J. Embryol. exp. Morph. 82, 67-95 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
(fj
Mutational analysis of patterning of oral structures
in Tetrahymena
II. A graded basis for the individuality of intracellular structural
arrays
By JOSEPH FRANKEL, E. MARLO NELSEN, JULITA
BAKOWSKA AND LESLIE M. JENKINS
Department of Biology, University of Iowa, Iowa City, Iowa 52242, U.S.A.
SUMMARY
The ciliary arrays of the oral apparatus of the ciliated protozoan Tetrahymena thermophila
each have their own unique 'pattern signature', which varies little so long as the number of
arrays remains the same. In this study, we analyse the consequence of increases in the number
of these arrays (membranelles) brought about by certain mutations. In oral apparatuses of
mutant cells, the addition of a membranelle is associated with specific alterations in at least
one of the other membranelles. The features that are altered include the relative lengths of
membranelles, the state of ciliation of basal bodies located at specific positions within these
membranelles, and the spatial configurations resulting from displacement of ciliary units
during late oral development. The final organization of each membranelle depends upon its
relative position along the length of the oral apparatus. This indicates that the membranelles
are not individually 'named' by the organism, and suggests that the unit of pattern organization
is the membranelle field as a whole. In the Discussion, we consider means for testing whether
the same underlying idea might also apply to multicellular systems, such as the vertebrate
limb, in which spatially ordered differences appear to be superimposed upon a fundamental
repeating pattern.
INTRODUCTION
Whenever a complex structure is made up of individually recognizable
elements, one may inquire whether these elements are formed independently
from each other or whether their formation is coordinated in some way. This
question was raised forcefully by Bateson (1892, 1894), and is commanding
renewed attention (e.g. Goodwin & Trainor, 1983; Holder, 1983). While this
issue typically is considered in the context of the development and evolution of
compounded structures such as vertebrate teeth or digits, it is equally applicable to any other set of structures in which three conditions are met: (1) the set
of structures is clearly distinguishable and well demarcated, (2) the elements of
the set differ qualitatively from each other, so that they may be recognized
individually without reference to their relative position in the whole, and (3)
there exists variation in the form and especially the number of the elements in
the set, so that one can monitor effects of changes in the composition of the set
68
J. FRANKEL AND OTHERS
on the individuality of the elements within that set. This third criterion is the
foundation of Bateson's analytical method, and of ours as well.
Although the question of individuality of the elements that constitute a larger
structure or set has been considered for multicellular structures, most commonly
the mammalian dentition and the pentadactyl limb, it can equally be raised for
any complex structure that fulfills the fundamental criteria listed above. An
intracellular organelle system, the oral apparatus (OA) of the ciliated
protozoan Tetrahymena thermophila, meets all of the requirements for addressing this question. Not only is it clearly demarcated from the surrounding
cortical topography, but its four elements, the undulating membrane (UM) and
the three membranelles, differ from each other in qualitative features that
allow easy recognition without reference to relative position within the OA as
a whole. The UM differs fundamentally from the membranelles in internal
architecture (e.g. Williams & Luft, 1968; Smith, 1982), while the three membranelles of OAs from wild-type cells manifest patterns of arrangement of basal
bodies ('sculpturing') that are highly distinctive for each membranelle and
virtually invariant among OAs (Williams & Bakowska, 1982; Bakowska, Frankel & Nelsen, 1982a). Finally, the crucial requirement for variation in number
of elements is satisfied by the existence of mutations that alter the number of
membranelles.
Fulfilment of the three conditions allows ascertainment of whether or not
individuality of the elements is maintained in the face of a change in their
number. Yet, whenever one uses mutants to carry out such a test, one must be
on guard against modifications of patterning that are idiosyncratic effects of
individual mutations. This can be controlled in a number of ways, all of them
utilized in this study: (a) using a single mutation, one can compare OAs in which
the number of pattern elements has been altered with other OAs from the same
culture in which the number of these elements is normal, to ascertain whether
modification of pattern is associated with modification in number; (b) one can
compare the pattern modifications associated with similar changes in number of
elements brought about by different mutations, to find out whether the modifications are the same or at least mutually consistent; (c) one can compare the
modifications of patterns associated with change in number of elements with
abnormalities associated with other changes in the elements, to ascertain
whether the modifications associated with alteration in number of elements are
unique; finally (d) one can determine whether the modifications of the patterns
are in fact spatially coordinated within the OA.
In this study, we will show that one cannot add a membranelle without altering
the pattern of at least one of the others. This result is consistent with earlier
observations on effects of subtraction of a membranelle by severe starvation
(Bakowska et al. 1982a) and is suggestive of coordination of development of all
of the membranelles of the OA through unified underlying mechanisms that
operate in a continuous manner throughout the membranelle field.
Gradation of intracellular patterns in Tetrahymena
69
MATERIALS AND METHODS
The mutant stocks employed in this study are IA-305 (mpD), IA-309 (mpCl),
IA-317 (mpC2), and IA-313 (big). Further information about the derivation of
these stocks is included in Table 1 of the preceding paper (Frankel, Jenkins,
Bakowska & Nelsen, 1984). All of the methods utilized in this study are
described in that paper.
RESULTS
I. Mutations that increase the number of membranelles
(a) Genetics
The mutations considered here include mpD and big, utilized in the preceding
study (Frankel et al. 1984), as well as two alleles at the mpC locus, mpCl and
mpC2. Both of these are single-gene recessive mutations, the former induced by
nitrosoguanidine and the latter by ethyl-methane-sulfonate (EMS) [see Table 1
in Frankel et al. (1984)]. mpC is located on chromosome 3L, following Bruns'
(1982) nomenclature.
(b) Phenotypes
The mpD (mp = 'membranellar pattern') mutation has been described briefly
in the previous paper (Frankel et al. 1984). At 28 °C, this mutation brings about
a modest enlargement of the OA without an increase in the number of oral
membranelles (Figs 1,4, this paper). Following transfer to 36-5 °C, OAs possessing four or five membranelles (Fig. 2) appear at a rate that suggests that all
predivision oral primordia formed after the temperature shift produce four or
five membranelles (Fig. 5). A study of oral development in mpD cells at 15-min
intervals after the temperature shift has shown that this is indeed true for an
overwhelming majority of oral primordia (Lansing, personal communication).
The mpD mutation is unique among the known mutations that affect oral
patterns in lacking branched membranelles or short membranelle fragments
located either anterior to the others or interposed among them. The vast majority
of mpD OAs are as regular as the ones shown in Figs 2 and 5.
The expression of the 4- or 5-membranelled phenotype of mpD at 36-5 °C is
accompanied by a substantial increase in length both of cells and of OAs while
cell width remains constant and OA width nearly so (Table 1). The ratio of
estimated oral surface area to cell surface area (Table 1, right column) is increased in mpD cells, especially at 36-5 °C, suggesting a possible differential
effect of this mutation on the size, mostly length, of the midbody oral primordium. This is quite different from the relationship in big cells (Frankel etal. 1984,
Table 2) in which enlargement of the OA typically fails to keep up with the still
greater enlargement of the cell.
70
J. FRANKEL AND OTHERS
The threshold temperature for expression of extra membranelles by mpD cells
has not been determined precisely, but is not likely to be much above 28 °C, since
approximately half of the OAs possessed four membranelles after growth of
mpD cells at about 30 °C for 12 h.
mpCl and mpC2 are partially-conditional mutations. Oral abnormalities are
expressed in 20 to 60 % of the mutant cells at 28 °C and in about 80 % of the cells
after 8h at 39-5 °C. Cell size and population growth rate remain approximately
normal at both temperatures.
The oral abnormalities in mpCl and mpC2 cells are very similar. OAs of both
mutants typically possess a short extra membranelle that is located far back in the
buccal cavity, as if it were a prey item in the process of being swallowed (Fig. 3).
Oral primordia all are of the midbody predivision type. Results of enumeration
of membranelles in oral primordia at stages late-4 and 5 (see Fig. 1C of the
preceding paper for diagrams of stages) in both mpCl and mpC2 at both 28 °C
and 39 • 5 °C indicates a rough agreement between the proportion of oral primordia
mpD-28°
mpD-36-5
Ml
mpC2
M1
M2
*
.j
mpD-28°
mpD-36-5
0
mpC2
Gradation ofintracellular patterns in Tetrahymena
71
Table 1. Dimensions of cells and of OAs* in wild-type and mpD
Oral dimensions^
Cell dimensions^:
A
f
Geno- Tempera- Length
type ture (°C)t (ixn)
Length
(jum)
Width
LW
C"m)
LW
(^m2)
1073
±108
1075
±97
9-8
±0-6
10-0
±0-5
6-6
±0-4
64-6
±6-5
6-4
±0-4
64-5
±5-2
1173
±113
10-7
±0-6
12-5
+ 0-8
7-3
±0-5
7-9
+ 0-7
78-6
±6-7
Width
WT
28°
45-7
±1-7
WT
36-5°
48-0
±1-9
23-5
±1-8
22-4
±1-7
mpD
28°
mpD
36-5°
50-1
±3-1
56-0
+ 3-5
23-4
±2-0
23-2
+ 2-7
1300
+ 184
98-5
+ 13-8
OALW
-5-cell
LW x 100
6-1%
± 0-8 %
6-0%
± 0-6 %
6-7%
± 0-7 %
7-6%
±1-0%
* OAs of cells in stages 1-3 of oral development. OAs all are 3-membranelled in WT
cultures, virtually all (23/25) are 3-membranelled in the mpD culture at 28 °C, virtually all
(23/25) are 4-membranelled in the mpD culture at 36-5 °C.
t Wild-type and mpD cultures were grown overnight on the same shaker at 28 °C, and
sampled simultaneously (cell densities: WT, 8600/ml; mpD, 8,200/ml). Both were then
shifted to 36-5 °C (bath temperature, 37 °C) and sampled 6 h after transfer (cell densities: WT,
44700/ml; mpD, 31000/ml).
XMeans ± standard deviation.
Figs 1-6. Photographs of silver-impregnated T. thermophila cells, either nondeveloping (Figs 1-3) or in stage 5 of oral development (Figs 4-6). All of these
photographs are printed at the same magnification, with the scale bar, shown in Fig.
1, indicating 10 fim.
Fig. 1. An mpD cell grown at 28 °C, with a mature OA. The undulating membrane
(UM) as well as the three membranelles (Ml, M2, M3) are labelled. Note the
sculptured right ends of the membranelles.
Fig. 2. An mpD cell 6h after a shift from 28°C to 36-5 °C, with a mature OA
possessing four membranelles (Ml, M2, M3, M4).
Fig. 3. An mpC2 cell grown at 29 °C, with a mature OA. M4 is located to the cell's
posterior left of the other three membranelles.
Fig. 4. An mpD cell grown at 28°C, with a stage-5 oral primordium (OP) at midbody. The three membranelles are almost rectangular in form, with no sculpturing
at their right ends. The anterior oral apparatus (OA) is everted onto the surface at
this stage, and the membranelles have transiently lost most of their sculpturing (cf.
Bakowskaefa/. 19826).
Fig. 5. An mpD cell, 6h after a shift from 28°C to 36-5 °C, with both midbody oral
primordium and anterior OA possessing four parallel membranelles.
Fig. 6. An mpC2 cell grown at 29 °C, with a both midbody oral primordium and
anterior OA possessing a small fourth oral membranelle. The fourth membranelle
has developed in rough alignment with the others, although at this stage its displacement has already begun.
72
J. FRANKEL AND OTHERS
with a small fourth membranelle posterior to the others (Fig. 6) and the proportion of completed OAs with a displaced supernumerary membranelle (Fig. 3).
We therefore tentatively conclude that the supernumerary membranelle first
develops as an ordinary but small fourth membranelle, which is then moved to
the left-posterior of the others during the final shaping of the buccal cavity.
In both mpCl and mpC2 cells, OAs are sometimes found with two small
posterior displaced membranelles, one behind the other. Small extra anterior
membranelles, or branched membranelles, are also observed but much less often
than extra posterior membranelles.
Cell and oral dimensions of mpC mutants appear similar to those of wild type.
The expression of an additional small membranelle in about one half of the OAs
of mpC2 cells at 29 °C makes it possible to find out whether, within a single
sample, there is a positive association between cell size and the presence or
development of a fourth membranelle. We found no evidence for such an
association (data not shown).
Finally, about 10 % of the OAs of cells homozygous for the big mutation
(Frankel et at. 1984) have four membranelles instead of the more common three.
II. Analysis of oral patterns
(a) General appearance of oral apparatuses with extra membranelles
Scanning electron micrographs of detergent-extracted preparations indicate
that the oral system of 4- and 5-membranelled mpD cells is expanded in a
coherent manner. The spacing of membranelles is unaltered. In many cases, the
anteriormost membranelle has the general appearance of a normal Ml, the
posteriormost that of a typical M3, while the intervening membranelles look
Table 2. Lengths of membranelles 1 and 2 in OAs* o/mpD cells
Total no. of
membraMem- nellesf in
branelle theOA
1
3
4
5
2
3
4
5
Length!
N.
r
12 13 14 15 16 17 18 19 20 21 22 Mean
18-15
2 6 7 8 3
1 17-79
1 3 5 4 4 1
1 16-71
1 1
4
2 13 11
1 2 6
1 1
3
5
2
5
1
4
1
2
1
13-52
15-24
15-71
Standard
deviation
±1-16
±1-65
±2-63
±0-78
±1-59
±2-28
* Combined data from OAs of three cultures, one grown at 28 °C, one at approximately
30 °C, and one at 36-5 °C.
t '3V2' membranelle intermediates are included in 4-membranelle class;
l
AW membranelle intermediates are included in 5-membranelle class.
tThe total number of basal body columns, including modified columns in the sculptured
region (see Frankel et al., 1984, for further explanation).
Gradation of intracellular patterns in Tetrahymena
73
more or less like M2 (Figs 9-12). The same is often true of 4-membranelled OAs
of big cells (Fig. 18). In mpC, on the other hand, casual inspection suggests a
normal oral organization to which is added a displaced posterior membranelle
that looks like a poorly organized version of M3 (Fig. 16).
(b) Effect of number of membranelles on the relative lengths of membranelles 1
and 2
Lengths of Ml and M2 were compared in 3-, 4-, and 5-membranelled OAs of
mpD cells. Ml is about the same length, and M2 tends to be longer, in 4membranelled OAs than in 3-membranelled OAs (Table 2; Figs 9,10,11,14 and
15). Only in 5-membranelled OAs is there some decrease in the length of Ml,
but probably not of M2 (Table 2; Fig. 12). Increase in number of membranelles
is clearly not achieved at the expense of a compensatory reduction in the size of
each membranelle.
The relationship of length of Ml and M2 in 3- and 4-membranelled OAs of
mpD cells is plotted in Fig. 7. The slopes of the best-fit regression lines for the
22 r
20
18
16
/
w
v
v
g
14
10
0
2
M-2
/ '
'
12
i
14
i
i
i
16
18
i
i
2(
Length of M2
Fig. 7. Mutual relationship of the length, assessed in terms of number of basal bodies,
of Ml and M2 in 3-membranelled ( • ) , 4-membranelled (T), and 5-membranelled
(V) OAs of mpD. Cases of '3£' (W) and '4|' (V) membranelled intermediates are also
shown. The solid and dashed lines indicate least-squares best-fit linear regressions for
the 3-membranelled (solid line) and 4-membranelled (dashed line) OAs respectively;
the four intermediate cases were not included in computing these regressions. The
inset (using the same axes) shows the regression lines projected to the ordinate; the
intercepts are nearly the same and are not significantly above the origin.
74
J. FRANKEL AND OTHERS
two sets of OAs do not differ significantly, but the adjusted means do differ
significantly and substantially. For any given length of Ml, M2 is longer in
4-membranelled cells than in 3-membranelled cells. The regressions are consistent with the conclusion of that the M2/M1 length ratio is constant within the
3-membranelle and 4-membranelle subgroups, but is greater in the latter subgroup than in the former. Much scantier data suggest similar a relationship in
OAs of big cells. The situation in mpC is uncertain.
(c) A gradient in sculpturing of membranelles
Increase in number of membranelles in OAs of mpD cells is associated with
systematic alterations in the sculpturing of the membranelles. These alterations
take the form of possible intermediates between the M-l and M-2 patterns, and
of a sequence of intergrades between the M-2 and M-3 sculpturing patterns. The
latter is both more prevalent and easier to interpret. We will explain these
intergrades with the aid of diagrams before presenting photographic and tabular
evidence. Fig. 8 shows 12 patterns observed at the right ends of membranelles.
Six of these, labelled 'M-l', 'M-2' and 'M-3' respectively, are identical to those
illustrated in figure 11 of the previous paper. Three of these six represent the
usual sculpturing patterns of the three membranelles (left), while the other three
show the extended variants (right). Between the M-2 and M-3 patterns three
successive intergrades are shown, both in the standard versions (left), and the
extended variants (right).
The first deviation from the M-2 pattern in the direction of the M-3 pattern is
the ciliation of the y basal body. This one change generates the ciliation pattern
observed in M3 of typical OAs, which gives the membranelle a long ciliated
'finger' projecting to the cell's anterior-right. All M-2,3 patterns are characterized by this easily diagnosed feature. If the ciliation of the y basal body is the
only clear deviation from the M-2 pattern, the pattern is designated M-2,3(A).
Even in some membranelles classified as M-2,3(A), there is a subtle shift such
that basal bodies 2b and 3b are moved more posteriorly. The angle a, formed by
the intersection of a line along basal bodies 2a-3a and a line along basal bodies
2b-3b, graphically registers the change that occurs with progressive displacement of basal bodies 2b and 3b (Fig. 8). This angle becomes less acute with
further displacement, until it approaches the obtuse angle seen in M-3 (Fig. 8).
As displacement increases, the 'hook' (highlighted by double lines in Fig. 8) that
is characteristic of the M-2 sculpturing pattern is moved to a position in which
basal body 3c is brought sufficiently close to basal body 4c to create the appearance of a hexagon made up of basal bodies 2b, 2c, 3c, 4c, 4b, and 3b. The gestalt
of a hexagon, combined with the ciliation of basal body y and the continued
presence of at least two complete columns of ciliated basal bodies to the left of
the hexagon, defines the M-2,3(B) sculpturing pattern.
The transition toward the M-3 end of the M-2,3 spectrum is associated with
further basal body displacement (increasing angle a) plus progressive loss of
Gradation of intracellular patterns in Tetrahymena
75
basal body columns at the left end of the membranelle. The most extreme
example of the M-2,3 transition is observed when the angle a approaches that
observed in M3 of typical OAs, and the loss of basal bodies is sufficiently great
Extended
Standard
M-l
ffif:
M-2
M-2,3(A)
TO
"\ y
M-2,3(B)
M-2,3(C)
a
^ » Q L -
b
M-3
Fig. 8. Patterns of sculpturing of membranelles. A uniform convention is used in all
12 diagrams. Closed circles indicate ciliated basal bodies, dashed circles indicate
unciliated basal bodies. The dashed lines connecting basal bodies into sets of three
indicate the basal body columns; in the sculptured regions these indicate probable
paths of displacement of basal bodies late in oral development (see figure 1C in
Frankel et al. 1984). Basal body columns are numbered from right to left (viewer's
left to right), while the small letters are used to identify the anterior (a), middle (b)
and posterior (c) basal body within each column, x and y indicate basal bodies
originally generated as a short fourth row. Solid lines forming angles and hexagons
are used to designate aspects of sculpturing, as explained in the text.
The diagrams on the left show normal sculpturing patterns of the membranelles,
while those on the right indicate the corresponding extended patterns, with the extra
displaced basal bodies shaded. For further explanation, see the text.
76
J. FRANKEL AND OTHERS
that only two complete ciliated columns are left, one (4a, 4b, 4c) forming the left
wall of the hexagon, the second (5a, 5b, 5c) to its left. This sculpturing pattern
is then designated M-2,3(C)
The M-2,3(C) sculpturing pattern is a near neighbour to the M-3 pattern,
because the only clear-cut difference between the two is the presence of a single
ciliated basal body, 5c, in the former and not in the latter. This small difference,
however, creates a large difference in subjective gestalt: ciliation of basal body
5c allows one to distinguish a short stretch of columns (4 and 5) at the left end
of the membranelle, while the removal of the 5c cilium makes the columns
disappear perceptually, since now only one complete column is left (4), and that
one is perceived as only a part of the hexagon. Hence, before the discovery of
the M-2,3 intergrades in 4- and 5-membranelled OAs, M3 was thought of as
being entirely sculptured (Bakowska et al. 1982<z)*and an incorrect inference was
drawn concerning the precise manner in which M3 becomes sculptured during
development (compare figure 28 of Bakowska, Nelsen & Frankel, 1982ft to Fig.
8 in this paper). It is the M-2,3 intergrades that permit recognition of the fact that
the M-3 sculpturing pattern is the M-2 pattern subjected to some additional
spatial deformation.
Each of the three M-2,3 intergrades can be drawn in a version with extended
sculpturing (right column of Fig. 8) simply by displacing basal body 4c posteriorly, as in the M-2 and M-3 extended-sculpturing patterns that were
described in section IIIc of the previous paper (Frankel et al. 1984). This opens
the hexagon out into an 8-membered ring. The sole inconsistency in the two-way
scheme in Fig. 8 is allowing the extended variants of the M-2,3(C) and M-3
patterns to retain columns up to no. 6 rather than up to no. 5 as in the standard
versions.
Before proceeding to the evidence, an apparent inconsistency of an entirely
different kind needs explanation. Numbers are used both for labelling of membranelles in anterior-to-posterior order, and for designation of sculpturing
patterns. To avoid confusion, numbers used in the anterior-to-posterior
enumeration of membranelles within an OA immediately follow the 'M' designation (e.g. M5), while numbers that refer to the patterns illustrated in Fig. 8 are
separated from the 'M' abbreviation by a hyphen (e.g. the M-3 pattern). Thus
when we state, for example, that 'M5 expresses the M-3 pattern', we mean that
the fifth membranelle in the anteroposterior sequence within an OA expresses
the M-3 pattern shown in Fig. 8, i.e. the pattern which would be observed in M3
of a normal 3-membranelled OA. We also note that even when the total number
of membranelles in an OA can be counted unambiguously, the sculpturing
patterns of some of these membranelles may be obscured; this is the reason why
the total numbers of membranelles that have been assessed for sculpturing
pattern differs for different membranelles in each category of OAs within Tables
3 and 5, and also accounts for the question-mark entries in Table 4.
Three-membranelled oral apparatuses of mpD cells reliably express the
Gradation of intracellular patterns in Tetrahymena
77
normal M-l, M-2, and M-3 sculpturing patterns in membranelles, 1,2, and 3
respectively (Table 3, Fig. 9).
In 4-membranelled OAs of mpD cells, the sculpturing of Ml is completely
normal (Fig. 10) or occasionally extended (Table 3). M2 is most commonly
normal (Fig. 10), but not infrequently modified (Table 3). One rather typical
modification is shown in Fig. 11. This and others like it may be M-l,2 intermediates, but we are uncertain about this inference because we have been unable
to work out an unambiguous transitional sequence. M3 characteristically expresses the M-2,3(A) sculpturing pattern (Table 3), sometimes with (Fig. 10) and
sometimes without (Fig. 11) an increase in the angle a (see Fig. 8). M4 displays
either a normal M-3 sculpturing pattern (Fig. 10), or a modified M-3 pattern with
disruption of the basal body hexagon (Fig. 11, Table 3).
In 5-membranelled mpD OAs, Ml exhibits the M-l sculpturing pattern (Fig.
13), except that basal body x may be missing (Fig. 12) or unciliated (Table 3).
Sculpturing of M2 is usually normal (Fig. 13), or, exceptionally, extended (Fig.
12). M3 expresses either the typical M-2 pattern (Fig. 12) or an M-2,3(A) pattern
in which the ciliation of basal body y is not accompanied by any increase in angle
a (Fig. 13). M4 typically exhibits the M-2,3(B) pattern (Fig. 12) but may express
an M-3 pattern with a surplus of unciliated basal bodies (Fig. 13). M5 ranges from
the M-2,3(C) or M-3 pattern (Fig. 12) to a small vestige with a few variably
arranged basal bodies, displaced to the cell's left (Fig. 13).
Comparison of Figs 12 and 13 and examination of Table 3 indicate that 5membranelled OAs exhibit considerable variation in sculpturing patterns of M3
through M5. Table 4, which summarizes the sculpturing patterns of each of the
eight 5-membranelled OAs that were examined, shows that this variation is not
random within OAs. The systematic nature of the variation is clearest if one
compares the four 5-membranelled OAs in which M3 expresses the M-2 pattern
with the four 5-membranelled OAs in which M3 expresses the M-2,3(A) pattern.
All four OAs of the former category have five well-developed membranelles all
lined up as in Fig. 12, and in all of these M4 expresses the M-2,3(B) sculpturing
pattern and M5 expresses the M-3 sculpturing pattern [M-2,3(C) in one case]. M3
may be considered to occupy a position at the centre of these OAs and thus
correspond to M2 in a 3-membranelled OA. In contrast, in the 5-membranelled
OAs in which M3 expresses the M-2,3(A) sculpturing pattern, M5 tends to be
located far to the posterior-left and in two cases is a small vestige (Fig. 13) and
M4 has a configuration close to that of a typical M3 (Table 4). These OAs are
best considered as transitional between 4-membranelled OAs and welldeveloped 5-membranelled OAs, with the consequence that M3 lies posterior to
the centre of the oral system. The systematic relationship between pattern and
relative position suggests that the sculpturing pattern is determined on a ratio
scale rather than by ordinal count (see Discussion).
While OAs that express intermediates between 4- and 5-membranelle patterns
are observed after growth of mpD cells at 36-5 °C, intermediates between typical
78
J. FRANKEL AND OTHERS
mpD
UM
Gradation of intracellular patterns in Tetrahymena
79
3- and 4-membranelle patterns are formed during growth of mpD at about 30 °C.
These might best be described as expressing a '32'-membranelle organization,
with the most posterior membranelle poorly organized and swept back toward
the oral opening (cytostome), as in typical mpC OAs (see below). In such OAs,
M3 usually expresses the M-2,3(B) sculpturing pattern (Fig. 14) rather than the
M-2,3(A) pattern exhibited by M3 of more fully developed 4-membranelled
OAs.
The M3 of the '3i'-membranelled OA shown in Fig. 14 is highly truncated at
its left end. While it might have developed that way from the start, the clear space
to its left suggests original formation of a larger membranelle followed by
subsequent resorption of some ciliary units. Supporting evidence for this conclusion is provided by the appearance, in other OAs from the set of mpD cells
Figs 9-12. Isolated OAs from mpD cells lysed following growth in PPY near 30°C
(Fig. 9) or at 36-5 °C (Figs 10-12). All photographs are oriented so that the cell's left
side corresponds to the viewer's right. The UM is thus to the viewer's left, the
membranelles to the viewer's right, with Ml always most anterior (up) and M3 most
posterior (down). Arrowheads refer to the state of ciliation of basal bodies, while
wavy arrows indicate basal body displacements involved in extended patterns. The
membranelles are individually labelled (Ml, M2, M3...) as is the anterior end of
the undulating membrane (UM). The posterior portion of the UM has commonly
been displaced in preparation. Scale bars indicate ljum.
Fig. 9. A typical 3-membranelled mpD OA. Virtually all ciliated basal bodies of the
three membranelles are visible, except for a few basal bodies of Ml which are
covered by a fold of the surface lamina (epiplasm). Most of the preparation is seen
in external view, with ciliated basal bodies visible as thick-walled rings. The normal
sculpturing pattern of the three membranelles is exhibited at the right (your left) ends
of the membranelles, with the M-l pattern, including the ciliated basal bodies x and
y, completely visible. The 5c basal body of M3 is present but unciliated (arrowhead).
Fig. 10. A typical 4-membranelled mpD OA. All ciliated basal bodies are visible,
except for a few at the left end of M2. Unciliated basal bodies at the right ends of the
membranelles are seen especially clearly in this preparation (compare with Fig. 8 for
identification). The x and y basal bodies are pointed out at all places where they are
present. Note that the y basal body is ciliated in all membranelles except M2, where
it is unciliated but more distinct than in the other preparations. Epiplasmic ridges
separating the membranelles are indicated by (r).
Fig. 11. A 4-membranelled mpD OA with modified M2 and M4. Ml is sculptured
as in the OA depicted in Fig. 10 (the y basal body in Ml is presumably concealed by
a flap of epiplasm). M2 is abnormally sculptured, and possesses one anomalous
'extra' fourth-row basal body (e), unique to this preparation. M3 has a ciliatedy basal
body, but otherwise is sculptured like M2 in the OA in Fig. 10. In M4, the six basal
bodies that normally form a hexagon are instead irregularly placed.
Fig. 12. An mpD OA withfivewell-developed membranelles. They y basal body is
indicated on allfiveof these: it is ciliated in Ml, M4, and M5, but not in M2 and M3.
The x basal body of Ml is missing. The sculpturing of M2 is extended by slight
posterior displacement of basal body 4c (wavy arrow). The arrow in M4 indicates
the 3c basal body at the end of the 'hook', which has been displaced to the posteriorleft to bring it close to the 4c basal body and thus form a basal-body 'hexagon' (cf.
Fig. 8).
80
J. FRANKEL AND OTHERS
Gradation of intracellular patterns in Tetrahymena
81
Table 3. Sculpturing of membranelles in OAs o/mpD
Pattern*
Total
/
*
no. of
M-l
M-2
M-2,3
A
K
^ ,
^
m'lles in
,—A—N ,
the OA m'lle Normal Normal Modified A B C
3
4f
5t
1
2
3
1
2
3
4
1
2
3
4
5
M-3
,
UnrecognizNormal Modified able
K
N
17(1)
26
2
17(1)
1
11(1)
9
15(3)
21
6
9
1
19(1) 4
1(1)
1
5$
7(1)
4
1
4(1)
4(1)
1
1
2
2
1
2
* The total number of membranelles exhibiting the pattern is indicated first; the number of
these (if any) that express the extended variant of the pattern (cf. Fig. 8) is indicated in
parentheses. Some 'modified' M-2 patterns may be M-l,2 intermediates. See Fig. 8 and the
text for explanation of the patterns.
t Two cases of '3W membranelles (displaced fourth membranelle) and two cases of lAVi
membranelles' (small fragment of a fifth membranelle) are included in the 4- and 5membranelled classes respectively.
$ The x basal body is missing in two of these OAs and is unciliated in one other.
Figs 13-16. Isolated OAs with intermediate or unusual configurations of membranelles, from mpD cells grown in PPY at 36-5 °C (Fig. 13) or near 30°C (Figs 14,
15) or from an mpCl cell grown in PPY at 36-5 °C (Fig. 16). The orientation of the
photographs j and the symbolic conventions, are the same as in Figs 9 to 12. Scale bars
indicate 1/im.
Fig. 13. An mpD OA with five membranelles, the fifth one unusually small. Epiplasmic ridges (r) separate the membranelles, including the fourth and fifth. The
complete sculpturing patterns of all the membranelles are visible. The x and y basal
bodies are labelled in M l , as is the y basal body in M3 and M4. The y basal body is
not visible in M2. Unciliated basal bodies are seen at the left end of M4 (large
arrowhead).
Fig. 14. An mpD OA with a displaced M4. Epiplasmic ridges (r) separate the
membranelles.
Fig. 15. A four-membranelled mpD OA with an interrupted M3. The zone of
presumed basal body resorption is indicated by the thick arrow. Note that the
epiplasm in this region is smooth, while ridges (r) demarcating membranelles are
visible anterior and posterior to the interrupted membranelle. The 4c basal body of
M3 (wavy arrow) has undergone extensive posterior displacement, generating an
extended M-2,3(C) pattern.
Fig. 16. An mpCl OA with a displaced M4. The epiplasmic ridge separating M3 and
M4 is either not present or obscured by overlying material. Note the ciliated 5c basal
body of M3 (arrowhead), which is characteristic of most mpC OAs with displaced
M4s.
82
J. FRANKEL AND OTHERS
Table 4. Sculpturing patterns in 5-membranelled oral apparatuses o/mpD cells
Sculpturing of: *
A
OAno.
Ml
M2
1
?
M-2
2t
M-r*
M-r*
eM-2
3
4
5
6
7
8*
M-2
?
M-l
mM-2
?
M-2
M-l1"
M-l
M-2
M-2
M-2
M3
M-2
M-2
M-2
M-2
eM-2,3(A)
M-2,3(A)
M-2,3(A)
M-2,3(A)
M4
M-2,3(B)
M-2,3(B)
eM-2,3(B)
M-2,3(B)
M-3
M-3,2(C)
?
M-3
M5
M-3
M-3
M-2,3(C)
mM-3
tiny
?
?
tiny
* Prefixes: e = extended, m = modified; Suffixes: ~x = basal body x absent; "* = basal body
x unciliated. A '?' indicates that the structure is obscured in whole or part,
t See Fig. 12.
t See Fig. 13.
Figs 17-18. Isolated OAs from an mpCl cell grown in PPY near 30 °C (Fig. 17) and
from a big cell grown in PPY at 28 °C. The orientation of the photographs, and the
symbolic conventions, are the same as in Figs 9-16. Scale bars indicate 1/xm.
Fig. 17. An mpCl OA with a somewhat less displaced M4, and a somewhat larger
M3, than in Fig. 16. M4 is partly obscured by overlying material.
Fig. 18. A big OA with four membranelles. Note the ciliated y basal body of M3.
Sculpturing of Ml is slightly reduced, while that of M2 and M3 is extended, with
posterior displacement of the 3b and 4c basal bodies in M2, and of the 4c basal body
in M3 (wavy arrows).
Gradation of intracellular patterns in Tetrahymena
83
grown near 30 °C, of gaps similar to those noted in M3 of the largest 3membranelled OAs (Frankel etal. 1984). An example is shown in Fig. 15. Thus,
the posterior zone of ciliary-unit regression may transgress into the next-to-last
membranelle of those OAs that possess more than three membranelles (see
Discussion).
While analysis of the effects of changes in the number of membranelles on the
spatial distribution of sculpturing patterns is carried out most readily on OAs of
mpD cells, data from OAs of big and mpC mutants are important for checking
whether these effects truly are related only to the number of membranelles. In
big clones, cells with 4-membranelled OAs are occasionally observed. These
OAs are generally similar to the 4-membranelled OAs of mpD cells (Fig. 18).
The mpCl and mpC2 mutations are of still greater importance, because in these
a substantial majority of the 4-membranelled OAs express the '3i'-membranelle
organization, with a small displaced M4. In such OAs, from both mpCl and
mpC2, Ml is generally normal, M2 is normal or modified such as in 4membranelled mpD OAs, while M3 most commonly expresses an M-2,3(C)
pattern (Fig. 16), but may also express an M-2,3(B) pattern with extreme
ciliary unit displacement as well as truncation of the left end of the membranelle
(Fig. 17) or a normal or modified M-3 pattern (Table 5). Gaps are sometimes
observed within M3, which are very similar to those occasionally observed in
OAs from mpD cells grown near 30 °C. M4 expresses either a poorly organized
version of the M-3 sculpturing pattern (Fig. 16), or else is made up of an
assemblage of a dozen or fewer basal bodies in an apparently chaotic array
(Table 5).
Table 5. Sculpturing of membranelles in OAs of mpC*
Pattern t
A
Total no.
i
—•
ofm'lles
M-l
M-2
M-2,3
K
K
in the
^
(—*—^ (
N ,
OA
m'lle Normal Normal Modified B
C
3
4§
1
2
3
13(4)
1
2
3
4
121|
25
(
A
^ UnrecogNormal Modified nizable
2
2
19
\
M-3
1(1)
8
1%
2
3
8
10
4
15
7
* Data from mpCl and mpC2 combined.
t Patterns are categorized and data presented as in Table 3. 'Unrecognizable' patterns
indicate chaotic arrays not clearly relatable to any of the regular patterns.
X Includes 3 OAs with side-by-side duplication of all or part of the M3 pattern.
§ Mostly '3V2' membranelled OAs with a swept-back fourth membranelle.
\ One of these is modified.
84
J. FRANKEL AND OTHERS
The similarity in the sculpturing patterns of mpC OAs and those of some
mpD OAs is striking (compare Figs 14 and 16). This similarity is dependent upon
a correspondence in the overall membranelle array; thus, when mpD and
mpC OAs both exhibit the '3|'-membranelle complement, the sculpturing
patterns are also alike. mpD differs from mpC in that mpD generates the '3£'membranelle organization only exceptionally, whereas mpCl and mpC2 do so
typically.
(d) The length of the undulating membrane is related to the number of
membranelles
In wild-type cells, the anterior end of the undulating membrane (UM) is
located next to the middle or anterior portion of M2 (Bakowska et al. 1982a).
38 r
V
36
V T
34
T
32
•
W
V
W •
T
T
O
£
30
60
28
26
24
22
12
14
16
Length of M2
18
Fig. 19. Mutual relationship of the length, assessed in terms of number of basal
bodies, of the UM and of M2 in mpD OAs with 3 ( • ) , 4 (T), '4£' (V) and 5 (V)
membranelles.
Gradation of intracellular patterns in Tetrahymena
85
This is true for OAs of pattern mutants as well, irrespective of the number of
membranelles (Figs 9-18). The UM thus borders on a greater number of membranelles in 4- and 5-membranelled OAs than in 3-membranelled OAs. If the
membranelles are evenly spaced, the UM should become longer as the number
of membranelles increases. This is indeed the case in the great majority of OAs
(Fig. 19). Comparison of the length of the UM in 3-, 4-, and 5-membranelled
OAs to the length of M2 in the same OAs suggests that within this range of oral
sizes the length of the UM is influenced more by the number of membranelles
than by their length.
DISCUSSION
A. The membranelle field is a unit of pattern organization in the oral apparatus
of Tetrahymena
The major conclusion of this study is that the striking individuality in number
and arrangement of basal bodies exhibited by each membranelle is not a
consequence of instructions unique to that membranelle; rather it is an outcome
of processes that are spatially coordinated within the oral field as a whole. The
existence of this coordination is revealed when the number of membranelles that
develop within the oral apparatus (OA) is other than the normal three. This had
been observed in 2-membranelled OAs of severely starved cells (Bakowska etal.
1982a), and is demonstrated in greater detail in 4- and 5-membranelled OAs of
mutant cells. Such OAs express systematic modifications of relative membranelle lengths, of patterns of membranelle sculpturing, and of configurations
resulting from localized resorption. Together these suggest that the unit of
organization is the membranelle field rather than the individual membranelles.
The inferred spatial coordination must come about through mechanisms
operating during oral development. Oral development has been described in
considerable detail for wild-type cells (Bakowska et al. 19826; Lansing, Frankel
& Jenkins, 1984), and is summarized briefly in the preceding paper (figure 1C
and accompanying text of Frankel etal. 1984). The coordination of membranelle
size and form can be explained as a result of four component processes that are
spatially organized within the oral field. One of these is involved in the initial
formation of oral membranelles, the other three in their subsequent remodelling.
The first of these processes is the formation of promembranelles through sideby-side alignment of basal body couplets during stages 3 and 4 (see figure 1C of
the preceding paper). The final length of each membranelle depends upon the
number of couplets that are initially aligned minus the number of basal body
columns (derived from these couplets) that are subsequently removed by resorption. Since spacing of columns is uniform and basal body resorption probably has
a differential effect only on the membranelle (s) located in the posterior portion
of the oral primordium (see Discussion of the preceding paper), the relative final
lengths of all but the posterior membranelle(s) will depend solely on where basal
86
J. FRANKEL AND OTHERS
Side 1
Side 1
Fig. 20. Effects of variation of length (A) and of number (B) of membranelles on the
relative length of membranelle 1 (Ml) and membranelle 2 (M2) in a simple triangular
model of membranelle-field organization. Three sizes of oral primordia, differing in
the length of side 1 (i, ii, and iii in diagram (A)) or of side 2 (i, ii, and iii in diagram
(B)) are presented. Lengths of Ml and M2 are indicated in both of these sets of
diagrams. For further explanation, see the text.
body couplets are located within the oral field when promembranelle alignment
takes place.
Observations on lengths of the anterior membranelles can be accommodated
within a simple two-dimensional model, shown in Fig. 20. In this model, the
portion of the oral field allocated to membranelle production is symbolized by
a right triangle, with side 1 (horizontal in Fig. 20) representing the length of the
anteriormost, longest membranelle (Ml), side 2 representing the right (viewer's
left) edge of the field at which promembranelles originate, and the hypotenuse
representing the line along which membranelle formation eventually terminates.
Membranelles posterior to Ml originate at equally spaced intervals along side 2,
and.extend.horizontally (i.e. parallel to side 1) out to the hypotenuse. Comparison of the model to the actual form of the developing membranelles (see
Gradation of intracellular patterns in Tetrahymena
87
figure 1C of the preceding paper, and light micrographs in both papers)
indicates that the model gives a realistic representation of certain aspects of
membranelle patterning, namely the equal spacing of membranelles and their
generation in an anterior-to-posterior sequence along the cell's right margin of
the membranelle field. Other aspects, notably the curvature of the membranelles, are ignored.
Within this model, modification of membranelle length without change in
number can be represented by a change in the length of side 1, with side 2
constant (Fig. 20A), and a modification in membranelle number without change
in the length of Ml can be represented by a change in the length of side 2, while
side 1 remains the same (Fig. 20B). In the former case the M2/M1 length ratio
should remain unaltered, while in the latter the M2/M1 length ratio should
increase as the length of side 2 (proportional to the number of membranelles)
increases. Within the relatively narrow range of membranelle lengths (15 to 22
basal bodies) and number (3 to 5) considered in this paper, both expectations are
fulfilled. The first expectation fares increasingly poorly as one departs from this
restricted range in membranelle length, due to non-linearity in the Ml versus M2
length relationship (see figure 9 and accompanying text in preceding paper).
The three processes that remodel membranelles, namely basal body resorption, ciliary regression and ciliary-unit displacement, all take place during late
oral development (late stage 5 and stage 6). They convert similar and nearly
rectangular membranelle prototypes into the diverse mature membranelles (see
figure 1C of the preceding paper). The spatial distribution of all three processes
can be described within the framework of the model, resulting in a schematic but
fairly accurate account of the spatial coordination of the organization of different
membranelles. First, the wedge-shaped posterior zone of basal body resorption
postulated in the preceding paper is assimilated into the model by converting it
into a family of parabolas whose position along side 1 is always the same, and
whose anterior extension (reciprocal of the distance between vertex and focus)
is proportional to the length of side 2 (Fig. 21). The position of the vertex of the
parabola is arbitrarily set at 0-49 of side 2 (measured from the posterior end), so
as to interrupt M3 in Fig. 21C and just miss it in Fig. 21D. Given these assumptions, the steady increase of a single parameter, the length of side 2 (roughly
interpretable as field length), leads progressively from the normal pattern (Fig.
21 A), to a somewhat less severe truncation of M3 together with a small and
displaced M4 (Fig. 21B), to a split M3 associated with an M4 expressing a nearnormal M-3 configuration (Fig. 21C), to a non-truncated M3 associated with an
M4 that resembles a normal M3 in size, position, and often also arrangement
(Fig. 21D). Comparison of these diagrams with the appropriate SEM
photographs (referred to on the right side of Fig. 21) indicates that this simple
model yields a reasonable approximation to the observations. What is particularly
significant is that the same model that accounts for a gap in the most-posterior
membranelle as it becomes longer (figure 22 of the preceding paper) can also
88
J. FRANKEL AND OTHERS
Sidel
Mli
M2
M3
A
3
Figures
9
B
3*6
14,16,17
C
3%
15
10,11,18
E
4V4
Ml
M2
M3
M4
M5
13
12
Fig. 21. Effects of aspects of the remodelling of membranelle-prototypes in membranellefieldsdiffering in the length of side 2. The diagrams (A) through (F) indicate
oral fields with a progressive increase in the length of side 2 and a corresponding
increase in number of membranelles (3-5). In each of these diagrams, the horizontal
lines represent the lengths of membranelles, with the segments situated within the
stippled regions indicating portions that are resorbed due to inclusion within the
parabola-shaped zone of ciliary-unit regression. The heavy vertical bar at the left
indicates the zone of M-2,3 transition in membranelle sculpturing patterns. Scanning
electron micrographs corresponding to each of these schematic patterns are listed on
the right. For further explanation, see the text.
generate a gap in the next-to-most posterior membranelle when the number of
membranelles is increased (Fig. 21C of this paper).
There are, however, some discrepancies among certain 5-membranelled OAs.
In mpD, M4 and M5 are sometimes larger and better developed than predicted
by the model (compare Fig. 21F to Fig. 12), whereas in the rare 5-membranelled
OAs of mpC the reverse is true: both M4 and M5 are highly truncated and even
Gradation of intracellular patterns in Tetrahymena
89
M3 shows signs of basal body resorption at its left end (data not shown). It is
possible that in mpC the zone of ciliary-unit resorption is sometimes expanded.
The geography of the other two processes involved in remodelling of M2 and
M3, namely localized ciliary regression and ciliary-unit displacement, can be
accommodated within the simplified model in much the same manner, i.e. by
fixing thresholds along the length of side 2. The anterior threshold is determined
by the anteriormost position at which basal body y remains ciliated, while the
posterior threshold is the level at which the degree of ciliary-unit displacement
is equivalent to that normally found in M3. As with the resorption threshold, the
procedure for assigning specific values is a posteriori and somewhat arbitrary: we
derive the anterior threshold from data presented in Table 4, setting it so as to
be located just anterior to M3 in Fig. 21E and just posterior to M3 in Fig. 21F.
This gives a value of 0-58 measured from the posterior end. The posterior
threshold is set at the relative location of M3 in normal 3-membranelled OAs,
i.e. 0-33. Thus the zone of the M-2,3 transition is between 0-58 and 0-33 along
the right margin of the membranelle field. We can then make predictions for
kinds of OAs other than those from which the thresholds were derived: M-2,3
transitions must be observed always, and only, between these thresholds, and
positions near the 'top' of the zone (e.g. 0-50) should manifest the M-2,3(A)
pattern, while positions near the 'bottom' (e.g. 0-40) should exhibit the M-2,3(C)
pattern or a M-2,3(B) pattern with considerable ciliary-unit displacement.
Again, comparison with the observations indicates that these predictions hold in
almost all cases.
The model just elaborated does little more than provide a schematic description of the observations. However, the nature of the description has a major
impact on the kind of mechanism that must be sought. In this case, describing the
final form of the membranelle as a conjoint effect of separate processes that are
controlled according to relative position along one dimension of a membranelle
field implies that there is no unique design for each membranelle. The nearly
invariant membranelle organization found in wild-type cells is thus not a
consequence of the existence of three discrete membranelle blueprints that are
selected in an anterior-to-posterior sequence. Rather, it appears to be due to
fairly tight control of a critical length, represented by side 2 of the model. So long
as this length remains the same, the relevant thresholds for ciliation, displacement, and resorption of basal bodies will retain the same position in relation to
the membranelles themselves, and the ensuing organization will remain constant. In this view, pattern homeostasis depends on a proper spatial relationship
of globally varying field properties with the separate events that generate serial
repeats, rather than on unique individualized designs of the repeated structures.
What is 'side 2' in the real oral primordium? It is tempting to conclude that it
is the length of the oral field or of some parameter directly proportional to that
length. The differential increase of measured length of mpD OAs at 36-5 °C,
a temperature at which the 4- and 5-membranelle phenotype is expressed, is
90
J. FRANKEL AND OTHERS
consistent with that idea. This conclusion cannot be wholly true, since the 3membranelle condition is substantially buffered against changes in field size, as
in most OAs of starved wild-type cells and of big and psm mutant cells. The
critical parameter that is represented by side 2 in the model is thus likely not to
be oral field length per se, but rather length of the segment of the right margin
of the membranelle field along which separate membranelles may be initiated.
This segment is lengthened in long oral primordia of mpD mutants, but generally
is not in the still longer oral primordia oipsmAl mutants. The reason for this
difference is unknown, although it might possibly be related to differences in the
geometrical orientation of developing membranelles. A detailed study of oral
development in mutants such as psmAl and mpD might help to clarify why oral
fields of apparently similar initial size and shape sometimes generate extra membranelles and sometimes only longer ones.
B. The dilate membranellefieldmay resemblefieldsin multicellular organisms
We will now inquire whether the determination of the form of membranelles
in the Tetrahymena oral apparatus (OA) bears a meaningful relationship to the
specification of elements of serially repeated structures of metazoa. Considered
as a series, the membranelle set is characterized by a small number of elements
in which differences in detail are superimposed on an otherwise repeating
pattern and in which the number of elements is normally tightly controlled but
can be altered by mutation. Certain metazoan series, such as the teeth and digits
of mammals, manifest similar general properties. The question is whether these
similarities are meaningful or only superficial.
A
4
A
A
A
T5
T4
T3
T
T2
\
/—V — r ~
Thresholds
Tl
TO
Pattern
Fig. 22. Two alternative ways of determining a periodic pattern, either via a
monotonic gradient with many thresholds (A) or via a periodic prepattern with a
single threshold (B). The three pattern-elements are shown as similar in (B), and
dissimilar in (A). For further explanation, see the text. Modified from Wilby & Ede
(1975).
Gradation of intracellular patterns in Tetrahymena
91
If the similarities of the ciliate and metazoan series are meaningful, we would
expect that the same model of pattern formation should apply to both. To start
this inquiry, we can ask whether and how two fundamentally different modes of
pattern specification might apply to the two situations. These two modes are
illustrated in Fig. 22 (modified from Wilby & Ede, 1975). In the first (Fig. 22A),
the elements of the pattern result from interpretation of a monotonically graded
property that conveys positional information (Wolpert, 1969). In this formulation, differently determined states (the 'non-equivalence' of Lewis & Wolpert,
1976) are specified by a reading of ranges of values of the positional signal, the
ranges being bounded by discrete thresholds (Lewis, Slack & Wolpert, 1977).
Periodicity and non-equivalence are thus acquired simultaneously and each
element is specified uniquely for its final pattern. In the alternative formulation
(Fig. 22B), the periodicity of the pattern is a consequence of an underlying
periodicity that is isomorphic to the pattern itself (i.e. a 'prepattern', Stern,
1968), and there is only a single critical threshold. This class of model, however,
generates periodicity but not non-equivalence, and 'secondary modifications'
must be added to make the repeating elements asymmetrical across the structure
as a whole (Goodwin & Trainor, 1983).
The specification of membranelles of the Tetrahymena oral apparatus clearly
is best accounted for by the second kind of model. The periodicity of membranelle formation is dictated by the stereotyped way in which promembranelles
initially develop, while the differences that appear in the finished membranelles
are determined separately, at a later stage of membranelle development. In the
specific model that was presented in the preceding section, the uniform spatial
intervals of membranelle formation reflect the prepattern, while the resorption
parabola and modelling thresholds (Fig. 21) represent conditions that operate on
the results of the prepattern to generate the unique features of the individual
membranelles.
The positional information conception (Fig. 22A) is inappropriate for membranelle formation because it cannot generate intermediate patterns. If this
conception were correct, changing the length of the membranelle field (i.e. side
2 in Figs 20, 21) should change neither the number nor the form of each membranelle. Instead, the system should regulate through a proportional change in
the width of each of the three membranelles5 in accordance with Wolpert's
(1969) 'French flag' rule. A proportionality of this general kind is indeed observed for the positioning of the oral primordium and other structures within the cell
surface as a whole (Frankel, 1974, 1984), but it clearly is inapplicable to the
details of development within an already established oral field of normal asymmetry.
If the positional information conception were known to be correct for the
specification of serially repeated structures in multicellular organisms, then
we could safely conclude that resemblances between the ciliate membranelle
series and metazoan series such as teeth or digits have no common basis. The
92
J. FRANKEL AND OTHERS
fundamental difference between the two types of series might then be ascribed to
differences in the units of which they are composed, i.e. subcellular structures in
the former and cells in the latter. But it is by no means certain that the positional
information model really is true for the multicellular examples. In the case of
vertebrate limb buds, excellent experimental evidence favouring a positional information model is available (Slack, 1977; Summerbell & Honig, 1982). Much of
this evidence, however, can also be reconciled with the alternative i d e a ' . . . that a
set of spatially distributed locally equivalent situations of the prepattern type is
being responded to in parallel with a range of unique readings for some other variable to give the graded properties' (Cooke, 1982, p. 102). Several theoretical
models for the generation of prepatterns in the limb bud have been proposed
(Wilby & Ede, 1975; Newman & Frisch, 1979; Goodwin & Trainor, 1983).
In our view, a careful analysis of how the form of the individual elements of
a series becomes modified as the number of these elements changes can make a
vital contribution to an evaluation of the positional information versus prepattern alternative. The positional information model shown in Fig. 22A, which
is characterized by unique thresholds that are programmed into the system,
cannot generate supernumerary elements unless the positional-signal profile is
altered to a U-shape. If this were so, the ensemble of elements would exhibit a
mirror-image pattern. On the other hand, the prepattern type of model shown
in Fig. 22B predicts the formation of extra elements as the field becomes wider,
and also allows for the generation of elements that are intermediate in form, the
specific nature of these intermediates depending on the nature of the 'unique
readings' that are superimposed on the prepattern.
To our knowledge, the most detailed investigation of the effect of changes in
the total number of elements of a series on the form of the individual elements
in Bateson's extensive inquiry into variation in repeated structures, carried out
nearly a century ago (Bateson, 1892,1894). Bateson's methods and conclusions
are best exemplified by his exhaustive analysis of variation in mammalian
dentition. Bateson found that when a supernumerary tooth is added to the end
of a series, or when a space normally occupied by three teeth is instead occupied
by four, several or all members of the series are altered so that they no longer
correspond to any member of the typical series. He therefore concluded that
' . . . the whole Series of Multiple Parts is bound together into one common
whole' (Bateson, 1892, p. I l l ) , with conservation of the 'general configuration
of the whole series' but not necessarily of the individual elements that make up
that series. These results and conclusions are strikingly paralleled by our own
independently conceived observations on ciliate membranelles. It is pertinent to
note that Bateson's views concerning the mammalian dentition have been
amplified and supported by subsequent biometrical analyses of normal dentition
(Van Valen, 1970; Lombardi, 1975), and also that Van Valen (1970) explains
these field properties by invoking prepatterns and gradients in a way that is at
least generically similar to our model for ciliate membranelles.
Gradation of intracellular patterns in Tetrahymena
93
Bateson also studied polydactyly in some detail (Bateson, 1894, Chapter XIII)
and found more complexity and variety than in teeth. He reported some clearcut cases of mirror duplication of sets of digits and even of lower arms, as in
'double hand' in humans; cases no. 492 and 495 in his book (Bateson, 1894) are
particularly clear examples. Such examples, as well as the limbs of certain
mutants such as duplicate in fowl (Landauer, 1956a), resemble the
experimentally-derived mirror-image limbs resulting from grafts of the posterior
polarizing zone to the anterior margin of the limb bud (Tickle, Summerbell &
Wolpert, 1975; Slack, 1977) and are in excellent accord with the positional
information model of Fig. 22A. However, as was noted by Bateson and confirmed in subsequent studies (reviewed in Hinchcliffe & Johnson, 1980, section
7.4), most cases of polydactyly do not involve such obvious mirror-image
duplication. Bateson found that the presence of supernumerary digits may be
associated with a reversal of digital asymmetry in some cases (most remarkably
in cats, see pp. 313-324 in Bateson, 1894), but in some other cases the formation
of extra digits occurs without obvious mirror imaging and with coordinated serial
modifications such as Bateson had seen in teeth (see especially his human cases
no. 485 and 510). A careful reinvestigation of examples of polydactyly by serial
addition, perhaps in mutants such as diplopodia of fowl (Landauer, 1956ft) or
luxate of mice (Carter, 1951) would be, as Wilby & Ede (1975) have suggested,
of particular interest. If what Bateson observed in paws of polydactylous cats
holds true generally, then apparent serial duplications might really be attenuated
mirror-image reversals like those observed following a graft of a weakened
posterior polarizing zone to the anterior margin of the chick wing bud (Smith,
Tickle & Wolpert, 1978). If, instead, the predominant pattern is that of tandem
repitition with intermediate forms, as in ciliate membranelles and mammalian
teeth, the existence of a periodic prepattern with 'secondary modifications'
would be strongly supported. There might then be deeper similarities in the
operation of developmental fields in unicellular and multicellular organisms.
The drawings were executed by Mary Thorson. The authors also thank Drs Anne W. K.
Frankel, Malcolm Maden, Stephen F. Ng, Dennis Summerbell, and Norman E. Williams, as
well as Mr Timothy Lansing, for their criticisms and comments, and Dr Marijo A. Readey for
pointing out the relevance of the mammalian dentition. Responsibility for any remaining
errors and misinterpretations remains our own. The research was supported by grant HD08485 from the U.S. National Institutes of Health.
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{Accepted 21 March 1984)