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GIANT CENTRIOLE FORMATION IN SCIARA
DAVID M. PHILLIPS
From the Whitman Laboratory, tile University of Chicago, Chieago. The author's present address
is tile Department of Anatomy, Harvard Medical Sehool, Boston
ABSTRACT
Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop
giant centrioles with 50-90 singlet tubules disposed in an oval array. Some 9-membered Do
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centrioles still may be seen in second instar spermatogonia. Each of these centrioles is asso- nlo
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ciated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles de
d
of second instar spermatogonia consist of 20-50 singlet tubules arranged in an oval, some- fro
m
times associated with an even larger secondary centriole. The more recently formed centriole h
inosfi de a pair its is tubules. distinguishable If a pair from of centrioles its partner at by right a concentric angles to banedac h of other electron-opaque is pictured as material a "T" ttp://rup
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formed by two cylinders, the secondary centriole is always the stem of the T; the primary ss.o
centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that rg
the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell /jcb/a
receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a rticle
new daughter centriole. A Golgi area of characteristic morphology is found in association -p
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with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole f/3
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may give rise to a daughter morphologically different from itself. Whether the daughter is a /1/7
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9-membered or giant centriole depends on the tissue type and stage of development. /10
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8
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Centrioles were familiar to early cytologists as The mode of centriolar perpetuation has been 8/7
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minute granules which could be identified at the particularly puzzling. Centrioles often are described .pd
focus of the astral rays after iron-hematoxylin or as self-replicating bodies; indeed, forming cen- f by g
crystal violet staining. They classically were impli- trioles generally are found only in close proximity ue
cated as functioning in cell division and flagellar to "mature" centrioles. But proof of any sort of st o
n
formation (17, 23, 28). The structure of centrioles, genetic continuity between "mother" and "daugh- 24
which are apparently present in all metazoan cells ter" centrioles has been very elusive since, in any Jan
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celaepcatbrlone of microscopy, division, has but, been beyoncdla rified this, by the modern use of "gmivuent ant" species, centrioles all the have centrioles been described. look alike. There- No ary 20
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techniques have failed to reveal much more about fore, when variations in centriolar form were en-
these apparendy indispensable cell components countered during a study of spermiogenesis in the
than already was assumed by many biologists half fungus gnat Sciara, it was felt that a unique oppor-
a century ago. The intractibility of centrioles to tunity was thereby provided for studying the
modern analytical techniques is due partially to extent to which differences in centriole morphology
their relatively small size and low number per cell were propagated from parent to daughter or-
that make electron microscopic studies rather ganelle.
arduous and cytochemical procedures extremely The somatic tissues of Sciara contain only 9-
difficult to apply and hence contribute to the lack membered centrioles (26); these differ slightly
of success of isolation attempts necessary for bio- from centrioles of other organisms, in that they are
chemical analysis. composed of nine doublet rather than the usual
73
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FmvaEs 1-4 Transversely or nearly transversely sectioned 9-membered centrioles adjacent to longi- df/3
tudinally cut giant centrioles in second instar Sciara spermatogonia. The doublet nature of the ccntriolar 3/1
elements can be most clearly discerned in Fig. .1 >( 105,000. /73
/1
0
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nine triplet tubules. The spermatogonial centrioles MATERIALS AND METHODS 820
8
of fourth instar larvae, however, differ markedly in /7
size and structure from the familiar 9-membered The strain of Sciara alihporpoc used in this study is 3.pd
centriole. These centrioles are composed of about dmeovneolgoepn ic, into either i.e., all the male eggs or all of female a given progeny female (22). f by g
a70n sohvoarlt , which evenly has spaced axes of singlet about tubules 1.0 and displaced 0.4 ~ (26), in Vuqhpeothne r whether the offsprintgh e mother are carries male or an femaX le chromosome depends uest on
itnr iplet contrast centrioles to the (10). 0.15 Though # diameter their morphoolf ogy typical 9- is wtiiotnh, thew avy female-determininwgi ngs, which is closely trait. A linked dominant to the muta- sex- 24 Jan
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nuonsutsiuca l, characteristics these giant ocfe ntrioles true centrioles: exhibit the they are diag- dtoe termining predict which locus females on the X will chromosome, bear female enables and which one ary 20
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found in pairs at right angles to one another; they will bear male progeny (6, 7). 3
are situated at the poles of the mitotic and meiotic Larvae were selected from matings of known female
spindles; and they are capable of serving as basal producers or male producers. Larval gonads (testes or
ovaries, depending on the culture) were fixed for 1-4
body of a flagellum whose tubule array reflects
hr in 2.5% glutaraldehyde buffered in 0.05 ~ Soren-
their unusual tubule pattern (26, 27).
son's phosphate buffer (pH 7), rinsed several times in
The present investigation was undertaken to
0.1 M phosphate buffer (pH 7.6), and postfixed for
determine the mode of origin of giant centrioles
1-2 hr in 1% OsO4 in 0.1 g phosphate buffer (pH
in Sciara, an organism containing mainly 9-mem- 7.6). Tissues were dissected in cold (0-4oC) fixative,
bared centrioles, with the hope of enlarging our and subsequent fixation was carried out in the cold.
comprehension of the general process of centriole Tissues were dehydrated in cold ethanol and em-
formation. bedded in Epon 812 according to Luft (21). Sections
74 THE LANRUOJ "rO CELL YGOLOIB • EMULOV 8~, 1967
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ERUGIFSecond instar 5 9-membered spermatogonium, centriole ,c (arrow) chromosome. and adjacent giaX nt 31,000. centriole at a pole of the mitotic spindle. rticle-p
d
f/3
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were cut on a Porter Blum MT-1 ultramicrotome, not exactly parallel to the long axis of the centriole, /1/7
stained in 3% aqueous uranyl acetate (12 hr), post- but are slightly tipped as is apparently the case in 3/1
0
stained with lead citrate (36), and examined with a 9-triplet centrioles (1, 10). In all cases where a 68
2
Siemens Elmiskop I. cross-sectional profile is seen, the section longi- 08/7
3
tudinally transects another centriole lying adjacent .p
d
RESULTS to the 9-membered centriole (Figs. 1-4). The f b
distance between the two sides of the longitudinally y gu
N ine-M embered C entrioles in Sciara sectioned centriole is considerably greater than the est o
n
Somatic tissues of araicS embryos and larvae diameter of the adjacent 9-membered centriole. 24
appear to possess only 9-membered centrioles. Al- The longitudinally cut centriole is, therefore, al- Jan
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gtheorumg h line most tissue of the are centrioles giant centrioloefs , male second we always instar pmrosotb ably certainly is composed larger oft han more its thanp artnnienre and tubules. thus ary 20
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find some 9-membered centrioles in these early 9-membered centrioles also are found at right 3
testis cells. In order to postitively identify a cen- angles to larger centrioles at the pole of the mitotic
triole as the 9-membered type, the centriole must spindle in second instar spermatogonial divisions
appear in cross-section or near cross-section. We (Fig. 5).
have observed cross-sectional profiles of 51 9-
Giant Centrioles of the Testis
membered centrioles in spermatogonia of second
instar larvae. In the most favorably transected Most of the centrioles of second instar larval
centriole (Fig. 1), the tubular components on one spermatogonia are giant centrioles similar to but
side of the centriole appear as doublets; the tubules smaller than those found in fourth instar larvae at
on the opposite side are sectioned obliquely. There- the onset of spermatogenesis. Giant centrioles of
fore the nine doublets of the centriole probably are second instar testis are composed of only 20--50
DIVAD M. PItILLIPS Giant eloirtneC noitamroF in araicS 75
tubules, in contrast to the 70 or more singlet trioles always occur in pairs in which one member
tubules making up the centrioles of larval testis of the pair is a primary and one a secondary cen-
just prior to pupation. These smaller giant cen- triole, and that they always bear the same geo-
trioles contain occasional doublet tubules similar to metric relationship to one another.
those comprising 9-membered centrioles of araicS In transverse sections of secondary centrioles, all
(Figs. 6 and 7), whereas the centrioles of fourth the tubules are occasionally seen in cross-section;
instar larvae contain only singlets. more often, however, the tubules of one side are
Two distinct types of smaller giant centrioles are seen in cross-section while the tubules of the op-
found in second instar larvae. In one type, a thin posite side are sectioned obliquely (Figs. 51 and
concentric band of electron opaque material lies 16). In longitudinal section the sides of secondary
inside the centriole tubules and is separated from centrioles are seen to diverge (Fig. 17). Therefore,
them by a distance of about 400 A (Figs. 9, I0 and secondary centrioles generally take the form of a
11). Viewed in cross-section these centrioles dis- bilaterally flattened, truncated cone. The form of
play oval (Figs. 9 and )01 or rectangular profiles. primary centrioles is less regular, but their sides are
This type of centrlole is easily distinguishable from also often divergent.
the other type which lacks this band of electron- In sections which cut a primary centriole trans-
opaque material (Fig. 8). When a centriole lacking versely, so that it appears as an ellipse, and which D
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a dense line is seen in cross-section, it is syawla cut the adjacent secondarycentriole longitudinally, wn
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adjacent to a longitudinally sectioned centriole in so that it appears as two parallel rods, the length of ad
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which the dense line is present (Figs. 6, 7, and 8). the longer axis of the ellipse is often less than the d fro
We shall refer to centrioles without a dense line as distance between the parallel sides of the adjacent m
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primary centrioles and those with a dense line as secondary centriole (Figs. 18-23). In some cases it ttp
secondary centrioles, for reasons which are given appears that, even if the primary centriole were ://ru
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tine xt. the Primardyi scussion centrioles of centriole seen formatioinn cross-section later in usu- the fblea ttened so long longitudinaasl ly, the distance the between greater the axis two would sides not of ress.org
ally are flattened slightly on the side which borders the adjacent secondary centriole. Therefore one /jcb
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ctehnet riole adjacent (Figs. longitudin6 ally and 8). When sectioned a secondary secondary cen- lmaursgte r caonndc lude presumably that theco nsists secondary of a greater centriole number is rticle-p
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triole is sectioned transversely, its primary cen- of tubules than the adjacent primary centriole. f/3
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triole partner is not in the plane of section and is Thus the change from 9-membered centrioles in /1
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not seen. testes of young larvae to giant centrioles in testes 3/1
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In a three-dimensional reconstruction of the of late fourth instar larvae occurs by a series of in- 68
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spatial relationship which the primary and sec- creases in number of tubules in each newly formed 08
/7
ondary centriole must have to each other (Fig. ,21 centriole. In late fourth instar larvae the number 3.p
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configuration I), it is clear why a section which of component tubules in centrioles varies. In fact, f b
cuts the secondary centriole in cross-section will during spermiogenesis the giant centrioles, which y gu
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not include the adjacent primary centriole. From still range in size, serve as basal bodies to giant st o
Fig. 21 it also is apparent that it is possible to flagella in which the number of tubules also varies n 2
4
transect the primary centriole longitudinally with- from about 60 to 90 (26, 27). Ja
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out cutting through the adjacent secondary cen- Fibrous material in varying amounts often is ua
triole. However, it is not possible to cut the sec- found inside giant centrioles (Figs. 31 and 17). ry 2
0
2
ondary centriole longitudinally without cutting The chemical nature of this material is not known. 3
through the primary centriole. Indeed, we have Ribosomes, endoplasmic reticulum, microtubules,
observed that whenever a longitudinally sectioned and mitochondria also occur in giant centrioles
centriole does not lie adjacent to another centriole (Figs. 11 and 14).
it is a primary centriole, i.e., does not have the
Behavior of Giant Centrioles during Mitosi.~
band of dense material (Figs. 31 and 14). From
the above considerations, especially the fact that The nuclear membrane remains almost entirely
each of 54 different transversely or nearly trans- intact throughout the spermatogonial divisions in
versely sectioned primary centrioles we have ob- .araicS To our knowledge, no other metazoa have
served was found to lie adjacent to a longitudinally been found in which the nuclear membrane does
cut secondary centriole, we conclude that cen- not break down during cell division although this
76 THE LANRUOJ FO CELL YOOLOIB • E~'VLOV 33, 1967
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6 FmVRES DNA 7 Most of the tubules which comprise the small giant centrioles of second instar spermato-
gonia are singlets; however, a few doublet tubules (arrows) can be discerned. Fig. 6, X 115,000; Fig. 7,
X l~,000.
FmVRE 8 Transversely sectioned primary centriole lying adjacent to longitudinally cut secondary
centriole. Second instar spermatogonium. X 160,000.
77
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SERUGIF 9--11 Transversely cut secondary centrioles in second instar spermatogonia. Secondary eentri-
oles can be identified by the thin concentric band of electron-opaque material which lies inside the cen-
triole tubules and is separated from them by a distance of about 400 A (arrows). rot, mitochondrion.
Fig. 9, X 114,000. Fig. 10, X 96,000. Fig. 11, X 90,000.
phenomenon is common among the protista (14, the centrioles. Microtubules directed towards the
,51 32). two pairs of centrioles can be seen in the cytoplasm
In prophase, microtubules extend from two of early prophase cells before tubules are discerni-
pairs of centrioles on either side of the nucleus into ble in the nucleoplasm. This suggests that spindle
the nucleoplasm (Fig. 24). These microtubules tubules are cytoplasmic in origin.
are never actually continuous with the tubules of During prophase, the tubules of the primary
78 T~E LANRUOJ FO Ct:LL YGOLOIB • EMULOV 38, 1967
~
~S at the opposite side of the nucleus. Since the
T IT
plane of section cuts $2 transversely, it misses the
primary centriole which lies next to it. However, a
--/o 02-
parallel section of the same cell (Fig. 24) transects
the primary centriole P2 whose tubules indeed, are,
directed towards S 1 and P1.
In metaphase and anaphase, as in prophase,
spindle tubules are not confluent with the tubules
which comprise the giant centrioles. In fact, very
°°OOooo °°Ooo ooOOOOooo few of the spindle tubules extend beyond the nu-
o o %% o( o ° ~ " " ~ % )q-co '
o o o oo o _/o clear membrane into the cytoplasm (Fig. 26).
°°°OOoooooooo% O°0 o Oo, o io o Ribosomes are abundant inside the nuclear mem-
Oo0ooo~ brane, but mitochondria and other cytoplasmic
o I II-e ° e* I I-/o
bodies are excluded (Fig. 27). Here, as in prophase,
the tubules of the two primary centrioles are par-
ooo oo°° °°OOoo Ooo oo°° °°°Ooo °
g~o o_ ~o ° ,2 oo o o allel to the long axis of the spindle; the tubules of
oo o_o the adjacent secondary centrioles are perpendicu-
o o% oO oo / lar to the spindle axis (Figs. 26 and 27). In telo- Do
b f ~). °°°OOOOO°°° wn
phase cells in which the cleavage furrow has lo
a
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started to form, this orientation of centrioles is lost. e
I _/o Ii Ii-e° d fro
C g* Centriole Number m http
/°-m A dividing cell clearly possesses four centrioles, ://ru
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two at each pole of the spindle (Fig 27.). Each re
/°-I I eql II daughter cell then receives two centrioles which ss.org
must then give rise to two more centrioles before /jcb
d )devresbh* OreveN(* ttwhoe panierxst of division. centrioles In are spermatogoniaslo metimes found interphase in close /article-p
proximity to each other (Figs. 28 and 29), and at df/3
ERUGIF 19 I and II are three-dimensional diagram- other times at some distance apart (Fig. 17). Al- 3/1
/7
marie representations of the two possible eonfigurations though a cell frequently is found to contain four 3/1
of one primary 1( °) and one secondary (2 °) eentriole centrioles, we have never observed more than four 06
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when one is adjacent and perpendieular to the other. centrioles in one cell. In six of the eight cases where 20
8
The two-dimensional profiles diagrammed in a, ,b ,e and we observed four centrioles in close proximity, all /73
d through are obtained eentrioles by disposed representative as in configuration planes of Is.e etion The fmoautre rial, centrioles as do prilmaacrkeyd a centriolesc.o ncentric In bandt he other of dense two .pdf by g
ibmayge s various diagrammed planes of in section ,e f, ,g through and h would centrioles be obtained in con- instances, and in all cases where the two pairs were uest o
figuration II, but none of these images are ever ob- some distance apart (Fig. 17), each pair had the us- n 2
4
tained. We, therefore, conclude that the actual rela- ual appearance of one primary and one secondary Ja
tionship of secondary to primary eentrioles is always centriole. When in close proximity to one another, nua
as diagrammed in configuration I. When sectioned, a the four centrioles are found in one of two specific ry 2
0
secondary eentriole without any primary partner could spatial relationships. In the first array (Fig. 28), 23
give an image such as g, and a lone primary centriole the four centrioles are in a row and the tubules of
could give an image such as f, and these are never ob- the two yradnoces centrioles are aligned with each
tained; therefore we believe that spermatogonial cen-
other. Although the secondary centriole may not
trioles of second iustar Seiara larvae always occur in
contain the concentric dense band, it can be
pairs.
identified by its relationship to the adjacent cen-
triole (see Fig. 12). In the other array (Fig. 29), the
centriole are orientated parallel to the future two secondary centrioles lie side by side and the
spindle axis while the secondary centriole is at tubules of the two primary centrioles aligned. When
right angles to its partner. This is illustrated by the four centrioles occur in close proximity, a Golgi
Fig. 25 in which the tubules of the primary cen- area of characteristic morphology often is asso-
triole P1 point towards the secondary centriole ciated with them (Figs. 28 and 29). This type of
DIVAD M. PHILLIPS Giant Centrlole Formation in Selara 79
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FIGURE 31 Longitudinal section through a primary eentriole. Fibrous material (m) is often associated -p
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with giant centrioles. Second instar spermatogonium. X 44,000. f/3
3
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FIGURE 41 Longitudinal section of a primary eentriole. Mitochondria (rot), ribosomes, and micro- /73
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tubules often occur in giant centrioles. Second instar spermatogonium. <) 53,000. 06
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2
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8
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Golgi zone rarely is observed except in association cores indicative of meiotic prophase (9, 24). Each 3
.p
with four centrioles in close proximity to each primary oocyte is connected to a polytene nurse df b
other. cell, also of germ origin (7), by a cytoplasmic y g
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Centrioles of Second Instar Larval Oogonia bridge (Fig. 30). We have not observed many st o
centrioles in these large cells; however, all the n 2
4
We have examined second instar larval oogonia centrioles which have been seen in cross-section Ja
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are either smaller giant centrioles or 9-membered u
lceses ntrioles extensively appear than to bes pertmhea togonia; same in bohtohwe ver, types the of centrioles. They occur both in polytene nurse cells ary 20
germ ceils. As in spermatogonia most of the cen- (Fig. 30), where they sometimes are misshapen 23
and thus probably degenerating (Fig. 31), and in
trioles are smaller giant centrioles. In a few cases
primary oocytes, where in two cases we have ob-
we have observed four giant centrioles in the same
served four giant centrioles in the same oocyte
cell. They always occur in the relationships de-
(Fig. 32).
scribed in the preceding paragraph. On one oc-
casion we observed a 9-membered centriole in an
oogonium. DISCUSSION
Centrioles of Fourth Instar Larval Oocytes Centriole Formation
By the late fourth instar (stage c, d, and e) (12) When found in pairs, centrioles of a wide range
all the oocyte nuclei contain the chromosomal of organisms are arranged so that the long axis of
80 TrIE LANRUOJ FO CELL YGOLOIB • EMULOV 38, 1967
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SERUGIF 51 DNA 61 When viewed in transverse section the tubules of one side of secondary eentrioles p
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are seen in cross-section while the tubules of the opposite side are sectioned obliquely. In Fig. ,51 arrows ss.o
indicate doublets. Second instar spermatogonia. Fig. ,51 X 10~,000; Fig. ,61 <) lll,000. rg
/jcb
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one is perpendicular to the long axis of the other (There are approximately three spermatogonial rticle
(10, 11, 20, 30, 34). Since centrioles are known to cell generations between second and fourth instar.) -p
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form at right angles to pre-existing centrioles Since we frequently observe primary centrioles f/3
3
(4, 8, 13, 25), 1 the perpendicular position of two adjacent and at right angles to larger secondary /1/7
3
mature centrioles may be a consequence of their centrioles (Figs. 18-23), we conclude that primary /1
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disposition during replication, although it also may centrioles represent the original, or mother, cen- 82
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have some other significance. In Sciara, the mor- trioles and that secondary centrioles are newly 8/7
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phological dissimilarity of the members of each formed daughter centrioles. .p
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pair enables us to demonstrate that pairs of cen- The correspondence of primary centrioles to f b
trioles are disposed in a specific configuration: if mothers and secondaries to daughters is borne out y gu
e
the two centrioles are pictured as a T formed of further by the finding that the giant centrioles st o
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two cylinders, the primary centriole is always the which lie next to transversely sectioned 9-mem- 24
top of the T and the secondary centriole intersects bered centrioles are secondary centrioles, i.e., they Ja
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ittr anseicn ts the midbdolteh. centrioles Therefore, of a in pair, any one section can diswthiinc- h dSiisnpclea y no the 9-membered band of dense centrioles material, are o(bFisges.r ved 1-4). in ary 20
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guish between the primary and secondary cen- fourth instar spermatogonia, spermatocytes, sper- 3
trioles by their spatial relationships. (See legend of matids, or sperm (26, 27), it is logical to assume
Fig. 21 for more detailed explanation.) that the giant centriole is the newly formed
Since the giant centrioles of spermatogonla of member of the pair.
second instar larvae are comprised of fewer tubules One wonders whether mother and daughter
than those of spermatogonia of fourth instar larvae, centrioles of other organisms are also orientated in
it is logical to assume that centrioles of increasing a specific way, but since all mature 9-membered
size are formed at each successive interphase. centrioles look alike, it is difficult to demonstrate
this relationship. When centrioles are forming,
1 Sorokin, S. P. Reconstructions of ciliogenesis in however, they are shorter and the tubules are
mammalian cells. Data in preparation. less clearly defined than those of mature centrioles.
DIVAD M. PHILLIPS Giant Centriole Formation in Sciara 81
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FIGURE 17 Two pairs of centrioles in an inteqahase spermatogonium. All four centrioles have been
sectioned longitudinally. Note that the primary and secondary centrioles bear a specific spatial relation-
ship to each other (configuration d of Fig. 1~). m, fibrous material. X 84,000.
82