Patterns of cellular proliferation during thyroid organo ...(Marchok & Herrmann, 1967 Zwaa; &n...

/ . Embryo/, exp. Morph. Vol. 48, pp. 269-286, 1978 2 6 9Printed in Great Britain © Company of Biologists Limited 1978

Patterns of cellular proliferation duringthyroid organo genesis

By MARYBETH S. SMUTS,1 S. ROBERT HILFER2 ANDROBERT L. SEARLS2

From the Department of Biology, Temple University, Philadelphia

SUMMARYThe 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. Pulselabeling with tritiated thymidine indicates that the gland's labeling index declines throughoutits development. Initially, the thyroid placode has a lower labeling index than the neighboringpharyngeal epithelium. An evaluation of the positions of pulse-labeled cells reveals that theevaginating thyroid grows by annexing cells from the pharyngeal epithelium. The olderevaginated regions of the gland exhibit the lowest labeling indices. The newly acquired regionsstill maintain higher labeling indices.

INTRODUCTION

The mechanism commonly invoked to explain invagination and evagination ofepithelial organs is the purse-string contraction model (Baker & Schroeder,1967). Yet this model alone cannot explain the invagination process for allepithelial organs. Contrary to the expected results of a purse-string contractionmodel, Hilfer (1973) observed that the greatest concentrations of apical micro-filaments were not in the areas of the most pronounced bending in the evaginatingthyroid. Wrenn & Wessells (1970), in analyzing oviduct tubular gland develop-ment, could not elicit duct elongation when DNA synthesis was partiallyinhibited. Spooner & Wessells (1972) obtained similar results; cytochalasin-treated salivary glands recovered only narrow clefts when colchicine was presentin the medium. Observations of this sort have led several workers to evaluatethe role of cell division within developing epithelial organs. Pictet, Clark,Williams & Rutter (1972) observed that, in the pancreas, cells are apicallyconnected while growth is generalized. They suggested that lobulation is causedby lateral pressure generated by dividing, adherent cells. Pourtois (1972)suggested that nasal placode invagination may be explained by increased cellularadhesions 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 basedupon population pressure generated by continuing cell division within a restrictedarea.

In the present investigation, the embryonic thyroid was used to determine iflocalized areas of cell divisions were influencing the gland's morphogenesis.Three questions were considered: (1) whether new cells were added to theprimordium and, if so, at what developmental stage; (2) whether the distributionof mitotic activity was random or localized; and, (3) whether the addition ofnew 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 correlationwith previous cytological and biochemical work (Shain, Hilfer & Fonte, 1972).The eggs were incubated in a Jamesway incubator at 37 °C. A window wasprepared in every egg (Zwilling, 1959) and all labeling was performed onwindowed eggs.

Areas and rates of cell division were investigated by counting the number ofcells 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 ofthe egg. Since stage 10-14 embryos were weakly labeled by this method, thethymidine was dripped onto these embryos through the window. The windowwas sealed and the eggs reincubated for either 1 h or, for continuous labeling,up to 24 h. The embryos were staged (Hamburger & Hamilton, 1951) justbefore injection and restaged before removal of the thyroid.

After incubation with tritiated thymidine the embryos were removed anddissected in medium 199 (GIBCO). The thyroids were fixed in 2-0% glutaral-dehyde buffered with Coleman's phosphate buffer (Coleman, Coleman &Hartline, 1969) for 20 min, washed in buffer, dehydrated, embedded in 60 °Cparaffin and sectioned at 5 jam. The sections were dipped in Kodak NTB-3emulsion, stored for 4 weeks at 4 °C, developed in D-19 (Kodak), and stainedwith hematein (Searls, 1967). A Wild microscope equipped with a drawing tubewas used to score sections for labeled and unlabeled nuclei.

A DNA analysis was performed on mechanically cleaned glands. It wasdifficult to dissect out cleanly early thyroid glands; therefore, analyses weremade only on glands of stage 14 and older. Five glands were pooled for DNAanalyses for each stage, 14 through 29. Samples at later stages varied from onepair of glands to five pairs. The Santoianni & Ayala (1965) fluorometric methodwas used since it is sensitive enough to measure the nanogram amounts of DNAthat the sample contained. Calf thymus DNA was used as the standard.

Cell proliferation in the thyroid 271

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1—t——

1

11 1

.. 43 72 90 120 240 264 288 336 360 408 450 480Hours 52 go 108

Stage 1114 20 24 26 36 37 38 40 41 43 44 45

Time of incubation with label (li)

Fig. 1. The pulse-labeling indices (percentage of labeled cells in the thyroid after1 h labeling with tritiated thymidine) plotted v. stage and time of embryonicdevelopment. Each point represents the mean labeling index of at least three thyroidsand the range is the standard deviation. The line, determined by linear regressionanalysis, 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 theposition of the primordiiim on the floor of the pharynx and by the closelyadhering cells that comprise the thyroid regions (Shain et al. 1972). Duringstages 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 thethyroid was drawn to show the location of labeled and unlabeled nuclei and ofmitotic figures. A nucleus was considered labeled if it had more than five silvergrains over it. From stage 11 to stage 16 a minimum of 50 nuclei per sectionwere 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 perthyroid 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 thymidinelabel was determined. At least three thyroids per stage were averaged to calculatethe labeling index. These percentages or labeling indices were then plottedagainst the stages of the embryos and hours of embryonic development (Fig. 1).Linear regression analysis indicated a constant decline in the labeling index

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M. S. SMUTS, S. R. HILFER AND R. L. SEARLS

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1 2 3 40

5 6 7 8 9 100

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120

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14 160

18 20

Fig. 2. Graphs representing the experimentally determined labeling percentagesobtained throughout a 20 h continuous labeling period. Ten microcuries oftritiated thymidine were injected in ovo every 4 h (injected times [0]). The points rep-resent the labeling indices obtained by counting labeled and unlabeled nuclei. Thesolid lines represent the hypothetical labeling curves calculated by the Okada plotmethod (Okada, 1967). (a) Initial injection at stage 16 during early thyroid evagi-nation. (b) Initial injection at stage 2.1, late in vesicle formation. (•) represents per-centages obtained after a single injection of radioisotope. (c) Initial injection atstage 4.1, when the thyroid contains mature follicles.


Table 1. Value for the cell cycle parameters for stage-21 labeling series

LabeledInflection Okada mitotic

points method methodestimates calculations estimates

Parameters (h) (h) (h)

Gz — — 2G2 + M 2 1-9 —

Ca + M+Gi 4-5 3-9 —Gt 2-5 2 —5 — 5-6 4

Q 95

throughout thyroid development, from 30-5% ±3-4 at stage 11, the placodeformative period, to 8-6 % ± 4-5 at stage 45, just prior to hatching.

The thyroid originates from the pharyngeal epithelium, but from stage 11 to21 the gland is consistently distinguished from the pharynx by a lower labelingindex. During stage 11, the average labeling index of the thyroid was 30-5 % ±3-4while 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 was19 % ± 4-5 in the thyroid and 15 % ± 1.7 in the pharynx. After separation of thethyroid from the floor of the pharynx, the pharyngeal epithelium was not scored.

(B) Continuous labeling

Continuous labeling experiments were performed to determine if the declinein the labeling index of the thyroid, between stage 11 and stage 45, was due toa decrease in the percentage of replicating cells. Several stages were chosen forthese experiments: stage 16, when the thyroid was an evagination of pseudo-stratified cells; stage 21, when the vesicle was almost completely closed; andstage 41, when the gland was mature.

Carefully staged batches of embryos were injected with 10 /̂ Ci of tritiatedthymidine at 0 time. Every half-hour an embryo was fixed for analysis. Sincethe thymidine appears to be available to the embryo for a short period of time(Marchok & Herrmann, 1967; Zwaan & Pearce, 1971), in order to ensure thatsufficient label was available, additional injections of label were given every 4 h.Injection times of less than 4 h decreased embryonic survival. The points inFig. 2a-c represent the percentages of labeled cells in thyroids injected at thegiven stages.

Within 2 h, cells that had incorporated label entered mitosis; therefore, 2 his the minimum time of G2. This increase in labeled cells is reflected by thesteeper slopes of the curves (a) and (b) (Fig. 2). The plateau portions of the curvesindicate that the maximum percentage of cells have been labeled and correspondto C


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 asfixed values to determine the parameters of the cell cycle. The simple graphicmethod 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 eachportion of the cell cycle could be determined (Janners & Searls, 1970). TheseOkada plot-estimated percentages are congruent with the experimentally derivedlabeling 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 determineinflection points, an Okada plot could be constructed using the previouslycalculated cell cycle times. The estimated percentages of labeled cells representedby the curve, corresponds to the experimentally derived points. The cellparameters are assumed to be values that best described the stage-41 cell cycle.

Quastler & Sherman (1959) observed that by varying the time betweeninjection of tritiated thymidine and sacrifice, an average cell cycle could bereconstructed. A cell cycle was estimated by monitoring the presence or absenceof labeled mitotic figures. This labeled mitotic method was used to check theparameters 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 embryofixed every half hour. The first labeled mitotic figure was observed 2 h afterinitiation 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 figureswere labeled at 5-5 h of incubation. At 7-5 h, 50% of the mitotic figures wereonce again unlabeled. The interval between the two points yielding 50%unlabeled mitotic figures was considered the S period and lasted 4 h. Table 1compares the cell cycle time obtained by the Okada plot method, and theQuastler & Sherman labeled mitotic method.

The continuous labeling results indicate that throughout the development ofthe thyroid there is little change in the length of the cell cycle, which remainsabout 9-5 h. The decrease in the labeling index appears to be due to a declinein 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 theneighboring pharynx. The proliferative index, that is the percentage of cellsactively dividing in one division cycle, was estimated to be 25 % for the stage-16thyroid. (If 25 out of 100 cells synthesize DNA and divide, at the end of theirdivision cycle 50 cells are labeled and 75 are unlabeled. A total of 125 cells will bepresent and 50 cells or 40 % of the cells are labeled.) The proliferative index fromstage 18 to 20 was estimated to be about 22%. From stage 21 to stage 23 theproliferative index was estimated to be 30%; the increase may be due to anentry into S of previously quiescent cells. The thyroid at this time changes froma vesicle to a solid sphere of cells. At stage 41, the proliferative index wasestimated to be 17%.


Table 2. DNA (nanograms) per gland

Gener- Prolif. Calculated:]: Determined§Stages Hours* ationsf index (%) DNA/gland DNA/gland

141719202324252627283031323335363738394041

5258708092108114120130140158168

176185216240264288231336360

012

345

67891112131417202225273032

252222303022222121212020201919181817171717

(34)43526382107

130157190230332397

478573965

16272 2653 7235 0968 16211 173

344272759595

111150173319564406441885922

1 37019423 9004 9308 72410 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 duringembryonic development is given in Table 2 and is represented by points inFig. 3. There was an exponential increase in the amount of DNA duringdevelopment, indicating that the use of the Okada plot method for analyzingthe cell cycle was appropriate.

The amount of DNA that would be expected in the gland at each stage couldbe calculated using the proliferative indices (see section B) and the determinedgeneration time. The amount of DNA that was determined experimentally tobe present in the thyroid at stage 14 (34 ng of DNA/gland) was used as theinitial value. Roughly 9-5 h after stage 14, the embryo has reached stage 17 indevelopment. Between stage 14 and stage 17 (about 10 h or one generationtime) 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 A N D R . L . S E A R L S

100 000 -

I I 1 I I I

100 200 300 400 500 Time (h)

Fig. 3. The amoun t of D N A per thyroid gland determined by fluorometric analysis, plotted in nanograms D N A v. hours of development. Each point represents an average of two determinat ions. The solid line represents the estimated increase in D N A per gland. The amoun t of D N A per gland at stage 14 (approximately 52 h) was used as the base number , since this was the earliest stage that the thyroids can be cleaned mechanically of adherent tissues. A generation t ime of 9-5 h and the proliferative indices obtained from the cont inuous labeling series were used in calculating the estimated increase in D N A , as seen in Table 2.

DNA in the gland should have increased by 25 %. The calculated amount of DNA 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 DNA, calculated in this way, are plotted as a continuous line in Fig. 3.


A R o o f o f pha rynx

Fig. 4. Camera lucida drawings of selected stages in thyroid development, drawn to the same scale. Each thyroid is divided into regions as described in the text. Average labeling indices (LI) are given for each region. Depicted are the thyroid and adjacent s tructures: (A) at stage 11, early in placode format ion; (B) at stage 15, during early evaginat ion; (C) at stage 17, close to the end of evaginat ion; (D) at stage 19, during vesicle format ion; and (E) at stage 21 , towards 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 cross-

section the thyroid appears suspended over the heart cavity (Fig. 5). By stage 11,


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 inthe adjacent pharyngeal epithelium and 51 % ± 3-6 in the epithelium that makesup the roof of the pharynx (Fig. 4 A).

Early evagination

One generation time, or 9-5 h after the placode is discernible, the thyroidexhibits a pronounced bend (Fig. 4 B). Two identations or grooves, one on eitherside of the base, form a circle in the basal surface of the gland. The sloping sidesof the organ are each bisected by another shallower groove, beyond which thethyroid extends for a short distance. The grooves were used as the naturallyoccurring boundaries for partition of the gland into sections for counting. Thegrooves, delineating region I from region II and region II from III, have two tofour cells located immediately above the indentations. These cells are alwaysunlabeled and seem to contain less apical cytoplasm. Region I has a labelingindex of 25%, region II has a labeling index of 26%, region III, 30%, and theadjacent pharynx has a labeling index of 35 %.

Late evagination

At stage 17, about one generation time later, the thyroid is horseshoe-shapedin cross-section. Evagination is not complete since thyroid cells extend beyondthe inturning shoulders (Fig. 6). The grooves are still present and the cellsimmediately above the grooves remain unlabeled. The labeling index in region Ihas now dropped to 15 % (Fig. 4C). Region II now occupies part of the base ofthe horseshoe-shaped gland and has a labeling index of 13 %. In regions I andII there is an obvious pseudostratification of the nuclei. The labeled nuclei areobserved in the layers nearest the cell base; whereas, all mitotic figures areobserved in the apical cytoplasmic area.

The sides of the thyroid between the second groove and the lateral marginsof the organ possess several smaller indentations that are separated from eachother by bulges about six cell diameters wide. Region III labels at 17 % andregion IV at 25 %. Region V, the area beyond the inturned shoulders of thegland, 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 dis-tribution of labeled nuclei. The thyroid's pseudostratification allows the glandto be divided into layers of nuclei. The first layer of nuclei at the basal sur-face of the thyroid is two nuclei thick. The middle layer is also two nucleithick and the apical level possesses at least one layer of nuclei and the apicalcytoplasm. During the first hour of labeling, 50 % of the labeled cells are locatedin the basal layer, 33 % of the labeled nuclei are located in the middle layer and15% are in the apical area. After the second hour of labeling, 33% of thelabeled nuclei are situated basally, 47 % are in the middle layer of nuclei and


20% of the labeled nuclei are located apically. The first labeled division figure isobserved 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 continuouslabeling (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 ofnuclei, 36%, and the apical layer, 13%. The nuclei incorporate thymidine inthe basal layer, migrate apically to divide and then return to their basal position.After 20 h of continuous labeling the interkinetic migration of labeled nucleistill continues and the majority of the labeled nuclei are occupying the basaland middle layers. Only nuclei synthesizing DNA appear to undergo inter-kinetic 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 basalregion that has a labeling index of 15 % (Fig. 4D). Region III and IV, formingthe sides of the vesicle, have labeling index of 19% and 20%. The cells ofregion V have a labeling index of 21 % and are not layered as obviously as thepseudostratified cells of regions I, II and III. The nearby pharynx has a labelingindex of 25%. By stage 21, the size and shape of the gland has changed verylittle; the lumen is reduced in size and the areas composing region V are almosttouching across a narrow duct (Fig. 8). Region V still possesses the highestlabeling index (26%) in the gland (Fig. 4E).

Continuous labeling was performed for stage 21-24, the period in which thevesicle closes and its lumen is nearly obliterated. One hour of labeling producedan 8 % labeling in the apical zone of nuclei. This figure increased to 19 % after2 h; 25 %, after 3-5 h, and finally 40 % after 5-5 h of continuous labeling. After5-5 h, the labeled nuclei accumulated noticeably in the apical zone and did notmigrate 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 towhich it is connected by a short, narrow stalk (Fig. 9). The labeling for allregions 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 dividingcells whose spindles are oriented in the direction of the stalk's length.

Bilobation

Stage 23 exhibits a dramatic shape change that marks the beginning ofbilobation. The central lumen has been reduced to a small space on the rightside of the dividing gland. When the two lobes are counted separately, the rightlobe with the remnant of the lumen has a labeling index of 27 % compared with


24 % for the smaller left lobe. Mitotic figures are randomly scattered in thecenter of the two lobes. The periphery of the gland is prominently outlined withpulse-labeled nuclei.

At stage 25, the gland assumes an elongated shape and remnants of stalkhang 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 thelarger right lobe and 24 % in the left lobe. The tips of the lobes possess thegreatest number of labeled cells.

The stage-26 gland is separating into two lobes (Fig. 11). The right lobe of thegland has a low labeling index of 18 %. The left lobe labels at 23 %. Both thelabeled 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 nopattern of labeling associated with the forming follicles.

Stage 45, 20 days of incubation, represents the period when the thyroid isfully matured and the follicles are formed (Fig. 12). The 8-6% ±4-5 labelingindex is the same throughout the gland. In the interior of the gland, the folliclesare 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 isrecognizable 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 fromthe adjacent pharyngeal area by its lower proliferative index and by its closelyadhering cells, x 250.Fig. 6. A median cross-section of a stage-17 thyroid exhibits an advanced state ofevagination, although thyroid cells extend beyond the inturning areas. Note theindentations (G) in the basal surface of the thyroid and the apical accumulationof cytoplasm (A). Pulse-labeled nuclei are more numerous towards the shouldersthan 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 open-ing 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 narrowstalk 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); thelumen (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 isoutline by labeled nuclei, x 250.Fig. 12. A portion of the thyroid at stage 45. The follicles possess labeled nuclei(arrows), x 640.


three cells in circumference but labeled nuclei are present in both areas. Whena mitotic figure is observed, the spindle is oriented parallel to the lumen of thefollicle.

DISCUSSION

Evidence obtained from this study proves that DNA is synthesized in thethyroid 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 synthesizingDNA. This low labeling index distinguishes the thyroid placode from theadjacent pharyngeal cells. Continuous labeling experiments indicate that thelow labeling index is due to a low proliferative index rather than a lengtheningof the cell cycle. In fact, a large percentage of cells in the developing thyroid doesnot appear ever to enter the S phase of the cell cycle.

Within the thyroid placode, the labeling index tends to increase from thecenter of the gland out to the pharyngeal epithelium. From stage 14 to stage 21shallow grooves form a total of five concentric circles (Fig. 13). During evagi-nation the pattern remains unchanged, with the lowest labeling index in thecentral region of the gland and the labeling index increasing in the regions awayfrom the center. The cells in region V, the area of the thyroid beyond the lastconcentric 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 hasa lower division rate than the mesenchyme intervening between the pharynxand the gland. The subsequent attenuation and breaking of the stalk may bedue to the expansion of this rapidly proliferating mesenchymal tissue. Celldivision in the stage-23 gland occurs mainly at the sides, hence, widening thegland. The right side with the lumen has a slightly higher labeling index thanthe left side. This higher labeling index is maintained until the gland lobes, sothat the right side is larger immediately following lobation.

Follicles begin to form when vascular and connective tissue cells invade thethyroid. 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 thefollicles.

With the exception of Pictet et al (1972) and Zwaan & Hendrix's (1973)studies, previous investigators of evagination have discounted the importanceof increases in cell number during morphogenetic shape change. This study givesrise 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 con-stant width with the circular groove acting as the placode's boundary. Incross-section, the thyroid cells immediately above the grooves are never found tobe labeled. These cells contain highly oriented bundles of microftlaments andmicrotubules parallel to the longitudinal dimension of the cells (Hilfer, 1973).The cells within the boundaries proliferate and pseudostratify. Growth within

Cell proliferation in the thyroid

Pharynx

Stage 13

Pharynx

283

Stage 15

Stage 17

Pharynx

Pharynx

Stage 20

Fig. 13. Diagrammatic representation of the events occurring during thyroidevagination. See text for explanation.


the groove occurs in height rather than in width, a fact that is evident when thestage 11 and stage 15 glands are compared. This suggests that an, as yet,undetermined force is holding the groove in place. Therefore, the dividing cellsin the placode are prevented from separating laterally, just as lens cells arerestricted to the area of contact with the optic vesicle (Zwaan & Hendrix, 1973).

The width of the organ increases through the incorporation of adjacentpharyngeal areas. The high rate of proliferation in the pharynx creates populationpressure there. The thyroid placode is situated in a curve at the base of thepharynx; this increased tension in the pharynx accentuates the curve. Thepharyngeal cells adjacent to the placode are pressed closer together so that theirlateral spaces are lost and their nuclei become longitudinally oriented. Thesecells form the newly added areas of regions II and III. They form the slopingsides of the thyroid and also acquire grooves on their basal surfaces.

At stage 17, after the thyroid has acquired more cells from the surroundingpharynx, the sides of the evaginating gland are steeper. The placode still formsthe base of the thyroid but the lateral regions first acquired are now part of thebase of the evaginated gland. The bulging of the thyroid in each new regioncreates an indentation to allow for the basal expansion of the area. Theindentation then acts as a hinge between the original placode and the newlyadded regions of the evaginating gland. Continued division pressure producesa tight, closed sphere with a central lumen.

The region V in the stage-17-20 gland does not show the basal location oflabeled nuclei. Many labeled nuclei appear near the apical surface of the thyroid.The tightly clustered nuclei are longitudinally oriented with respect to the lumensurface of the gland but appear almost horizontal to the pharynx. Continueddivisions 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 thevesicle. The additions of these new cells to the thyroid will close the opening andproduce a stalk.

At stage 21, this hollow sphere of cells begins to become a solid ball. Inter-kinetic migration ceases at this stage; the nuclei migrate to the apical surfaceto divide but do not return to their basal position. The thyroid becomes a multi-layered sphere of cells whose central lumen is reduced by increased thicknessof the walls. Most of the labeling occurs in the basal layer at the apex and sidesof 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 attributableto previously quiescent cells entering the division cycle. Since the cells in thecenter of the gland are those that remained after rounding up for division, theyare the original proliferating population. The labeled cells around the sides ofthe gland are possibly the previously unlabeled cells that are now dividing;hence, the gland acquires its lobed shape by the asymmetrical additions to itssides.

Recently, the thyroid has been provoked into premature evagination in a con-


traction medium (Hilfer, Young & Fithian, 1974; Hilfer, Palmatier & Fithian,1977). Through the use of ATP, a stage-14 thyroid will become as evaginated asa stage-16 gland within 20 min. This precocious evagination confirms theobservation that the early thyroid annexes pharyngeal areas in the process ofevagination. Since in the contraction medium the thyroid evaginates andincreases in size within 20 min, this rapid size increase cannot be due to celldivision.

Thus, this study answers the questions posed in the introduction: (1) newcells are continually being added to the thyroid either by cell division orannexation of adjacent pharyngeal cells; (2) cell division is random although atsome 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 Assistant-ships and Fellowships.

This paper represents a portion of a dissertation submitted to the graduate faculty ofTemple University in partial fulfillment of the requirements for the Ph.D. degree.

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(Received 29 September 1977, revised 21 July 1978).

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