/ . Embryo/, exp. Morph. Vol. 48, pp. 269-286, 1978
Printed in Great Britain © Company of Biologists Limited 1978
269
Patterns of cellular proliferation during
thyroid organo genesis
By MARYBETH S. SMUTS, 1 S. ROBERT HILFER 2 AND
ROBERT L. SEARLS 2
From the Department of Biology, Temple University, Philadelphia
SUMMARY
The changes in rate and location of cellular proliferation were analyzed to determine if
localized areas of cell division were influencing shape changes in the chick thyroid. Pulse
labeling with tritiated thymidine indicates that the gland's labeling index declines throughout
its development. Initially, the thyroid placode has a lower labeling index than the neighboring
pharyngeal epithelium. An evaluation of the positions of pulse-labeled cells reveals that the
evaginating thyroid grows by annexing cells from the pharyngeal epithelium. The older
evaginated regions of the gland exhibit the lowest labeling indices. The newly acquired regions
still maintain higher labeling indices.
INTRODUCTION
The mechanism commonly invoked to explain invagination and evagination of
epithelial organs is the purse-string contraction model (Baker & Schroeder,
1967). Yet this model alone cannot explain the invagination process for all
epithelial organs. Contrary to the expected results of a purse-string contraction
model, Hilfer (1973) observed that the greatest concentrations of apical microfilaments were not in the areas of the most pronounced bending in the evaginating
thyroid. Wrenn & Wessells (1970), in analyzing oviduct tubular gland development, could not elicit duct elongation when DNA synthesis was partially
inhibited. Spooner & Wessells (1972) obtained similar results; cytochalasintreated salivary glands recovered only narrow clefts when colchicine was present
in the medium. Observations of this sort have led several workers to evaluate
the role of cell division within developing epithelial organs. Pictet, Clark,
Williams & Rutter (1972) observed that, in the pancreas, cells are apically
connected while growth is generalized. They suggested that lobulation is caused
by lateral pressure generated by dividing, adherent cells. Pourtois (1972)
suggested that nasal placode invagination may be explained by increased cellular
adhesions in the center and cell growth at the periphery of the placode. Similarly,
1
Author's address: Biology Department, Wheaton College, Norton, Massachusetts 02766,
U.S.A.
2
Author's address: Department of Biology, Temple University, Philadelphia, Pennsylvania,
19122, U.S.A.
l8
EMB 48
270
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
Zwaan & Hendrix (1973) formulated a model to explain lens invagination based
upon population pressure generated by continuing cell division within a restricted
area.
In the present investigation, the embryonic thyroid was used to determine if
localized areas of cell divisions were influencing the gland's morphogenesis.
Three questions were considered: (1) whether new cells were added to the
primordium and, if so, at what developmental stage; (2) whether the distribution
of mitotic activity was random or localized; and, (3) whether the addition of
new cells could affect the shape of the organ as it developed.
MATERIALS AND METHODS
Rhode Island Red chicken embryos were used in order to allow correlation
with previous cytological and biochemical work (Shain, Hilfer & Fonte, 1972).
The eggs were incubated in a Jamesway incubator at 37 °C. A window was
prepared in every egg (Zwilling, 1959) and all labeling was performed on
windowed eggs.
Areas and rates of cell division were investigated by counting the number of
cells that incorporated tritiated thymidine. Ten microcuries of tritiated thymidine
(New England Nuclear Corp., specific activity 20 Ci/mole) in 0-1 ml of Hanks'
solution were injected into the yolk through a hole drilled at the blunt end of
the egg. Since stage 10-14 embryos were weakly labeled by this method, the
thymidine was dripped onto these embryos through the window. The window
was sealed and the eggs reincubated for either 1 h or, for continuous labeling,
up to 24 h. The embryos were staged (Hamburger & Hamilton, 1951) just
before injection and restaged before removal of the thyroid.
After incubation with tritiated thymidine the embryos were removed and
dissected in medium 199 (GIBCO). The thyroids were fixed in 2-0% glutaraldehyde buffered with Coleman's phosphate buffer (Coleman, Coleman &
Hartline, 1969) for 20 min, washed in buffer, dehydrated, embedded in 60 °C
paraffin and sectioned at 5 jam. The sections were dipped in Kodak NTB-3
emulsion, stored for 4 weeks at 4 °C, developed in D-19 (Kodak), and stained
with hematein (Searls, 1967). A Wild microscope equipped with a drawing tube
was used to score sections for labeled and unlabeled nuclei.
A DNA analysis was performed on mechanically cleaned glands. It was
difficult to dissect out cleanly early thyroid glands; therefore, analyses were
made only on glands of stage 14 and older. Five glands were pooled for DNA
analyses for each stage, 14 through 29. Samples at later stages varied from one
pair of glands to five pairs. The Santoianni & Ayala (1965) fluorometric method
was used since it is sensitive enough to measure the nanogram amounts of DNA
that the sample contained. Calf thymus DNA was used as the standard.
Cell proliferation in the thyroid
271
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Hours
52 go 108
Stage 1114 20 24 26
i
240 264 288
1
1
336 360
36 37 38
40 41
Time of incubation with label (li)
1
1
1
408
450 480
43
44
45
Fig. 1. The pulse-labeling indices (percentage of labeled cells in the thyroid after
1 h labeling with tritiated thymidine) plotted v. stage and time of embryonic
development. Each point represents the mean labeling index of at least three thyroids
and the range is the standard deviation. The line, determined by linear regression
analysis, indicates that the labeling indices decline with the age of development.
RESULTS
(A) Pulse-labeling
From stage 11 to stage 23 the thyroid boundaries were determined by the
position of the primordiiim on the floor of the pharynx and by the closely
adhering cells that comprise the thyroid regions (Shain et al. 1972). During
stages 21-23, in which the thyroid pinches off from the floor of the pharynx,
the stalk was counted as part of the thyroid. Every third section through the
thyroid was drawn to show the location of labeled and unlabeled nuclei and of
mitotic figures. A nucleus was considered labeled if it had more than five silver
grains over it. From stage 11 to stage 16 a minimum of 50 nuclei per section
were counted and at least three sections per thyroid were scored. After stage 17,
at least 100 nuclei per section were counted and an average of 11 sections per
thyroid were scored. From stage 26 to stage 45 every tenth section was drawn.
An average of 400 nuclei were scored per section.
In each gland, the percentage of cells incorporating the tritiated thymidine
label was determined. At least three thyroids per stage were averaged to calculate
the labeling index. These percentages or labeling indices were then plotted
against the stages of the embryos and hours of embryonic development (Fig. 1).
Linear regression analysis indicated a constant decline in the labeling index
18-2
272
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
50
40
30
20
10
J
0
80
I
I
I
I
I
I
t
1
10
12
0
14
16
18
20
0
12
0
14
16
0
18
20
14
16
0
18
20
(b)
70
~
60
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o
i 40
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W
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J
L
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9
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(c)
40
30
20
10
0
0
1
2
3
4
0
5
6
7
8
9 10
12
0
0
Time of incubation with label (h)
Fig. 2. Graphs representing the experimentally determined labeling percentages
obtained throughout a 20 h continuous labeling period. Ten microcuries of
tritiated thymidine were injected in ovo every 4 h (injected times [0]). The points represent the labeling indices obtained by counting labeled and unlabeled nuclei. The
solid lines represent the hypothetical labeling curves calculated by the Okada plot
method (Okada, 1967). (a) Initial injection at stage 16 during early thyroid evagination. (b) Initial injection at stage 2.1, late in vesicle formation. (•) represents percentages obtained after a single injection of radioisotope. (c) Initial injection at
stage 4.1, when the thyroid contains mature follicles.
Cell proliferation in the thyroid
273
Table 1. Value for the cell cycle parameters for stage-21 labeling series
Parameters
Gz
G2 + M
Ca + M + G i
Gt
5
Q
Labeled
Inflection
Okada
mitotic
points
method
method
estimates calculations estimates
(h)
(h)
(h)
—
2
4-5
2-5
—
—
1-9
3-9
2
5-6
95
2
—
—
—
4
throughout thyroid development, from 30-5% ±3-4 at stage 11, the placode
formative period, to 8-6 % ± 4-5 at stage 45, just prior to hatching.
The thyroid originates from the pharyngeal epithelium, but from stage 11 to
21 the gland is consistently distinguished from the pharynx by a lower labeling
index. During stage 11, the average labeling index of the thyroid was 30-5 % ±3-4
while the average labeling index of the pharynx was 51 % ± 3-6. During stage 23,
when the thyroid is nearly separated from the pharynx, the labeling index was
19 % ± 4-5 in the thyroid and 15 % ± 1.7 in the pharynx. After separation of the
thyroid from the floor of the pharynx, the pharyngeal epithelium was not scored.
(B) Continuous labeling
Continuous labeling experiments were performed to determine if the decline
in the labeling index of the thyroid, between stage 11 and stage 45, was due to
a decrease in the percentage of replicating cells. Several stages were chosen for
these experiments: stage 16, when the thyroid was an evagination of pseudostratified cells; stage 21, when the vesicle was almost completely closed; and
stage 41, when the gland was mature.
Carefully staged batches of embryos were injected with 10 /^Ci of tritiated
thymidine at 0 time. Every half-hour an embryo was fixed for analysis. Since
the thymidine appears to be available to the embryo for a short period of time
(Marchok & Herrmann, 1967; Zwaan & Pearce, 1971), in order to ensure that
sufficient label was available, additional injections of label were given every 4 h.
Injection times of less than 4 h decreased embryonic survival. The points in
Fig. 2a-c represent the percentages of labeled cells in thyroids injected at the
given stages.
Within 2 h, cells that had incorporated label entered mitosis; therefore, 2 h
is the minimum time of G2. This increase in labeled cells is reflected by the
steeper slopes of the curves (a) and (b) (Fig. 2). The plateau portions of the curves
indicate that the maximum percentage of cells have been labeled and correspond
to C
274
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
The time of the first labeled mitotic figures, the maximum number of cells in S,
and the locations of inflection points on graphs (a) and (b) (Fig. 2) were used as
fixed values to determine the parameters of the cell cycle. The simple graphic
method of Okada (1967) was used to calculate the times for G1} G2 and M, and S,
Table 1. Using these parameters, the percentage of cells estimated to be in each
portion of the cell cycle could be determined (Janners & Searls, 1970). These
Okada plot-estimated percentages are congruent with the experimentally derived
labeling indices for the stage 16 and 21 continuous labeling series (Fig. 2a, b).
Although the stage-41 series did not have enough collection points to determine
inflection points, an Okada plot could be constructed using the previously
calculated cell cycle times. The estimated percentages of labeled cells represented
by the curve, corresponds to the experimentally derived points. The cell
parameters are assumed to be values that best described the stage-41 cell cycle.
Quastler & Sherman (1959) observed that by varying the time between
injection of tritiated thymidine and sacrifice, an average cell cycle could be
reconstructed. A cell cycle was estimated by monitoring the presence or absence
of labeled mitotic figures. This labeled mitotic method was used to check the
parameters of the cell cycle obtained by the Okada plot method. At stage 21,
a batch of embryos was injected at 0 time and incubated for 8 h with an embryo
fixed every half hour. The first labeled mitotic figure was observed 2 h after
initiation of labeling, which indicated that G2 was a minimum of 2 h long.
After 3-5 h of incubation half the mitotic figures were labeled. All mitotic figures
were labeled at 5-5 h of incubation. At 7-5 h, 50% of the mitotic figures were
once again unlabeled. The interval between the two points yielding 50%
unlabeled mitotic figures was considered the S period and lasted 4 h. Table 1
compares the cell cycle time obtained by the Okada plot method, and the
Quastler & Sherman labeled mitotic method.
The continuous labeling results indicate that throughout the development of
the thyroid there is little change in the length of the cell cycle, which remains
about 9-5 h. The decrease in the labeling index appears to be due to a decline
in the percentage of replicating cells.
A thyroid from the stage-16 series that was continuously labeled had 40%
labeled cells after 10 h of incubation, in contrast to 100% labeled cells in the
neighboring pharynx. The proliferative index, that is the percentage of cells
actively dividing in one division cycle, was estimated to be 25 % for the stage-16
thyroid. (If 25 out of 100 cells synthesize DNA and divide, at the end of their
division cycle 50 cells are labeled and 75 are unlabeled. A total of 125 cells will be
present and 50 cells or 40 % of the cells are labeled.) The proliferative index from
stage 18 to 20 was estimated to be about 22%. From stage 21 to stage 23 the
proliferative index was estimated to be 30%; the increase may be due to an
entry into S of previously quiescent cells. The thyroid at this time changes from
a vesicle to a solid sphere of cells. At stage 41, the proliferative index was
estimated to be 17%.
275
Cell proliferation in the thyroid
Table 2. DNA (nanograms) per gland
Stages
Hours*
Generationsf
Prolif.
index (%)
Calculated:]:
DNA/gland
Determined§
DNA/gland
14
17
19
20
23
24
25
26
27
28
30
31
32
33
35
36
37
38
39
40
41
52
58
70
80
92
108
114
120
130
140
158
168
176
185
216
240
264
288
231
336
360
0
1
2
3
4
5
6
7
8
9
11
12
13
14
17
20
22
25
27
30
32
25
22
22
30
30
22
22
21
21
21
20
20
20
19
19
18
18
17
17
17
17
(34)
43
52
63
82
107
130
157
190
230
332
397
478
573
965
1627
2 265
3 723
5 096
8 162
11 173
34
42
72
75
95
95
111
150
173
319
564
406
441
885
922
1 370
1942
3 900
4 930
8 724
10 840
* From Hamburger & Hamilton (1951).
•f Obtained by dividing the number of hours elapsed since stage 14 by 9-5, the generation
time.
% Calculated from the generation time and the proliferative index as described in this
paper.
§ Determined by the ftuorometric method of Santoianni & Ayala (1965).
(C) DNA analysis
The amount of DNA per gland was determined using a fluorometric assay
(Santoianni & Ayala, 1965). The amount of DNA found at various stages during
embryonic development is given in Table 2 and is represented by points in
Fig. 3. There was an exponential increase in the amount of DNA during
development, indicating that the use of the Okada plot method for analyzing
the cell cycle was appropriate.
The amount of DNA that would be expected in the gland at each stage could
be calculated using the proliferative indices (see section B) and the determined
generation time. The amount of DNA that was determined experimentally to
be present in the thyroid at stage 14 (34 ng of DNA/gland) was used as the
initial value. Roughly 9-5 h after stage 14, the embryo has reached stage 17 in
development. Between stage 14 and stage 17 (about 10 h or one generation
time) the proliferative index was calculated to be 25 %; therefore the amount of
276
M . S. S M U T S , S. R . H I L F E R
AND
R. L. S E A R L S
100 000 -
I
I
100
1
200
I
I
I
300
400
500
Time (h)
Fig. 3. T h e a m o u n t of D N A per thyroid gland determined by fluorometric analysis,
plotted in n a n o g r a m s D N A v. h o u r s of development. E a c h point represents a n
average of t w o determinations. T h e solid line represents t h e estimated increase in
D N A per gland. T h e a m o u n t of D N A per gland at stage 14 (approximately 52 h)
was used as the base n u m b e r , since this was the earliest stage t h a t the thyroids can
be cleaned mechanically of adherent tissues. A generation time of 9-5 h a n d the
proliferative indices obtained from the c o n t i n u o u s labeling series were used in cal­
culating the estimated increase in D N A , as seen in Table 2.
D N A in the gland should have increased by 25 %. The calculated amount of
D N A per gland at stage 17 (43 ng) compares favorably with the experimentally
derived amount of 42 ng. The length of time from stage 17 to all of the older
stages in Table 2 was calculated from the incubation times given for the normal
staging in Hamburger & Hamilton (1951). The proliferative index that is given
for each stage in Table 2 is the percentage by which the D N A was calculated to
increase during the next generation time of 9-5 h. Thus, a value of 107 ng
calculated for stage 24 represents a 30 % increase over the value of 82 ng at
stage 23. The amounts of D N A , calculated in this way, are plotted as a con­
tinuous line in Fig. 3.
Cell proliferation
A
in the thyroid
277
R o o f of pharynx
Fig. 4. C a m e r a lucida drawings of selected stages in thyroid development, d r a w n to
the same scale. E a c h thyroid is divided into regions as described in the text. Average
labeling indices (LI) are given for each region. Depicted are the thyroid a n d adjacent
structures: (A) at stage 11, early in placode f o r m a t i o n ; (B) at stage 15, during early
evagination; (C) at stage 17, close to the end of evagination; (D) at stage 19, during
vesicle f o r m a t i o n ; a n d (E) at stage 2 1 , t o w a r d s the end of vesicle formation.
(D) Regions of cellular
proliferation
The pulse labeling and D N A determination data indicate that the thyroid is
increasing in cell number while undergoing its morphogenetic shape changes.
Since this study emphasizes the influence that cellular proliferation exerts on
shape changes, a detailed account of where new cells are added to the thyroid
is necessary. These observations are used to evaluate the contribution that cell
divisions make to the shape of the gland (Fig. 4).
The placode stage
The floor of the pharynx bends into the precardial cavity so that in crosssection the thyroid appears suspended over the heart cavity (Fig. 5). By stage 11,
278
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
this early placode has a width of 12 cell diameters in median cross-section.
The thyroid exhibits a labeling index of 30-5 % ± 3-4, compared to 39 % ± 4 in
the adjacent pharyngeal epithelium and 51 % ± 3-6 in the epithelium that makes
up the roof of the pharynx (Fig. 4 A).
Early evagination
One generation time, or 9-5 h after the placode is discernible, the thyroid
exhibits a pronounced bend (Fig. 4 B). Two identations or grooves, one on either
side of the base, form a circle in the basal surface of the gland. The sloping sides
of the organ are each bisected by another shallower groove, beyond which the
thyroid extends for a short distance. The grooves were used as the naturally
occurring boundaries for partition of the gland into sections for counting. The
grooves, delineating region I from region II and region II from III, have two to
four cells located immediately above the indentations. These cells are always
unlabeled and seem to contain less apical cytoplasm. Region I has a labeling
index of 25%, region II has a labeling index of 26%, region III, 30%, and the
adjacent pharynx has a labeling index of 35 %.
Late evagination
At stage 17, about one generation time later, the thyroid is horseshoe-shaped
in cross-section. Evagination is not complete since thyroid cells extend beyond
the inturning shoulders (Fig. 6). The grooves are still present and the cells
immediately above the grooves remain unlabeled. The labeling index in region I
has now dropped to 15 % (Fig. 4C). Region II now occupies part of the base of
the horseshoe-shaped gland and has a labeling index of 13 %. In regions I and
II there is an obvious pseudostratification of the nuclei. The labeled nuclei are
observed in the layers nearest the cell base; whereas, all mitotic figures are
observed in the apical cytoplasmic area.
The sides of the thyroid between the second groove and the lateral margins
of the organ possess several smaller indentations that are separated from each
other by bulges about six cell diameters wide. Region III labels at 17 % and
region IV at 25 %. Region V, the area beyond the inturned shoulders of the
gland, has the same labeling index as that of the adjacent pharyngeal epithelium,
30%.
The continuous labeling from stage 16 to 19 was used to calculate the distribution of labeled nuclei. The thyroid's pseudostratification allows the gland
to be divided into layers of nuclei. The first layer of nuclei at the basal surface of the thyroid is two nuclei thick. The middle layer is also two nuclei
thick and the apical level possesses at least one layer of nuclei and the apical
cytoplasm. During the first hour of labeling, 50 % of the labeled cells are located
in the basal layer, 33 % of the labeled nuclei are located in the middle layer and
15% are in the apical area. After the second hour of labeling, 33% of the
labeled nuclei are situated basally, 47 % are in the middle layer of nuclei and
Cell proliferation in the thyroid
279
20% of the labeled nuclei are located apically. The first labeled division figure is
observed at 2 h of labeling and is situated in the apical cytoplasm of region I.
At 5 h of labeling, the basal nuclear region possesses only 23 % of all labeled nuclei,
the middle layer 50% and the apical portion 27%. After 20 h of continuous
labeling (Fig. 7), the thyroid is fully evaginated and 48 % of its cells are labeled.
The basal layer of nuclei possess 51 % of the labeled nuclei, the second layer of
nuclei, 36%, and the apical layer, 13%. The nuclei incorporate thymidine in
the basal layer, migrate apically to divide and then return to their basal position.
After 20 h of continuous labeling the interkinetic migration of labeled nuclei
still continues and the majority of the labeled nuclei are occupying the basal
and middle layers. Only nuclei synthesizing DNA appear to undergo interkinetic migration since the majority of unlabeled nuclei remain in the basal layer.
Vesicle formation
By stage 19 the thyroid has developed into a vesicle with a wide lumen.
Regions I and II are faintly recognizable as bulges and form the widened basal
region that has a labeling index of 15 % (Fig. 4D). Region III and IV, forming
the sides of the vesicle, have labeling index of 19% and 20%. The cells of
region V have a labeling index of 21 % and are not layered as obviously as the
pseudostratified cells of regions I, II and III. The nearby pharynx has a labeling
index of 25%. By stage 21, the size and shape of the gland has changed very
little; the lumen is reduced in size and the areas composing region V are almost
touching across a narrow duct (Fig. 8). Region V still possesses the highest
labeling index (26%) in the gland (Fig. 4E).
Continuous labeling was performed for stage 21-24, the period in which the
vesicle closes and its lumen is nearly obliterated. One hour of labeling produced
an 8 % labeling in the apical zone of nuclei. This figure increased to 19 % after
2 h; 25 %, after 3-5 h, and finally 40 % after 5-5 h of continuous labeling. After
5-5 h, the labeled nuclei accumulated noticeably in the apical zone and did not
migrate back to their basal positions in the thyroid.
Stalk separation and closure
The thyroid during stage 23 is pinching off from the floor of the pharynx to
which it is connected by a short, narrow stalk (Fig. 9). The labeling for all
regions of the thyroid is nearly the same at 19% ±4-5, except for the stalk,
which has a lower labeling index of 15 %. The short stalk does- contain dividing
cells whose spindles are oriented in the direction of the stalk's length.
Bilobation
Stage 23 exhibits a dramatic shape change that marks the beginning of
bilobation. The central lumen has been reduced to a small space on the right
side of the dividing gland. When the two lobes are counted separately, the right
lobe with the remnant of the lumen has a labeling index of 27 % compared with
280
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
Cell proliferation in the thyroid
281
24 % for the smaller left lobe. Mitotic figures are randomly scattered in the
center of the two lobes. The periphery of the gland is prominently outlined with
pulse-labeled nuclei.
At stage 25, the gland assumes an elongated shape and remnants of stalk
hang from the floor of the pharynx or form a little cap on the thyroid (Fig. 10).
The area bridging the two lobes has a labeling index of 21 % versus 20 % in the
larger right lobe and 24 % in the left lobe. The tips of the lobes possess the
greatest number of labeled cells.
The stage-26 gland is separating into two lobes (Fig. 11). The right lobe of the
gland has a low labeling index of 18 %. The left lobe labels at 23 %. Both the
labeled nuclei and the mitotic figures are oriented towards the center of the lobes.
Follicle formation
By stage 35, vascular and connective tissue elements have invaded the thyroid.
The labeling index of 18 % is equal in all parts of the thyroid and there is no
pattern of labeling associated with the forming follicles.
Stage 45, 20 days of incubation, represents the period when the thyroid is
fully matured and the follicles are formed (Fig. 12). The 8-6% ±4-5 labeling
index is the same throughout the gland. In the interior of the gland, the follicles
are at least six cells in circumference; while at the periphery the follicles are only
FIGURES
5-10
Fig. 5. Pharyngeal region of a pulse-labeled embryo at stage 11. The thyroid is
recognizable on the floor of the pharynx at the level of the second pharyngeal arch.
The thyroid (Thy) is suspended over the pericardial cavity and is distinguished from
the adjacent pharyngeal area by its lower proliferative index and by its closely
adhering cells, x 250.
Fig. 6. A median cross-section of a stage-17 thyroid exhibits an advanced state of
evagination, although thyroid cells extend beyond the inturning areas. Note the
indentations (G) in the basal surface of the thyroid and the apical accumulation
of cytoplasm (A). Pulse-labeled nuclei are more numerous towards the shoulders
than toward the center of the primordium. x 250.
Fig. 7. After 20 h of continuous labeling beginning when the gland was at stage 16,
the thyroid is completely evaginated (stage 22). Only 48 % of its nuclei are labeled,
whereas the mesenchyme and pharyngeal epithelium are 100% labeled, x 250.
Fig. 8. At stage 21 the walls of the thyroid vesicle approach each other and the opening to the pharynx is restricted to a narrow duct. Pulse labeled, x 250.
Fig. 9. The stage-23 thyroid is still connected to the floor of the pharynx by a narrow
stalk of epithelial cells. Note the pulse labeling in the stalk and in the mesenchyme.
x250.
Fig. 10. The stage-25 thyroid possesses remnants of the stalk as a small cap (A); the
lumen (arrow) is also visible. The gland is outlined by pulse-labeled nuclei, x 250.
Fig. 11. The stage-26 gland is separating into two lobes, and its dumb-bell shape is
outline by labeled nuclei, x 250.
Fig. 12. A portion of the thyroid at stage 45. The follicles possess labeled nuclei
(arrows), x 640.
282
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
three cells in circumference but labeled nuclei are present in both areas. When
a mitotic figure is observed, the spindle is oriented parallel to the lumen of the
follicle.
DISCUSSION
Evidence obtained from this study proves that DNA is synthesized in the
thyroid at all stages of its development, from stage 11 to stage 45. However,
only a small portion of cells in the newly formed thyroid are actively synthesizing
DNA. This low labeling index distinguishes the thyroid placode from the
adjacent pharyngeal cells. Continuous labeling experiments indicate that the
low labeling index is due to a low proliferative index rather than a lengthening
of the cell cycle. In fact, a large percentage of cells in the developing thyroid does
not appear ever to enter the S phase of the cell cycle.
Within the thyroid placode, the labeling index tends to increase from the
center of the gland out to the pharyngeal epithelium. From stage 14 to stage 21
shallow grooves form a total of five concentric circles (Fig. 13). During evagination the pattern remains unchanged, with the lowest labeling index in the
central region of the gland and the labeling index increasing in the regions away
from the center. The cells in region V, the area of the thyroid beyond the last
concentric circle, have a labeling index similar to that of the adjacent pharynx.
At stage 23, the stalk connecting the thyroid to the floor of the pharynx has
a lower division rate than the mesenchyme intervening between the pharynx
and the gland. The subsequent attenuation and breaking of the stalk may be
due to the expansion of this rapidly proliferating mesenchymal tissue. Cell
division in the stage-23 gland occurs mainly at the sides, hence, widening the
gland. The right side with the lumen has a slightly higher labeling index than
the left side. This higher labeling index is maintained until the gland lobes, so
that the right side is larger immediately following lobation.
Follicles begin to form when vascular and connective tissue cells invade the
thyroid. There are labeled nuclei and mitotic figures present in the follicle.
Follicular size appears to be increased by cell division, contrary to Hopkins'
(1935) conclusion, that the growth in size is predominantly by fusion of the
follicles.
With the exception of Pictet et al (1972) and Zwaan & Hendrix's (1973)
studies, previous investigators of evagination have discounted the importance
of increases in cell number during morphogenetic shape change. This study gives
rise to a model which emphasizes the role of cell division in shaping the organ.
During the early states of development, the thyroid placode maintains a constant width with the circular groove acting as the placode's boundary. In
cross-section, the thyroid cells immediately above the grooves are never found to
be labeled. These cells contain highly oriented bundles of microftlaments and
microtubules parallel to the longitudinal dimension of the cells (Hilfer, 1973).
The cells within the boundaries proliferate and pseudostratify. Growth within
283
Cell proliferation in the thyroid
Pharynx
Stage 13
Pharynx
Stage 15
Pharynx
Stage 17
Pharynx
Stage 20
Fig. 13. Diagrammatic representation of the events occurring during thyroid
evagination. See text for explanation.
284
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
the groove occurs in height rather than in width, a fact that is evident when the
stage 11 and stage 15 glands are compared. This suggests that an, as yet,
undetermined force is holding the groove in place. Therefore, the dividing cells
in the placode are prevented from separating laterally, just as lens cells are
restricted to the area of contact with the optic vesicle (Zwaan & Hendrix, 1973).
The width of the organ increases through the incorporation of adjacent
pharyngeal areas. The high rate of proliferation in the pharynx creates population
pressure there. The thyroid placode is situated in a curve at the base of the
pharynx; this increased tension in the pharynx accentuates the curve. The
pharyngeal cells adjacent to the placode are pressed closer together so that their
lateral spaces are lost and their nuclei become longitudinally oriented. These
cells form the newly added areas of regions II and III. They form the sloping
sides of the thyroid and also acquire grooves on their basal surfaces.
At stage 17, after the thyroid has acquired more cells from the surrounding
pharynx, the sides of the evaginating gland are steeper. The placode still forms
the base of the thyroid but the lateral regions first acquired are now part of the
base of the evaginated gland. The bulging of the thyroid in each new region
creates an indentation to allow for the basal expansion of the area. The
indentation then acts as a hinge between the original placode and the newly
added regions of the evaginating gland. Continued division pressure produces
a tight, closed sphere with a central lumen.
The region V in the stage-17-20 gland does not show the basal location of
labeled nuclei. Many labeled nuclei appear near the apical surface of the thyroid.
The tightly clustered nuclei are longitudinally oriented with respect to the lumen
surface of the gland but appear almost horizontal to the pharynx. Continued
divisions in region V, which has the highest labeling index of all the regions,
forces the shoulders together during stage 19 and narrows the opening of the
vesicle. The additions of these new cells to the thyroid will close the opening and
produce a stalk.
At stage 21, this hollow sphere of cells begins to become a solid ball. Interkinetic migration ceases at this stage; the nuclei migrate to the apical surface
to divide but do not return to their basal position. The thyroid becomes a multilayered sphere of cells whose central lumen is reduced by increased thickness
of the walls. Most of the labeling occurs in the basal layer at the apex and sides
of the gland, but labeled cells also are found at the lumenal surface of the gland.
The proliferative index at this stage increases to 30 %. This increase is attributable
to previously quiescent cells entering the division cycle. Since the cells in the
center of the gland are those that remained after rounding up for division, they
are the original proliferating population. The labeled cells around the sides of
the gland are possibly the previously unlabeled cells that are now dividing;
hence, the gland acquires its lobed shape by the asymmetrical additions to its
sides.
Recently, the thyroid has been provoked into premature evagination in a con-
Cell proliferation in the thyroid
285
traction medium (Hilfer, Young & Fithian, 1974; Hilfer, Palmatier & Fithian,
1977). Through the use of ATP, a stage-14 thyroid will become as evaginated as
a stage-16 gland within 20 min. This precocious evagination confirms the
observation that the early thyroid annexes pharyngeal areas in the process of
evagination. Since in the contraction medium the thyroid evaginates and
increases in size within 20 min, this rapid size increase cannot be due to cell
division.
Thus, this study answers the questions posed in the introduction: (1) new
cells are continually being added to the thyroid either by cell division or
annexation of adjacent pharyngeal cells; (2) cell division is random although at
some stages of development there is a higher proliferative rate in certain areas;
and, (3) the new acquisitions of cells play a role in shaping the thyroid.
Supported by N.S.F. grant no. 70-00580 and by Temple University Research Assistantships and Fellowships.
This paper represents a portion of a dissertation submitted to the graduate faculty of
Temple University in partial fulfillment of the requirements for the Ph.D. degree.
REFERENCES
P. C. & SCHROEDER, T. E. (1967). Cytoplasmic filaments and morphogenetic
movement in the amphibian neural tubes. Devi Biol. 15, 432-450.
COLEMAN, J. R., COLEMAN, A. W. & HARTLINE, E. (1969). A clonal study of the reversible
inhibition of muscle differentiation by the halogenated thymidine analog 5-bromodeoxyuridine. Devi Biol. 19, 527-548.
HAMBURGER, V. & HAMILTON, H. (1951). A series of normal stages in the development of the
chick embryo. /. Morph. 88, 49-92.
HILFER, S. R. (1973). Extracellular and intracellular correlates of organ initiation in the
embryonic chick thyroid. Amer. Zool. 13, 1023-1038.
HILFER, S. R., YOUNG, B. J. & FITHIAN, E. M. (1974). In vitro evagination of thyroid primordia
in muscle contraction medium. J. Cell. Biol. 63, 137a.
HILFER, S. R., PALMATIER, B. Y. & FITHIAN, E. M. (1977). Precocious evagination of the
embryonic chick thyroid in ATP-containing medium. J. Embryol. exp. Morph. 42, 163-175.
HOPKINS, M. L. (1935). Development of the thyroid gland in the chick embryo. /. Morph. 58,
585-604.
JANNERS, M. Y. & SEARLS, R. L. (1970). Changes in the rate of cellular proliferation during
the differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing.
Devi Biol. 23, 136-165.
MARCHOK, A. C. & HERRMANN, H. (1967). Studies of muscle development. Devi Biol. 15,
129-155.
OKADA, S. (1967). A simple graphic method of computing the parameters of the life cycle of
cultured mammalian cells in the exponential growth phase. /. Cell Biol. 34, 915-916.
PICTET, R. L., CLARK, W. R., WILLIAMS, R. H. & RUTTER, W. J. (1972). An ultrastructural
analysis of the developing embryonic pancreas. Devi Biol. 29, 436-467.
POURTOIS, M. (1972). Morphogenesis of the primary and secondary palate. In Developmental
Aspects of Oral Biology (ed. H. G. Slavkin & L. A. Bavetta), ch. 5, pp. 81-108.
QUASTLER, H. & SHERMAN, F. G. (1959). Cell population kinetics in the intestinal epithelium
of the mouse. Expl. Cell. Res. 17, 420-438.
SANTOIANNI, P. & AYALA, W. (1965). Fluorometric,okramicroanalysis of deoxyribonucleic
acid in human skin. / . invest. Derm. 45, 99-103.
SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick
limb bud. / . exp. Zool. 166, 39-50.
BAKER,
19
EMB 48
286
M. S. SMUTS, S. R. HILFER AND R. L. SEARLS
W. G., HILFER, S. R. & FONTE, V. G. (1972). Early organogenesis of the embryonic
chick thyroid. Devi Biol. 28, 202-218.
SPOONER, B. S. & WESSELLS, N. K. (1972). An analysis of salivary gland morphogenesis: Role
of microfilaments and microtubules. Devi Biol. 27, 38-54.
WRENN, J. T. & WESSELLS, N. K. (1970). Cytochalasin B: Effect upon microfilaments involved
in morphogenesis of estrogen-induced glands of oviduct. Proc. natn. Acad. Sci., U.S.A. 66,
904-908.
ZWAAN, J. & HENDRIX, R. W. (1973). Changes in cell and organ shape during early development of the ocular lens. Amer. Zool. 13, 1039-1099.
ZWAAN, J. & PEARCE, T. L. (1971). Cell population kinetics in the chicken lens primordium
during and shortly after its contact with the optic cup. Devi. Biol. 25, 96-118.
ZWILLING, E. (1959). A modified choriollantoic grafting procedure. Trans. Bull. 6, 115-116.
SHAIN,
(Received 29 September 1977, revised 21 July 1978).