Download Cilia in Cardiac Differentiation

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J. VLTRASTRUCTURERESEARCH29, 470-484 (1699)
Cilia in Cardiac Differentiation
JOHN E. RASH1, JERRY W. SHAY2, AND JOHN J. BIESELE
D~,,~artment of Zoology, The University of Texas at Austin, Austin, Texas 78712
Received April 14, 1969, and in revised form May 22, 1969
In a study of chick heart development, approximately 700 cilia were observed in
random and serial sections from differentiating myoblasts, myocytes, fibroblasts
and fibrocytes following glutaraldehyde-osmium fixation. Additional cilia
were observed in hearts of embryonic and adult lizards, mice, and rabbits. The
diplosomal "9 + 0" cilia, often observed completely enclosed in the cytoplasm,
were revealed to be present in most nonmitotic cells but were never observed in
mitotic cells (P=0.001). Because the number of cells increases greatly during
early cardiogenesis and because most of the cells were revealed to possess a single
abbreviated cilium each, the deposition of cilia presumably occurs. Therefore,
we suggest that the blockage of mitosis usually associated with the initiation
of cytodifferentiation can be correlated with the formation of cilia and may be
mediated by the transformation of the mitotic centrioles into ciliary basal bodies.
The presence of cilia in a tissue has long been correlated with epithelial origin of the
cells (3, 9). Recent reports, however, have demonstrated the presence of cilia in
virtually every organ and tissue, with the possible exception of blood. Increasingly
apparent is the correlation that cilia are present at some time in virtually all embryonic
tissues, regardless of derivation. Following the report of a single cilium in heavily
irradiated embryonic rat heart (32), a systematic investigation was completed concerning the possible prevalence and distribution of cilia during the normal course of
development in the chick (stages 3 to 15) and in other representative higher vertebrates. Because of the very limited nature of the previous reports concerning cardiac
cilia (19, 23, 24), an attempt was made to establish the ultrastructure, the mode of
development, and the funticon of these nonmotile cilia.
MATERIALS AND METHODS
Embryonic and adult cardiac and presumptive cardiac tissue was excised and placed in
2.5 % glutaraldehyde buffered with 0.1 M S6renson's buffer (27). After 1 hour the tissue was
1 Present address: Finney-Howell Cancer Research Laboratory, Johns Hopkins Hospitals, Baltimore, Maryland 21205.
2 Present address: Department of Physiology and Cell Biology, The University of Kansas, Lawrence, Kansas, 66044
CILIA IN CARDIAC DIFFERENTIATION
471
rinsed twice in buffer, postfixed in buffered 1% OsO~, rinsed twice in distilled water, and
placed in 0.5 % uranyl acetate for 16-20 hours. After rinsing in distilled water, the tissue
was dehydrated in successive ethanol concentrations, transferred through two changes of
acetone, and placed in successive concentrations of plastic diluted with acetone. The final
plastic mixture (10 % Epon, 20 % Araldite, 70 % DDSA with one drop of DMP-30 accelerator per milliliter of plastic) was evacuated, stored overnight at 30°C, and then polymerized
at 80°C for 8-12 hours. Random and serial sections were cut with glass and diamond knives
in a Sorvall Mt-1 ultramicrotome, spread with toluene vapors, picked up on uncoated
300-mesh or 75 x 300-mesh copper grids, poststained for 1-5 minutes with Reynolds' lead
citrate (25), and examined on a Siemens Elmiskop I operated at 60 kV.
OBSERVATIONS
In an examination of presumptive and differentiating heart tissue from embryonic
chicks, over 700 single cilia were observed. Though mitosis is extremely rapid throughout early heart formation, cilia were limited to nonmitotic cells ( P = 0.998). In contrast, dividing cells were never observed to possess cilia.
The earliest detected cilia were observed in isolated cells of the epiblast and hypoblast of the preprimitive streak stage (Figs. 1 and 2). Although this is a stage with
little detectable tissue or cellular differentiation, the observed cilia are presented
because they closely resemble those found in later stages and because they were from
the areas of future heart development (7).
As the mesodermal layer is formed during the primitive streak stage, numerous
cells in the presumptive heart regions were observed to possess single cilia. In stages
with progressively delimited heart rudiments, cilia were more numerous, several
often being included in a single field of view (Fig. 3). The four obliquely sectioned
cilia indicate the abundance of cilia in the presumptive cardiac mesoderm at the
6-somite stage. Two of the cilia do not project into the intracellular space, and a
third is sectioned through the basal body and associated cytoplasmic vesicle or sheath.
The presumptive myocardial rudiments (Fig. 4), which develop from the lateral
plate mesoderm, possess cells with internal and external cilia, though cilia (arrow)
projecting into the intraembryonic coelom appear to be more numerous. At the
9-somite stage, approximately 1 hour before the initiation of contraction, internal
cilia are more numerous. High magnification reveals a 2-~t cilium (Fig. 5), completely
enclosed in the cytoplasm. The primary basal body, with its enclosed 700 • vesicle,
is surrounded by Golgi lamellae and radiating microtubules, closely resembling the
centrosomal regions of nonciliated cells. The second basal body is not included in the
plane of section, but is indicated by the dense material aligned with the primary basal
body. The enclosed vesicles of cilia (Fig. 6 a-c) vary from 300 to 1450 A in diameter
and were occasionally observed in the ciliary shaft (Fig. 6c).
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RASH, SHAY, AND BIESELE
Cilia were not restricted to undifferentiated cells, for very often a cilium was
observed to project from definitive myocytes (Fig. 7), the basal bodies often closely
associated with the Z bands. Serial sections from myocardial cells of a 12-somite
chick ventricle (Figs. 8-11) reveal selected regions of a cell with a very abbreviated
cilium completely enclosed within the cytoplasm. The insets (Figs. 8b, 9b, 10b, and
11 b) are higher magnifications of the basal body and ciliary shaft. Cross sections of
several myofibrils (M) clearly identify this as developing cardiac myocyte. The myofibril cross sections and the mitochondria, which may be used as markers to indicate
approximate tissue intervals, confirm the calculated ciliary length of approximately
0.5/~. The primary basal body with its denser "plate" (Fig. 8), indicates that the section
is from the region of the junction of the shaft and basal body. Additional sections
reveal the enlarged base of the ciliary shaft as it enters the cytoplasmic sheath (Fig. 9),
the thin middle of the shaft (Fig. 10), and the ciliary tip (Fig. 11). Only 3 microtubule doublets remain at the tip, one pair clearly connected to the ciliary membrane
by a dense "stalk" (Fig. 11 b, arrow). These linear elements appear to be continuous
from base to tip, and may connect with the dense satellite material (Fig. 8b). The
brevity of the cilium may indicate an early stage in cilium development.
Cross sections of numerous cilia reveal varying configurations, most commonly
" 9 + 0 " or " 8 + 0 " (Fig. 12). The peripheral doublets (8 or 9?) are concentrically
arranged and lack the "arms" which Gibbons (10) ascribed to the A subfibers of
motile cilia. Dense material (arrows) connects the microtubule pairs with the ciliary
membrane and apparently exists as a continuous linear element from basal satellites
to the ciliary tip. Portions of this linear element may be interrupted by intervening
material, which is often encountered near the base of the ciliary shaft (Figs. 8b, 13,
14, and 15). Several images of microtubule circumflexion (i.e., "hairpin") at the ciliary
tip (Fig. 16) resembled images presented by Satir (29) of central microtubule circumflexion in Eliptio. The apparent absence of central tubules in cardiac cilia (Figs.
8-12) and the extended region of 300 A separation of the circumflexed tubules
(Fig. 16), however, indicates that the continuous tubules comprise subfibers of adjacent doublets, though the subfibers involved have not been identified.
Recognition of immature or developing cilia proved to be considerably more
difficult than anticipated. Often images were observed (Fig. 17a) which closely
resembled the various stages of ciliary development advanced by Sorokin (31-32),
Sorokin and Adelstein (33), and Dingle and Fulton (8). A nonadjacent serial section
of the same "immature cilium" (Fig. 17b), however, clearly revealed a complete shaft
FIG. 1. Two cilia (arrows) in preprimitive streak epiblast. Note oblique section of annulate lamellae
(AL ref. 16) in continuity with rough endoplasmicreticulum, x 19,000.
FIG. 2. Cilium on ventral surface of preprimitive streak hypoblast, x 11,000.
FIG. 3. Four cilia in precardiac mesenchymeof 6-somite chick, x 11,000.
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lying in a deep "well" in the cytoplasm. Although the second basal body is no longer
included in the plane of section, the mitochondria, yolk platelets, vacuoles, endoplasmic reticulum, and plasma membranes clearly establish the serial nature of the
sections.
The epicardium, which forms from the parietal layer of lateral plate mesoderm,
possesses cilia at all stages. Two truncated cilia (Fig. 19) were observed in the developing fibrocytes of the recently formed epicardium of the 15-somite chick. No
collagen fibers were observed in this region of the epicardium, although isolated
fibers were observed as early as the 12-somitage stage. The fibrocyte cilia, although
formed early in development, were also observed in the 12-day mouse embryo
(Fig. 19), hatchling chick, adult chickens, adult rabbit (Fig. 20), and an adult lizard
(Fig. 21). The cilia of fibroblasts and fibrocytes were observed to be very similar to
the cilia of myoblasts and myocytes, demonstrating the diplosomal, "9 + 0" configuration. A brief survey of the distribution of the 200 fibrocyte cilia observed, revealed
them to be restricted to nonmitotic cells (probability of chance occurrence=0.08)
in the hearts of embryonic and adult chickens, lizards, mice, and rabbits. In addition
to the cilia of cardiac myocytes and fibrocytes, Dr. S. R. S. Rangan of Johns Hopkins
Hospital confirmed the presence of diplosomal cilia in differentiating myocytes in
cultured chick striated muscle (Fig. 22).
DISCUSSION
Our investigations of differentiating chick heart muscle have revealed the presence
of numerous single cilia in muscle and connective tissue. Mitotic figures encountered
in the tissue were often observed to possess centrioles but never to possess cilia. In
obvious contrast were the nonmitotic cells, which often exhibited a single cilium. The
two basal bodies, "diplosome, (1, 2, 6), with associated microtubules and Golgi
lamellae, closely resemble the two centrioles in the centrosomes. Additional free
centrioles, however, were never observed in the ciliated cells. Utilizing serial sections,
six apparent "centrioles" in nonmitotic cells subsequently proved to be basal bodies
of cilia, indicating a high cilia-to-centriole ratio. Further, of 700 ciliated myocytes
and fibrocytes, none was observed to possess additional centrioles or more than one
cilium. Assuming a mitotic rate of 1.5 % in the early chick heart (7) and random
Fro. 4. Mesocardial rudiment of 6-somite chick heart. Note the double layer of cells, one cell with a
truncated cilium (arrow). x 5000.
FIG. 5. Semioblique longitudinal section of an enclosed cilium in 9-somite chick ventricle. Note the
700 A vesicle in the first basal body. G, Golgi apparatus, x 38,000.
Fro. 6. Vesicles (300 A to 1450 •) in base and shaft of cilia from myoblasts and fibroblasts. (a)
x 43,000, (b) x 52,000, and (c) x 34,000.
FIG. 7. Cilium near Z band in 10-somitechick myocyte, x 12,000.
CILIA IN CARDIAC DIFFERENTIATION
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RASH~ SHAY, AND BIESELE
distribution of cilia in mitotic and nonmitotic cells, 10 of the 700 cilia would be
expected in dividing cells. However, none was observed. According to the chi-square
test, chance alone cannot account for the observed restriction of cilia to nonmitotic
cells (P=0.002). Additional data from 11 of 12 consecutive serial sections of the
12-somite chick ventricle permitted additional conclusions. In the volume-equivalent
of 7 cells, no free centrioles were observed, though 7 were to be expected. However, 8
cilia were observed, suggesting that chance alone cannot account for the absence of
centrioles (P--0.01). We therefore suggest that virtually all the nonmitotic cells at
this stage possess a single cilium and lack additional free centrioles.
An analysis of many micrographs suggests that the cilia in cardiac myocytes and
fibrocytes possess the diplosomal basal complement and have a 9 + 0 cross sectional
configuration. However, the possibility of a normal 8 + 1 configuration is not eliminated, for the microtubule doublets are often disrupted on one side. Because only one
8 + 1 cilium was observed, we shall tentatively attribute it to atypical formation or to
the central displacement of a peripheral doublet, similar to the displacement demonstrated by Allen (1) in retinal neurons.
The peripheral doublets, arranged perpendicular to any radius, lack the " a r m s "
attributed by Gibbons (10) to motile cilia. Dense material connecting the doublets
to the ciliary membrane appears in serial sections and may form a linear element
continuous from the basal satellites to the ciliary tip. Near the base of some very
short cilia, a bulb-like swelling is observed, apparently formed by additional dense
material interrupting the dense linear "stalk". The possible role of this material in a
mechanism of incorporation of new subunits into forming microtubules should be
considered, for no definitive information is available on ciliary microtubule growth
[but see Ringo (26)]. No function as yet can be surmised for the vesicle often observed
within the basal body and shaft of the cilia, but apparently it is characteristic of the
cilia from myoblasts and myocytes of cardiac and striated muscle (19) as well as other
diplosomal cilia (8).
The inherent difficulties of inferring three-dimensional structure and developmental sequences from two-dimensional, static micrographs have prompted us to
utilize serial sections in most designations of three-dimensional structure. Indeed,
numerous figures were encountered that closely resembled the stages of cilium
formation proposed by Sorokin (31, 32), Sorokin and Adelstein (33), and Dingle and
Fulton (8). Serial sections, however, revealed that the supposed sections of immature
stages were, in actuality, truncations of complete cilia. In addition, the use of "median
FIGS. 8-11. Serial sections of a very short cilium in a ventricular myocyte (M) of a 12-somite chick.
Linear elements (arrows) apparently are continuous from tip of cilium to "satellites" near basal body.
(a) x 12,000 and (b) x 70,000.
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RASH, SHAY, AND BIESELE
longitudinal sections", as claimed by Dingle and Fulton (8), may contribute to the
confusion, especially since "recognition" of median sections has been reported far
more often than is predicted (20). The designation as "median longitudinal section"
due solely to the presence of apparent central tubules is considered particularly
unreliable, for figures containing three sets of peripheral doublets closely resemble
figures with two sets of peripheral doublets plus central tubules. Further, longitudinal
serial sections of 9 + 0 cardiac cilia frequently demonstrated such "median" images.
Therefore, we suggest that previous reports designating developmental sequence and
three-dimensional structure of apparently forming cilia must be considered unproved.
However, it should be emphasized that the serial sections presented in this report do
not contradict the models of ciliogenesis of Sorokin (31, 32), Sorokin and Adelstein
(33), or Dingle and Fulton (8), but appear to favor the Sorokin model (32).
The significance of the recently discovered cardiac cilia may extend far beyond
their mere presence in a new tissue. Cilia, once attributed solely to epithelial tissues
(2, 9), have now been observed in virtually all vertebrate organs and tissues, with the
major exception of blood (30). It appears unlikely that motility or sensory function
can be attributed to the single cilia enclosed in embryonic bone, myocytes, or fibrocytes. It seems equally unlikely that they are of "accidental" circumstances (2, 39).
Further, the discovery of a few isolated cilia in the preprimitive streak stages, both in
epiblast and hypoblast, cannot be explained by any of the presently available hypotheses, except possibly by invoking a "leaky" control mechanism and a normal
propensity for centriole transformation. We have demonstrated, however, a correlation between the possession of cilia and the interphase stage of the mitotic cycle.
Therefore, it seems relevant to note that the abrupt transformation from mitotic
replicative tissue to nonmitotic structuring tissue is correlated with the disappearance
of centrioles and the formation of cilia. A function related to the mitotic cycle, tissue
determination, and cellular differentiation is indicated.
FIG. 12. (a) Enclosed cilium of 15-somite chick ventricle, x 25,000. (b) Higher magnification, revealing
microtubule pairs (8 or 9?) connected to sheath by a linear element (arrows). x 200,000.
Fro. 13. Semi-oblique longitudinal section of diplosomal cilium with enlarged area near base (see
Fig. 12). Note continuity of ciliary microtubules with first basal body and the oblique sectioning of
additional peripheral doublets (arrows). × 50,000.
FIG. 14. Section through bulbous base of cilium, linear elements obscured by amorphous material.
Note disruption of 9+0 pattern, x 70,000.
FIG. 15. Cross section of atypical cilium, possibly a developing "ciliary bud". The microtubule pairs
apparently are characteristic of these developing cilia (see Fig. 12b), and should be contrasted with
microtubule singlets at the tips of certain mature cilia (29). x 85,000.
FIG. 16. Microtubules continuous in "hairpin" arrangement, implying opposite or no polarity in
opposed microtubules, x 80,000.
F~G. 17. Serial sections of a "mature" cilium. The diplosome (a) is associated with an apparent vesicle.
Only one basal body is observed in the nonconsecutive serial section (b). [According to Byers (5),
hypothermia of this sample resulted in the ribosome crystallization.] x 11,000.
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RASH, SHAY, AND BIESELE
In 1898 Henneguy (13) and von Lenhossek (17) independently observed that cilia
and flagella are formed upon transformation of centrioles into basal bodies, with
subsequent growth of the shaft. They also suggested that this transformation is
(one of) the control mechanism(s) for the regulation of mitosis and differentiation.
Many have subsequently supported this interpretation, but Stubblefield and Brinkley
(35) were able to induce the proposed centriole-basal body transformation in fibrocytes by blocking mitosis with colcemid. We have interpreted this as suggesting that
the transformation may be a result and not necessarily a cause of the blockage of
mitosis.
The role of mitosis in regulating tissue differentiation has been considered by
several investigators. Przybylski and Blumberg (22) have suggested that as striated
muscle differentiates, myoblasts are produced by a mitotic "stem-line," but that the
differentiating myocytes are nonmitotic. Holtzer (14), Bischoff and Holtzer (4), and
Stockdale and Holtzer (34) have suggested that mitosis must cease before fusion of
myoblasts can occur. Bischoff and Holtzer (4) thus consider myoblast fusion as the
initial and regulating event in the differentiation of striated muscle. Because cardiac
myoblasts never fuse, some mechanism other than cell fusion (4) must be the regulator
of differentiation in this tissue. If this is true, what mechanism blocks mitosis to allow
tissue differentiation, and does this control mechanism occur in other cell types such
as chondrocytes, fibrocytes, or cardiac myocytes?
For several reasons, the heart appears to be an excellent tissue for examining the
hypothesis of Bischoff and Holtzer (4), especially in view of the earlier hypothesis of
Henneguy (13) and von Lenhossek (17). The heart must become functional very early
in development, at a time when it possesses less than 1/10,000 of its adult mass. If all
mitosis were to cease before differentiation could occur, and if the heart were to start
beating very early in development, it would be relegated to a very small size. Conversely, if all cells were mitotic until the final number of heart cells were produced,
and if mitotic cells were undifferentiated, heart activity could not occur until late in
development. Both of these possibilities would be fatal to the embryo. It is not
surprising, therefore, to find that there appear to be two types of cells in the heart:
(a) nondifferentiated, dividing cells which may comprise the tissue-producing "clone"
FIG. 18. Two cilia (arrows) in fibroblasts of the developing pericardium (15-somite). x 65,000.
FIo. 19. Cilium in developing fibrocyte of 12-day mouse ventricle. Collagen fibers (C) and muscle
subunits are evident, x 17,000.
Fie. 20. Fibrocyte cilium of an adult rabbit heart. The basal bodies are oriented perpendicularly.
x 19,000.
F~G. 21. Cardiac fibrocyte cilium of an adult anole. Collagen fibers (arrows) between cells indicates
cell to be fibrocyte, x 19,000.
FIG. 22. Partially enclosed cilium in nonmitotic myocyte (myotube stage) from tissue culture of chick
striated muscle. By courtesy of Dr. S. R. S. Rangan. × 28,000.
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RASH, SHAY, AND BIESELE
of Wainrack and Sotelo (37), and (b) differentiating, nonmitotic cells produced
throughout development to meet the ever-increasing demands on the heart. It should
be noted, however, that Manasek (18) has observed the division of differentiating
and "differentiated" cardiac muscle cells. It can be assumed, therefore, that mitosis
and differentiation are not mutually exclusive activities and that some mechanism
other than myofibril deposition or cell fusion must be the primary mediator of
differentiation. In myocytes, mitotic activity and the possession of cilia apparently
are mutually exclusive activities. The restriction of cilia to nonmitotic differentiating
cells may then constitute evidence for the hypothesized control mechanism and appears to support the hypotheses of Holtzer (14), Henneguy (13), and von Lenhossek
(17). It thus appears that mitotic inhibition, which is under nuclear control, is somehow mediated by or results in the observed centriole-basal body transformation as an
alternate pathway for centriole activity. The mitotic cycle may thereby be delayed,
awaiting replacement of the mitotic centriole, either by ciliary resorption or basal
body replication. The undiscounted possibility of a resorption of cilia would provide
a semireversible control mechanism, which would be able to respond to excessive
demands on the heart or to possible tissue damage. Wilson and McWorter (38)
have suggested that if "stripping-off" the cilia from epithelia results in the burst of
mitotic activity noted by Pinkus (21), many of the "liberated" basal bodies would
reenter the mitotic cycle, triggering cell division. (Wilson and McWorter (38) suggest
that this mechanism may be important in wound repair and surface regeneration.)
Although Ringo (26) has demonstrated the retraction of cilia (flagella) and the transformation of the basal bodies into mitotic centrioles in Chlamydomonas, no one has
demonstrated a similar reversible resorption of cilia in vertebrates. Although it may
be assumed that the lack of reports of cilia in the adult vertebrate heart may indicate
a possible resorption of cilia and possible return to centriole-mediated activities,
other possibilities must be considered.
Because we have observed numerous single cilia in adult heart fibrocytes and have
encountered no free centrioles in adult cardiac myocytes, we support an additional
(or alternative) hypothesis. If cilia were to exist in large numbers of adult heart
myocytes, one would expect to see them only very rarely, for the volume of the adult
cardiac muscle cell is at least 100 to 1000 times the volume of differentiating cardiac
myoblasts, and the size of the cilium is unchanged. In addition, the cilia would be
totally enveloped in the tissue and therefore camouflaged by the parallel mitochondria
and myofibrillar substructures. Thus, the lack of reports of cilia in adult heart
myocytes is hardly surprising. However, since cilia are readily observed in the much
smaller fibrocytes of adult hearts, it seems reasonable to suggest the possibility of
finding cilia in adult cardiac myocytes, even if it requires extended searches in innumerable serial sections.
CILIA IN CARDIACDIFFERENTIATION
483
In view of the relatively minute knowledge concerning the possible role of 9 + 0
and 8 + 1 diplosomal cilia in the control or regulation of mitosis and differentiation,
it would appear to be desirable to determine mitotic rates and centriole-cilia ratios in
various differentiating and adult tissues. Embryonic tissue appears to be the tissue of
choice in most instances, primarily because cell determination and tissue differentiation are occurring, the tissue volumes are very small, and the tissue is readily available.
Additional differentiation of certain tissues occurs late in development in several
tissues (bone epiphyses, gonads, etc.) and information from these sources would be
particularly informative.
Recently, Trelstad et al. (36) have reported that "each cell of dermatome, sclerotome, and myotome apparently possess one cilium", though no attempt was made to
correlate this with mitotic activity or regulation of differentiation. With this in mind,
we contemplate a comparative investigation of differentiating myocytes and fibrocytes
of cardiac and striated muscle in reptiles, birds, and mammals.
Most of the research was accomplished at the University of Texas at Austin with partial
support by U.S. Public Health Service research grant number GM-15875-04 from the
National Institute of General Medical Sciences, training grant number 5T01 GM-00337
from the National Institute of General Medical Sciences, and Research Career Award
number 5-K6-CA-18366 from the National Cancer Institute.
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