development is independent of the parent animal. The same developmental
processes giving rise to the form of the bud may persist in the maintenance of
the adult form. Should this be the case, the adult form would be expected to
possess a dorso-ventral axis of polarity at right angles to its disto-proximal axis.
The most likely place to begin the analysis is the growth region of the mature
animal, since it is here that the dorso-ventrally oriented tentacle pattern is
formed during bud morphogenesis. Campbell (1965), on the basis of his counts
of mitotic figures in Hydra littoralis, has suggested that hydra do not possess
localized growth regions. However, Burnett (1966) has shown in H. pseudoligactis, the species used in this study, that mitotic activity is greatest in the
subhypostomal region known as the growth region. Burnett's mitotic data substantiate the concept of a growth region postulated by Tripp (1928) and later
confirmed by Brien & Reniers-Decoen (1949), Burnett (1959), and Burnett &
Garofalo (1960) for other species of hydra. It may be that the growth regions of
various species vary in extent and, therefore, in mitotic activity. Should this be
the case, in species with relatively diffuse growth regions, mitotic activity along
the body column may appear to be relatively uniform. However, the presence
of a subhypostomal growth region in H. pseudoligactis has been well established.
Consequently, the ability of the growth region of hydra to regenerate tentacles
was utilized. Although regenerated tentacles exhibit more synchrony in their
order of appearance than do developing bud tentacles, the relative positions
of the regenerated tentacles form a recognizable pattern identical with the bud
tentacle pattern. In addition, the dorso-ventral axis of the regenerated tentacle
pattern coincides with the dorso-ventral axis of the bud tentacle pattern. Neither
the pattern nor the coincidence of the dorso-ventral axes of the two patterns
appears to alter as the animals age chronologically. If the growth region consists
of a ring of homogeneously active cells, the observed results would be impossible.
It seems more likely that the cells of the growth region proximal to the base of
each tentacle are more active than the intervening cells. There must also be a
differential in the activity of these cells regulating the relative order of appearance of the regenerated tentacles. This seems the only possible explanation for
the polarized positioning of regenerated tentacles. In other words, the dorsoventral polarity observed in the bud tentacle pattern persists in the growth
region of mature animals.
2. The dorso-ventral axis of the budding region
In view of the normal growth pattern of hydra—that is, by the disto-proximal
displacement of cells along the length of the animal—the dorso-ventral polarity
of the growth region may well extend down the body of the animal. The only
Dorso-ventral axis in Hydra
71
distinguishing morphogenetic feature of the budding region is its ability to
produce buds. The presence of a dorso-ventral axis of polarity of the budding
region could possibly be detected by its role in the site of bud initiation and this
is the case. The first bud produced by a recently detached animal is initiated on
the dorsal side of the animal. The site of consecutive buds, while initially
alternating between the dorsal and ventral sides of the animal, soon appears
to be forming randomly relative to the dorso-ventral axis of the parent animal.
Measurement of the distance between consecutive buds shows that buds tend
to form opposite the position of the preceding bud. Excision of the regions of the
parent animal distal to the budding region does not alter the position of consecutive buds at least for short periods of time. These animals are unable to
feed so budding ceases shortly after excision. Excision of the parental peduncle,
while having no effect on the relative positions of consecutive buds, increases
the number of buds forming at a level proximal to the level of the preceding bud.
It appears that the peduncle may be inhibiting bud initiation in the proximal
budding region. Thus, the dorso-ventral polarity of the parent animal appears
to regulate the site where the first bud is initiated, whereas the position of subsequent buds is regulated by the position of the preceding buds. However, if
budding animals are starved until budding ceases and re-fed until budding resumes, the position of the first bud formed appears to be regulated by the dorsoventral polarity of the parent animal rather than by the position of the last bud
formed during starvation. The presence of a dorso-ventral axis of polarity at the
level of the growth and budding regions of mature animals demonstrates a dorsoventral polarity of the animals. The growth regulatory effect of this polar axis
is seen in the formation of a bilaterally symmetrical dorso-ventrally polarized
bud tentacle pattern, in the orientation of the regenerated tentacle pattern and
in the determination of the site of the first bud produced after maturation
or starvation of adult animals. The possible mechanics of the action of the dorsoventral axis of polarity in bud initiation, development and maturation will be
discussed in terms of a model of bud development.
D. A model of bud morphogenesis
The following model of bud morphogenesis is based on Burnett's (1961,1966)
growth model for hydra, and the relationships expressed in Figs. 19 and 20 are
also based on Burnett's model. As shown in these figures, the highest levels of
stimulator and inhibitor activity are found in the growth region. In non-budding
animals (Fig. 19), both of the growth factors form a bipolar gradient with the
lowest levels of activity at the distal and proximal extremities of the animal.
Although the level of stimulator exceeds that of the inhibitor in the budding
region, peduncle, and basal disc, it is only in the budding region that the relative
level of stimulator is sufficient to initiate growth. In budding animals (Fig. 20)
the gradients of growth factors produced by the developing bud are super-
72
R. V. BAIRD & A, L. BURNETT
Relative amounts of growth factors
0
1
2
3
*
4
Tentacles
Growth reg. "
Gastric reg. -
Budding reg. -
Peduncle
Basal disc
No growth I
Growth
Fig. 19. Profile of the hypothetical distribution of growth factors
in a non-budding animal.
Relative amounts of growth factors
0
1 2
3
4
Tentacles
""~~^^^
"^v^
Stimulator
Inhibit
^~^"-^^^
""
Growth reg. ~
\
Gastric reg. -
)
Distal inhibition
by bud
Developing bud
Budding reg. ~
/ ' /
Peduncle
/
/
Basal disc
No growth I
Growth
Fig. 20. Profile of the hypothetical distribution of growth factors
in a budding animal.
Dorso-ventral axis in Hydra
73
imposed on the gradients of growth factors in the parent animal. Since the
stimulator is considered to be a non-diffusable factor, growth in the budding
region is restricted to the developing bud. However, inhibitor diffuses from the
developing bud into the adjacent tissues of the parent, thereby exhibiting an
inhibitory effect in the immediate area. The growth-stimulating and growthinhibiting factors invoked have been isolated from hydra (Lesh & Burnett,
1964; L. Davis, personal communication) and have the following theoretical
characteristics:
(1) The stimulator is produced primarily in the hypostome and forms a bipolar
disto-proximal gradient.
(2) The stimulator induces cell division or differentiation.
O
Fig. 21. Hypothetical distribution of net stimulator activity in hydra.
(3) The inhibitor is produced by dividing cells; therefore the greatest amount
is in the subhypostomal growth region.
(4) The inhibitor diffuses proximally and distally in the animal and leaks
from the animal; therefore the stimulator to inhibitor ratio varies at each level
of the animal.
(5) The inhibitor represses the activity of stimulator.
(6) Growth and differentiation of the animal are the result of the net activity
of stimulator.
In the model to be presented, various ratios of stimulator and inhibitor have
been given arbitrary values ranging from 1 to 4 consistent with a net activity
of stimulator (Fig. 21). A value of 1 represents the net activity of stimulator in
the tentacles, peduncle and basal disc. Growth has virtually ceased in these
regions. A value of 2, characteristic of the proximal portion of the tentacles and
of the budding region, represents minimal growth. A value of 3, which characterizes the tentacle bases distal to the growth region and the gastric region,
represents a moderate level of net stimulator activity, and the growth region,
with the highest growth rate, has a value of 4. The dorso-ventral axis of polarity
will be assumed to represent a dorso-ventral gradient of net stimulator activity.
74
R. V. BAIRD & A. L. BURNETT
1. Bud initiation and body formation
Prior to bud initiation, the net stimulator activity of the budding region has
a uniform value of 2 (Figs. 21, 22a). As a result of the dorso-ventral gradient
of stimulator activity originating in the growth region, there is a dorsoventral gradient of cell growth with the highest rate on the dorsal side of the
animal. Consequently, there is a proximal displacement of gastric region cells
(growth value of 3) into the dorsal region of the budding zone (Fig. 22b). This
creates a two-dimensional growth differential at the distal budding region and
Fig. 22. A model of bud initiation and body formation. Each diagram represents
the parent animal at the level of the initiating bud. Disto-proximal displacement of
the initiating bud relative to the parent animal is indicated by the bars in the side
of the diagrams. Hypothetical levels of net stimulator activity are proportional to
the numbers in each diagram, (a) The budding region prior to bud initiation, (b)
The first step in bud initiation: disto-proximal displacement of gastric region cells
(net stimulator activity of 3) into the budding region (net stimulator activity of 2)
as a result of the dorso-ventral polarity of the parent animal, (c) The growth
differential established in {b) gives rise to the disto-proximal axis of the bud. (d)
Inhibition of growth at the distal tip of the bud by increased amounts of inhibitor
produces a bilaterally symmetrical, dorso-ventrally polarized bud with the primordia
of the first tentacles.
results in a localized area of relatively accelerated growth (Figs. 20, 22 c). As
growth progresses, the inhibitor produced, although diffusing from the area,
also accumulates in the central portion of the growing tissues. This reduces the
net activity of stimulator centrally and effectively subdivides the growing region
into two lateral growth regions (Fig. 22 d) which, in turn, give rise to the first
two tentacles. The continued increase of inhibitor at the distal tip of the initiating
bud ultimately completely suppresses stimulator activity, thereby forming the
primordial hypostome. During this process, the initiated tissues are moving
proximally along the budding region and this increases the differential of net
stimulator activity. The cross-sectional views of the level of the parent animal
at the junction of the gastric and budding regions where bud initiation occurs
are represented in Fig. 23. It should be noted that this area does not become
displaced proximally during bud initiation and development, but rather consists
of a specific level of the animal through which cells move disto-proximally.
Prior to bud initiation, the net activity of stimulator is uniform at this level and
Dorso-ventral axis in Hydra
75
represents the disto-proximal transitional zone from a net stimulator value of 3
to a net activity of 2 (Fig. 23 a). As a result of the greater net stimulator dorsally
and distally to the budding region, cells from the gastric region with a higher
net stimulator activity are displaced proximally into the transition zone between
the gastric and the budding region (Fig. 23 b). This creates a stimulator, and hence
growth, differential on the dorsal side of the animal. The resulting increased
growth produces inhibitor which diffuses circumferentially from the localized
growth region and decreases the net stimulator activity of the surrounding
tissues (Fig. 23 c). As the initiating bud is displaced proximally, the inhibitor
3/2
3/2
3 3/2
3/2
3/2
3/2
3/2
i
3/2
3/2
Fig. 23. A model of the regulation of the site of consecutive buds. Each diagram
represents the junction of the gastric region (net stimulator activity of 3) and the
budding region (net stimulator activity of 2). Hypothetical differences in net stimulator activity are proportional to the numbers in each diagram, (a) Prior to bud
initiation, (b) Disto-proximal displacement of gastric region cells into the budding
region, (c) Inhibitor produced by the new growth center diffuses laterally, reducing
the net stimulator activity, (d) Progressive lateral diffusion of inhibitor, (e) Lateral
diffusion of inhibitor from the developing bud results in a growth differential on
the opposite side of the animal. (/) The growth differential established gives rise to
a bud. (g, h) Inhibitor diffuses laterally from the newly initiated bud and the process
is repeated.
produced by the growing tissues of the bud continues to diffuse laterally
(Fig. 23 d), ultimately creating a differential on the opposite side of the animal
(Fig. 23 e, f) which becomes the growth center of the next bud (Fig. 23 g). The
inhibitor produced by this growth center again diffuses laterally and the process
is repeated (Fig. 23 h). Should the diffusion of inhibitor around the lateral aspects
of the parent animal not be symmetrical, the next bud formed would not be
exactly 180° from the preceding bud. It would, however, be on the opposite
side of the animal and this is consistent with the experimental observations.
76
R. V. BAIRD & A. L. BURNETT
Initially, the region of growing tissues of the bud would be characterized by a
high net stimulator activity, but, as more inhibitor is produced, the net stimulator activity is reduced in a disto-proximal fashion along the bud. As the
amount of inhibitor increases, stimulator activity would be inhibited and morphological differentiation of the body column would take place. Basal disc
formation physically isolates the bud from the parent animal and is followed by
detachment of the bud. With this model, bud initiation results from the interaction of the disto-proximal and dorso-ventral gradients of the growth factors:
the dorso-ventral axis of the parent animal giving rise to the disto-proximal axis
of the bud and the dorso-ventral axis of the bud being determined by the distoproximal axis of the parent animal. Bud initiation would then consist of the
induction of a dorso-ventrally polarized disto-proximally growing localized
region of cells. In effect, the complete development of the bud is programmed
into the earliest stage of bud initiation. The observed ability of isolated buds to
complete their development normally reflects the efficiency of this inductive
process. In a sense, the early stage of an initiated bud consists of a pre-pattern
for the entire morphogenetic process of bud development.
2. Tentacle formation
Returning to tentacle development, note the two lateral regions in Fig. 22d
with the highest net activity of stimulator. Both foci of growing cells are surrounded by a symmetrical distribution of growth factors. Initially, the highest
rate of growth is at the center of each focus and the increased surface area results
in a localized outpushing of the body wall. As growth continues, diffusion of
the resulting inhibitor increases the amount of inhibitor distally on the tentacle
primordia. Once the level of inhibitor is high enough to suppress the activity of
stimulator in the distal regions of the tentacles, distal growth ceases, the tissues
differentiate and the growing tissues become restricted to a ring surrounding the
base of the tentacles. Referring again to Fig. 226?, the differential between the net
stimulator activity on the dorsal and ventral sides of the bud regulates the relative time of their further development. As the inter-tentacular interval increases,
the stimulator differential dorsally and ventrally would increase and the development of tentacle number three mid-dorsally begins before and is followed by the
mid-ventral development of tentacle no. 4. There are two possible factors determining the development of the fifth and sixth tentacles in the dorso-lateral
intervals: one is the greater net activity of stimulator dorsally relative to the
ventral side of the bud and the other is the continued increase in the intertentacular distance which would permit a greater loss of inhibitor. In other
words, the mid-points between the two lateral and single mid-dorsal tentacles
have a greater net stimulator activity. The dissymmetry in the order of appearance of the fifth and sixth tentacles may reflect a dissymmetry in net stimulator
activity between the dorso-lateral aspects of the developing bud. Since the dorsal
and ventral sides of the bud are essentially identical but out of phase, if additional
Dorso-ventral axis in Hydra
11
tentacles form they develop later and, for the same possible reason as the fifth
and sixth tentacles, diachronically. Tentacle formation generally ceases once six
tentacles have developed and form a distinctive pattern at the early six-tentacle
stage: two mid-lateral, three dorsal tentacles, and one ventral tentacle. As the
bud continues its development, the six tentacles become equally spaced around
the periphery of the hypostome. It would appear that the number and final
spacing of the tentacles represent a dynamic equilibrium between the size of the
system, the efficiency of the stimulator, and the loss by leakage or diffusion of
the inhibitor.
3. Bud maturation
Recently detached buds are characterized by their relatively small size and
by the indistinct junction between the peduncle and adjacent distal region of the
animals. During the period preceding onset of blastogenesis by these animals,
they increase in size and the junction between the peduncle and budding region
becomes well defined. It would appear that during this phase of development
the disto-proximal and dorso-ventral gradients of stimulator and inhibitor are
progressing towards the dynamic equilibrium characterizing the mature
animal.
E. The dorso-ventral axis in other hydroids
Although this thesis deals primarily with the growth pattern of hydra, it
seems reasonable to anticipate that the developmental phenomena described for
hydra could be extended to other, especially closely related, forms. For example,
the colonial hydroids are generally characterized by regular patterns of growth
(Hyman, 1940). The spatial relationships of the hydranths, hydrocauli and
hydrocladi exhibit a disto-proximal polarity, and in many instances appear also
to reflect a dorso-ventral polarity of developmental activity. Apparent dorsoventrality is especially evident in some of the forms which branch in one place.
For example, the hydranths of Pennaria and Plumularia are on the dorsal
surface of each hydrocladus. The hydrocladi, in turn, are formed alternately
on opposite sides of the hydrocaulus. In view of the developmental role of the
dorso-ventral axis of hydra, the same polar axis may well exist and play a morphogenetic role in the colonial hydroids.
Since preliminary histological observations in progress indicate that the dorsoventral axis of polarity is not correlated with qualitative differences in cell types,
further work is under way investigating the possibility of quantitative differences.
v. SUMMARY
The developmental relationship between bud morphogenesis and the parent
animal was analysed by following bud development subsequent to each of five
experimental treatments of the parent animals.
(1) Excision of the region of the parent animal distal to the budding region
78
R. V. BAIRD & A. L. BURNETT
demonstrates that: (a) the tentacle pattern and body form of all buds produced
are normal; (b) the number of initiated buds is not altered; (c) the number of
detached buds is reduced.
(2) Excision of the region of the parent animal proximal to the budding
region demonstrated that (a) the tentacle pattern and body form of all buds
produced are normal; (b) the number of initiated buds is not altered; (c) the
number of detached buds is not altered.
(3) Isolation of the budding region demonstrated that: (a) the tentacle pattern
and body form of all buds produced are normal; (b) the number of initiated
buds is not altered; (c) the number of detached buds is not altered.
(4) Polarity reversal of the budding region demonstrated that: (a) distoproximal polarity reversal results in a dorso-ventral reversal of the bud tentacle
pattern; (b) polarity reversal of the budding region may persist.
(5) The development of isolated buds demonstrated that: (a) the tentacle
pattern and body form of all buds are normal; (b) the polar phenomena of all
buds are normal; (c) the maturation process is extended but complete.
On the basis of the results of these experiments, the conclusion is drawn that,
once bud initiation has occurred, bud morphogenesis proceeds independently
of parental regulation. It is proposed that this occurs because the factors regulating body form, the tentacle pattern, and the two axes of the bud are produced by the developing bud.
The relationship of bud morphogenesis to maintenance of the adult form
was analysed in two groups of experiments. Both groups of experiments were
designed to determine if the dorso-ventral axis of the bud persisted after
bud maturation.
(1) The effect of the dorso-ventral axis of polarity at the level of the growth
region: (a) regenerated tentacles on mature animals form a pattern identical with
the bud tentacle pattern; (b) the dorso-ventral axis of the regenerated tentacle
pattern coincides with the dorso-ventral axis of the bud tentacle pattern;
(c) chronological ageing has no effect upon the pattern of regenerated tentacles
or its polar axis.
(2) The effect of the dorso-ventral axis of polarity at the level of the budding
region: (a) the first bud formed by recently detached animals appears on the
dorsal side of the animals; (b) consecutive buds tend to form opposite the preceding bud rather than alternating between the dorsal and ventral sides of the
parent animal; (c) following a non-budding period, the incidence of dorsal buds,
as budding resumes, increases irrespective of the position of the last bud formed
prior to the non-budding phase; (d) the position of consecutive buds is not
altered by excision of the regions of the parent animal proximal or distal to the
budding region.
On the basis of the results of these experiments, the conclusion is drawn that
the dorso-ventral axis of the bud persists at the level of the growth and budding
region after bud maturation.
Dorso-ventral
axis in Hydra
79
The hypothesis is proposed that the growth regulatory effect of the dorsoventral axis of polarity is demonstrated by the formation of a bilaterally symmetrical, dorso-ventrally polarized bud tentacle pattern, by the orientation of
the regenerated tentacle pattern and by the determination of the site of the
first bud produced following maturation or starvation of adult animals.
A possible mechanism of the action of the dorso-ventral axis of polarity in
bud initiation, development and maturation is discussed in terms of a model of
bud development.
RESUME
Observations sur la decouverte d'un axe dorso-ventral chez VHydre
Les relations intervenant au cours du developpement entre la morphogenese
du bourgeon et l'animal parent, ont ete analysees en suivant le developpement
du bourgeon a la suite de cinq types de traitements experimentaux des parents.
(1) L'excision de la region du parent distale a la zone de bourgeonnement
demontre que: (a) les caracteres des tentacules et la forme du corps de tous les
bourgeons sont normaux; (b) le nombre des bourgeons formes n'est pas modifie;
(c) le nombre de bourgeons detaches est reduit.
(2) L'excision de la region du parent proximale a la zone de bourgeonnement
demontre que: (a) les caracteres des tentacules et la forme du corps de tous les
bourgeons produits sont normaux; (b) le nombre de bourgeons formes n'est
pas modifie; (c) le nombre de bourgeons detaches n'est pas modifie.
(3) L'isolement de la zone de bourgeonnement a demontre que: (a) les
caracteres des tentacules et la forme du corps de tous les bourgeons produits
sont normaux; (6) le nombre de bourgeons formes n'est pas modifie; (c) le
nombre de bourgeons detaches n'est pas modifie.
(4) L'inversion de la polarite de la zone de bourgeonnement a demontre que:
(a) l'inversion de polarite disto-proximale produit une inversion dorso-ventrale
des caracteres des tentacules du bourgeon; (b) l'inversion de polarite de la zone
de bourgeonnement peut persister.
(5) Le developpement de bourgeons isoles a demontre que: (a) les caracteres
des tentacules et la forme du corps de tous les bourgeons sont normaux;
(b) les phenomenes de polarite de tous les bourgeons sont normaux; (c) le processus de maturation est allonge mais complet.
Sur la base des resultats de ces experiences, on tire la conclusion suivante:
une fois que le developpement des bourgeons a commence, leur morphogenese
se deroule independamment d'une regulation parentale. On suggere qu'il en est
ainsi parce que les facteurs assurant la regulation de la forme du corps, des
caracteres des tentacules et des deux axes du bourgeon sont produits par le
bourgeon en cours de developpement.
Les relations entre la morphogenese du bourgeon et le maintien de la forme
adulte ont ete analysees dans deux groupes d'experiences realises tous deux dans
80
R. V. BAIRD & A. L. BURNETT
le but de determiner si l'axe dorso-ventral du bourgeon persiste apres la maturation de celui-ci.
(1) Effets de l'axe de polarite dorso-ventral au niveau de la zone de croissance: (a) les tentacules regeneres sur des animaux murs ont des caracteres
semblables a ceux des bourgeons; (b) l'axe dorso-ventral de la structure du
tentacule regenere coincide avec l'axe dorso-ventral de la structure du tentacule
du bourgeon; (c) l'age chronologique n'a pas d'action sur les caracteres des
tentacules regeneres ni sur leur axe polaire.
(2) Effets de l'axe de polarite dorso-ventral au niveau de la zone de bourgeonnement: (a) le premier bourgeon forme par des animaux recemment detaches
apparait sur leur face dorsale; (b) des bourgeons consecutifs tendent a se
former a l'oppose des precedents plutot qu'en alternance entre les faces dorsale
et ventrale du parent; (c) a la suite d'une periode sans bourgeonnement, l'apparition de bourgeons dorsaux, quand le bourgeonnement reprend, s'accroit
sans rapport avec la position du dernier bourgeon forme avant la phase de nonbourgeonnement; (d) la position des bourgeons consecutifs n'est pas modifiee
par l'excision des regions du parent proximales ou distales par rapport a la zone
de bourgeonnement.
Sur la base des resultats de ces experiences, on tire la conclusion suivante:
l'axe dorso-ventral du bourgeon persiste au niveau de la zone de croissance
et de bourgeonnement apres la maturation du bourgeon. On propose une
hypothese selon laquelle l'effet regulateur de croissance de l'axe dorso-ventral
de polarite est mis en evidence par la formation d'un plan de structure du tentacule du bourgeon, polarisee dorso-ventralement et a symetrie bilaterale, par
1'orientation de la structure du tentacule regenere et par la determination de
l'emplacement du premier bourgeon produit a la suite de la maturation ou du
jeune d'animaux adultes.
On discute, en termes d'un modele de developpement du bourgeon, d'un
mecanisme possible de l'action de l'axe de polarite dorso-ventral dans l'apparition, le developpement et la maturation du bourgeon.
REFERENCES
BRAEM, F. (1910). Die ungeschlechtliche Fortpflanzung. Biol. Zbl. 30, 367-79.
BRIEN, P. & RENIERS-DECOEN, M. (1949). La croissance, la blastogenese, l'ovogenese
chez
Hydra fusca (Pallas). Bull. Biol. Fr. Belg. 82, 293-386.
BRIEN, P. (1955). La signification des cellules interstitielles des hydres d'eau douce et le
probleme de la reserve embryonnaire. Biol. Bull. Fr. Belg. 89, 259-325.
BURNETT, A. L. (1959). Histophysiology of growth in hydra. / . exp. Zool. 140, 281-342.
BURNETT, A. L. (1961). The growth process in hydra. /. exp. Zool. 146, 21-84.
BURNETT, A. L. (1962). The maintenance of form in hydra. In Twentieth Growth Symposium
'Regeneration' (ed. D. Rudnick), pp. 27-52. London: Ronald Press.
BURNETT, A. L. (1966). A model of growth and differentiation in hydra. Am. Nat. 100,
165-90.
BURNETT, A. L. & GAROFALO, M. (1960). Growth pattern in the green Hydra, Chlorohydra
viridissima. Science, N.Y. 131, 160-1.
Dorso-ventral axis in Hydra
81
R. D. (1965). Cell proliferation in Hydra: an autoradiographic approach. Science,
N.Y. 148, 1231-2.
DAVIS, R. P. (1965). An experimental analysis of the establishment and expression of organismal polarity in hydra. Unpublished doctoral thesis, Cornell University.
HAACKE, W. (1879). Zur Speciesunterscheidung in der Gattung Hydra. Zool. Anz. 2.
HERTWIG, R. (1906). Uber Knospung und Geschlechtsentwicklung von Hydra fusca. Biol.
Zbl. 26, 469-508.
HYMAN, L. H. (1930). Taxonomic studies on the hydras of North America. II. Trans. Am.
microsc. Soc. 49, 322-33.
HYMAN, L. H. (1940). The Invertebrates, vol. i, pp. 403-7.
KANAJEW, J. (1930). Zur Frage der Bedeutung der interstitiellen Zellen bei Hydra. Arch.
EntwMech. Ml, 736-59.
KOELITZ, W. (1910). Morphologische und experimentelle Untersuchungen an Hydra. Arch.
EntwMech. 31, 197-257.
LESH, G. & BURNETT, A. L. (1964). Some biological and biochemical properties of the
polarizing factor in Hydra. Nature, Lond. 204, 492-3.
LOOMTS, W. F. & LENHOFF, H. (1956). Growth and sexual differentiation of hydra in mass
culture. /. exp. Zool. 132, 555-74.
RULON, O. & CHILD, C. M. (1937). Observations and experiments on developmental pattern
in Pelmatohydra oligactis. Physiol. Zool. 10, 1-13.
TRIPP, K. (1928). Regenerationsfahigkeit von Hydren in den verschiedenen Korperregionen
nach Regenerations- und Transplantationsversuchen. Z. wiss. Zool. (Korshelt Festband),
132, 476-525.
WEIMER, B. R. (1927). Use of Nile blue sulfate as a vital stain on hydra. Biol. Bull. 52, 219-22.
WEIMER, B. R. (1928). The physiological gradient in hydra. Physiol. Zool. 1, 183-230.
WIGGLESWORTH, V. B. (1954). The Physiology of Insect Metamorphosis, pp. 105-9. Cambridge
Monographs in Experimental Biology, I.
YAO, T. (1945). Bud induction by developing hypostome. /. exp. Biol. 21, 155-60.
CAMPBELL,
{Manuscript received 27 June 1966)
JEEM 17