THE DEVELOPMENT OF BASAL BODIES IN PARAMECIU.

II*
BY RUTH V. DIPPELL
DEPARTMENT OF ZOOLOGY, INDIANA UNIVERSITY, BLOOMINGTON

Communicated by T. M. Sonneborn, August 2, 1968

Basal bodies or centrioles occur in the cytoplasm of almost all animals and
lower plants and are implicated in the formation of flagella, cilia, and certain
sensory structures and in the organization of the mitotic apparatus. Electron
microscopic studies have shown that the structural pattern of these two types of
organelles is almost identical from Protozoa to man, but the origin and develop-
ment of this pattern remain largely unknown. Previous investigators, searching
for early developmental stages, have observed at the site where the new structure
will develop either a sphere of dense, amorphous material or a young organelle
shorter than the adult but nevertheless possessing most or all of the features
characterizing the mature pattern. Intermediate stages have rarely been de-
tected. The present paper, based on observations reported in a previous ab-
stract,' provides evidence for these stages and establishes for the kinetosome
(ciliary basal body) of Paramecium a mode of development that may be expected
also to apply to basal bodies and centrioles of other organisms.
Methods.-Paramecium aurelia, stock 51, was grown at maximal rate in baked lettuce
medium2 at 270C. Success in detecting the earliest stages of basal body formation was
based ul)on recognition of three facts. First, the new ciliary basal body arises at right
angles and in close Iproximnity to the anterior portion of an existing basal body (Figs. 2,
7, 8). Second, basal body proliferation occurs first and mainly within a zone encom-
passing the middle third of the body (Fig. 1). Third, maximal basal body production is
reached within a 20-min period beginning approximately 50 min prior to completion of
cell division (ef. Ehert and DeHaller's stage D).3 At this time the cell has begun to
broaden anteriorly but as yet shows no sign of a cleavage furrow. Single cells at this
stage were fixed in 1.5%o veranol-acetate-buffered glutaraldehyde (except where noted),
washed in buffer, I)ostfixed in 1%7 buffered osmium, dehydrated, and oriented in Epon
to yield both longitudinal and cross sections through developing kinetosomes. Sections
were stained with uranyl acetate and lead citrate and observed with a Hitachi HIT-1i1A
electron microscol)e.
The illature Oryanelle. Like the mammalian centriole and the ciliary and
flagellar basal bodies of other eukaryotes, the basic architecture of the Parame-
cium basal body consists of a cylinder whose "wall" is composed of 27 microtu-
bules arranged in nine equally spaced groups of three (Figs. 3, 4, 7). The nine
triplet sets run parallel to the long axis of the basal body, but each set is pitched at
an approximate 40° angle to the cylinder surface; the outermost tubule of each
triplet is designated C, the innermost A. Individual tubules are approximately
200-230 A in diameter, have a globular subunit structure comparable to the tu-
bule composition in other organisms,4 5 and are surrounded by an electron-dense
fibrous matrix. A and B of each triplet continue into the cilium to form one of
its nine doublet sets (Fig. 2) but C terminates at the basal plate. In the proximal
portion of the basal body, the triplets assume a more acute angle with respect to
the cylinder surface, and connections become evident between the A tubule of
one triplet and the C tubule nearest to it in an adjacent triplet (Figs. 4, 7).
461
462 ZOOLOGY: R. V. DIPPELL PROc. N. A. S.

FIGS. 1-18.-See text for descriptions.

Abbreviations: (nbb) new basal body; (ibb) immature basal body; (abb) adult basal body;
(a) axosome; (has) hub and spokes; (f) "foot"; (L) A-C linkers; (kf) kinetodesmal fiber;
(It) lateral tubules; (gd) generative disc; (s) spacers.
Magnifications: Fig. 1, X 600; Figs. 12, 16, 17, approx. X 50,000; Fig. 18, X68,000;
Fig. 5, X70,000; Figs. 3, 6, X120,000; Figs. 2, 4, X130,000; Figs. 7-11, 13-15, X140,000.

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FIG. L.-Silver-stained, ventral surface of an early dividor, showing region (between bars) of
maximal basal body proliferation; gullet and developing cleavage plane at arrow.
FIG. 2.-Relation of new basal body to lateral tubules and adult basal body. Note "hairpin
loop" of fibrous lumen component (arrows).
FIG. 3. (Collidine-buffered fixation).-Cross section of upper portion of adult basal body
(viewed from cell exterior) showing triplets and peripheral fiber (at arrow).
FIG. 4.-Cross section through proximal end of adult basal body in gullet (viewed from cell
interior).
FIG. 6.-30 A-thick double strands (arrows) in central mass, associated with large, dense
bodies.
FIG. 7.-Appendages of the adult basal body: kf and It indicated. Note new basal body
anterior and at right angles to old.

Within the proximal cylinder lumen, the most prominent structure is the hub-
and-spoke which is present both in basal bodies and centrioles and consists of nine
delicate fibers radiating from a central hub to the "foot"6 of each of the nine A
tubules (Figs. 4, 5). The complex of hub, nine spokes, and nine triplet tubules is
commonly called a "cartwheel." In Paramecium, the hub frequently appears bi-
or tripartite and the spokes segmented and interconnected by fibers. Above this
structure a slender filament courses the inner rim of the lumen in contact with
VOL. 61, 1968 ZOOLOGY: R. V. DIPPELL 463

the nine A tubules,7 visibly corresponding to the "internal helix" of the centriole6
(Fig. 3). The center of the lumen contains groups of dense bodies, approxi-
mately 300 A in diameter, alternately (helically?) disposed within a ramifying
fibrous matrix (Figs. 2, 6, 16). Two parallel threads (Fig. 6) (or a single hairpin
loop? (Fig. 2)), each approximately 30 A wide, appear to spiral through the
matrix and to make contact at definite points with the large, dense bodies. Pre-
liminary light and electron microscope studies, combining nuclease digestion and
autoradiography, indicate that these bodies contain RNA.
Basal bodies of Protozoa frequently also bear various appendages. In Para-
mecium, two serve as important landmarks to pinpoint the site of basal body
origin: a broad, "kinetodesmal fiber" whose roots derive from the three antero-
right triplets (Fig. 7) and a linear set of four or five laterally directed tubules,
originating across the anterior base of the basal body and sloping upward toward
the animal's left side (Figs. 7, 8). The nascent basal body will arise in precise
juxtaposition to these two markers (cf. Stage 1 in text).
Basal Body Development.-Stage 1, the generative disc: Basal body synthesis
starts with the appearance of a flat disc of dense, fibrous material near the left
side of the kinetodesmal fiber and immediately anterior to an adult basal body
and its set of lateral tubules. The disc is circular in outline, slightly smaller than
the mature basal body, and shows no internal organization. A distance of
several hundred angstroms intervenes between it and the adult basal body.
Serial sections of this region were examined particularly for connections between
the old and new structures. Although no microtubular connections were found,
the fine fibrous material surrounding the triplets appeared confluent with that of
the disc.
Stage 2, formation of a ring of "singlets": Looking anteriorly and viewing the
following developmental stages from the posterior side of the disc, one observes
that microtubule assembly always begins in the upper right quadrant, adjacent to
the kinetodesmal fiber (Fig. 9). Individual tubules then develop consecu-
tively (Figs. 10, 11) at equally spaced intervals of approximately 400 around the
disc rim until a circle of nine tubules is completed. Seen from the posterior sur-
face, the first few additions occur counterclockwise to the original tubule but later
stages suggest that they may also form in a clockwise direction. Conceivably,
the spacing is accomplished by short fibers or connectives observed between
adjacent tubules in the developing ring (Fig. 11), a suggestion consistent with
the fact that once the ring is established, the connectives disappear. These
tubules constitute the inner or A tubules of the nine triplet sets in the adult basal
body. Addition of disc material appears to accompany this stage.
Stage 3, formation of "doublet" sets: When six to seven single tubules are pres-
ent, a second round of tubule proliferation is initiated, again in the upper right
quadrant. This round may not always be sequential. Figure 13 shows that the
second or B tubule develops as an outgrowth from a single site on the first, start-
ing as a short stub which elongates, arcs into a "C," and establishes contact at a
second site on the singlet approximately 900 counterclockwise from its point of
origin. The B tubule is therefore an incomplete structure, sharing a quarter of
its boundary with A.
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When tubule elongation begins is not known precisely, but in stage 4 the na-
scent basal body is still less than 70 m1A long, as shown by its restriction to a single
serial section (Fig. 12b).
The disc which is uniformly dense in stage 1 becomes hollowed out by stage 4.
A circular outline vaguely resembling a cartwheel hub is frequently observed in
its center between stages 2 and 4. Wispy, short projections are occasionally
associated with it or one or more of the A tubules, possibly suggesting a forming
hub-and-spoke (cf. Discussion).
Stage 4, triplet formation: A third round of tubule production is initiated
before completion of the second and follows a similar developmental pattern with
the third or C set of microtubules growing from and to corresponding sites on the
tubules of the second ring. At the same time, the new basal body begins to
elongate rapidly, a fact probably accounting for reports that in adult basal bodies
and centrioles the C tubule sometimes appears to be shorter at its proximal end
than the other two tubules of a triplet.
Slightly later, a fully organized hub-and-spoke structure is recognizable in the
young basal body (Fig. 14), which by now is approximately 100 m/A long but still
oriented at right angles to the old one (Fig. 2). It differs from a mature basal
body, however, in the steeper pitch of its triplets (Fig. 15), which still lack the
A-C linkers, "internal helix," organized central mass, vesicular and fibrous com-
ponents associated with the cartwheel, and the accessory outer appendages.
Occasionally the earliest sprouting of linkers is seen at this stage. On the other
hand, cytoplasmic microtubules such as are often found associated with mature
basal bodies are also often found associated with new ones at this stage (Fig. 15).
Although never confluent with the basal body microtubules, like the latter they
originate from a dense pad of material that is linked by fibrous bridges to a basal
body (Fig. 15). From this stage on, a distinct layer of relatively dense material
forms a cover across the distal (probably the growing) end of the new basal body
(Figs. 2, 1 6).
As tubule elongation proceeds, the old and new basal bodies move apart, and
the new begins its upward tilt toward the cell surface. During maximal basal
body proliferation, three or four successive generations of basal bodies may be
FIGS. 8-15.-Early basal body development: all cross sections of forming basal bodies
appear at right angles to the plane of the figure and to the adult basal body. FIG. 8, site of
origin. For orientation, this section is cut perpendicular to that in Fig. 2, passing approxi-
mately through the area designated "It." The new basal body will form away from the
reader, i.e., anterior and at right angles to the lateral tubules and adult basal body. FIG. 9,
formation of the first tubule. FIG. 11, an almost-completed ring of A tubules. FIG. 12, three
consecutive (700 A) serial sections through the developing basal body area: only the middle
section contains the forming basal body (incomplete doublet ring); 1(c), the most anterior
section. FIG. 13, doublet formation; a B tubule forms at arrow. FIG. 14, proximal
end of a new basal body (as in Fig. 2). FIG. 15, distal part of a new basal body; arrow indicates
"pad" from which cytoplasmic microtubules develop in association with the new basal body.
FIG. 16.-New basal body with distal "cover," approximately one-third grown. Tilting
has begun. Note also distribution of dense bodies in adult.
FIG. 17.-New basal body, approximately three-fourths grown, with cap: cell surface
appears at upper right.
FIG. 18.-Four generations of basal bodies: note that the most immature one is itself
associated with a new basal body. An organized central mass is absent from immatures.
466 ZOOLOGY: R. V. DIPPELL PROC. N. A. S.

present within a single cortical unit" (Fig. 18). The "genealogical" relations
among the basal bodies of such a group vary, apparently in relation to the dis-
tances separating them. Shortly before the new basal body surfaces, its elonga-
tion is completed and a definite cap appears on it (Fig. 17). A dense apical
granule or axosome develops at the cap center, from which at least one of the
central pair of ciliary tubules originates (Figs. 2, 18). The "internal helix" may be
present, but the central lumen contains only small amounts of fibrillar material
with no recognizable double-stranded core. Development of this central mass
continues up to the time of ciliogenesis.
Discussion.-(1) Development: If typical basal bodies and centrioles develop
similarly in most organisms, as seems likely, the almost universal failure of other
investigators to find early developmental stages now becomes understandable.
In Paramecium, stages preceding development of the complete singlet ring were
much less frequently observed than later stages. This implies that tubule as-
sembly must be rapid. Hence, events prior to triplet organization could easily
be missed in material less suitable than Paramecium, in which thousands of basal
bodies arise almost synchronously in known positions at a recognizable stage of
the cell cycle. Only one clearly defined early stage of presumed basal body for-
mation has been demonstrated in other material, viz., a ring of nine single tubules
with hub-and-spokes in Chlamydomonas.8 All other reports9-11 of early stages
are relatively late ones possessing triplets and representing stages in elongation,
except possibly the very short centrioles in the fern Marsilea, which show an
obscure nine-pronged structure and tubule pattern.12
The source of the basic ninefold symmetry is currently attributed to the hub-
and-spokes,6 mainly because such symmetry has been seen in even the shortest
centrioles, which, however, already had at least a ring of single microtubules.
In Paramecium, on the other hand, there is no evidence of radial architecture
within the generative disc prior to the appearance of the first few tubules. A
definitive hub-and-spokes has not been found until near completion of the doublet
ring. Between these two stages, we occasionally see what might be a de-
veloping hub-and-spokes, as mentioned earlier. Evidence that these central
structures are not essential for tubule organization is also shown by their absence,
at any observed stage, from otherwise typical basal bodies in the cirri and mem-
branelles of the ciliate Euplotes,'3 from giant centrioles in Sciara,"4 and from the
Haemoproteus axoneme'5 which, moreover, develops in the absence of a basal
body. These observations suggest that attribution of an organizing role to the
hub-and-spokes may be placing the "cartwheel before the horse." Information
provided in the present paper suggests an alternative mechanism of developing
the ninefold symmetry, viz., use of transient spacers for determining tubule
distribution.
Concerning other aspects of basal body development, there is even less ground
for sound speculation. It is possible to invoke processes of self-assembly and
cytotaxis'6 to explain these developments, since the latter are specifically localized
in relation to existing structures. It appears, however, that little would be
gained by extending interpretations in our present state of ignorance.
VOL.- 61, 1968 ZOOLOGY: R. V. DIPPELL 467

(2) Origin: The Chatton-Lwoff hypothesis,17 based on light microscope ob-

servations, that "one kinetosome is always generated by division of another" has
not been supported by electron microscope studies. However, their conclusion
of genetic continuity between kinetosomes (basal bodies) is currently being re-
vived and modified, based on evidence for the occurrence of DNA in basal
bodies.'8, 19
Regardless of the presence or function of basal body DNA, it is clear that the
existing organelle is involved in the origin of a new basal body. The precise
position and orientation of the latter, with respect to the former, is determined by
the existing organelle and not by the surrounding geography: a young basal
body still immature and beneath the surface (and therefore having a different
relationship to the surrounding geography than that of an adult) can nevertheless
be associated with a nascent basal body (Fig. 18); and, when it is, the nascent
body bears the same relationship to the maturing body as a new basal body bears
to an adult at the cell surface (Fig. 2). We have also found that, during regen-
eration of amputated regions, the "naked," cut surface acquires new basal bodies
only in regular juxtaposition to existing ones at the cut edge and only at the time
of normal basal body production preceding fission.
This ordered physical association between the old and new basal body is traced
to the generative disc, which assumes its prescribed position before any or-
ganized structure appears in it; it is never found elsewhere in the cell or at any
other stage in the cell cycle. The only clue thus far to the relationship between
the disc and the existing basal body is the fine fibrous material which accumulates
around the base of the latter during the prefission period, concentrating more on
the side where the disc will arise and appearing, in places, to connect with similar
material comprising the disc. Fibrous connections have also been seen between
small "clouds" of dense material (resembling that of the disc) and clusters of
surrounding, nascent centrioles in differentiating rat tracheal epithelium.'0 The
"centriolar plaque" (which in yeast gives rise, in the absence of a centriole, to a
small, intranuclear spindle) also bears a superficial resemblance to the disc.20
On the other hand, possible intermediates between existing and new basal
bodies and centrioles presumably are lacking in instances of apparent de ovo
origin of these organelles.10' 15, 21 -In certain of the cases, and in formation of the
yeast centriolar plaque, the nucleus may be directly implicated in microtubular
formation and organization. However, we can only surmise what the relation-
ship between these situations and those involving regular association of old and
new structures may be. Localization of pertinent DNA in the organelle rather
than the nucleus may constitute the underlying basis of difference; and allocation
of the DNA to the organelle may in turn be related to the persistence and/or
function of basal bodies or centrioles throughout the life cycle. In addition, the
difference may be correlated, as Sonneborn has proposed, with the requirement
for definite localization and orientation of new organelles in cells like Parame-
cium. Beisson and Sonneborn, for example, reported abnormal swimming in
cells containing experimentally inverted rows of basal bodies.22 Since the whole
reaction system of the cell-swimming, mating, feeding, etc., involves cilia that
468 ZOOLOGY: R. V. DIPPELL PROC. N. A. S.

develop from basal bodies, positioning of the new by the old structures becomes a
mechanism that doubtless would have positive selective advantage in evolution.
This rationale may apply as well to other cells with precisely placed cilia, flagella,
and centrioles.
The author wishes especially to thank Mr. Gary W. Grimes for expert technical as-
sistance, Mr. Steve Lindle for photographic services, Dr. T. M. Sonneborn for invaluable
discussions and sustained encouragement, and Dr. K. R. Porter for stimulating an interest
in this project.
* Supported in part by contract COO-235-52 of the Atomic Energy Commission, grant GM

15, 410-01 of the USPHS, and grant E81 of the American Cancer Society to T. M. Sonneborn.
Contribution no. 816 from the Department of Zoology, Indiana University.
1 Observations reported by R. V. Dippell during the 1967 Autumn Meeting of the Academy
(abstract in Science, 158, 527 (1967)).
2 Sonneborn, T. M., J. Expt. Zool., 113, 87 (1950).
3 Ehret, C. F., and G. DeHaller, J. Ultrastruct. Res. (Suppl.), 6, 1 (1963).
4Ledbetter, M., and K. R. Porter, Science, 144, 872 (1964).
6 Ringo, D. L., J. Ultrastruct. Res., 17, 266 (1967).
6 Stubblefield, E., and B. R. Brinkley, in Formation and Fate of Cell Organelles, ed. K.
Warren (New York: Academic Press, 1967).
7Gibbons, I. R., and A. V. Grimstone, J. Biophys. Biochem. Cytol., 7, 697 (1960).
8 Randall, Sir John, T. Cavalier-Smith, A. McVittie, J. R. Warr, and J. M. Hopkins, in
Control Mechanisms in Developmental Processes, ed. K. Warren (New York: Academic
Press, 1968).
9 Gall, J. G., J. Biophys. Biochem. Cytol., 10, 163 (1961).
10 Dirksen, E. R., and T. Crocker, J. Microscopie, 5, 629 (1966).
" Bradbury, P., and D. Pitelka, J. Microscopie, 4, 805 (1965).
12 Mizukami, I., and J. Gall, J. Cell Biol., 29, 97 (1966).
13 Dippell, R. V., and G. Grimes, unpublished data.
14 Phillips, D. M., J. Cell Biol., 33, 73 (1967).
11 Bradbury, P. C., and W. Trager, J. Protozool., 15, 89 (1968).
16 Sonneborn, T. M., these PROCEEDINGS, 51, 915 (1964).
17 Lwoff, A., Problems of Morphogenesis in Ciliates (New York: John Wiley and Sons, Inc.,
1950).
18 Smith-Sonneborn, J., and W. Plaut, J. Cell Sci., 2, 225 (1967).
19 Randall, Sir John, and C. Disbrey, Proc. Roy. Soc. (London), Ser. B, 162, 473 (1965).
20 Robinow, C. F., and J. Marak, J. Cell Biol., 29, 129 (1966).
21 Schuster, F., J. Protozool., 10, 297 (1963).
22 Beisson, J., and T. M. Sonneborn, these PROCEEDINGS, 53, 275 (1965).