AMER. ZOOL., 14:603-618 (1974) .
Non-budding Hydra: Form Regulation and Bud Induction
LINDA L.
BRINKLEY
Department of Developmental and Cell Biology, University of California,
Irvine, California 92664
SVNOPSIS. Form regulation and bud induction were studied in a non-budding strain
of Cliloroliydra viridissitna. Regeneration at a cut surface in a column piece with an
existing hydranth was observed and found to be dependent on the column length
Another aspect of form regulation, formation and control of supernumerary tentacles,
was investigated by grafting. Supernumerary tentacle formation in long polyps can be
suppressed by implants of hypostomal or siibhypostomal tissue.
Non-budding hydra can be induced to bud by implanting small pieces of normal
tissue into their columns. The cellular basis of this process was investigated by means
of grafting, radioautography, and histological methods. N'o differences in the proportions or appearances of the cell t\pes were observed between non-budding and normal
animals. However, induced buds have higher proportions of interstitial cells and their
deiivatives (nerves and nematoblasts) than do normal buds. Many of these interstitial cells and derivatives originate from cells in the grafted implant. Normal tissue
from which interstitial cells have been previously removed will not induce buds in
non-budding hydra.
The non-budding syndiome is probably related to a deficiency in interstitial cell
differentiation. If nerve cells are involved in bud initiation and form regulation, these
results suggest interstitial cells of non-budding hydra are unable to transform into
sufficiently active and/or numerous nerve cells to control those processes.
INTRODUCTION
A non-budding hydra with abnormal
form was isolated from a fertilized egg and
described by LenhofE (1965; LenhofF et al.,
1969). The animal grows to giant proportions and forms supernumerary tentacles
and hydranths. In addition, grafts of normal tissue cause buds to be initiated in
the non-budding tissue. This paper reports
the results of experiments designed to investigate form regulation and the process
of bud induction in this strain of hydra.
Only a few investigators have made use
of abnormal hydra to investigate developThe author wishes to thank Dr. Richard D.
Campbell for his invaluable guidance and assistance during the course of this work.
The work reported herein was supported by
U.S.P.H.S. Graduate Training Grant HD-00347, an
NDEA Predoctoral Fellowship to L. B. Moore
(Brinkley), NSF Research Grant and NIH Research Development Award 1-K04-GM42595 to R.
D. Campbell.
Present address of the author is Department of
Oral Biology, Laboratory of Oral Histology, School
of Dentistry, The University of Michigan, Ann
Arbor, Michigan 48104.
603
mental processes. Schulz and Lesh (1970)
and Lesh-Laurie (1971) obtained hydra
similar in appearance to the Lenhoff animal from heat-shocked cultures. Another
abnormal strain was reported which develops a very long peduncle and was studied developmentally by Brien and ReniersDecoen (1952) and by Haynes et al.
(1964). In an attempt to study the budding
process through genetic dissection of its
events, several other non-budding strains
were obtained from sexual crosses (Moore
and Campbell, 1973b). All non-budding
strains examined possessed a common syndrome which apparently accompanies the
inability to bud: a lengthened embryonic
period, plasticity of form often characterized by supernumerary structures, and the
ability to be induced to bud by implantation of normal tissues. The information
gained in the present study on the cellular
basis of the non-budding phenotype and
the process of bud induction may apply
to these non-budding strains as well.
Two aspects of form regulation in nonbudding hydra were explored, polarity of
604
LINDA L. BRINKLEY
regeneration and regulation of supernumerary tentacle placement by grafts of nonbudding and normal tissue. Non-budding
animals show abnormal regeneration. When
a monopolar individual is bisected, the
apical portion usually regenerates a head,
producing a bipolar animal. The basal
portion produces a multipolar animal. It
was found that the structure regenerated
at a cut surface is dependent on the size
of the piece. Tentacle placement was
studied by testing the ability of grafts of
various parts of the body column of nonbudding animals to suppress supernumerary tentacle formation. There is a direct
relation between the original column position of the implant and its effect on the
development of supernumerary tentacles
in the host. Hypostomal tissue was most
effective with more proximal tissues being
less so. Part of the abnormality of the
non-budding strain may be seated in the
hypostomal region. This in turn may be
related to the hydra's inability to bud since
budding involves the early development
of a hypostome.
Grafts labeled with vital markers including tritiated thymidine provided information on the cellular basis of both
the phenomenon of bud induction in the
non-budding strain and the non-budding
phenotype itself. It was found that many
interstitial cells move from the normal tissue and appear in the young induced buds;
nerves and nematoblasts derived from these
normal interstitial cells are also present.
The data from these experiments suggest that the developmental lesions in at
least one non-budding strain are spread
throughout the hydra body rather than
localized. The disability of non-budding
animals appears to be an organizational
one. The animals lack the ability to influence cell behavior and thus maintain normal form and initiate budding. The lesion
underlying the non-budding phenotype is
probably associated with interstitial cells,
possibly involving altered interstitial cell
competencies. This could explain the pleiomorphic expression of the basic lesion
since interstitial cells are thought to give
rise to a number of other cell types.
MATERIAL AND METHODS
Cultures of Lenhoff's non-budding strain
and the parent normal Chlorohydra viridis
(Lenhoff, 1965) were obtained from Dr.
Howard M. Lenhoff. Both aposymbiotic
(white) and symbiotic (green) forms of
both strains were used. Stock cultures were
maintained in "M" solution by the culture
methods of Lenhoff and Brown (1970).
Experimental animals were removed from
the stock culture and maintained separately prior to use.
For regeneration experiments, column
pieces of various lengths were cut from the
distal part of an animal, allowed to extend, and measured. All specimens used
were originally bipolar, so that each cut
piece then possessed one hydranth at the
time of transsection. Regeneration was
observed at the cut surface.
Grafting
Tentacle placement. Abnormal bipolar
animals were used as hosts and donors.
Bipolar host hydra free of supernumerary
tentacles were produced by isolating pieces
of body column of non-budding animals
and allowing them to regenerate for 3
days. Donor pieces from aposymbiotic animals were taken from the following body
regions and were approximately 0.25 mm
in length: (i) hypostome, (ii) hypostome
including surrounding tentacle ring, (iii)
subhypostomal column, and (iv) central
body column (midway between hypostomes). In addition, normal (parental)
strain hydra also served as donors of hypostomal tissue for some experiments.
Donor pieces were grafted into host columns midway between the two host hydranths. The host was punctured and the
donor grafts were inserted into the wound
so that the most basal surface of the donor
ring of tissue contacted the host tissue.
The donor piece thus formed a lateral
projection from the host. Donor tissue was
held in place with glass needles until the
graft healed. As controls, some hosts were
punctured by glass needles but received
no implant: other control animals were
unoperated. Control and experimental
605
NON-BUDDING HYDRA
animals were observed the day after grafting and were monitored for 7 days for the
formation of supernumerary tentacles. During this time they were not fed, but the
culture medium was changed daily.
Bud induction. Hydra were placed in
paraffin-lined petri dishes filled with "M"
solution and cut transversely, and reciprocal pieces were grafted together.
Two methods were used to mark the site
of healing. One method was to graft the
bottom of a green hydra to the top of a
white hydra, or vice versa (Browne, 1909).
The color boundary in grafts of green and
white hydra marks the site of healing. This
boundary remains distinct for a week or
so, after which the algae invade into the
white portion. The other method was to
mark the epithelio-muscular cells of one
hydra donor with India ink as follows. One
day before grafting, the hydra were allowed
to attach to the bottom of a clean dish.
While holding the animal by its tentacles
with forceps, a micropipette (10 ^ bore)
containing India ink was pressed against
the hydra, and a sudden jet of ink forced
out using a mouth pipette. The ink particles were forced into the tissue, and some
were phagocytized and retained in vacuoles
within the epithelial cells (Campbell,
unpublished).
Isotopic labeling
Five to ten hydra starved for 24 hr were
placed in petri dishes and each animal
was fed 1 to 3 Artemia nauplii. After 15
to 30 min, 0.1 jxl tritiated thymidine
(methyl-labeled, Sp. Act. 1.9 c/mM, 1 me/
ml, Schwarz Bio Research Co., Lot no.
2001) was injected into the gastric cavity
using a motor driven 10-yu.l syringe connected to a fine polyethylene tube inserted
through the hydra's mouth. Three hours
after injection, animals were transferred
through four changes of medium and were
immediately used for grafting.
Separation of cells by maceration
Whole hydra or pieces of hydra were
placed on a gelatin-coated slide, excess
medium was drawn off, and a drop of
macerating fluid added (glycerol: glacial
acetic acid: water = 1:1:3) (Bode, personal communication, modified from Haller, 1886). The slide was covered with a
petri dish to prevent evaporation and the
tissue allowed to macerate for 1 to 2 hr.
Then the cells were teased apart using
glass needles and the slide was examined
with phase contrast microscopy or was prepared for autoradiography by adding a
small drop of 40% formalin and allowing
the slide to dry. Prior to autoradiography
the slide was briefly rinsed in tap water
to remove glycerol. All cell types were
readily identifiable using this method
(Bode et al., 1972).
A utoradiography
Slides were dipped in Kodak NTB-3
liquid emulsion, dried, exposed at 4 C for
1 to 2 weeks, developed in Dektol, and
mounted in Permount. The slides were
examined using phase microscopy and cells
were identified and counted according to
the criteria of David (1973). Cell counts
were always normalized against the number of epithelial cells.
Nitrogen mustard treatment
Trituration of Mustargen-HCl (Merck,
Sharp and Dohme, Lot #1031K) was dissolved in distilled water to make a 1%
stock solution. Animals to be treated were
placed in 10 ml of "M" solution in petri
dishes, and nitrogen mustard stock solution was added to bring its final concentration to 0.1%. Animals were treated for
10 min, then rinsed three times in large
volumes of medium. The proportion of
interstitial cells was counted beginning at
clay 4 after treatment, by macerating individual animals and counting cell types.
RESULTS
Form regulation
Regeneration. Table 1 shows the frequency with which pieces of different sizes,
containing an existing hydranth, regenerated normally (base) or abnormally (hy-
606
LINDA L. BRINKLEY
TABLE 1. Regeneration in a non-budding hydra strain.
Structure regenerated at the
proximal (basal) surface of
an isolated distal tissue piece
Length of
piece (mm) *
Sample size
% Bases
% Hydranths
0-1
1-2
2-3
3-5
17
22
24
8
83
42
17
0
17
58
83
100
* The length was measured after cutting.
dranth), as a function of tissue length.
Length refers to the maximal extent to
which the pieces would extend shortly
after they were cut out. The majority of
pieces less than 2 mm long after cutting
regenerated normally, while those longer
than 3 mm formed hydranths. Thus, the
regeneration polarity of the apical portion
of the column depends on the length of
the piece, with the shorter column segments regenerating quite normally.
Influence of tissue implants on supernumerary tentacle formation. The ability
of various parts of the body column of the
non-budding animals to suppress supernumerary tentacle formation was assessed
by implanting various body regions into
non-budding bipolar hosts.
Hypoitome (o2)
Hrpostome and tentocks (89)
Subhyposfomal Column (lU)
|
i
Central body Column (llo)
Intact host^ no implonfi (221)
tinctured hosts, no implant! (331)
% Amman forming supernumerary tentacles
FIG. 1. Effects of implanting various body regions
of non-budding animals on formation of supernumerary tentacles. Pieces of tissue 0.25 mm long
were implanted in the middle of bipolar Lenhoff
(1965) strain non-budding hydra ranging from 1
to 10 mm in length. Donor tissue was taken from
one of four body regions indicated. Untreated hosts
and hosts punctured for operation but receiving
no implant were used as controls. Ordinate indicates the per cent of hosts forming supernumerary
tentacles within 7 days. Numbers in parentheses
denote how many of each type of graft were made.
607
NON-BUDDING HYDRA
.^.Xypostome (2)
Subhypostomal column (52)
Central body column (37)
Intact hosts, no implant (KQ)
Punctured hosts, no implant (95)
Hypa>lome<36)
3
1
Hypoitome and tentoclei (39)
Subhypostomal column (S3)
Central body column (68)
Intact hosts, no implant (116)
Punctured hoit^na implant (140)
Hyposlome (24)
Hypostome and tentoclei (16)
Subhypostomol column (7)
Central body column (ll)
Intact hosts, no implants C
Punctured controls (22)
B
"TIT
TiT
StT
~sr
"TO"
T0-
-Rx>
% Animab forming, supernumerary tentacles
FIG. 2. Formation of supernumerary tentacles in
hosts o£ different sizes with implants of non-budding tissue. These data are arranged according to
host size. The body regions and controls are labeled
according to the plan shown in Figure 1.
Figure 1 illustrates the effects of implants on supernumerary tentacle formation. The body regions tested and the graft
plan are shown in this Figure. Both the
untreated and punctured controls formed
supernumerary tentacles in about 65% of
the cases. An implanted tissue taken from
the vicinity of a hydranth reduced the
appearance of supernumerary tentacles to
about 35% of the cases. Central body
column tissue implants were not effective
in inhibiting tentacle formation.
Figure 2 depicts the results examined in
terms of host size as well as donor tissue
type. Punctured controls showed only small
variation from intact controls. In general,
the percentage of control animals, both intact and punctured, which form supernumerary tentacles increased with increasing
column length. About 45% of the control animals developed supernumerary ten-
tacles at the smallest host size (1 to 4 mm);
the percentage increased to 76% in hosts
4 to 7 mm long, and to 95% in hosts 7 to
10 mm long. Hypostome, with or without
tentacles, was the most effective region in
inhibiting supernumerary tentacle formation in hosts of all sizes, but the magnitude
of its effects decreased with increasing host
size. Thus, implanted hypostome completely suppresses supernumerary tentacles
in small hosts (1 to 4 mm long). In longer
hosts (4 to 7 mm) 17% of the animals
with hypostomal implants developed such
tentacles, while in still longer hosts (7 to
10 mm), 67% formed supernumerary tentacles. Tissue implants taken from the subhypostomal body column also reduced
supernumerary tentacle formation, but
showed the same variation with host size
that is seen when hypostomal implants
were considered. Central body column was
608
LINDA L.
BRINKLEY
WO,
90
80
\
(4-7)
(7-10)
30
4(1-4)
40>
FIG. 3. Effects of tissue implants on supernumerary
tentacle formation viewed as inhibition and stimulation relative to control values. The ordinate
represents the per cent of hosts inhibited or stimulated to produce supernumerary tentacles. The
abscissa represents the body position schematically.
Data are taken from Figure 2 and normalized relative to intact controls. Host sizes are given in
parentheses after each curve.
609
NON-BUDDING HYDRA
not effective in influencing supernumerary
tentacle formation in hosts 4 to 7 or 7 to
10 mm long. However, in smaller hosts, 1
to 4 mm, animals with central body column
implants developed supernumerary ten-
tacles 20% more often than did controls.
One can thus view implanted tissue as
being inhibitory or stimulatory to supernumerary tentacle formation; the distribution of this inhibitory quality throughout
100)
LJ
Normol
3 Non-budding lenhoff strain
.s
5
I
8
"S
JO
Interstitial
Nematoblast
Nemotocyte
Nerve
Glandular
Cell types
FIG. 4. Proportions of cell types in normal and mals and 5336 cells from 13 hydra of the nonnon-budding strain animals. Count totals were budding strain. (From Moore and Campbell,
5126 cells in 15 normal (but without buds) ani- 1973a.)
610
LINDA L. BRINKLEY
the hydra body is shown in Figure 3.
There is a direct relation between the column position of an implant's origin and
its effect on the development of tentacles.
Hypostomal tissue is most inhibitory.
This gradation is more apparent in short
hosts. Here, central column tissue even
appears stimulatory to supernumerary tentacle formation.
Similar grafting experiments were performed implanting hypostomal tissue from
normal animals into non-budding hosts 4
to 7 mm long. In 24 grafts, 67% of the
hosts receiving normal implants formed
supernumerary tentacles, whereas in hosts
of the same size receiving Lenhoff strain
implants, 17% formed supernumerary tentacles (Fig. 2).
During these experiments, 63% of the
implanted normal hypostomes separated
from the host column in the form of a
small bud. This graft separation was never
observed with implanted hypostomes taken
from non-budding donors. Supernumerary
tentacles formed on 67% of hosts with normal implants whether or not their grafts
remained attached. When the graft separated, 60% of the animals subsequently
formed supernumerary tentacles; if the implants remained on the host, 78% formed
supernumerary tentacles.
Whether or not the normal hypostomal
implants remain attached to their hosts,
they are not as effective as hypostomal implants from non-budding animals in suppressing supernumerary tentacle formation
in non-budding hosts (Figs. 1, 2).
tions were also studied in normal buds
and in buds induced to form in non-budding hydra by normal tissue grafts. Buds
studied were in one of three developmental
stages. Stage I included early bud development, from the time the bud appeared as
an outpocketing of the parental body wall
until the axis was somewhat elongated
but still without tentacle rudiments. Stage
II was composed of buds with tentacle
rudiments. Stage III included buds with
elongating tentacles.
Figure 5 gives the relative density of
each cell type found in normal and induced buds in stages I, II and III. Interstitial cells are more abundant in induced
buds than in normal buds at all stages.
Nematoblasts and nematocytes are also
generally more abundant in induced buds
than in normal buds during the course
of bud development. Nerve cell distribution does not follow this pattern; only at
early stages (I) do induced buds contain
more (ca. 90%) nerve cells than normal
buds. The distribution of glandular cells
also follows this developmental pattern.
Interstitial cells and nematocytes are the
only types which constitute a consistently
higher proportion of the cell population
of induced buds as compared to normal
buds. Considering the changes in the pattern of distribution of cell types during
the course of bud development, the increased amounts of interstitial cells and
nerve cells found in early induced buds
may also be important to the question of
normal bud induction.
Bud initiation in the non-budding strain
Column position of induced buds in relaCell populations
tion to the composition of the graft. Buds
Normal and non-budding strain animals. can occur in three places in grafts of norThe proportion of the cell types in macer- mal and non-budding tissue: in the nonated preparations both of the non-budding budding tissue, at the graft junction, or
hydra and of normal animals which carried in normal tissue. Observations on bud inno buds was determined (Fig. 4). No dif- duction in grafts of non-budding and norferences in the proportions of each cell mal tissue suggested that a relationship
type were observed; therefore, the non- might exist between the relative amounts
budding phenotype does not seem to be a of normal and non-budding tissue and the
reflection of any major change in the rela- position of the first bud initiated. This
tive proportions of the principal cell types. hypothesis was tested by making grafts
Normal and induced buds. Cell popula- composed of various proportions of norBud induction
Gil
NON-BUDDING HYDRA
100,
1—J Normal
•ilhijl Induced
Stage III
50
100
Stage II
50
• • • ; • • • • ; • • • • ;
I:::::::::::::
&;=;
::::::::::::::
":::*::::::::
;;;•;;;••;•;•;
100,
Stage I
50
Interstitial
Nematoblpst
Nerve
Nematocyte
Glandular
Cell types
FIG. 5. Proportions of cell types in young normal
and induced buds of different developmental
stages as defined in text. Count totals were: Stage
I, 1157 cells from 4 normal buds and 1320 cells
from 4 induced buds; Stage II, 632 cells from 2
normal buds and 2378 cells from 5 induced buds;
and Stage III, 1806 cells from 6 normal buds and
1712 cells from 4 induced buds. (From Moore and
Campbell, 1973a.)
612
LINDA L. BRINKLEY
TABLE 2. 1-'osiiion of first
buds.
Site of first bud (%)
Fraction of normal
tissue in graft*
V2
'/H/2
14
n
In normal
strai n tissue
At
graft junction
In non-budding
strain tissue
13
23
11
77
43
23
39
9
17
91
• The apical portion of all grafts was non-budding tissue; the basal portion was composed of
normal tissue. All grafts were observed for 7 days.
mal and non-budding stained tissue. In
Table 2 the site of the first bud in a
grafted animal is correlated with the
amount of normal tissue in the graft. In
all grafts the apical portion was composed
of non-budding strain tissue and the basal
portion of normal tissue. If only onequarter or less of the animal is normal
tissue, the first bud is most likely to be
initiated in the non-budding tissue.
Effects of temporary normal tissue grafts
on bud induction. The basal halves of
green normal hydra were grafted to equal
sized white, apical halves of non-budding
animals. Normal tissue ectoderm was
marked with India ink in addition to the
natural gastrodermal cell marker (algae).
Half of the grafted animals were cut apart
at the graft site, as distinguished by the
algal boundary, after either 1, 4, 8, 12, 24,
or 36 hours, 2 or 4 days. The remaining
grafted individuals were left intact and
observed as controls. Both isolated nonbudding apical halves and intact controls
were fed daily and their budding behavior
noted over a 3-week period. Budding did
not occur in any apical halves of nonbudding animals which were separated
from the normal half sooner than 4 days
after grafting. Three out of 18 non-budding
halves which were separated on the fourth
day after grafting later formed one bud;
none of these three induced buds contained algal or ink marked cells. One of
the induced buds later formed buds itself.
Cell migrations during bud induction.
Normal tissue was labeled with tritiated
thymidine 3 hr prior to grafting and cell
movements were analyzed by radioautography. The per cent of cells labeled in
control animals sampled at the time of
grafting was as follows: epithelial cells,
14%; interstitial cells, 75%; nematocytes
and nerves, 0%; nematoblasts, 37%.
Grafts were made between apical portions of green, non-radioactive, non-budding strain hydra, and basal portions of
tritiated, white normal tissue. The amount
of normal tissue in the grafts varied. In
15 grafts, normal tissue comprised less than
one-quarter of the total animal and in
six grafts the amount of normal tissue was
one-quarter to one-half of the animal.
These composite animals were fed and observed daily. When a bud appeared, it was
cut off and macerated; the stages of buds
removed varied from I—III. The remaining
portions of the parent animals were separated into the original non-budding and
normal portions and macerated. Thirteen
of the 15 grafts with a small amount of
normal tissue formed buds in the nonbudding regions, and five of the six grafts
with a larger amount of normal tissue
produced buds at the graft junction. All
grafts were intact for approximately the
same amount of time (2 to 3 days) prior
to budding.
Buds induced in non-budding tissue. The
relative abundance of different cell types
found labeled in induced buds of various
stages is shown in Figure 6. Epithelial cells
were almost entirely unlabeled at all
stages. The proportion of interstitial cells
and nerve cells labeled increased during
bud development; however, the proportion
of nematoblasts labeled remained fairly
constant (18 to 21%) at all stages considered. Only three types of cells from the
labeled normal tissue were found in induced buds: interstitial cells, nematoblasts,
and nerve cells.
613
NON-BUDDING HYDRA
Bud stage I
20
iq
Bud stage II
20
J
10
0.2
.
0
0
.
Bud stage 1
20
0
0
0
Epithelial
Interstitial
Nematoblast
Nematocyte
Nerve
Glandular
Cell types
FIG. 6. Proportions o£ cell types labeled in induced
buds sampled at different stages. Number of cells
counted: Stage I, 1320 cells from 4 buds; Stage II,
2378 cells from 4 buds; and Stage III, 1712 cells
from 4 buds. (From Moore and Campbell, 1973a.)
G14
LINDA L.
BRINKLEV
50I
I—INon-budding apical
;ii!!:ilNormal basal half
Fbrenls of bud stage I
25
iiiill
50,
Parents of bud stage II
LfiiL
0
. 0 .
, n
0
.
0
50|
fbrenls of bud stage I
25
0
Epithelial
Interstitial
Nematoblasr
.
JL
0
Nematocyte
Nerve
Glandular
Cell types
FIG. 7. Proportions of cell types labeled in the
parental tissues of induced buds. Grafts weie intact an average of 2 days. Number of cells counted:
Stage I, 1778 cells from 3 non-budding apical halves;
1850 cells from 4 normal basal halves; Stage II,
1631 cells from 4 non-budding apical halves; 1733
cells from 4 normal basal halves; and Stage III,
1878 cells from 4 non-budding apical halves; 1450
cells from 3 normal basal halves. (From Moore
and Campbell, 1973a.)
615
NON-BUDDING HYDRA
The parent tissues of the induced buds
were also examined for labeled cells. Figure 7 shows the proportions of the various
cell types which were labeled in both the
non-budding apical halves (originally unlabeled) and in the normal basal halves
(originally labeled). Parent tissues were
orouoed according to the stage of the bud
removed from them. All normal basal tissue still contains labeled interstitial and
epithelial cells, but the percentages are
reduced from the original control values.
This is to be expected as cells are constantly being sloughed and replaced in the
course of normal hydra growth. Nematoblasts and nerve cells are also labeled, having differentiated from labeled interstitial
cells during the course of the experiment.
Some normal labeled cells are also found
in the apical non-budding tissue; interstitial cells, nematoblasts and nerves were
present, but essentially no epithelial cells.
Buds induced at the graft junction. Buds
developing at the graft junction were also
examined (Fig. 8/4). These composite
buds contained normal (white) and nonbudding (green) tissue. The pattern of cell
types labeled was similar to that found in
buds produced in non-budding tissue, with
two modifications. The composite buds
contained a few labeled epithelial cells
and an increased percentage of labeled
nematoblasts. Parental tissues of these buds
(Fig. 8B) also show the same pattern of
labeled cell types as do the parental tissues
of buds formed in the non-budding tissue.
The labeled cell types found in all induced buds, regardless of their position,
and in the apical non-budding parental
tissue support the conclusion that epithelial cells do not migrate. Substantial numbers of interstitial cells, nematoblasts, and
nerves were found in non-budding tissue;
of these cell types, only interstitial cells
are thought to be migratory. The presence
of the two non-migratory cell types, nema-
Compoiite buds slooe III
FWents of buds stags III
100.
I
[Non-budding apical hall
5Normal basal hall
.P.O.
Epilheliol
Intent'fal
Nemalobhst Nematocyte
Nerva
Epilhelid
Glandular
Interstitial
Nerrahblast^hniatocyte
N«rv«
•0 0
Glandular
Cell rytws
FIC. 8A. Proportions of cell types labeled in buds
formed at the graft junction (composite buds) .
2123 cells from 5 buds were counted. Buds were
removed an average of 2 days after grafting. All
buds were in stage III. FIG. SB. Proportions of
cells labeled in parental tissues of buds formed at
the graft junction. 1922 cells from 5 non-budding
apical halves and 2069 cells from 5 normal basal
halves were counted. (From Moore and Campbell,
1973a.)
G16
LINDA L. BRINKLEY
toblasts and nerve cells, is probably the
result of the in situ differentiation of normal interstitial cells that have moved into
the non-budding tissue.
Effects of the elimination of interstitial
cells on the bud-inducing capacities of
normal tissue. Diehl and Burnett (1964)
have shown that treatment with nitrogen
mustard destroys interstitial cells while the
hydra remains intact. This effect is similar to that of X-irradiation treatment
employed by Brien and Reniers-Decoen
(1955). Using nitrogen mustard, experiments were conducted to see if bud induction by normal tissue could occur in the
absence of interstitial cells.
Normal animals were treated with nitrogen mustard and cell counts were made
on maceration preparations of animals prepared 4, 7, and 9 days later. Interstitial
cells comprised 0.5%, 0.1%, and 0.0% of
the cell populations on days 4, 7, and 9,
respectively. Interstitial cells make up about
12% of the total cell population in normal, untreated hydra. No nematoblasts
were found in any sample. The relative
proportions of the other cell types remained essentially unchanged.
Grafts were made with treated tissue
taken after each of the time intervals.
Grafts were made by combining treated,
normal hydra tissue with equal-sized pieces
of untreated non-budding or untreated
normal tissue. Both types of grafts were
made in reciprocal ways, varying which of
the two tissues was in the apical position.
The animals were fed and observed daily
for 2 to 4 weeks.
Table 3 indicates the number of grafts
of each type that budded. There are no
major differences between grafts made at
different times after nitrogen mustard
treatment. Also, it did not matter which
of the two tissue types was apical in the
reciprocal graft arrangements. Grafts which
contained untreated normal hydra tissue
showed extensive budding while the other
grafts did not.
DISCUSSION
The hypostomes of animals of the nonbudding strain differ from hypostomes of
normal animals in several ways. They do
not always form first. Building supernumerary hydranths, tentacles form first in
a somewhat disorganized pattern, then a
hypostome develops in their midst. After
the hypostome develops, the tentacles become more organized into a whorl around
the hypostome. Observations on regeneration indicate the hypostome of non-budding animals does not seem to be able to
exert its influence over more than 2 mm
of body column (Table 1). Interestingly,
this is about the size of a normal hydra.
Beyond 2 mm a new hypostome organizes
at the cut surface. The hypostomes of nonbudding hydra also differ from normal hypostomes in that they do not separate from
TABLE 3. Grafts of nitrogen mustard-treated hydra which budded.
Fraction of animals which budded
Days after nitrogen mustard treatment
Graft combination*
Non-budding, untreated
Normal, treated
Normal, treated
Non-budding, untreated
Normal, untreated
Normal, treated
Normal, treated
Normal, untreated
4
7
9
Total
1/30
1/13
0/8
3.9%
0/12
0 °1
8/8
79 %
8/8
6/12
4/6
8/13
"
/o
63 %
• The apical portion of the graft is written above the line; the basal portion below it for all
graft combinations.
NON-BUDDING HYDRA
their hosts when grafted into bipolar animals as do normal hydranths. Also, hypostomes of non-budding animals are more
effective than normal ones at suppressing
supernumerary tentacle formation when
grafted into non-budding hosts. The hypostomes of non-budding animals are not
abnormal in a sLraightfoi ward way. They
do not show a simple loss of ability to
control hydranth formation along their
body column; rather, the impairment is
manifested in more subtle alterations.
Results reported here and elsewhere
(Moore and Campbell, 1973a) imply that
bud induction in non-budding strains of
hydra is mediated by migratory interstitial
cells. During bud induction there is extensive migration of interstitial cells from the
implanted normal tissue into the induced
regions. Bud initiation can occur and continue even after the original inducing tissue has been secondarily removed. Normal
tissue loses its ability to induce buds
when its interstitial cells are eliminated.
Thus, the developmental defect underlying
the non-budding phenotype probably is associated with a property of the interstitial
cells, presumably their ability to differentiate. Hydra of the non-budding strain
examined have normal numbers of interstitial cells and their derivatives. Thus,
the developmental lesion must affect a
rather specific characteristic of interstitial
cells which is associated with bud initiation. Schaller (1971) found that nerve cells
accumulate locally just prior to budding
and suggested that a surge of nerve cell
differentiation or activity may be responsible for bud initiation. Since nerve cells
arise only from interstitial cells (Brien and
Reniers-Decoen, 1955; Burnett and Diehl,
1964), this suggestion is consistent with
the observations (Brien and ReniersDecoen, 1955; Diehl and Burnett, 1965)
that elimination of interstitial cells by
X-irradiation or nitrogen mustard also
eliminates the ability of a hydra to initiate
new buds. If it is true that nerve activity
initiates budding, our observations suggest
that the interstitial cells of the non-budding
hydra are unable to transform into nerves
which are sufficiently active or numerous
617
to initiate budding. As hypostomal regions
are known to contain large nerve cell populations, this might also account for the
abnormal behavior of non-budding hypostomes seen in regeneration and grafting
experiments.
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