AMER. ZOOL., 22:35-^16 (1982)
Pattern Determination in Hypotrich Ciliates1
GARY W. GRIMES
Department of Biology, Hofstra University, Hempstead, New York 11550
SYNOPSIS. The hypotrich ciliates possess a highly localized ciliature arranged in a predictable asymmetric and polarized fashion. They also possess a large repertoire of developmental alternatives, including morphogenesis of ciliature prior to fission, as well as
during regeneration and reorganization. They also undergo a cystment process in which
all visible ciliature dedifferentiates during cyst formation but redifferentiates upon emergence from the cyst. These organisms thus are highly suitable for analysis of cell patterning
in eucaryotic unicells. Analysis of various types of experimentally induced cortical anomalies reveals that the final pattern of the cortex in these ciliates is determined by at least
three distinct informational systems. Two of these systems are inherited cytotactically; one
is independent of the presence of visible structure and provides information for the polarity
and asymmetry of developmental fields as well as serving as a potential initiation site for
a complete set of cortical structures, whereas the second is dependent upon visible ciliature
and provides local information for structure-type and polarity of new ciliary structures.
The third informational system, developmental assessment, operates coordinately with
the other two and generates the final ciliary pattern of the morphostatic ciliate. Currently
the molecular bases of each of these informational sets remain unknown.
INTRODUCTION AND HISTORICAL
PERSPECTIVE
These characteristics of ciliates have
been exploited in various ways to analyze
The ciliated protozoa have been utilized cell patterning. The classic grafting experwidely in studies of cell patterning, and iments of Tartar (1961) on the heterotrich,
they continue to serve as ideal model sys- Stentor, illustrate the ciliates' utility for that
tems for such studies (e.g., Aufderheide et type of experiment and demonstrate that
al., 1980). The reasons for this are many, transplants of portions of the cortex could
but I will emphasize only those which I have major morphogenetic significance.
consider to be of the most general signifi- The genetics has been most extensively
cance in studies of intracellular patterning. developed and utilized for studies of patThe first characteristic of major signifi- terning in the hymenostome ciliates, Parcance is that the ciliates possess highly or- amecium (Sonneborn, 1963, 1964, 1970,
dered polarized and asymmetric arrays of 1975), and more recently, Tetrahymena (Ng
cilia which can be visualized and studied and Frankel, 1977; Frankel, 1979; Aufby standard techniques. The second note- derheide et al., 1980). The important studworthy characteristic is size; many species ies by Sonneborn (1963), Beisson and Sonare sufficiently large to perform precise neborn (1965), and later by Ng and
microsurgical alterations and grafts. The Frankel (1977), serve as the basis and
third major characteristic is the ability to proof of the concept of "cytotaxis," i.e.,
perform classical Mendelian genetics on that preformed structure plays a role in
selected species, thus enabling the investi- determining orientation and organization
gator to determine if phenotypic differ- of newly developing structure.
ences result directly from genie differA far broader question that remains unences between cells {e.g., Sonneborn,
1963). Thus, in ciliates, unlike most meta- answered by these earlier studies is: "What
zoan systems, the question of whether dif- is the informational system(s) for intracelferent genotypes account for different lular determination of positioning, assemcellular phenotypes can be answered bly, and patterning of cellular components?" Ultimately, what we really want to
unequivocally.
know is what determines the spatial fate
(intracellular localization) of molecules in
1
From the Symposium on Principles and Problems
the cell. I present to the reader a review of
of Pattern Formation in Animals presented at the Annual Meeting of the American Society of Zoologists, my work on patterning in hypotrich ciliates which addresses these questions (see
27-30 December 1980, at Seattle, Washington.
35
36
GARY W. GRIMES
Feeding +
Encystment
Prefission
Development
I Excystment
MCP
Hi d
1 Typical Singlet
II d
1 Typical Singlet
1 Typical Singlets
Fic. 1. Typical singlet structure and developmental alternatives. A. Ventral surface of a typical morphostatic
singlet cell illustrating the three major ventral ciliary groups: oral apparatus (AZM and UM), FVT cirri, and
marginal cirri (MC). Typical asymmetry and polarity of the entire set of organelles indicated by arrows.
Developmental alternatives include: prefission morphogenesis (iia—d); regenerative morphogenesis (ia-e);
physiological reorganization (ma-d); and cystment (n'a, b). In all types of morphogenesis, the first primordium
PATTERNING IN UNICELLS
37
Aufderheide et al., 1980 for a more comprehensive review of ciliate development).
Each of these four major groups of ciliature is derived from distinct primordia
which are initiated in a constant, predictTHE ORGANISM
able sequence (Fig. 1). The oral primorI have chosen highly similar ciliates of dium (OP) is the first to be initiated, and
the family Oxytrichidae (Oxytricha fallax, generally (see exceptions below) gives rise
Stylonychia mytilus, Pleurotricha lanceolata) as only to new oral structures. The frontoa focus of my research because they pos- ventro-transverse primordium (FVTP) is
sess certain characteristics which allow us formed next and a complete set of FVT
to answer specific questions regarding the cirri differentiate within it. This process is
control of cell patterning in these unicells. followed by initiation of primordia for the
Some of these characteristics are: (i) they marginal cirral rows (marginal cirral pripossess a highly asymmetric and polarized mordia, MCP), in which existing cirri withset of compound ciliary organelles, each of in the rows disaggregate, basal bodies prowhich also has a characteristic polarity and liferate, and individual cirri later
asymmetry; (ii) this set of ciliary organelles redifferentiate within the same row. The
develops in a predictable and repeatable dorsal primordia form last within existing
manner; (Hi) they can undergo encyst- rows in a manner similar to that of the
ment-excystment processes (all ciliary MCP.
structures are absent in the resting cyst);
Significantly, this sequence of primor(hi) they can acquire and maintain multiple dium initiation (i.e., OP followed by FVTP,
sets of ciliary organelles in various geo- MCP, and dorsal primordia) is the same
metrical configurations; (v) they respond for all types of morphogenetic processes
to injury by initiating a morphogenetic se- which these ciliates undergo. In division,
quence to "repair" the cell (regeneration); two sets of primordia develop which difand (vi) they are large enough to allow us ferentiate into a full ciliary set for the two
to perform precise microsurgical opera- fission products (Fig. 1, wa-d). However,
tions.
during regenerative morphogenesis (inThe typical set of compound ciliary or- duced by trauma; e.g., microsurgery, Fig.
ganelles is comprised of four major groups 1, ia-e), physiological reorganization (inof ciliature—the oral apparatus (OA), mid- duced by starvation, Fig. 1, ma-d), or exventral cirri, marginal cirral rows (MC), cystment morphogenesis (see below), only
and dorsal ciliary rows (Fig. 1, A). The OA one set of primordia forms, but the seis composed of the adoral zone of mem- quence of development of that set is the
branelles (AZM) and undulating mem- same as that in prefission morphogenesis.
branes (UM) localized in the antero-left The result of these types of morphogenetquadrant of the ventral surface. The re- ic processes is (with exceptions to be dismaining mid-ventral ciliature is designated cussed below) the establishment of cells
as the fronto-ventro-transverse cirral field with a normal single complete set of cilia(FVT). On both the left and right margins ture.
of the cell is a row of marginal cirri (MC).
Starvation of these cells also induces enThe typical dorsal surface is comprised of cystment, a process in which cells undergo
six longitudinal rows of short cilia.
a profound dedifferentiation and form a
to form is the oral primordium (OP; ib, iia, in a) which develops as a localized random array of basal bodies
which later organize into membranelles of the AZM. The next primordium to form is the fronto-ventrotransverse primordium (FVTP) for the mid-ventral cirri (ic, iib, iiib). This primordium is followed by initiation
of the marginal cirral primordia (MCP) within existing rows (id, iic, iiic). These processes are followed by
maturation of ciliary organelles. The result of prefission morphogenesis is two typical singlet cells (iid), and
of reorganization and regeneration, one typical "repaired" singlet cell (ie, iiid). In iva, b, starvation induces
encystment, where all ciliary structures dedifferentiate. The mature cyst (iva) thus possesses no ciliature, but
upon refeeding, redifferentiation of an entire ciliary complement occurs (iub).
38
GARY W. GRIMES
Doublet
Typical
Singlet
Typical
Singlet
Regeneration
Typical
Singlet
Longitudinal
Cutting
Typical
Singlet
Regeneration
Hi 9
Typical
Singlet
FIG. 2. Local patterning and developmental assessment. ia-g. From a typical homopolar doublet (ia), cells
can be obtained which possess supernumerary rows of MC located mid-dorsally (ib at arrows). These rows
can be lost by migration to the cell's right margin (ic) with subsequent regulation to a typical singlet (id).
These rows also can be propagated cytotactically asexually (ib, c), but they are lost as a result of encystment
and excyslment (if, g; after Grimes, 1976 and Grimes and Hammersmith, 1980). iia-d. Effects of folding on
polarity of MC rows. The polarity of both MC rows in a typical singlet cell is the same (arrows in iiz). As the
result of longitudinal cutting (lib) and folding, the MC row on the left is inverted (iic; after Grimes and
40
GARY W. GRIMES
Typical
ivo
iv b
'v
c
Inverted
Ivd
Membranelles
Fic. 3. Global patterning and assembly (in all figures, relative polarity and asymmetry of the overall pattern
of the ciliature is indicated by arrows). ia-g. Fate of standard homopolar doublets (ia). When fed, these cells
undergo asexual propagation true to type (ib), and also undergo encystment and excystment true to type
(ib-d). However, when the cells are cut longitudinally, separating the two ventral surfaces (ie) the resultant
cells are typical singlets (if, g; after Grimes, 1973c). iia-d. Folding of right fragments not only results in
inversion of MC rows (see Fig. 2, iia-d), but also inversion of the entire original posterior half of the cell.
The result of the subsequent regenerative morphogenesis is the formation of two mirror-imaged primordial
fields (iic) which differentiate into a deficient, mirror-imaged doublet, both halves of which possess the same
PATTERNING IN UNICELLS
protective "shell" around the cytoplasm
(Fig. 1, iva, b). Cells remain viable for
years in this state without feeding, and
when induced, redifferentiate and excyst,
resuming a typical asexual cycle of growth
and division. The important aspect of cyst
formation for our purposes involves the
extent of this dedifferentiation. Exhaustive ultrastructural analysis of mature cysts
has demonstrated that all ciliary organelles
and associated structures (cilia, kinetosomes, microtubules, fibers, etc.) are resorbed during encystment (Grimes, 1973a,
b; Hammersmith, 1976). The resting cyst
thus possesses none of the typical visible
organelles associated with the cortex of
vegetative cells. This observation provides
an essential analytical tool to our approach
to certain questions of cell patterning.
The complex structure of these cells,
when coupled with their impressive repertoire of developmental potential, make
these organisms highly suitable for analysis
of cell patterning. The experimental analyses and their interpretations are presented in the following sections.
T H E EXPERIMENTAL APPROACH
The approach to the question of patterning control in these ciliates is straightforward; the inherent biological flexibility
of the organism is used to study the effects
of various perturbations on the system.
These perturbations can be of many kinds;
e.g., additions to cells (effectively grafting),
deletions from cells (by microbeam irradiation or microsurgery with needles), or
creation of axial conflicts (either naturally
or experimentally induced). Rather than
belabor the "experimentology" of these
analyses, I will consider the interpretations
first and provide the data which lead to
those conclusions. In summary, we conclude that at least three distinct informational systems (all of which are extranuclear) control the final form and pattern of
39
the ciliate cortex; these are considered individually below.
Short-range pattern determination based
upon pre-existing visible structure
This type of "cytotaxis" was convincingly
demonstrated by Beisson and Sonneborn
(1965) who studied inverted ciliary rows in
Paramecium. Their work illustrated that inverted rows were inherited sexually and
asexually, independent of changes in nuclear genotype or fluid cytoplasm. Perhaps
the easiest way to visualize this informational system is that the pre-existing structure serves as a patterning guide for the
new structure; consequently, the new
structure possesses the same geometry.
This informational system can be illustrated with hypotrich ciliates in several
ways, but is best exemplified by marginal
cirral rows which in effect can be "transplanted" onto the mid-dorsal region of the
cell (Fig. 2 ib, c). Significantly, even though
the structures are positioned in a totally
"foreign" cellular environment (i.e., on the
dorsal surface) they undergo morphogenesis true to type and are inherited in subsequent generations (Grimes, 1976). Thus
the developmental fate of primordia produced by marginal cirri is not determined
exclusively by cellular position, but also by
the pre-existing structure within which the
primordia originate.
The cyst stage in hypotrich ciliates allows
us to conclude that this informational system is based exclusively upon visible preexisting structure. When cells possessing
these rows of atypically located marginal
cirri are encysted and subsequently excysted, the excysted cells invariably lack the
supernumerary rows of cirri (Fig. 2, «e-g;
Grimes and Hammersmith, 1980). This
observation illustrates that when MC rows
are present, their propagation is cytotactic
with regard to developmental fate for both
structural type and polarity; however,
L'Hernault, 1979). After regeneration, the row retains its inversion (iid; Grimes el at., unpublished), ma—g.
Regeneration in non-folded longitudinal fragments. Both left (mb, d, f) and right longitudinal fragments
(me, e, g) are capable of undergoing regeneration resulting in typical singlet cells (ii'if, g). In both cases, the
missing MC row is replaced by an extension of the OP into the area (arrows in md and me), a process never
observed when mature MC are present in those areas (after Grimes and Adler, 1978).
PATTERNING IN UNICELLS
41
when they are absent (as in the cyst) the
information for cytotactic propagation is
lost. The inevitable conclusion is that this
short-range cytotactic informational system is based solely upon directed patterning by pre-existing visible structure.
The analogy to ciliary rows in Paramecium can be extended further by looking
at cases where inversions of these MC rows
can be induced. When cells are cut longitudinally, the right fragments occasionally
fold completely, creating cells which can
undergo regeneration and possess an inverted row of marginal cirri on their left
margin. Analysis of inverted MC rows illustrates that their primordia undergo development in an inverted manner, and
that cirri derived from these primordia are
also structurally inverted (Fig. 2, iia-d;
Grimes and L'Hernault, 1979; Grimes et
al., unpublished). This illustrates not only
that the morphogenetic fate of primordia
produced by marginal cirral rows is structure-type dependent, but also that the polarity of the newly derived structures is
determined by the orientation of the row
in which the primordium arises.
cortical organelles, as well as the polarity
and asymmetry of those organellar sets is
functional within the cell. Additionally,
this global informational system is independent of the visible ciliature and is inherited cytotactically; i.e., if lost, it cannot
be replaced. The evidence for the existence of such an inherited system of patterning is multifold, and I shall consider
three examples here.
The first example of this informational
system is the inheritance pattern of homopolar doublets. These are cells which possess two complete sets of ciliary organelles,
180° apart, and have a common structural
asymmetry; in essence, they are Siamese
twins fused back-to-back with common cytoplasm and nuclei. Homopolar doublets
reproduce sexually and asexually true to
type, fulfilling the requirements for cytotaxis (Fig. 3, ia, b). One crucial difference
between this type of cytotactic phenomenon and that described in the preceding
section is indicated by the effect of encystment and excystment on the phenotype;
whenever doublets are encysted and subsequently excysted, they do so true to type,
independent of cellular volume, and more
Global patterning based upon an
significantly, independent of the presence
ultrastructurally unidentifiable
of visible ciliature (Fig. 3, ic, d; Grimes,
molecular architecture of the cell
1973c). When cells are created with multiple
sets of organelles (monsters), the cells
Other perturbations of the cell cortex
also
pass
through encystment and excystwhich involve alterations of the number of
sets of ciliature, as well as the polarity and ment with the same number of sets of corasymmetry of those sets of ciliature, lead tical organelles (Hammersmith, 1976). Mito conclusions which differ significantly crosurgical experiments on encysting
from those presented in the preceding sec- doublets have illustrated that a localized
tion. The general conclusion to be derived informational set exists on each ventral
from the following observations is that a cortical surface which can serve as the iniglobal informational system for patterning tiation site for an entire set of ciliary organwhich determines the number of sets of elles. This information is localized (i.e., can
polarity (iid; after Grimes and L'Hernault, 1979). iiia—g. Fate of mirror-imaged doublets. These cells are
propagated asexually true to type (ma, b). When these cells encyst and then excyst, they also do so true to
type (iiib-d). However, when the cells are longitudinally-cut, dividing the two mirror-imaged halves of the
doublet (me), the subsequent regeneration results in two singlet cells, one an apparently normal singlet (tug),
the other a singlet which possesses an asymmetry reversal pattern for the arrangement of the complete set of
ciliary organelles (mf; contrast to results in ie—g above), iv. Organization of structures in mirror-imaged
doublets in Hi above. The polarity and asymmetry of the cirri in the FVT group is identical in both the
standard-symmetry (ira; and reversed-symmetry [nsb]) halves of the mirror-imaged doublets (individual dots
represent basal bodies and dense lines represent associated microtubular bundles). However, even though the
membranelles in the standard-symmetry half are normal (ivc; each dot represents a basal body), each membranelle within the AZM in the symmetry-reversed half is inverted (ivd; after Grimes et al., 1980).
42
GARY W. GRIMES
Stage
Lased
Primordia
Present
i
Primordia
Removed
Effect on Development
Portion
of OP
1. None; i.e., typical division.
1. Development
OP
stops.
2. No cytokinesis.
All of
OP
ii
3. OP reforms hours later in typical
position followed by typical
prefission development
1. Continued development
cytokinesis.
All of
OP
i
and
2. Posterior fission product lacks
an OA.
1. Development proceeds for
prospective anterior fission product.
OP
FVTP
B
All of OP
and FVTP
for
prospective
posterior
fission
product
ii
2. Development
stops for prospective
posterior fission product.
3. Nuclei attempt division.
4. No cytokinesis.
5. OP reforms hours later in typical
position, followed by typical prefission
development
FIG. 4. Effects on prefission development of laser microbeam irradiation of primordia. Cells are sensitized
prior to irradiation by vital staining with Bismark Brown-Y. Irradiation of ciliary structures with 1-2 piM laser
spot results in blebbing of the cortex at the site of irradiation. TEM analysis of irradiated cells reveals that
irradiated ciliary organelles and associated structures are completely removed (Grimes, unpublished).
be separated from another informational
set; Fig. 3, ie, f), but it is subdividable (i.e.,
a portion of it can serve as an initiation
site; Grimes, 1973c). Such an informational system thus possesses many properties
of an "embryonic field," but must be considered different because it is cytotactically
inherited. I have previously named this cytotactically inherited localization the "determinative region" (Grimes, 1973c).
Additional information contained in the
"determinative region" is revealed by analysis of cells in which axial conflicts are created. This can be accomplished by the
same experiment which leads to the inversion of a marginal cirral row (the result of
such an experiment is a fully-folded right
fragment, see above). Not only is the marginal row of cirri inverted, but so is the
entire original posterior end of the cell.
The resultant regenerative morphogenesis
from such a cytogeometrical alteration is
impressive; rather than a single set of primordia, two sets of primordia form, one
on either side of the original wound margin. The one on the right side possesses a
typical asymmetry, whereas the one on the
folded left side (formerly postero-right of
the cell) is arranged as its mirror-image;
both possess a common antero-postero polarity (Fig. 3, Ma-d; Grimes and L'Hernault,
1979). This mirror-imaged arrangement
apparently is obtained because the folded
portion retains its original asymmetry, but
undergoes a polarity reversal to conform
to the non-folded region to its right. These
results indicate (i) that the determinative
region is subdividable, (ii) that the asymmetry is stable even after drastic rearrangement, and (in) that polar and lateral
PATTERNING IN UNICELLS
43
axes are determined independently. Un- these mirror-image doublets through cystfortunately, these fragments are incapable ment processes shows that the configuraof asexual propagation (presumably be- tion is not dependent upon the presence
cause of deficient oral apparatuses) and of visible ciliature, because these cells excannot be analyzed through cystment pro- cyst true to type (Fig. 3, mb-d).
cesses.
In this section, I have illustrated some
Similar conclusions are obtained by anal- inheritable phenotypic differences among
ysis of a different type of mirror-imaged cells of the same genotype which cannot
doublet which apparently can be obtained be explained by "short-range" pattern deby similar means; i.e., folding of left lon- termination based upon visible ciliary
gitudinal fragments. These cells possess a structure. However, the two types of inmirror-image structure reciprocal to that formational systems already discussed are
described above; the wound margin rep- insufficient to explain the final cell pattern.
resents the right side of the cell rather than We still must explain what determines the
the left side (Fig. 3, ma). Because these positioning and fate of individual primorcells possess functional oral apparatuses dia and the resultant mature structures of
and are capable of feeding, they undergo the morphostatic cell; this problem is disvegetative propagation, and do so true to cussed in the next section.
type (Tchang Tso-Run et al., 1964; Tchang
Tso-Run and Pang Yan-bin, 1977; Grimes Developmental assessment: Interactions
et al., 1980). On the folded side, both the among cortical components
final pattern of the ciliature and the arAlthough the two previously discussed
rangement of the primordia are mirror- informational systems contribute signifiimaged (Fig. 3, iiia, b); thus, the final cantly to cell patterning, the final detailed
pattern on this side is the result of the re- pattern of a morphostatic cell clearly is a
versed asymmetry of an entire developmental result of a detailed sequence of complex
field (presumably the "determinative re- morphogenetic events. Our observations
gion"). However, when we look at the de- on primordium development allow us to
tailed structure of the component organ- conclude that the sequence of development
elles {e.g., cirri and membranelles), we find (OP, FVTP, MCP, dorsal) is invariant but
that no individual ciliary structure is mir- that the site of origin and source of new
ror-imaged; rather, all ventral cirri are structures can vary drastically. Thus, the
identical, and the oral membranelles in the exact developmental fate of a given prisymmetry reversed half are inverted (Fig. mordium is not an inherent property of
3, ir/a-d; Grimes et al., 1980). The signif- that primordium, but rather a function of
icant conclusion to be derived from this other, more global properties of the cell
observation is that the global patterning of cortex, such as the stage and position of
the ciliature is determined independently other cortical primordia and/or mature
from the individual assembly events; i.e., structures. I shall refer to these multiple
the final pattern is not simply the sum of (reciprocal?) interactions among primorthe individual steps of assembly of the cil- dium as intracortical communication, or
iature.
developmental assessment.
The conclusion that the sequence of priThis conclusion, and the significance of
the determinative function of this global mordia initiation is invariant can be
patterning mechanism, can be emphasized tested directly with laser microbeam irrafurther in two ways. When the two halves diation experiments, in which portions of
of these mirror-imaged doublets are mi- (or entire) primordia can be removed at
crosurgically separated, both maintain different stages of prefission development.
their inherent asymmetry; thus, the mir- When the various permutations of these
ror-image configuration is not the result of experiments are performed, one basic rule
interactions between two fields on one ven- emerges; individual primordia proceed
tral surface (Fig. 3, me—g; Grimes et al., through development not according to a
unpublished). Additionally, analysis of cellular "time clock," but rather according
44
GARY W. GRIMES
to the stage and position of the preceding
primordium within its developmental field
(Grimes, unpublished). Thus, for example, if only a portion of the OP is removed
prior to initiation of the FVTP, then development proceeds normally (Fig. 4, i). If
all of the OP is removed, then development does not proceed but rather, after
substantial delay, the sequence is reinitiated by OP formation (Fig. 4, it). However,
if the OP is completely removed after
FVTP initiation, the development continues and the cell divides, even though the
posterior daughter cell lacks an oral apparatus (Fig. 4, Hi). When the next stage
of development is analyzed similarly, removal of OP and FVTP (for only the posterior fission product) prior to MCP initiation results in continued development in
the anterior product's field, but cessation
of development for the posterior fission
product. A delay in cell division results,
followed by initiation of a new OP and normal development (Fig. 4, iv). Thus, the cell
apparently must start at zero (within a primordial set) and cannot proceed without
the successful "completion" of the preceding stages.
Even though the sequence of primordium initiation is invariant, it is clear that
the developmental fate of certain individual primordia can be highly variable. The
primordia have been granted names on
the basis of their fate during prefission
morphogenesis; however, the developmental potential of some individual primordia far exceeds the manifestation
expressed during typical prefission
morphogenesis. When longitudinal fragments are created microsurgically, most
fragments do not fold as in the previously
described examples; instead, most cells
undergo a regenerative morphogenetic sequence which results in a typical singlet
cell. Detailed analysis of the morphogenetic sequence in these fragments reveals
that the morphogenetic manifestations of
individual primordia are a function of the
precise location of other mature structures
and/or primordia on the cell cortex. This
conclusion is best exemplified by observing
the changes in OP behavior; when mature
marginal cirri are absent on either margin,
the OP participates in the formation of
those MCP, even though the OP never participates in their formation when the mature marginal cirri are present (Fig. 2, a a f; Grimes and Adler, 1978). It appears,
therefore, that at least this primordium is
"aware" of its neighbors, and exhibits specific morphogenetic manifestations as a response to the status of those neighbors.
(This analysis is substantiated further by
results obtained by studying excystment
morphogenesis. In the total absence of
pre-existing ciliary structures, all ventral
ciliary primordia are derived from a single
initial primordium [Hammersmith, unpublished].) These data, besides illustrating
the multi-potency of certain primordia,
also are consistent with the previously
presumed function of a "determinative
region," i.e., to serve as the potential site
of initiation for a set of ciliature.
In addition to recognition of cortical deficiencies by primordia via an intracortical
communication system, this system can
"recognize" supernumerary structures,
and correspondingly regulate the number
of structures to conform to a typical phenotype. For example, when supernumerary rows of marginal cirri are located on
the cell's right margin (instead of on the
dorsal surface as described above), the
ventralmost rows undergo morphogenesis
true to type as expected, but their development is aberrant and the typical marginal structures never form (Grimes, 1976).
Thus, within a few cell cycles these supernumerary rows are lost, apparently a function of "developmental assessment" occurring during morphogenesis (Fig. 2, zb-d).
The results of this developmental assessment can be visualized directly by observing morphogenesis in live mirror-imaged doublets (described above) with
Nomarski optics. A full set of FVTP is
formed in each half of the doublet, but at
a certain stage following cirral segregation
and during migration of those cirri to their
final positions on the cell, a large number
of the ciliary structures are resorbed, resulting in significantly less than two complete sets of ventral ciliature (Grimes, unpublished). The cortex of these ciliates is
thus capable of extensive regulation, both
PATTERNING IN UNICELLS
in the direction of compensation of deficiencies, as well as downward regulation of
supernumerary cortical organelles. Available data suggest that this morphallactic
regulation, as well as the detail of the developmental manifestations of individual
primordia appears to be based upon the
exact position and stage of other structures
on the cell surface, in addition to the nutritional status of the cell.
45
expanded far beyond the narrow interpretation which generally has been given to it.
Hopefully, we will soon have the technology to visualize such structure. Most likely,
the structure of the "determinative region"
in these ciliates is a specific, stable, and inheritable organization of macromolecules
somewhere in the cortex of the organism.
The third informational system apparently is not cytotactic in nature, but operates coordinately with the above cytotactic
systems to provide the final pattern of the
DISCUSSION
ciliature, and we can consider this to be
Taken together, these data illustrate that "developmental assessment" or intracortithe final pattern of ciliature in the hypo- cal communication. The temporal order of
trich cortex is determined by at least three the developmental sequence is apparently
informational systems. The most obvious fixed; specific primordia form in an invarsystem is determination by organization of iant sequence within their "set" of primorpre-existing visible structure; examples dia. Nonetheless, the detailed fate and dewhich illustrate this include the self-deter- velopmental manifestations of individual
mination of developmental fate and polar- primordia are a function not only of which
ity of marginal cirral rows during asexual structures are present, but also the arreproduction. (These examples are ho- rangement of those structures.
mologous to the inheritance of polarity of
CONCLUSIONS
ciliary rows in the hymenostome ciliates,
Paramecium and Tetrahymena.) This type of
The question of what determines the fiinformational system appears to be the nal form and pattern of the ciliature of
most exacting and least flexible; thus, only hypotrich ciliates obviously cannot be anminor deviations can be tolerated.
swered easily, and we do not yet have that
The second informational system also is answer. What we have been able to demself-determinative (inheritable) but is based onstrate by our work on these ciliates is
upon what I have called an ultrastructural- that the final pattern of visible ciliary strucly unidentifiable organization of the cell tures in a unicell is not governed simply by
cortex (e.g., the "determinative region"). a sequence of gene switches, but also by
This informational system serves as a the intracellular localization of gene prod"counter," (i.e., it determines the numbers ucts. Thus, new cell products do not enter
of sets of structures), as well as a determi- a randomly organized environment, but
nant of the polarity and asymmetry of rather a highly ordered molecular lattice
those sets of structures. I emphasize here which aids in the determination of the orthat I consider this information system to ganization of new structure. Such a conbe cytotactic in nature; new structures are clusion matches well those of investigators
organized under the influence of pre-ex- analyzing the role of egg cytoplasm in deisting structure. Such a consideration is a velopment. The grey crescent in amphibfunction of my personal perspective of ians (Curtis, 1960), the anterior determiwhat "structure" is. If asked to describe nant in Smittia (Kalthoff et al., 1977) and
thoroughly the structure of a cell at a given polar granules in Drosophila (Ilmensee et
point in time, I would feel obliged to pro- al., 1974) all represent cases of intracelluvide coordinates of the location of each lar localizations of morphogenetic signifimolecule within the cell. If one considers cance. These cytoplasmic localizations
cell structure broadly as the molecular orga- have not been demonstrated to be inhernization of the cell (i.e., the three-dimen- itable, although present data do not presional localization of molecules within the clude that such localizations might be incell), then the concept of "cytotaxis" can be herited through the germ line in a manner
46
GARY W. GRIMES
similar to that observed in the ciliates. Ultimately, we all wish to understand the
biochemical interactions among gene
products which result in the complex phenomenon described in this paper, and
technological advances of the future might
allow this goal to be realized.
incomplete doublets of Oxytricha fallax. Genet.
Res. Cambridge 27:213-226.
Grimes, G. W. and J. A. Adler. 1978. Regeneration
of ciliary pattern in longitudinal fragments of the
hypotrichous ciliate, Stylonychia. J. Exp. Zool.
204:57-80.
Grimes, G. W. and S. W. L'Hernault. 1979. Cytogeometrical determination of ciliary pattern formation in the hypotrich ciliate Stylonychia mytilus.
Develop. Biol. 70:372-395.
ACKNOWLEDGMENTS
Grimes, G. W. and R. L. Hammersmith. 1980. Analysis of the effects of encystment and excystment
A large portion of this research was
on incomplete doublets of Oxytricha fallax. J. Embryol. Exp. Morph. 59:19-26.
funded by the NSF; this manuscript was
G. W., M. E. McKenna, C. M. Goldsmithprepared with the support of NSF grant Grimes,
Spoegler, and E. A. Knaupp. 1980. Patterning
PCM79-08992 to G.W.G. The author
and assembly of ciliature are independent progratefully acknowledges the editorial assiscesses in hypotrich ciliates. Science 209:281-283.
tance of J. Frankel, C. M. Goldsmith- Hammersmith, R. L. 1976. The redevelopment of
heteropolar doublets and monster cells of OxySpoegler, E. A. Knaupp-Waldvogel, R. L.
tricha fallax after cystment. J. Cell Sci. 22:563Hammersmith, G. E. Dearlove, S. Grimes,
573.
and S. W. L'Hernault during the prepa- Ilmensee, K. and A. P. Mahowald. 1974. Transplanration of this manuscript. Illustrations
tation of posterior pole plasm in Drosophila. Induction of germ cells at the anterior pole of the
were prepared by Robin Lazarus.
egg. Proc. Nat. Acad. Sci. U.S.A. 71:1016-1020.
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