Address for correspondence : Hans R. Bode, Department of Developmental and Cell. Biology, University of California at Irvine, Irvine, California 92717, U.S.A. ...
J. Cell Set. 24, 31-50 (1977) Printed in Great Britain
31
CONSTRAINTS ON THE RELATIVE SIZES OF THE CELL POPULATIONS IN HYDRA ATTENUATA HANS R. BODE,* KRISTINE M. FLICK AND PATRICIA M. BODE Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92717, U.S.A.
SUMMARY The steady-state relative population sizes of the several cell populations in Hydra attenuata were examined. In contrast to the constant average population size ratios between groups of animals, these ratios vary within limits between individual animals within a group. By maintaining animals on different feeding regimes (number of shrimp larvae ingested per day), the steady-state population size ratios were altered. The kinds of changes that occurred in these ratios suggest where controls may be operating to maintain the steady-state population sizes.
INTRODUCTION
Hydra fed 5-8 shrimp larvae daily will double in number of animals, and thereby in total tissue mass, every 3-4 days (see e.g., David & Campbell, 1972)- Over hundreds of asexual generations the relative sizes of the several types of cell populations, as well as the total number of cells per animal, remain almost constant (Bode et al. 1973). Thus, as the tissue mass increases exponentially, the several cell populations composing it are growing exponentially in constant proportion to one another. Some of the cell types are not capable of cell division, and those that are have cell cycles of varying lengths, some differing by as much as a factor of three (David & Campbell, 1972 ; Campbell & David, 1974; David & Gierer, 1974). As described earlier (Bode, 1973), these facts suggest that controls exist to maintain the steady state of the relative population sizes during continuous growth. There is some evidence that a given steady state can be altered by environmental conditions. The size as well as the total number of cells per animal is directly related to the number of shrimp larvae ingested daily (Bisbee, 1973 ; Bode et al. 1973), and is inversely related to the temperature of the culture medium (Park & Ortmeyer, 1972 ; Bisbee, 1973 ; Bode et al. 1973). There is also a suggestion that the relative population sizes may change with different feeding regimes (Bode et al. 1973). In this report we present evidence that (1) the population size ratios of individuals within a group show some variation in contrast to the constant average values of these ratios between groups of animals, and (2) the steady-state relative population sizes are very sensitive to the amount of food ingested. A description of the changes that • Address for correspondence : Hans R. Bode, Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92717, U.S.A.
32
H.R. Bode, K. M. Flick and P. M. Bode
occur at the different feeding regimes suggests where controls maintaining the steady state may be operating. MATERIALS AND METHODS Culture methods Hydra attenuata were used for all experiments. The stock culture of animals was maintained in either ' M ' solution (Lenhoff& Brown, 1970), or in a medium consisting of I X I O " ' M CaClj plus 1-25 x IO~ 5 M Na2 EDTA in spring water (Arrowhead) at 20 ± 1 °C. They were fed nauplii of Artemia salina daily and washed 6-8 h after feeding. Animals maintained on specific feeding regimes were fed as follows. For all regimes up to 10 shrimp larvae per day, the designated number of shrimp were placed daily on the tentacles of the animal. Animals fed 20 were simply flooded with large numbers of shrimp of which on average they ingested 20. Those fed 30 shrimp per day were treated as were those fed 20 except they were fed every 16 h instead of every 24 h. Cell nomenclature For cells in the body column the cell classification scheme of David (1973) was used, and extended for nematoblasts and nematocytes as follows. Three stage3 of nematoblast development were categorized as early, middle and late. Early nematoblasts were similar to little i-cells except that they contained a small ( ~ one tenth the diameter of the cell) grey-white spot or bubble, which was the first indication of nematocyst development. In middle nematoblasts the developing nematocyst consisted of a large (~ half the diameter of the cell) grey-white spot of indeterminate shape. In the late nematoblast the final shape of the nematocyst capsule was present as well as some material within the capsule. Nematocytes were defined as cells in which the final structures found within the nematocyst capsule were well developed. The nematocytes in the tentacles were classified as to type as described by Bode & Flick (1976). Quantitation of cell types Cell types of the body column. Tentacles and buds were removed from either individual or groups of animals. The remaining body column(s) was macerated into single cells and a portion of the cells prepared for measurement of the cell composition according to the method of David (1973). The total number of cells per animal was determined with a Neubauer cell counter and phase optics ( x 400). The number of cells of each cell type was determined from the total number of cells per animal and a general cell composition obtained by classifying 1000 cells by type. In some experiments more extensive data for the intermediates and products of interstitial cell differentiation were obtained. 1500-2500 cells of these cell types plus the big interstitial cells (Bi) were classified by type. The number of cells of each cell type per animal was derived from a simple proportionality : No. of cells of type X per animal No. of cells of type X in the cell composition
No. of B, cells per animal No. of B, cells in the cell composition"
The number of B, cells per animal was obtained as described above. Nematocytes in the tentacles. The total number of nematocytes in the tentacles per animal and the fraction of the total represented by each of the 4 nematocyte types were measured as previously described (Bode & Flick, 1976). Number of stenoteles per tentacle in live animals. An animal was placed in a drop of culture solution on a glass slide and gently flattened with a coverslip so that it was immobile, yet still intact. With phase optics ( x 400) all the stenoteles on the surface of a tentacle facing the objective were recognizable and were counted. The stenoteles of 3-6 tentacles were counted and a mean number of stenoteles per half tentacle obtained. After counting, the animal was returned to a culture dish. One hour later it appeared normal and was capable of feeding. The
Cell populations of Hydra
33
Jl
15
10
1
3
6
5
10 14 18
28
15 20
1
3
5
0
4
8
11 15 19 23
42
15
10
1 3
5 7 1 9 17 Fraction of total cells, %
72
Fig. i. Distribution of percentages of the total cells represented by a particular cell type of 50 animals for 12 cell types : A, mucous ; B, gland; c, big I-cell; D, epithelial; E, nematocytes (body); F, nerve; G, little I-cell; H, nematoblast; I, holotrichous isorhiza; j , stenotele ; K, atrichous isorhiza; L, desmoneme. Values for the upper 8 cell types are expressed as fractions of the total cells in the body column, while the lower 4 are expressed as percentages of the total nematocytes in the tentacles. Dashed lines (O O) represent the expected normal distnbution calculated as described by Zar (1974). Each distribution of values was tested for normality using the chi-square goodness of fit test (Zar, 1974).
CEL 24
H. R. Bode, K. M. Flick and P. M. Bode
34
number of stenoteles per half tentacle was measured every 2-3 days for the extent of the experiment. Only stenoteles are readily counted with this procedure. Desmonemes were too numerous, and the 2 types of isorhizas too difficult to distinguish with this method. RESULTS
Variation in cell population sizes among individual animals The constancy of the relative population sizes of the several cell types has been based on the comparison of the cell composition of different groups of animals (Bode et al. 1973). Such averages may mask wide individual variations. To determine what variations occur from animal to animal, the cell composition of 50 individuals was measured. To obtain uniform animals the fifty were fed 6 shrimp larvae per day and maintained at 20 °C for 3 weeks before analysis. The percentage of the total cells in Table 1. Mean cell composition and mean total cells of the body column and tentacles Sample
Mean
Standard deviation
Coefficient of variation*
(A) Mean cell comp. Cell type
of the body column 0-30 Epithelial 2i-8f 6-5 2-1 0-16 i3'0 Big interstitial 180 019 Little interstitial 3-4 0-17 Nematoblast 5'7 33-1 2-8 i-o Nematocyte (body) o-3S 1-2 4-0 0-30 Nerve i-o 0-24 Gland 4'i 0-37 i-o 2-7 Mucous Mean nematocyte Desmoneme oos (B) 4-4 87-3t 6-2 30 0-48 comp. of the Atrichous isorhiza 2 0 071 2-8 tentacles Holotrichous isorhiza 2-O Stenotele 37 o-54 Region (C) Total cell 17 IOO 79200 § 0-22 number Body column 189OO 0-36 Nematocytes in the tentacle 52100 • Coefficient of variation = (standard deviation-;-mean). t Mean value expressed as percentage of total cells in body column plus hypostome. X Mean value expressed as percentage of total nematocytes in tentacles. § Mean value expressed in total cells.
the body column represented by a particular cell type was calculated from the cell composition for each of the 50 animals using the nomenclature of David (1973) as detailed in Materials and methods. The nematocyte composition of the tentacles of each animal was measured separately. These data are presented in Fig. 1 and Table 1. Of those cell types that on average comprise more than 10 % of the total cells in the body column (Table 1), the variation in percentage of total cells is 2-2-5-fold for
Cell populations of Hydra
35
each cell type (Fig. 1), except for the desmonemes, for which it is much less. For each of the cell populations which make up less than 10% of the total, the variation is greater, as is clear from their range of values (Fig. 1) and their coefficients of variation (Table 1). These variations are in large part due to the small numbers measured. Of importance is that the several cell populations of an individual animal have widely differing mean values, while each population varies within a limited range. Another measure of the relative uniformity of values for a given cell type is that the distribution of values for each cell type but one, the atrichous isorhizas, is normal (see legend of Fig. 1). 1
1
1
1
1
1
1
B
A
15 -
o
0
j 10
•
-
{
-
\ \
o
J
\
0 7
5
?
o
1
J
t
'°\ \
a
I
i
\
-
o \
1 o
1
\
1
I
\
/
p
^n
20
60
h
I
100 20 Total cells, x10 3
1
60
i
i
, 100 >„ n
Fig. 2. Distribution of (A) total cells per body column and (B) total nematocytes in the tentacles of fifty animals. Dashed lines (O O) represent the expected normal distribution calculated as described in the legend of Fig. 1.
In the same experiment the variation among individuals in the total number of cells per body column and the total number of nematocytes was also measured, and presented in Fig. 2. Two- to three-fold differences in the number of total cells per body column and 3-4-fold differences in the total nematocytes occurred. The differences were not due to errors in the measurement of total cells or total nematocytes as these were no larger than 15%. The larger variation in total nematocyte values is also reflected in a coefficient of variation that is nearly twice that for the total cells in the body column (Table 1). Because both distributions are normal, the differences are also not attributable to 2 subpopulations of animals within the group. These results suggest that the total number of nematocytes per animal may be less stable than the relative population sizes of the 4 nematocyte types, especially for the desmonemes. Additional data obtained from groups of animals over a period of time 3-2
36
H. R. Bode, K. M. Flick and P. M. Bode
support this view (Table 2). Though there were 2-fold differences in the total number of nematocytes (Table 2 A), the ratios of the nematocyte types to one another varied much less. For example, the desmoneme: atrichous isorhiza ratio was always 6-7:1 and the desmoneme: holotrichous isorhiza ratio was 10-15:1. However, the ratios are not completely invariant, as they can be altered by changing the environment of the animals. The data in Parts A and B of Table 2 were gathered about 4 years apart and in different locations, though the animals in Part B are direct descendants of those in Part A. The fraction of holotrichous isorhizas dropped from 6 to 2 % and the proportion of desmonemes rose slightly from 79 to 83 %. Zumstein (1973) also observed changes in these ratios with time. Table 2. Fluctuations in the nematocyte composition of the tentacles Fraction of total nematocytes, S Tentacle No. of nematocytes Group Expt. animals animals A
Atrichous Holotrichous Desmonenes isorhizas isorhizas Stenoteles
1
20
306
820
II-O
2
20
787
131
57 67
3 4 5 6 7 8 9
20
792
136
61
i'i
20
32-9 32-8 3S-o 419
20 20
26-7 24-5
n-6 I3-4 10-9
5-5
20
80-5 78-2 787 78-2
149
5i
20
349
802
6-3
20
42-4
76-9
107 127
2-5 3-2 4-0 18 27
81
2-3
10
20
287
780
12-3
7-3
u
20
7-1
2-4 3-2
20
I5'i
'3
20
27-3
4-8 46
14
20
22-O
75-6 75 -o 8o-o 77-3
138
12
262 229
306 ±64
7*5 ± 19
24-9 393 27-3
Mean B
/o
is
3i
16
3°
17 18
10
29
Mean
5'2
67
1-2
i-4
5i
61
3'8 3-5
127 ±1-4
6-i ± i-o
2 7±
84-0 86-8
n-3 9'3
i-3
3-4
2-1
i-8
U S 141
2 0
296
820 804
17
4-4 37
30 3 ±6 3
833 ± 2 8
n-6±2-o
i-8±O4
33
117 128
11
± 1 1
Error of the mean value is the standard deviation.
Since variations in the total number of nematocytes exist from animal to animal, as well as from group to group of animals, there may well be fluctuations within the same animal over a period of time. This question is not readily examined since cell number measurements usually require killing the animal. However, for one type of nematocyte, the stenotele, it was possible to make periodic measurements on a live animal as described in Materials and methods. The average number of stenoteles per half tentacle was measured every 2-3 days for more than 4 months for each of 18 animals. In 4 of 9 animals maintained at 6
Cell populations of Hydra
37
shrimp per day the number of stenoteles declined with time. Whether this reflects a biological change or damage accumulated during the repeated measurements is not known. In the other 5 the number of stenoteles per half tentacle fluctuated about a mean value as shown for 4 animals in Figs. 3 and 4. The fluctuations about the mean were small (~ 2-fold) as in Figs. 3 A and 4A or large (~ 3-4-fold) as in Fig. 4B. The nature of the fluctuations were also variable. Some showed aperiodic increases or drops, while others (Fig. 3B) showed something akin to periodic fluctuations or an oscillation. We have been unable to correlate the period of the oscillation with any 1
i ,1,1'"
,
t t
TTT fir,* 1
1
r
tl
I
20 10
20
I 1 _L4i_ 30 40 50 60 70 80 100 Time after beginning of measurements,days
120
140
Fig. 3. Fluctuations in the number of stenoteles per half tentacle with time. Each figure (A, B) represents the data for a single animal maintained on 6 shrimp per day for about 4 months. Each value is the mean number of stenoteles counted on half of each of 3-6 different tentacles. Error bars represent the average deviation of the mean. The dotted line is the average of all the mean values obtained for an animal.
events in the differentiation of nematocytes from interstitial cells. Similar trends for animals maintained at 3 shrimp per day were found, though as a group the mean values were somewhat lower. The number of stenoteles could vary 4-fold from animal to animal on any given day, but the variation from tentacle to tentacle within the same animal was usually about 50%. The general conclusions from all of these measurements are that the relative population sizes of the several cell types within the body column and separately within the tentacles can vary by factors of 2-5 from animal to animal within a group, but that the mean values between groups remain reasonably constant, consistent with the earlier findings (Bode et al. 1973). The total number of nematocytes per animal
H. R. Bode, K. M. Flick and P. M. Bode
1 '
A
140 -
,
t * I^
100
I(
60
-
20 1
1
f
260
,1
i
140
1 ..
•
" f
+
i
•I
1
i
1
••4--j
••••
't
100 60
i
B
220 180
1
( - • -
|
\
1 1 -
-
-
:
1 •
{
1
-
20 1
10
20
1
30
40
t
114
50
60
70
80'
100
120
j" 140
Time after beginning of measurements, days
Fig. 4. Fluctuations in the number of stenoteles per half tentacle with time. Each figure (A, B) represents the data for a single animal maintained on 6 shrimp per day for about 4 months. Each value is the mean number of stenoteles counted on half of each of 3-6 different tentacles. Error bars represent the average deviation of the mean. The dotted line is the average of all the mean values obtained for an animal.
varies more widely than the total number of cells and for at least one type of nematocyte there are 2-4-fold fluctuations in number in an animal with time. Changes in relative population sizes with changes in feeding regimes
Separate groups of animals were placed on different feeding regimes, receiving o, 1, 3, 6, 10, 20, or 30 shrimp per day. Changing the feeding regime changes the size and the budding rate. Since steady-state conditions for the total cells per animal and for the relative cell population size were necessary for these studies, animals were maintained on the several regimes until steady state was achieved before measurements were made. The budding rate, which is a direct function of the amount of food ingested (Bode, unpublished results), served as a convenient measure. Steady-state budding rates were reached within 10 days for all feeding regimes. Thereafter, groups of animals on each of the feeding regimes were analysed at intervals of 7—9 days starting on day 11 after the beginning of the experiment. The number of cells of each cell type in the body column and the total nematocytes in the tentacles were determined for each sample and are presented in Table 3. Though steady-state budding rates for each feeding regime were reached within
Day+
Ecto
Endo
Bi
Li
enb
A
rnnb
lnb +ncb
Cell type in body column ner
gl
muc
* Total body cells
Total nematocytes in tentacles
+ Day : day after beginning of feeding regime when measurements made. Ten animals were used for each measurement. Abbreviations : Ecto, epithelio-muscular cell ; Endo, digestive cell ; Bi, big interstitial cell ; Li, little interstitial cell ; enb, early stage nematoblast ; rnnb, middle stage nematoblast ; lnb, late stage nematoblast ; ncb, nematocytes in the body column ; ner, nerve cell ; gl, gland cell ; muc, mucous cell.
Fee* regime
I
Table 3 . Effect of feeding regime on the cell composition
4o
H. R. Bode, K. M. Flick and P. M. Bode
10 days, animal size and the relative population sizes did not reach a steady state in all cases, as is apparent in Table 3. Animals maintained on the extreme feeding regimes, o and 30 shrimp per day, never reached a steady state as some or all populations declined greatly throughout the course of the experiment. This was not unexpected in starving animals, and we, as well as others (Otto, personal communication), have observed that overfed animals eventually undergo bizarre morphological changes and die. Animals maintained at the next most extreme conditions, 1 and 20 shrimp, also may not have reached steady state, but the declines in most of the cell populations were much more gradual. Animals on the other 3 regimes, 3, 6 or 10 shrimp per day, were in a steady state. To examine the effects of the amount of food ingested daily on the relative population sizes, we have compared the means of the three sample values for each of the feeding regimes 1 through 20 and the last value for o and 30 with one another. The first and most obvious effect is an increase in animal size with increasing number of shrimp ingested. This is indicated by the cell composition on days 11 and 12 (Table 3) as a directly proportional increase in the number of epithelial cells (ectodermal and endodermal cells) per animal with increasing number of shrimp. However, with increasing time, the extreme feeding regimes (o and 30) show a decrease in that number. The ectodermal and endodermal epithelial cells for most of the feeding regimes remain in a 1:1 ratio. The marked differences between the 2 populations in animals maintained on o or 1 shrimp per day are most likely due to the difficulty in recognizing the endodermal cells at these feeding regimes. They would be low in food granules which distinguish them from the ectodermal epithelial cell. A convenient way to describe changes in relative population sizes is to relate a particular cell population to the epithelial cell population (defined as half the sum of the ectodermal plus endodermal cells). The two epithelial cell types make up the 2 tissue layers and, therefore, provide the matrix in which other cell populations reside. Changes in the ratio of any cell type to epithelial cells graphically represent increases or declines in the density of that particular cell. Three different kinds of changes were observed (Fig. 5). The populations of gland cells, mucous cells and nerve cells, decline relative to the epithelial cells as the number of shrimp ingested increases above one shrimp per day. In striking contrast, the ratio of total nematocytes to epithelial cells first rises 10-fold and then declines 20-fold over the range of feeding regimes. The 3 classes of nematoblasts, which are transient populations that provide a sensitive measure of the rate of nematocyte production, all exhibit the same patterns of change as the nematocytes. The data for the middle nematoblasts are shown in Fig. 5B. The remaining pair of cell types, the single big i-cells and single little i-cells, require a more extensive consideration. One population of great interest are the multipotent interstitial cells. The class of cells morphologically distinguishable as big interstitial cells, presented in Table 3, contains multipotent stem cells as well as cells committed to nematocyte differentiation (David & Gierer, 1974). Lumping these functionally different cells together obscures the effects of increased feeding on the relative population size of the stem cells. In macerates, big i-cells occur most often singly or in pairs, less frequently as nests of 4
Cell populations of Hydra
41
and occasionally as groups of 3, 6, or 8. David & Gierer (1974) have shown that the multipotent stem cells are among the single cells and pairs, while all larger nests are committed to nematocyte differentiation. However, a sizeable fraction of the pairs are probably also committed to nematocyte production. In measuring the cell composition, the big i-cells were classified by nest size, of which a condensed version is presented in Table 4. The decline in the fraction of nest sizes 3-8, which were committed
0-6
1
0-4
0-2
ii
1
1
20
1-6
1-2
0-8
0-4
rial 3
6
10
30
Feeding regime (No. of shrimp ingested daily)
Fig. 5. Ratios of the population sizes of several cell types to the epithelial cell population size. The data for calculating the ratios were taken from Tables 3 and 4, as described in the text. For each feeding regime the epithelial cell population was defined as one half the sum of the epithelio-muscular cell and digestive cell populations. Each histogram represents one cell type identified as follows. In A : | ° 0 |, gland cell; I *» I, mucous cell; ^ | , single big i-cell. And in B, I I. single little i-cell; Y. .], nerve cell i Y//X, middle nematoblast; \X$], nematocyte. Error bars represent the standard deviation of the mean.
3670
771 780
190
1380
1790 2290
2990 1400 2670
2650 2710 1080
4980
1270
612
3240 1480 1760
3210 1950 3270
4210
3 950
1930
4240 2280 1200
6 6 6
10
10
20
20
20
30 30 30
10
2040
224
0-30
o-34 0-37 o-34 0-47 o-33
O-II
0-13 0-09
O-I2 0-09 0-06
O-II
0-15 0-13
o-is 0-25 O-23
0-44
O-23
0-40 0-41 0'43 0-41 0-36 0-40
O-I9 O-2I
o-5S 0-46
0-16
O-22 O-22
O-II O-2O O-2O
3—8/2'j' 1
4280 2090 1400
3400
S47O
452O
2920 3220
2O6O 2 030 2 2OO
1850 1880 1680 2850 2980 2480 3000 1980 1730 734O
284
798
272O 2 38O I53O 2I4O 239O 471 250O
3O8O I9OO l68O
258O I 89O
255°
27
I84
546
> I*
A
306
I33O 1070
r
2890 1680
6660 7860 3870 6840
475°
34io 10100 5 300
3880
437O 6080
5560
5400
3880
1880 1250 333 3910 3910
N.
063 0-72 087
o-88
068 0-70
o-73 o-55 c-68
0-49 0-51 0-51
o-57
OS4
053
017
037 0-28
O-I2
030
0-32
0-32
o-45
0-27
049
0-51 0-49
0-57 0-47 0-46 o-43
0-52
o-53
008
0-47 0-48 o-43
ois
C29
> l/£t
0-92
A
Ratios
0-85
0-71
i/2f
Little interstitial cells No. of each nest size per animal (nest size)
• > 1: all nests with more than one little i-cell. f 2 : sum of all nests.
o-59 0-59
3860
347
o-54 o-6i
054
0-40
7800 7320 3170
0-50
389S 6670 049
0-44
O'33
o-34
0-41
049
0-29 0-32 0-52
0-50
o-33
O-29 032
2/Sf
Ratios
028
7290
8330 4360 5 33O
3 39°
5800 4280
10600
734 936 659
1090 506
1250 1090 1230
o-33 0-37
3 35°
636
1 840 1690 1360
871 1 210 1250
3 3 3
1
1
3670
0-26
3830
1280 942 613
2900 2100 1990
1620 1240 1230
1
132
0-48 0-47
62
991
0
2430
267 198 26
7°5 3J7 44
0
1435 475
i/2f o-S9
0
2
3-8
2
1
Feeding regime
No. of each nest size per animal (nest size)
A
Big interstitial cells
Table 4. Distribution of nest sizes of big and little interstitial cells
to
•
•
-
to
Cell populations of Hydra
43
to nematocyte formation, parallels the decline in numbers of nematoblasts and of nematocytes in the tentacles with increasing number of shrimp (see Table 3, Fig. 5). The fraction of pairs has a similar behaviour, being maximal when maximal numbers of nematocytes were produced, and declining at those feeding regimes where fewer nematocytes were made. In contrast, the single big i-cells are not correlated with nematocyte production at all. A more accurate measure of the behaviour of the ratio of the multipotent stem cell population with respect to the epithelial cells would be
30 Feeding regime (No. of shrimp ingested daily )
Fig. 6. Ratios of the population sizes of several cell types to the single big i-cell population size. The data were taken from Tables 3 and 4 as described in the text. Cell types identified as follows : • , single little i-cell; HI, nerve cell; 0 , middle nematoblast; §|, nematocyte. Error bars represent the standard deviation of the mean.
to compare the ratios of single big i-cells with epithelial cells as shown in Fig. 5 A. This ratio remains reasonably constant over the range 1-20 shrimp per day, declining at both extremes. The single little i-cell population is of interest because it is probably a measure of the nerve cell precursor population. Little i-cells, which occur in nests of 8, 16 or 32 cells, have been considered to be early intermediates in nematocyte differentiation
44
H. R. Bode, K. M. Flick and P. M. Bode
(David, 1973). Other nest sizes including some single little i-cells are thought to be parts of these larger nests broken down during the maceration procedure. However, in the hypostome where i-cell differentiation is almost solely into nerves (Bode et al. 1973; David & Gierer, 1974), a very high fraction of the little i-cells occur as single cells (Smith & Bode, unpublished results). Hence, the single little i-cells, though to some extent a mixed population can be considered as a fair measure of nerve cell production. This is consistent with the greater ratios of single little i-cells found at Table 5. Nematocyte composition of the tentacles Fraction of total nematocytes, % Feeding regime
Total nematocytes*
o o o
3600 1100 360
i i i
13200 8500 7900 10300 15600 11900 33 500 20400 22000
Atrichous isorhizas
Holotrichous isorhizas
Stenoteles
2'5
129 26-7 33'3
75-9 665 64-2
107
82-0
103 14-4 17-5
o-6
7-1
o-i
12-6
9"5
O-2 o-i o-i
n-4
73'0
747 78-9 78-2 796 86-5
6-8
16-0 II-2
77
4-3 4'3
o-i
6-5
84-6 846
8-8 138
836 823
129 141
20 20 2O
10500 6600 3100
766 77'5
22-6 211 141
0-4 o-i o-i
9100 6400 2100
76-5 777 80-5
23-1 22'0
o-i o-i
HI HI
CO CO CO
O OO
17600 18100 17800
M
91 12-7
82-2
18-8
57 92
o-i
830
o o o
6 6 6
Desmonemes
03 o-i
35 36 o-6 1-2
36 O-2 0-3 0-7
• Data from Table 3.
feeding regimes with relatively few nematocytes (o, 20, and 30) as presented in Table 4. As shown in Fig. 5 B, the relative size of this population is reasonably constant up to 10 shrimp per day and then declines. Unlike the middle nematoblasts and nematocytes which exhibited the same patterns of change with increasing feeding, the nerve cell precursor population and the nerve cells show somewhat different patterns. Since the multipotent interstitial cells, their differentiation intermediates, and their product cells are all closely related, changes in their relative population sizes may yield clues as to how these populations are controlled. In this case the ratio of each cell type to the single big i-cell, the multipotent stem cell, was calculated and is presented in Fig. 6. In general the patterns are similar to those observed for the
Cell populations of Hydra
45
epithelial cell ratios. The ratios of middle nematoblasts and nematocytes increase from low to middle level feeding regimes and decline sharply at high numbers of shrimp. As feeding ranged from 1 to 30 shrimp per day, nerve cells declined slightly with respect to the i-cells, whereas the single little i-cells were more or less constant. The one marked difference is the very large ratio of nerves and single little i-cells to single big i-cells in starving animals. Finally, of those i-cells committed to nematocyte differentiation, the effects of feeding regime on the relative population sizes of the 4 types of nematocytes among themselves was examined. Measurements of the nematocyte populations in the tentacles were made as part of the general analysis of the cell composition and are presented in Table 5. Most striking is that in contrast to the very large changes in the total numbers of nematocytes (~ 10-fold from 6 to 30 shrimp), the ratios of the nematocyte types change much less. The fraction of the total represented by desmonemes remains essentially constant from 1 to 30 shrimp. The only changes are in the fraction of atrichous isorhizas, which rise approximately i-5-fold, and a decrease in stenoteles over the range of feeding regimes, 1-30 shrimp. The sharp decrease in stenoteles in animals fed 20 and 30 shrimp is attributable to the very large numbers discharged upon contact with the daily offering of 50-150 shrimp. The 3-fold decline in stenoteles from 1 to 10 shrimp per day, however, cannot be explained in this manner since the number of stenoteles discharged in capturing 10 shrimp represents no more than 5 % of the entire complement (Smith, Oshida & Bode, 1974) and would be replaced daily by new incoming stenoteles (Bode & Flick, 1976). For reasons unknown the numbers of holotrichous isorhizas were low throughout these experiments. The ratios in the starved animals change strongly but simply reflect a condition where nematocyte synthesis has virtually ceased (see Table 3). The increase in the fraction of stenoteles could be due to movement of those mounted initially on the body column into tentacles due to tissue displacement (see Campbell, 1967). The same mild changes with respect to the different feeding regimes were also found for the 4 types at the late nematoblast stage. Though great changes take place in total number of nematocytes produced, the relative ratios of the several nematocyte types are much more stable.
DISCUSSION
Earlier observations have shown that the several populations of cells in the continually growing hydra remain in constant proportion to one another over hundreds of asexual generations (Bode et al. 1973). These measurements were made on groups of animals that received 3-12 brine shrimp larvae daily. By examining animals maintained on 6 shrimp larvae per day we found that though the mean values of the population sizes were constant for groups of animals, there was some variation from individual to individual within a group. The fraction of the total cells made up by each population varied over a 2-3-fold, occasionally 5-fold, range. Also, the stenotele population in individual animals varied in size with time over a 3-month period. Not only was there variation about the mean value of a population size, but the
46
H. R. Bode, K. M. Flick and P. M. Bode
relative sizes of the several cell populations could be altered. By maintaining animals on different feeding regimes, a number of different steady states with respect to relative population sizes were obtained. However, the range of steady states one could achieve by varying the level of food intake was limited. Animals maintained on the extreme regimes, o and 30 shrimp larvae per day, never reached a steady state and eventually disintegrated. Animals at the next most extreme conditions, 1 and 20 shrimp larvae per day, showed a gradual decline in at least some of the cell populations by the end of the experiment, 26-28 days. These results indicate that for a hydra to grow, reproduce asexually, and maintain itself, an exact set of ratios of cell population sizes is not necessary. The animal can tolerate a limited range of steady states of relative cell population sizes. Also for any given steady state it can tolerate at least 2-fold variations about a mean value for the population of any cell type. The animal is normal and healthy under all but the extreme conditions. Despite the observed allowable variations, the fact that for a given level of feeding the mean values of the relative population sizes remain constant over many generations presents a formidable problem. In a culture of well fed hydra the tissue mass of the culture, and therefore the population of each cell type, is expanding continuously at an exponential rate. If all cells were simply in the mitotic cycle and each had the same cell cycle time, then maintaining the several population sizes in constant proportion to one another would be trivial. However, only some of the cell populations divide and their cell cycle times differ (David & Campbell, 1972 ; Campbell & David, 1974 ; David & Gierer, 1974). Other cell populations arise through differentiation (e.g. Slautterback & Fawcett, 1959). The i-cell population is in part undergoing division to maintain itself, and in part differentiating into other cell types (David & Gierer, 1974). Our interest is to determine if there are any controls operating to keep the cell populations in register, or if the constancy is due merely to an averaging of independent reactions to environmental influences. The feeding regime data provide some support for the view that each cell population behaves independently of other populations with respect to size. The steady state population size of the epithelial cells and the single big i-cells, both populations in the mitotic cycle, increased with increasing levels of feeding, whereas the other populations did not. At 11-12 days when the budding rate had reached a steady state in all feeding regimes, the number of epithelial cells was directly proportional to the number of shrimp ingested per day. However, by the end of the experiment (26-28 days) the extreme regimes showed a decrease in the epithelial cell population, although the budding rate remained unchanged. Except for the starving animals, this is not understood. At feeding regimes of 1-20 shrimp larvae per day the single big i-cell population remained roughly in constant proportion to the epithelial cell population. On the basis of this experiment it is not possible to tell if this is due to coordination between the 2 cell types, or whether both are merely responding to the increased feeding in exactly the same manner. However, earlier work (Bode, Flick & Smith, 1976) suggests that a control could be operating here. The big i-cell population was reduced to 1-2% of
Cell population of Hydra
47
normal with hydroxyurea, which had no effect on the epithelial cells. If there were no means to 'measure' their own density in the tissue, either with respect to themselves or the epithelial cells, one would have expected the number of i-cells to remain at the reduced level. However, the i-cell numbers did recover to control levels. This indicates that there is some homeostatic mechanism governing the big i-cell population size or the big i-cell:epithelial cell ratio. The third cell type of the body column whose population is in the mitotic cycle is the gland cell (Challoner, 1973). For any feeding regime other than starvation, the absolute number of cells remained approximately the same. This population appears to increase at a fixed rate and apparently does not respond to either increased nutrients or its dilution with respect to an expanded epithelial cell population. Up to this point a culture of hydra has been considered as a set of cell populations that are expanding continuously at an exponential rate. However, steady-state adult animals remain relatively constant in size. Cells are sloughed at the extremities : the tentacles, hypostome and basal disk (Campbell, 1967). At feeding regimes above 2 shrimp larvae per day most of the excess tissue is removed by budding (Campbell, 1967). This is a dynamic situation in which all cells are continuously displaced toward the head, the foot, or into a bud. Formation of the buds and the specialized structures at the extremities are controlled by pattern-forming and morphogenetic processes. These processes regulate the shape and size of the animal as well as the relative proportions of the various body regions to one another. Hence, they impose limits on the size of the cell populations in the mitotic cycle. Since the cell populations that arise by differentiation are formed in a regional pattern, i.e. their differentiation is position-dependent, it is likely that their kind and size is also affected by these pattern-regulating processes. The most clear-cut example of this concerns the ectodermal epithelial cells. As they are displaced apically on to the tentacles they differentiate into non-dividing battery cells, and when displaced basally into the foot they form non-dividing foot gland cells (Campbell, 1967). Though these 2 population sizes were not measured, they form distinctive parts of the overall morphology, and appear to remain in constant proportion to the parent mitotic epithelial cell population. The mucous cells which comprise most of the endoderm of the hypostome are another example. The origin of this population is unclear. They are capable of cell division (Challoner, 1973), but also arise by differentiation as they reappear at the regenerating apical tip 2 days after decapitation (Bode et al. 1973), which removes the entire population. The size of this population was unaffected by the level of feeding and is probably affected by pattern-regulating processes governing the hypostome. The remaining family of cell populations to be considered are the differentiation products of the i-cells. In steady-state animals 60 % of the i-cells undergo division while the remaining 40 % differentiate (David & Gierer, 1974). Of those differentiating a constant fraction form nerves and the rest nematocytes. The 4 types of nematocytes are also formed in constant ratios to one another. These ratios are for the whole animal and mask the fact that the type of differentiation an i-cell undergoes is dependent
48
H. R. Bode, K. M. Flick and P. M. Bode
upon its position along the body column. The fraction of i-cells committed to nerves is close to 100% in the hypostome and basal disk, while it is only 10% in the body column (David & Gierer, 1974). For 2 of the nematocyte types, the frequency of i-cell commitment to desmonemes is 5-fold higher than to stenoteles in the gastric region, whereas in the peduncle commitment to stenoteles is twice that to desmonemes (Bode & Smith, 1976). As with the epithelial cells, pattern-regulating processes may influence i-cell differentiation behaviour, and thus, the population sizes. The steady-state ratios within this family and the differentiation behaviour of the i-cell population are not immutably fixed. Instead they can be altered in several ways, some of which suggest controls affecting these populations. In the hydroxyurea experiment described above the reduced i-cell population recovered to normal due to a shift in its division:differentiation ratio from 60:40 to 70:30 until the normal icell density or population size was achieved (Bode et al. 1976). Hence the fraction of i-cells committed to differentiation is a function of the i-cell density. The type of differentiation an i-cell undergoes is, in one case at least, subject to feedback from the product cell population. A reduction of the stenotele nematocyte population to 10% of normal leads to a specific increase of i-cells committed to this differentiation pathway (Zumstein & Tardent, 1971 ; Zumstein, 1973 ; Smith, Nadeau & Bode, submitted for publication). Altering the environmental conditions also affects the differentiation behaviour of the i-cells. Changes in level of feeding resulted in different nerve: nematocyte ratios. In striking contrast, the ratios among the 4 nematocyte types were fairly constant, even though the total number of nematocytes varied 10-fold over the several feeding regimes. Part of the observed nerve: nematocyte ratio changes may be attributable to changes in the relative sizes of the body regions, as illustrated by the starving animals as an extreme example. After 30 days without food, the head and foot comprise a much larger part of the total tissue than in fed animals (Cupp & Bode, unpublished results). I-cells in these animals undergo nerve cell differentiation to the exclusion of nematocyte differentiation which is typical of the head, foot and their subjacent regions (David & Challoner, 1974). However, this argument does not explain the drastic decrease in differentiation products at the higher feeding levels. As a second example, hydra will undergo sexual reproduction under the appropriate external conditions. In this state i-cells will form one or the other (or both in hermaphroditic species) type of gamete, sometimes to the exclusion of nematocyte production (Brien, 1961 ; Burnett & Diehl, 1964). What emerges is a variety of constraints on the several cell populations which serve to maintain their sizes, although not strictly with respect to one another under all conditions. Under the conditions examined the gland cell population appears to maintain its size unaffected by anything external to it (except starvation). The epithelial cells respond directly to increased feeding, but this population and its derivatives may vary only within a range governed by the pattern-regulating and morphogenetic processes. The i-cells and their family of differentiation products are apparently
Cell populations of Hydra
49
subject to a number of constraints. The most stringent of controls may be that regulating the density of the i-cells with respect to the epithelial cells. I-cell differentiation, and thus the numbers of differentiation products, are affected by the i-cell density, feedback from the differentiation products, location in the body column, proportions of the body, as well as some environmental parameters. The result of such a network of controls is that a hydra can grow, reproduce asexually and maintain itself within a range of steady states of population sizes. We thank Richard Campbell, David Rubin, and Marcia Yaross for their comments on the manuscript. This research was supported by a grant from the National Institutes of Health (HD 08086). REFERENCES BISBEE, J. W. (1973). Size determination in Hydra: The roles of growth and budding. J. Embryol. exp. Morpk. 30, 1-19. BODE, H. (1973). Humoral influences in Hydra. In Humoral Control of Growth and Differentiation (ed. J. LoBue & A. S. Gordon), pp. 35-58. New York : Academic Press. BODE, H.,
BERKING, S., DAVID, C. N.,
GIERER, A.,
SCHALLER, H.
& TRENKNER, E. (1973).
Quantitative analysis of cell types during growth and morphogenesis in Hydra. Wilhelm Roux Arch. EntwMech. Org. 171, 269-285. BODE, H. & FLICK, K. (1976). Distribution and dynamics of nematocyte populations in Hydra aUenuata.J. Cell Sci. 21, 15-34. BODE, H., FLICK, K. & SMITH, S. (1976). Regulation of interstitial cell differentiation in Hydra attenuata. I. Homeostatic control of interstitial cell population size. jf. Cell Sci. 20, 29—46. BODE, H. R. & SMITH, G. S. (1976). Regulation of interstitial cell differentiation in Hydra attenuata. II. Correlation of the axial position of the interstitial cell with nematocyte differentiation. Wilhelm Roux Arch. EntwMech. Org. (in Press). BRIEN, P. (1961). Etude d'Hydra pirardi (nov. spec). Origine et repartition des nematocystes. Gametogenese. Involution postgam6tique. Evolution reversible des cellules interstitielles. Bull. biol. Fr. Belg. 95, 301-364. BURNETT, A. L. & DIEHL, N. A. (1964). The nervous system of Hydra. III. The initiation of sexuality with special reference to the nervous system. J. exp. Zool. 157, 237-249. CAMPBELL, R. D. (1967). Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement. J. Morph. 121, 19—28. CAMPBELL, R. D. & DAVID, C. N. (1974). Cell cycle kinetics and development of Hydra attenuata. II. Interstitial cells. J. Cell Sci. 16, 349-358. CHALLONER, D. (1973). Cell Kinetics in Hydra attenuata (Pallas). M.Sc. Thesis, University of Newcastle-upon-Tyne. DAVID, C. N. (1973). A quantitative method for maceration of Hydra tissue. Wilhelm Roux Arch. EntwMech. Org. 171, 259-268. DAVID, C. N . & CAMPBELL, R. D. (1972). Cell cycle kinetics and development of Hydra attenuata. I. Epithelial cells. J. Cell Sci. 11, 557-568. DAVID, C. N. & CHALLONER, D. (1974). Distribution of interstitial cells and differentiating nematocytes in nests in Hydra attenuata. Am. Zool. 14, 537-542. DAVID, C. N. & GIERER, A. (1974). Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. .7. Cell Sci. 16, 359-375. LENHOFF, H. M. & BROWN, R. D. (1970). Mass culture of hydra : an improved method and its application to other aquatic invertebrates. Lab. Animals 4, 134-154. PARK, H. D. & ORTMEYER, A. B. (1972). Growth and differentiation in Hydra. II. The effect of temperature on budding in Hydra littoralis. J. exp. Zool. 179, 283-288. SLAUTTERBACK, D. B. & FAWCETT, D. W. (1959). The development of cnidoblasts of hydra. An electron microscope study of cell differentiation. J. biophys. biochem. Cytol. 5, 441-452. SMITH, S., OSHIDA, J. & BODE, H. (1974). Inhibition of nematocyst discharge in hydra fed to repletion. Biol. Bull. mar. biol. Lab., Woods Hole 147, 186-202. 4
CEL
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ZAR, J. H. (1974). Biostatistical Analysis. Englewood Cliffs, New Jersey: Prentice-Hall. ZUMSTEIN, A. (1973). Regulation der nematocyten produktion bei Hydra attenuata Pall. Willielm Roux Arch. EntwMech. Org. 173, 294-318. ZUMSTEIN, A. & TARDENT, P. (1971). Beitrag zum Problem der Regulation der nematocyten Produktion bei Hydra attenuata Pall. Rev. suisse Zool. 78, 705-714. {Received 16 March 1976 -Revised 30 September 1976)
