Patterns of cellular proliferation during thyroid organo genesis

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1 /. Embryo/, exp. Morph. Vol. 48, pp , Printed in Great Britain Company of Biologists Limited 1978 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 (197), 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 2766, U.S.A. 2 Author's address: Department of Biology, Temple University, Philadelphia, Pennsylvania, 19122, U.S.A. l8 EMB 48

2 27 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 2 Ci/mole) in -1 ml of Hanks' solution were injected into the yolk through a hole drilled at the blunt end of the egg. Since stage 1-14 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-% glutaraldehyde buffered with Coleman's phosphate buffer (Coleman, Coleman & Hartline, 1969) for 2 min, washed in buffer, dehydrated, embedded in 6 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.

3 Cell proliferation in the thyroid J 3 u -3 o I 2 T! " il _ TT J[. ^ ff i«j lii l i x T 1 1 t Ml II i i i Hours 52 go 18 Stage Time of incubation with label (li) 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 5 nuclei per section were counted and at least three sections per thyroid were scored. After stage 17, at least 1 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 4 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 T i i 18-2

4 M. S. SMUTS, S. R. HILFER AND R. L. SEARLS J I I I I I I t (b) ~ 6 I 5 o i 4 ~o 3 3 ^ 2 W W 1 J L J I I I I I (c) Time of incubation with label (h) Fig. 2. Graphs representing the experimentally determined labeling percentages obtained throughout a 2 h continuous labeling period. Ten microcuries of tritiated thymidine were injected in ovo every 4 h (injected times []). 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.

5 Cell proliferation in the thyroid 273 Table 1. Value for the cell cycle parameters for stage-21 labeling series Labeled Inflection Okada mitotic points method method estimates calculations estimates Parameters (h) (h) (h) G z 2 G 2 + M Ca + M+Gi G t Q 95 throughout thyroid development, from 3-5% ±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 3-5 % ±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 1 /^Ci of tritiated thymidine at 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 G 2. 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

6 274 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 G 1} G 2 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, 197). 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 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 G 2 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, 5% of the mitotic figures were once again unlabeled. The interval between the two points yielding 5% 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 4% labeled cells after 1 h of incubation, in contrast to 1% 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 1 cells synthesize DNA and divide, at the end of their division cycle 5 cells are labeled and 75 are unlabeled. A total of 125 cells will be present and 5 cells or 4 % of the cells are labeled.) The proliferative index from stage 18 to 2 was estimated to be about 22%. From stage 21 to stage 23 the proliferative index was estimated to be 3%; 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%.

7 Cell proliferation in the thyroid 275 Table 2. DNA (nanograms) per gland Gener- Prolif. Calculated:]: Determined Stages Hours* ationsf index (%) DNA/gland DNA/gland (34) * 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 1 h or one generation time) the proliferative index was calculated to be 25 %; therefore the amount of

8 276 M. S. SMUTS, S. R. HILFER AND R. L. SEARLS 1 - I I 1 I I I Time (h) Fig. 3. The amount of DNA per thyroid gland determined by fluorometric analysis, plotted in nanograms DNA v. hours of development. Each point represents an average of two determinations. The solid line represents the estimated increase in DNA per gland. The amount of DNA 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 time of 9-5 h and the proliferative indices obtained from the continuous labeling series were used in calculating the estimated increase in DNA, 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 DNA was calculated to increase during the next generation time of 9-5 h. Thus, a value of 17 ng calculated for stage 24 represents a 3 % 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.

9 Cell proliferation in the thyroid 277 A Roof of pharynx 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 structures: (A) at stage 11, early in placode formation; (B) at stage 15, during early evagination; (C) at stage 17, close to the end of evagination; (D) at stage 19, during vesicle formation; and (E) at stage 21, towards the end of vesicle formation. (D) Regions of cellular proliferation The pulse labeling and DNA 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,

10 278 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 3-5 % ± 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, 3%, 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, 3%. 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, 5 % 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

11 Cell proliferation in the thyroid 279 2% 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 5% and the apical portion 27%. After 2 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 2 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 2%. 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 4 % 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

12 28 M. S. SMUTS, S. R. HILFER AND R. L. SEARLS

13 Cell proliferation in the thyroid % 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. 1). The area bridging the two lobes has a labeling index of 21 % versus 2 % 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, 2 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 5-1 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 25. 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 25. Fig. 7. After 2 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 1% labeled, x 25. 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 25. 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. x25. Fig. 1. 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 25. Fig. 11. The stage-26 gland is separating into two lobes, and its dumb-bell shape is outline by labeled nuclei, x 25. Fig. 12. A portion of the thyroid at stage 45. The follicles possess labeled nuclei (arrows), x 64.

14 282 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

15 Cell proliferation in the thyroid 283 Pharynx Stage 13 Pharynx Stage 15 Pharynx Stage 17 Pharynx Stage 2 Fig. 13. Diagrammatic representation of the events occurring during thyroid evagination. See text for explanation.

16 284 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-17-2 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 3 %. 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-

17 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 2 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 2 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 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 BAKER, P. C. & SCHROEDER, T. E. (1967). Cytoplasmic filaments and morphogenetic movement in the amphibian neural tubes. Devi Biol. 15, 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, HAMBURGER, V. & HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, HILFER, S. R. (1973). Extracellular and intracellular correlates of organ initiation in the embryonic chick thyroid. Amer. Zool. 13, 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, HOPKINS, M. L. (1935). Development of the thyroid gland in the chick embryo. /. Morph. 58, JANNERS, M. Y. & SEARLS, R. L. (197). 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, MARCHOK, A. C. & HERRMANN, H. (1967). Studies of muscle development. Devi Biol. 15, 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, PICTET, R. L., CLARK, W. R., WILLIAMS, R. H. & RUTTER, W. J. (1972). An ultrastructural analysis of the developing embryonic pancreas. Devi Biol. 29, 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 QUASTLER, H. & SHERMAN, F. G. (1959). Cell population kinetics in the intestinal epithelium of the mouse. Expl. Cell. Res. 17, SANTOIANNI, P. & AYALA, W. (1965). Fluorometric,okramicroanalysis of deoxyribonucleic acid in human skin. /. invest. Derm. 45, SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick limb bud. /. exp. Zool. 166, EMB 48

18 286 M. S. SMUTS, S. R. HILFER AND R. L. SEARLS SHAIN, W. G., HILFER, S. R. & FONTE, V. G. (1972). Early organogenesis of the embryonic chick thyroid. Devi Biol. 28, SPOONER, B. S. & WESSELLS, N. K. (1972). An analysis of salivary gland morphogenesis: Role of microfilaments and microtubules. Devi Biol. 27, WRENN, J. T. & WESSELLS, N. K. (197). Cytochalasin B: Effect upon microfilaments involved in morphogenesis of estrogen-induced glands of oviduct. Proc. natn. Acad. Sci., U.S.A. 66, ZWAAN, J. & HENDRIX, R. W. (1973). Changes in cell and organ shape during early development of the ocular lens. Amer. Zool. 13, 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, ZWILLING, E. (1959). A modified choriollantoic grafting procedure. Trans. Bull. 6, (Received 29 September 1977, revised 21 July 1978).