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Follicle formation in the embryonic chick thyroid. III. Initiation of follicle formation
TISSUE & CELL 1979 ll(4) 727-740 Puhlishrd by Longmrn Group Ltd. Printed in Great Britain.
S. ROBERT HILFER
FOLLICLE FORMATION IN THE EMBRYONIC CHICK THYROID. III. INITIATION OF FOLLICLE FORMATION ABSTRACT. It has been proposed that the follicular spaces in the thyroid form by either the coalescence of intracellular droplets or by separation of cell apices by secretion into the extracellular space. On the basis of examination of thyroid primordia in early chick embryos this study provides evidence that in the chick, at least, follicle formation conforms to the second model. The first indications of chanp in the chick thyroid is the appearance of interdigitations of the cell apices. These interdigitations form microvilli as the two surfaces become separated and the follicular space is established. Vesicles with two types of contents can be identified in proximity with the cell surface during follicle formation, but it is not clear if either the dense particulate or the more electronlucid materials that they contain actually enter the follicular space. Neither removal of the pituitary gland by decapitation nor inhibition of collagen synthesis and a concomitant failure of the invasion of capsular mesenchyme prevents the initiation of normal follicle formation.
recently on the basis of electron microscopic observations (Shepard, 1967, 1968; Calvert, 1972; Calvert and Pusterla, 1973; Cordier et al., 1976; Olin et al., 1970; Remy et al., 1977). The other view, also based on electron microscopy, holds that microvilli first form by interdigitations of plasmalemmae at the point of incipient follicle formation. The follicular space then forms by secretion of material between the two surfaces, thus separating the interdigitated microvilli (Hilfer, 1964; Hilfer et al., 1968; Fujita and Tanizawa, 1966; Michel-Bechet et al., 1973 ; Cau et al., 1976; Tice et al., 1977). The present study of thyroid follicle formation was undertaken to provide more information on the initial opening of the follicular space. The results show that tight junctions and associated interdigitated membranes exist in the embryonic gland before vesicles of any appreciable size are formed. Operations on the embryo in ova and manipulation of the primordium in vitro have shown that neither pituitary hormone nor thyroid mesenchyme is needed for the initiation of follicle formation.
Introduction INVESTIGATIONS on the initiation of thyroid follicle formation in chicken (Hilfer, 1964; Fujita and Tanizawa, 1966), rat (Calvert, 1972; Calvert and Pusterla, 1973 ; Ishikawa, 1965), human (Olin et al., 1970; Shepard, 1968), mouse (Cordier et al., 1976) and rabbit (Michel-Bechet et al., 1973; Roques et al., 1972) in general are in agreement on the later stages of follicle formation. However, differences in interpretation exist on the origins of the follicular space. Two basically different mechanisms have been proposed to account for the formation of an extracellular space between the follicular cell apices. It was proposed on the basis of light microscopic observations that large, intracellular colloid droplets in the apices of adjacent cells coalesce and become extracellular (Bradway, 1929; Waterman and Gorbman, 1935). This point of view has been supported more
Department of Biology, Philadelphia, PA 19122.
Temple
University,
Received 3 January 1979. Revised 20 July 1979. 127
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Materials and Methods Rhode Island Red (Hardy’s Hatchery, Essex, Mass.) and White Leghorn (Shaw’s Hatchery, West Chester, PA) embryos were staged according to the development of the pharyngeal region based upon the normal stages of Hamburger and Hamilton (1951).
thyroids fixed at stages 30-36 for this study. The complete experimental data on the development of the thyroid in the absence of the pituitary is reported elsewhere (Hilfer and Searls, 1979). Only initiation of follicle formation will be considered in this paper. Results
Electron microscopy Thyroids from normal embryos at stages 2836, from decapitated embryos at the same stages, and from organ cultures of stage 25 thyroids in culture for l-3 days were fixed for 15 min or 1 hr, depending on size, at 20°C in 0.1 M phosphate buffered 2% glutaraldehyde, pH 7.2-7.4. The samples were washed in phosphate buffer, postfixed in cold 1 % osmium tetroxide, and embedded in Araldite. Sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. Thick sections were stained with Azure II-methylene blue (Richardson et al., 1960) and photographed with a Zeiss photomicroscope. Organ culture Primordia were removed from stage 25 Rhode Island Red embryos and trimmed of extraneous tissue. Some of the glands were incubated for 5 min in ice-cold trypsin (2.0% Bactotrypsin, Difco, Detroit, Mich.) after which the capsule was removed by gentle pipetting through a capillary pipette of approximately the same bore diameter as the primordium. Either this pure epithelial component of the gland or the intact primordium was placed on a Millipore THWP filter (Bedford, Mass.), covered with agar and incubated at the airmedium interface with Nutrient Medium 199 supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY). The incubator was gassedwith a mixture of 95 y0 02/S % Con. Some cultures were treated with the drugs cc,&-dipyridyl (10 pg/ml) and L-azetidine-2carboxylic acid (20-100 pg/ml) to interfere with collagen synthesis. The methods were described in more detail in Hilfer and Pakstis (1977). Hypophysectomy Embryos missing their pituitary were prepared by the method of Fugo (1940). Embryos in ovo were decapitated at stages 12-14 by pinching with forceps and the
Mature follicles in the chicken embryo are similar in structure to those of other vertebrates. They consist of cells arranged as a single layer around a central cavity, the follicular space, which contains the colloid. The cells exhibit an apical-basal polarity but tend to be cuboidal rather than columnar as they are in mammals. The basal cytoplasm contains extensive cisternae of rough endoplasmic reticulum and the nucleus tends to be positioned at the basal margin of the cell. The Golgi region is supranuclear and the apical cytoplasm contains vesicles of various sizes, some of which contain particulate material of the same texture as the extracellular colloid. Frequently the cells have cilia with two centrioles at their bases. The apical plasmalemma contains many microvilli with a core of microfilaments and flaplike extensions adjacent to the cell junctions. The lateral plasmalemmae are modified near the apex as junctional complexes, in section consisting typically of alternating adherent and occluded zonular junctions and one or more desmosomes. These characteristics of mature follicle cells will be used in the identification of developing follicles. All of the characteristics do not become visible at the same developmental stage. The sites of future thyroid follicles first become recognizable in both Rhode Island Red and White Leghorn embryos after 7 days of incubation, at stage 30 (Hamburger and Hamilton, 1951). During this stage, only the earliest, or incipient, follicles are identifiable; during subsequent development the thyroid contains a mixture of incipient follicles and follicles with increasingly larger lumina. The same pattern of follicles with an enlarging follicular space interspersed between early stages of follicle formation is seen in embryos that are hypophysectomized. A similar temporal sequence in the state of follicular development is found in organ cultures but the appearance of both incipient and more mature follicles occurs earlier than in ova.
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Follicles formed in organ culture in the same developmental sequence as in ovo whether or not mesenchyme was present and whether or not the primordia were treated with drugs that interfere with collagen synthesis. The apicobasal polarity of the thyroid epithelial cells is recognizable by stage 29, before mesenchyme invades (Fig. I), as a result of the formation of fibril-filled channels between rows of epithelial cells (Hilfer and Pakstis, 1977). The cellular polarity becomes even more pronounced when the epithelial primordium is penetrated by mesenchymal cells and capillaries late in stage 29. As a result, the epithelial mass becomes subdivided into a number of cords or plates approximately two cells thick, which are bounded by connective tissue partitions (Fig. 2). At these stages the cells exhibit extensive interdigitation (Fig. 3) but such folds of the cell surface differ in several ways from the follicular surfaces of later developmental stages. The surface projections vary in size and shape, do not contain a core of microfilaments, and the cells do not have tight junctions in the vicinity of the folds. The apical cell surface and the initiation of follicles Microvilli The first profiles that can be recognized as exclusively apical specializations are found in stage 30 embryos, or in stage 25 thyroids after 18 hr in organ culture (Figs. 4-S). The apical surfaces are interdigitated as coarse microvilli of almost uniform diameter; these microvilli contain a core of microfilaments (50-70 8, diameter) that are oriented parallel to the microvillar axis (Fig. 4B). The region of interdigitation is delimited by a specialized junction that rings the microvilli. The interdigitated surfaces vary in size and in whether the two surfaces are closely apposed or are separated by a small space. The differences in size are taken to represent differences in the plane of section through the cell apices whereas the differences in the amount of extracellular space probably represent slight differences in developmental age. Of the many images in which the plasma membranes of the microvilli are separated by distances of just a few hundred angstrom, only a few were cut to show longitudinal profiles of microvilli lying parallel to each
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other (Fig. 4a). As many as 15 interdigitated microvilli can be found in a section through a single region of interdigitation. The outer leaflet of the microvillar plasmalemmae contains added density that fills the narrow gaps between microvilli (Fig. 4b). Sections at different angles in planes perpendicular to the plane of this figure would result in a variety of arrangements of the microvilli. Some of these must be represented in Figs. 5-8. Whereas the sections through the plane of Fig. 4 pass through the apices of only two cells, either two or three cells participate in the interdigitations in other profiles. When two cell apices are involved and the microvilli cut longitudinally, the microvilli either extend the same distance into a pocket in the cytoplasm of each cell apex (Fig. 5) or the extracellular space is excentrically located in the cell apices (Fig. 6). Sections through three cells in which the microvilli are cut mostly longitudinally have a somewhat three-cornered appearance with the microvilli seeming to cross (Fig. 7). Sections at some angle to the longitudinal axis of the microvilli should produce a mixture of cross and tangential sections of microvilli (Fig. 8). Sections through all of these planes should pass through the adherent junctions more or less in cross-sections. In contrast, a crosssection through microvilli oriented as in Fig. 4 should give the impression either that the microvilli are within the cytoplasm or the apical plasmalemma should be included in a grazing section (Figs. 1 I, 12). The second type of profile that is seen at thyroid cell apices in embryos older than stage 30 and after 1 day in organ culture consists of small, microvillous-filled spaces between the apical cell surfaces. These spaces are at the limit of resolution with the light microscope (Fig. 9). These small follicles (Fig. 10) tend to have a greater diameter than the incipient follicles as a result of the larger extracellular space. Some of the variability in shape and in number of microvilli undoubtedly is due to the plane of section relative to the center or the edge of the follicular space. The microvilli appear to be directed toward the center of the follicular space and to cross. The microvilli are the same size and shape as in the initial stages of closely interdigitated microvilli. Since sections are not found that contain only longitudinal profiles of microvilli, it is likely that
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Fig. 1. Light micrograph of the subcapsular region of a thyroid from a stage 29Rhode Island Red embryo. Mesenchyme is not visible beneath the surface of the primordium; yet channels (arrows) exist between these epithelial cells. x 1350. Fig. 2. Light micrograph of a portion of a thyroid from a stage 30 Rhode Island Red embryo. Erythrocytes (E) serve as markers for the capillaries and fibroblasts that have invaded from the capsule (C). The epithelial cells are aligned as double rows (cords) and small islands (plates). x 560. Fig. 3. Electron micrograph of small portions of several cells from the thyroid of a White Leghorn embryo at stage 29. The cells are highly interdigitated but lack specialized junctions. x 26,000. Fig. 4. Electron micrograph of two cell apices in a stage 25 thyroid primordium that was stripped of its capsule and placed in organ culture for 18 hr. (a) The apposed surfaces of the epithelial cells possess a regular pattern of folds that are recognizable as microvilli (M). The interdigitated region is delimited by an extensive adherent junction. Interdigitations of the sort illustrated in Fig. 3 also are visible (arrow). The cytoplasm apical to the nucleus (N) contains parallel Golgi cisternae (G), multivesiculate bodies (MVB), and vesicles with dense (D) and lucent (L) contents. A portion of a centriole is visible in the lower cell. x 32,500. (b) Enlargement of the region in Fig. 4a labelled B. The microvilli and adjacent cortical cytoplasm contain microfilaments. x 82,200. (c) Enlargement of the region in Fig. 4a labelled C. The particulate material in the dense vesicles shows no apparent order. x 82,000. Fig. 5. Junctional region of two cells from an intact stage 25 thyroid after 24 hr in culture. The section passed through only a few microvilli, all cut longitudinally. Centrioles and vesicles with pale contents are in close proximity to the microvilli. x 33,000. Fig. 6. Junctional region of two cells from a stage 25 thyroid after 24 hr in culture medium containing 50 pg/ml LACA. The mixture of tangentially and longitudinally sectioned microvilli seems to occupy a depression in only one cell surface. A cilium (C) in a cytoplasmic pocket is visible in cross-section. x 22,500. Fig. 7. The junctional region of three cells in a stage 25 thyroid which was cultured with its capsule for 18 hr. Most of the microvilli were cut at an angle, but microfilaments are visible in their cores. The involvement of three cells in this incipient follicle is recognized on the basis of three regions of adherent junctions. x 26,000. Fig. 8. Apical region of three cells from a normal Rhode Island Red thyroid at stage 35 (8) days). Most of the microvilli were cut in cross-section with portions of two cilia (C) lying among the microvilli. Two centrioles (arrows) are present in the cell at the lower right. Many small vesicles with contents of different densities surround the Golgi region at the lower left. x 31,000. Fig. 9. Light micrograph of a small portion of the central region of a stage 32 Rhode Island Red thyroid (7+ days). This represents the earliest time at which follicles (F) can be positively identified by light microscopy. Cilia are visible as line lines within the follicular spaces. x 1260. Fig. 10. Small follicle from a stage 35 Rhode cell on the right appears to loop out of and back villi or flaps of cytoplasm are associated with the contains many vesicles with both electron-dense
Island Red thyroid. The cilium of the into the plane of section. Long microjunctional zones. The apical cytoplasm and lucid contents. x 17,000.
Fig. 1I. A small follicle in a stage 25 thyroid that was fixed after 3 days in nutrient medium containing 50pg/ml LACA. Although the microvilli may seem to be within the cell, the participation of two cell apices is evident from the interruption of the adherent junction (arrow). Portions of two cilia are visible, one in the follicular space and the other within a cytoplasmic pocket. The cytoplasm contains a large number of vesicles with dense contents. x 20,000.
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the orientation of the microvilli changes as the extracellular space expands. The microvilli adjacent to the junctional region frequently have a shape and length reminiscent of the marginal cytoplasmic flaps in mature follicles (Fig. 10). Follicles in this size range could be sectioned in the plane of the junctional zone, thus giving the impression that the follicular space is intracellular (Fig. I I), similar to those of the initial stages of follicle formation described above. Microvilli become a less prominent feature of the follicular space in later stages of development (Figs. 12, 14). Cilia A common characteristic of the earliest detectable follicles and a means of identifying incipient follicles is the presence of centrioles near the apical cell surface or of a cilium lying between the interdigitated microvilli. Sections frequently pass through a centriole or cilium in one cell of the incipient follicle and often the second cell also is sectioned through a part of either a centriole or cilium (Figs. 5, I I, 13). Usually a single centriole is visible but in a few sections a second centriole lies in the plane of section. Cilia commonly are included within pockets in the cytoplasm or extend into the follicular space (Figs. 8, 10, I I). The central pair of tubular subunits frequently is absent (Figs. 6, 10). The preponderance of cell apices that possess either centrioles or cilia for each cell participating in an early stage of follicle formation indicates that these structures are a constant feature of the follicles. Centrioles and cilia are seen as frequently at later stages in larger follicles (Figs. 12, 14). The sampling problems with the larger structures make it difficult to ascertain how many cells comprising each follicle possess a cilium, however. Cytoplasmic
Polarizution
organization
qf‘organelles
Polarization of cytoplasmic organelles is not as distinct at early developmental stages as it is at later stages because the endoplasmic reticulum is sparse (Fig. 4). The cells tend to be polygonal, with small portions of the cytoplasm of one cell inserting between the bulk of adjacent cells. Thus, it is difficult to trace one cell from base to apex in thin sections. Many of the apical cell regions appear trun-
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cated, with two faces meshing with another row of cells. The nuclei tend to be basal or to one side. A few short channels of endoplasmic reticulum lie in the basal cytoplasm near the nucleus. An extensive network of rough endoplasmic reticulum is not seen until much later stages (10 days, Hilfer, 1964). Golgi lamellae and vesicles invariably are supranuclear and sometimes form more than one stack of channels. The apical cytoplasm contains vesicles of different sizes and densities, microtubules, and microfilaments. Apical vesicles During the initial stages of follicle formation, the apical cytoplasm contains large numbers of vesicles with contents that preserve differently. Basically, they fall into two main groups; those with dense, particulate contents and those with little electron density. The vesicles with dense contents tend to have a greater size range than the others. For reasons that are not obvious, the contents stain more intensely in cells from organ culture than in thyroids from embryos i/z ovo but the cells contain comparable numbers of vesicles under all conditions that were examined. The particulate contents do not appear to be crystalline (Fig. 4~). Material of the same particle size and density is found in vesicles of various sizes and shapes (Figs. 4, 6, II, 12, 17). A few vesicles with material of similar density, primarily in cells in culture, also contain membrane whorls or smaller vesicles (Figs. 4, 6). It is possible that these vesicles represent dense bodies (primary lysosomes) and autophagic vacuoles (secondary lysosomes). However, without appropriate identification, the possibility cannot be excluded that the contents represent a secretory product destined to enter the follicular space. Dense vesicles are less numerous in the cells of larger follicles and tend to be smaller (Figs. 14, 15). The vesicles with pale contents exhibit less variation in size and are present in larger numbers between the Golgi lamellae and the cell surface than the dense vesicles. In contrast to the dense vesicles, some pale vesicles lie close to the plasmalemma and appear continuous with the plasma!emma from the earliest stages of follicle formation (Figs. 5. 8, 10, 12). The pale vesicles contain fine material that stains much less intensely than the particulate material of the dense vesicles
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(Figs. 14c, 1%). It is possible that some of the contents are lost during tissue preparation, especially since there is some variation in the amount of material found in different vesicles. The number of vesicles in follicle cells at different stages of development shows no apparent change.
common along lateral surfaces of follicle cells at later stages. Other specialized junctions are associated with follicles having larger extracellular spaces. Incipient desmosomes are first seen in follicles with small lumina, sometimes as plaques without tonofilaments (Fig. 16).
Cell junctions
Microfilaments
Extensive adherent junctions are a prominent feature of the early stages in follicle formation. The junctions surrounding the region of microvillar interdigitation consist ofrelatively straight, parallel membranes separated by a gap of approximately 200 A and with increased density along the inner membrane leaflet (e.g. Figs. 4-7). Irregular interdigitations characteristic of prefollicular stages (Fig. 3) often exist peripheral to the adherent junction-microvillar complex (Fig. 4a). lnterdigitations of this sort also are
Microfilaments (SO-70 A in diameter) form a core of parallel filaments within the microvilli from the time the microvilli first appear (Figs. 4-7). The bundle is oriented parallel to the longitudinal axis of the microvillus. Additional microfilaments are found in the apical cytoplasm apparently continuous with the core filaments. In larger follicles these apical filaments tend to be visible as bundles that penetrate somedistance into thecell body (Fig. 17). Additional filaments in follicle cells at all stages lie in the cortical cytoplasm in no
and microtubules
Fig. 12. (a) Another follicle which appears superficially to be within a single cell, found in a normal stage 35 White Leghorn embryo. Adjacent sections show that this is a tangential section of a follicular space between at least two cells. A small amount of fine, filamentous material is present within the follicular space and the cytoplasm contains vesicles with lucent and dense contents. x 19,750. (b) Enlargement of the upper right corner of (a). The membrane of a dense vesicle is continuous with the plasmalemma (arrow) and several vesicles with finer material (L) lie close to the surface. The figure also contains a profile of a microtubule (M) and microfilament (MF). x 59,000. Fig. 13. A small follicle in a thyroid from a normal embryo. Microtubules are associated with the centrioles
stage 30+ Rhode Island Red (C) in both cells. x 14,100.
Fig. 14. A larger follicle from a normal stage 35 White Leghorn embryo. (a) The microvilli are a less prominent component of the follicular space than in smaller follicles and the space contains more of the fibrillar and granular components. A large membranous whorl with filamentous contents is also visible. The junctional regions possess cytoplasmic ‘flaps’ extending into the follicular space but no new specialized attachment site is visible along the lateral cell surfaces. The section passed close to the surface of a cilium (C). x 13,600. The lumen contains clumps of dense granules with radiating filaments (G), enlarged in (b). The apical cytoplasm contains pale vesicles (L) with fine filaments that are enlarged in (c). (b) and (c) x 57,000. Fig. 15. Part of a follicle in the thyroid of a White Leghorn embryo that was decapitated at stage 14 and fixed at stage 36 (10 days). (a) Although the follicular space is smaller than in the previous illustration, the space contains a greater concentration of dense material (arrows, enlarged in (b)). The apical cytoplasm contains a few small vesicles with dense contents (D) and paler vesicles with filamentous contents (L), enlarged in (c). The base of a cilium is included in the section. (a) x 19,000; (b) and (c) x 66,500. Fig. 16. Junctional region of a larger follicle at stage 35. (a) The density on the cytoplasmic side at the desmosome is not clearly organized. The follicular space contains fine strands and clumps of dense granules. x 65,500. (b) Enlargement of several clumps of dense granules within the same follicular lumen. x 80,000.
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Fig. 17. A smaller follicle from a stage 25 thyroid primordium after culture for 24 hr without its mesenchymal component. Microfilaments of the microvilli and of the cortical cytoplasm are especially prominent, perhaps as a result of the plane of section. Microtubules are associated with the centrioles in the cell to the left. Some vesicles with dense contents lie close to the plasmalemma. x 36,600.
apparent orientation with respect to the surface. The identification of microfilaments depends to a large extent upon the plane of section since they cannot be recognized in cross-section. Since sufficient landmarks do not exist for determining the orientation of follicle cells in thin sections at early stages, the question of the distribution of microfilaments cannot be answered from the present study. Microtubules can be found at all levels within follicle cells, from the earliest developmental stage. Frequently tubules are seen close to the apical cell surface (Figs. 6, 12-15, 17). In only some of these is a centriole in the
vicinity (Figs. 13, 17), with the microtubules radiating from it. In the other sections, the orientation ranges from perpendicular to the apical surface to parallel with it. The follicular space In incipient follicles, the extracellular space is limited to a few hundred angstrom between the microvillar surfaces (Figs. 4-7). The extracellular space contains fine material that probably is a continuation of the plasmalemma1 surface coat (Fig. 4b). In follicles with a slightly larger extracellular space, the expanded lumen has a density equivalent to
THYROID
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the contents of the paler apical vesicles (Figs. 1l-15). Almost all of the material that is electron dense is associated with microvillar surfaces and consists of fine strands (Fig. 12b). Follicles with larger lumina than these contain similar fine strands and packets of dense particles (Figs. 14b, 16). In these larger lumina the particulate material is not confined to the surface of microvilli but forms loose strands throughout the extracellular space (Figs. 1417). The amount of extracellular material varies more or less with the size of the follicular lumen, from these loose meshworks (Figs. 1Sb, 16b) in smaller follicles to the dense accumulations that fill the lumina of mature follicles. A small number of follicles with larger lumina are seen as early as stage 35 (S$ days) in both normal and decapitated embryos. Cultures of stage 25 (4f5 days) thyroids, whether treated with drugs or incubated with or without the mesenchymal capsule, form a few larger follicles as early as 24 hr after explantation. Substantial numbers of these larger follicles do not appear before IO days in ova (Stage 36) or 48 hr in vitro. Even in these older thyroids many immature stages of follicle formation are found among the larger follicles. The follicular lumina change in shape from flattened spaces between two cell apices through cavities with scalloped margins to the rounded lumina of mature follicles. The intermediate sized follicles tend to have the irregularly shaped lumina with contents of intermediate packing of the particulate material. These follicular lumina also contain accumulations of membranes either as single layers (Fig. 15) or as multiple layers (Fig. 14). It is not known if these membrane profiles are formed during expansion of the follicular space or result from inadequate fixation of the follicular contents (Hasty and Hay, 1978).
Discussion A difference of opinion has arisen over the way in which thyroid follicle formation is initiated. Two basically different mechanisms have been proposed to describe the formation of the extracellular space. A possible reason for the different points of view is interpretation of electron microscope images. The obvious dangers in interpreting isolated
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images of thin sections in terms of threedimensional structure, when the plane of section cannot be determined, has been pointed out in previous studies. Thus, a section that appears to show an intracellular droplet may in reality be a section cut parallel to an apical surface that contained a depression. Furthermore, there are dangers in using an older embryonic stage and selecting images to construct a developmental sequence without checking the earlier stages, as was done by Cordier et al. (1976). Calvert and Pusterla (1973) ‘intracellular canaliculi’ are associated with plasmalemmae and may actually be tangential sections of extracellular spaces. Apparently, serial sections did not reveal any droplets large enough to correspond with the cytoplasmic droplets of light microscopy. Light microscopic images that have been interpreted as fusion of apical colloid droplets (Bradway, 1929; Waterman and Gorbman, 1935) probably represent the later period of expanding follicular size during which the small follicular space takes on a scalloped appearance (Fujita and Tanizawa, 1966; Hilfer, 1964, 1969; Hilfer and Hilfer, 1966). In this study thyroids were investigated under several conditions that allowed images of the first stages of follicle formation to be obtained in the absence of later stages. Under these conditions the largest number of profiles consisted of interdigitated coarse microvilli that were continuous with the cell surface. The few profiles that gave the superficial appearance of being intracellular on closer inspection either contained a tangentially sectioned adherent junction or such junctions appeared at the margin of the extracellular space when such ‘follicles’ were traced through adjacent sections. Thus, in the chick embryo, interdigitations of the apical plasmalemma associated with specialized junctions preceded the formation of any sizeable droplets in the apical cytoplasm. The absence of large droplets from the cell apex at early stages appears to be characteristic of the rat thyroid also (Tice et al., 1977). There is one welldocumented case of follicle formation proceeding from intracellular lumina (Remy et al., 1977). In porcine thyroid cells large, intracellular vesicles containing colloid can be demonstrated before multicellular follicles are formed. It is possible that the mechanism of follicle formation differs in different species. An alternate possibility is that
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isolated cells from mature follicles in culture differ in their morphogenetic pattern when reforming follicles as compared with the process in the early embryo. Processes similar to the opening of the follicular lumen occur in other developing organs. Some organs begin as solid primordia which secondarily hollow out. Examples include cavitation of the caudal portion of the chick neural tube (Criley, 1969) formation of bile canaliculi in the liver (Wood, 1965), and hollowing of nephric tubules (Saxen and Wartiovaara, 1966). In the chick embryo the esophagus at first is an open tube but becomes occluded only to open again before hatching commences (Allenspach, 1964, 1966). These processes of cavity formation have been suggested to occur either by erosion of the cell apices or by secretion of material into the extracellular space at the bases of coarse microvilli. In all cases the cell apices are joined by tight junctions, the apical membranes interdigitate, and the apical cytoplasm contains numerous small vesicles. In some cases, the cells have been reported to contain increased numbers of lysosomes (Saxen and Wartiovaara, 1966; Wood, 1965). Opening of the esophagus before hatching also corresponds to a time of increased activity of lysosomal enzymes (Wilson and Allenspach, 1974). At present experimental evidence is lacking on the method by which the extracellular spaces expand. The images of early thyroid follicles, showing the complete range from no extracellular space between interdigitated membranes to round follicular spaces, give the impression that expansion is gradual. It is possible that two types of material are secreted during follicle formation, based upon the difference in appearance of the extracellular lumen at early stages and at later stages of development. The electron-lucent contents of the small follicles that are found at early developmental stages slowly changes to a particulate component in the larger follicular spaces. Vesicles with two types of contents are seen in the apical cytoplasm at these stages. Many of the small vesicle have electron-lucent contents. Larger, irregularly shaped vesicles with a dense, particulate content are present in the apical cytoplasm of small follicles but are present in much reduced numbers in the apical cytoplasm of cells in larger follicles. We suggested pre-
HILFER
viously that these dense bodies might be lysosomes (Hilfer, 1964; Hilfer et al., 1968) but the appropriate cytochemistry to demonstrate acid hydrolases has not been done. The increased number of dense bodies and concomitant increase in acid phosphatase activity during reopening of the developing esophagus (Wilson and Allenspach, 1974) is suggestive of a similar process during expansion of the follicular space and resulting in the scalloped cell surfaces. It seems odd, however, for lysosomal enzymes to be secreted into the follicular space. Perhaps some or all of the dense material in the larger vesicles of the smaller follicles is an early form of colloid. This question will not be resolved until the pathway of synthesis and secretion is followed by labelling techniques. The types of junctions that form between early follicle cells may differ in chicken and rat. In the rat (Tice et al., 1977) it has been shown by freeze-fracture and tracer studies that occluded junctions form around the earliest interdigitated membranes. The earliest junctions consist of only a few furrows constructed of incompletely fused particles. Yet they exclude the tracer, horseradish peroxidase, and must be functional in preventing at least some loss of follicular contents. In the chick embryo, the simplest interdigitations and smallest follicles are associated only with adherent junctions. Whether or not these junctions are sufficient to contain the material in the extracellular space has not been tested so far. Only the larger follicles have a junctional complex consisting of occluded zones and desmosomes in addition to adherent zones. The question of what event causes initiation of follicle formation remains unanswered. Follicles are not found until mesenchyme invasion occurs at stage 30 (Shain et al., 1972). In the absence of mesenthyme or in the presence of inhibitors of collagen synthesis that prevent formation of the fibrillar material within the epithelial component of the gland, follicle initiation occurs normally (Hilfer and Pakstis, 1977). Thus, neither the mesenchyme nor the presumed epithelial collagen that precedes its invasion can be responsible for initiation of follicle formation. Similarly, TSH need not be present since follicle formation proceeds in the absence of the pituitary. It is not clear, however, if some pituitary hormones might
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be provided by the yolk in ovo in decapitated embryos or by the serum in organ culture. At present it appears that the control is intrinsic to the epithelial component of the primordium, itself.
Acknowledgements This work was supported by grant no. PCM 77 17686 and its predecessors from the National Science Foundation.
References ALLENSPACH,A. L. 1964. Experimental analysis of closure and reopening of the esophagus in the developing chick. J. Morph., 114, 287-302. ALLENSPACH, A. L. 1966. The reopening process of the esophagus in the normal chick and the crooked neck dwarf mutant. J. Embryol. exp. Morph., 15, 67-76. BRADWAY,W. 1929. The morphogenesis of the thyroid follicles of the chick. Ann?. Rec., 42, 157-167. CALVERT, R. 1972. Electron microscopic observations on the contribution of the ultimobranchial bodies to thyroid histogenesis in the rat. J. Amt., 133, 269-288. CALVERT, R. and PUSTERLA,A. 1973. Formation of thyroid follicular lumina in rat embryos studied with serial fine sections. Gen. camp. Endow.. 20, 584-597. CAU. P., BECHET-MICHEL, M. and FAYET, G. 1976. Morphogenesis of thyroid follicles in virro. Adv. Amt.. 52, l-66.
CORUIER, A. C., DENEF, J.-F. and HAUMONT, S. M. 1976. Ultrastructure
of the thyroid
in dwarf mice. J. Annt.,
146. 339-357.
CRILEY, B. B. 1969. Analysis of the embryonic sources and mechanisms of development of posterior levels of chick neural tubes. J. Morph., 128, 465-502. Fucjo, N. W. 1940. Effects of hypophysectomy in the chick embryo. J. exp. Zool., 85, 271-297. FWITA. H. and TANIZAWA, Y. 1966. Electron microscopic studies on the development of the thyroid gland of chick embryo. Amt. Entwickl.-Gesch.. 125. 132-151. HAMBURGER,V. and HAMILTON, H. 1951. A series of normal stages in the development of the chick embryo. J. Morph.,
88, 49-92.
HASTY, D. L. and HAY, E. D. 1978. Freeze-fracture studies of the developing cell surface. IT. Particle-free membrane blisters on glutaraldehyde-fixed cornea] fibroblasts are artefacts. J. Cell Biol., 78, 756-768. HILFER, S. R. 1964. Follicle formation in the embryonic chick thyroid. I. Early morphogenesis. J. Morph,. 115. 135-152.
HILFER. S. R. and HILFER, E. K. 1966. Effects of dissociating agents on the fine structure of embryonic chick thyroid cells. J. Morph., 119. 217-231. HII.FER, S. R., TSZARDL. B. and HILFER, E. K. 1968. Follicle formation in the embryonic chick thyroid. Il. Reorganization after dissociation. Z. Zellforsch. mikrosk. Amt., 92, 256-269. HILFER, S. R. and PAKSTIS, G. L. 1977. Interference with thyroid histogenesis by inhibitors of collagen synthesis. J. Cell Biol., 75, 446-463. HILFER, S. R. and SEARLS,R. L. 1979. Differentiation of the thyroid in the hypophysectomized chick embryo, De~a. Biol. (submitted). ISHIKAWA, K. 1965. Electron microscopic studies of the thyroid gland of the rat in embryonic life. Okajimas Fol. Amt. Jap.. 41, 295-31 I. OLIN. P.. EKHOLM, R. and ALMQVIST,S. 1970. Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology, 87, 1000-1014. MICHEL-BECHET, M., CAU, P., FAYET, G. and COURRIER, R. M. 1973. Etude ultrastructurale de la morphogenese du follicule thyroiden: reorganisation des cellules thyroidiennes de port en culture. C. Y. h&d SPanc. Acad. Sci. Paris., 277, 1029-1032. R~MY, L., MICHEL-BECHET, M., CATALDO, C., BOTTINI, J., HOVSEPIAN, S. and FAYET, G. 1977. The role of intracellular lumina in thyroid cell for follicle morphogenesis itr vitro. J. UNrtrtrsttrccf. Res., 61, 243-253. RICHARDSON,K. C., JARETT, L. and FINKE, E. M. 1960. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Tech&., 35, 313-323. R~Q~J~s, M. J., TORRESANI, M., BEQUET, M. and LISSITSKY, S. 1972. Relationship between thyroglobulin synthesis, iodine metabolism and hisrogenesis in the developing rabbit fetal thyroid gland. ~ornp. Endowi%,
19, 457-472.
SAXON,L. and WARTIOVAARA,J. 1966. Cell contact 271 290.
and cell adhesion
during tissue organization.
J. Cancc,r. I,
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HILFER
SHAIN, W. C., HILFER, S. R. and FONTE, V. G. 1972. Early organogenesis of the embryonic chick thyroid. I. Morphology and biochemistry. DA. Biol., 28, 202-218. SHEPARD, T. H. 1967. Onset of function in the human fetal thyroid: biochemical and radioautographic studies from organ culture. J. clin. Endocrin. Metab., 21, 945-958. SHEPARD, T. H. 1968. Development of the human fetal thyroid. Gen. camp. Endocrin., 10, 174-184. TICE, L. W., CARTER, R. L. and CAHILL, M. C. 1977. Tracer and freeze-fracture observations on developing tight junctions in fetal rat thyroid. Tissue & Cell, 3, 395-417. WATERMAN,A. and GORBMAN, A. 1935. Development of the thyroid gland of the rabbit. J. exp. Zoo/., 132, 509-53s. WILSON, J. R. and ALLENSPACH, A. .L 1974. The role of acid phosphatase and fi-glucuronidase in reopening and remoulding of the developing chick esophagus. Devl. Biol., 41, 288-300. WOOD, R. L. 1965. An electron microscope study of developing bile canaliculi in the rat. Anat. Rec., 151, 507-530.
S. ROBERT HILFER
FOLLICLE FORMATION IN THE EMBRYONIC CHICK THYROID. III. INITIATION OF FOLLICLE FORMATION ABSTRACT. It has been proposed that the follicular spaces in the thyroid form by either the coalescence of intracellular droplets or by separation of cell apices by secretion into the extracellular space. On the basis of examination of thyroid primordia in early chick embryos this study provides evidence that in the chick, at least, follicle formation conforms to the second model. The first indications of chanp in the chick thyroid is the appearance of interdigitations of the cell apices. These interdigitations form microvilli as the two surfaces become separated and the follicular space is established. Vesicles with two types of contents can be identified in proximity with the cell surface during follicle formation, but it is not clear if either the dense particulate or the more electronlucid materials that they contain actually enter the follicular space. Neither removal of the pituitary gland by decapitation nor inhibition of collagen synthesis and a concomitant failure of the invasion of capsular mesenchyme prevents the initiation of normal follicle formation.
recently on the basis of electron microscopic observations (Shepard, 1967, 1968; Calvert, 1972; Calvert and Pusterla, 1973; Cordier et al., 1976; Olin et al., 1970; Remy et al., 1977). The other view, also based on electron microscopy, holds that microvilli first form by interdigitations of plasmalemmae at the point of incipient follicle formation. The follicular space then forms by secretion of material between the two surfaces, thus separating the interdigitated microvilli (Hilfer, 1964; Hilfer et al., 1968; Fujita and Tanizawa, 1966; Michel-Bechet et al., 1973 ; Cau et al., 1976; Tice et al., 1977). The present study of thyroid follicle formation was undertaken to provide more information on the initial opening of the follicular space. The results show that tight junctions and associated interdigitated membranes exist in the embryonic gland before vesicles of any appreciable size are formed. Operations on the embryo in ova and manipulation of the primordium in vitro have shown that neither pituitary hormone nor thyroid mesenchyme is needed for the initiation of follicle formation.
Introduction INVESTIGATIONS on the initiation of thyroid follicle formation in chicken (Hilfer, 1964; Fujita and Tanizawa, 1966), rat (Calvert, 1972; Calvert and Pusterla, 1973 ; Ishikawa, 1965), human (Olin et al., 1970; Shepard, 1968), mouse (Cordier et al., 1976) and rabbit (Michel-Bechet et al., 1973; Roques et al., 1972) in general are in agreement on the later stages of follicle formation. However, differences in interpretation exist on the origins of the follicular space. Two basically different mechanisms have been proposed to account for the formation of an extracellular space between the follicular cell apices. It was proposed on the basis of light microscopic observations that large, intracellular colloid droplets in the apices of adjacent cells coalesce and become extracellular (Bradway, 1929; Waterman and Gorbman, 1935). This point of view has been supported more
Department of Biology, Philadelphia, PA 19122.
Temple
University,
Received 3 January 1979. Revised 20 July 1979. 127
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Materials and Methods Rhode Island Red (Hardy’s Hatchery, Essex, Mass.) and White Leghorn (Shaw’s Hatchery, West Chester, PA) embryos were staged according to the development of the pharyngeal region based upon the normal stages of Hamburger and Hamilton (1951).
thyroids fixed at stages 30-36 for this study. The complete experimental data on the development of the thyroid in the absence of the pituitary is reported elsewhere (Hilfer and Searls, 1979). Only initiation of follicle formation will be considered in this paper. Results
Electron microscopy Thyroids from normal embryos at stages 2836, from decapitated embryos at the same stages, and from organ cultures of stage 25 thyroids in culture for l-3 days were fixed for 15 min or 1 hr, depending on size, at 20°C in 0.1 M phosphate buffered 2% glutaraldehyde, pH 7.2-7.4. The samples were washed in phosphate buffer, postfixed in cold 1 % osmium tetroxide, and embedded in Araldite. Sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. Thick sections were stained with Azure II-methylene blue (Richardson et al., 1960) and photographed with a Zeiss photomicroscope. Organ culture Primordia were removed from stage 25 Rhode Island Red embryos and trimmed of extraneous tissue. Some of the glands were incubated for 5 min in ice-cold trypsin (2.0% Bactotrypsin, Difco, Detroit, Mich.) after which the capsule was removed by gentle pipetting through a capillary pipette of approximately the same bore diameter as the primordium. Either this pure epithelial component of the gland or the intact primordium was placed on a Millipore THWP filter (Bedford, Mass.), covered with agar and incubated at the airmedium interface with Nutrient Medium 199 supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY). The incubator was gassedwith a mixture of 95 y0 02/S % Con. Some cultures were treated with the drugs cc,&-dipyridyl (10 pg/ml) and L-azetidine-2carboxylic acid (20-100 pg/ml) to interfere with collagen synthesis. The methods were described in more detail in Hilfer and Pakstis (1977). Hypophysectomy Embryos missing their pituitary were prepared by the method of Fugo (1940). Embryos in ovo were decapitated at stages 12-14 by pinching with forceps and the
Mature follicles in the chicken embryo are similar in structure to those of other vertebrates. They consist of cells arranged as a single layer around a central cavity, the follicular space, which contains the colloid. The cells exhibit an apical-basal polarity but tend to be cuboidal rather than columnar as they are in mammals. The basal cytoplasm contains extensive cisternae of rough endoplasmic reticulum and the nucleus tends to be positioned at the basal margin of the cell. The Golgi region is supranuclear and the apical cytoplasm contains vesicles of various sizes, some of which contain particulate material of the same texture as the extracellular colloid. Frequently the cells have cilia with two centrioles at their bases. The apical plasmalemma contains many microvilli with a core of microfilaments and flaplike extensions adjacent to the cell junctions. The lateral plasmalemmae are modified near the apex as junctional complexes, in section consisting typically of alternating adherent and occluded zonular junctions and one or more desmosomes. These characteristics of mature follicle cells will be used in the identification of developing follicles. All of the characteristics do not become visible at the same developmental stage. The sites of future thyroid follicles first become recognizable in both Rhode Island Red and White Leghorn embryos after 7 days of incubation, at stage 30 (Hamburger and Hamilton, 1951). During this stage, only the earliest, or incipient, follicles are identifiable; during subsequent development the thyroid contains a mixture of incipient follicles and follicles with increasingly larger lumina. The same pattern of follicles with an enlarging follicular space interspersed between early stages of follicle formation is seen in embryos that are hypophysectomized. A similar temporal sequence in the state of follicular development is found in organ cultures but the appearance of both incipient and more mature follicles occurs earlier than in ova.
THYROID
FOLLICLE
FORMATION
Follicles formed in organ culture in the same developmental sequence as in ovo whether or not mesenchyme was present and whether or not the primordia were treated with drugs that interfere with collagen synthesis. The apicobasal polarity of the thyroid epithelial cells is recognizable by stage 29, before mesenchyme invades (Fig. I), as a result of the formation of fibril-filled channels between rows of epithelial cells (Hilfer and Pakstis, 1977). The cellular polarity becomes even more pronounced when the epithelial primordium is penetrated by mesenchymal cells and capillaries late in stage 29. As a result, the epithelial mass becomes subdivided into a number of cords or plates approximately two cells thick, which are bounded by connective tissue partitions (Fig. 2). At these stages the cells exhibit extensive interdigitation (Fig. 3) but such folds of the cell surface differ in several ways from the follicular surfaces of later developmental stages. The surface projections vary in size and shape, do not contain a core of microfilaments, and the cells do not have tight junctions in the vicinity of the folds. The apical cell surface and the initiation of follicles Microvilli The first profiles that can be recognized as exclusively apical specializations are found in stage 30 embryos, or in stage 25 thyroids after 18 hr in organ culture (Figs. 4-S). The apical surfaces are interdigitated as coarse microvilli of almost uniform diameter; these microvilli contain a core of microfilaments (50-70 8, diameter) that are oriented parallel to the microvillar axis (Fig. 4B). The region of interdigitation is delimited by a specialized junction that rings the microvilli. The interdigitated surfaces vary in size and in whether the two surfaces are closely apposed or are separated by a small space. The differences in size are taken to represent differences in the plane of section through the cell apices whereas the differences in the amount of extracellular space probably represent slight differences in developmental age. Of the many images in which the plasma membranes of the microvilli are separated by distances of just a few hundred angstrom, only a few were cut to show longitudinal profiles of microvilli lying parallel to each
129
other (Fig. 4a). As many as 15 interdigitated microvilli can be found in a section through a single region of interdigitation. The outer leaflet of the microvillar plasmalemmae contains added density that fills the narrow gaps between microvilli (Fig. 4b). Sections at different angles in planes perpendicular to the plane of this figure would result in a variety of arrangements of the microvilli. Some of these must be represented in Figs. 5-8. Whereas the sections through the plane of Fig. 4 pass through the apices of only two cells, either two or three cells participate in the interdigitations in other profiles. When two cell apices are involved and the microvilli cut longitudinally, the microvilli either extend the same distance into a pocket in the cytoplasm of each cell apex (Fig. 5) or the extracellular space is excentrically located in the cell apices (Fig. 6). Sections through three cells in which the microvilli are cut mostly longitudinally have a somewhat three-cornered appearance with the microvilli seeming to cross (Fig. 7). Sections at some angle to the longitudinal axis of the microvilli should produce a mixture of cross and tangential sections of microvilli (Fig. 8). Sections through all of these planes should pass through the adherent junctions more or less in cross-sections. In contrast, a crosssection through microvilli oriented as in Fig. 4 should give the impression either that the microvilli are within the cytoplasm or the apical plasmalemma should be included in a grazing section (Figs. 1 I, 12). The second type of profile that is seen at thyroid cell apices in embryos older than stage 30 and after 1 day in organ culture consists of small, microvillous-filled spaces between the apical cell surfaces. These spaces are at the limit of resolution with the light microscope (Fig. 9). These small follicles (Fig. 10) tend to have a greater diameter than the incipient follicles as a result of the larger extracellular space. Some of the variability in shape and in number of microvilli undoubtedly is due to the plane of section relative to the center or the edge of the follicular space. The microvilli appear to be directed toward the center of the follicular space and to cross. The microvilli are the same size and shape as in the initial stages of closely interdigitated microvilli. Since sections are not found that contain only longitudinal profiles of microvilli, it is likely that
730
HILFER
Fig. 1. Light micrograph of the subcapsular region of a thyroid from a stage 29Rhode Island Red embryo. Mesenchyme is not visible beneath the surface of the primordium; yet channels (arrows) exist between these epithelial cells. x 1350. Fig. 2. Light micrograph of a portion of a thyroid from a stage 30 Rhode Island Red embryo. Erythrocytes (E) serve as markers for the capillaries and fibroblasts that have invaded from the capsule (C). The epithelial cells are aligned as double rows (cords) and small islands (plates). x 560. Fig. 3. Electron micrograph of small portions of several cells from the thyroid of a White Leghorn embryo at stage 29. The cells are highly interdigitated but lack specialized junctions. x 26,000. Fig. 4. Electron micrograph of two cell apices in a stage 25 thyroid primordium that was stripped of its capsule and placed in organ culture for 18 hr. (a) The apposed surfaces of the epithelial cells possess a regular pattern of folds that are recognizable as microvilli (M). The interdigitated region is delimited by an extensive adherent junction. Interdigitations of the sort illustrated in Fig. 3 also are visible (arrow). The cytoplasm apical to the nucleus (N) contains parallel Golgi cisternae (G), multivesiculate bodies (MVB), and vesicles with dense (D) and lucent (L) contents. A portion of a centriole is visible in the lower cell. x 32,500. (b) Enlargement of the region in Fig. 4a labelled B. The microvilli and adjacent cortical cytoplasm contain microfilaments. x 82,200. (c) Enlargement of the region in Fig. 4a labelled C. The particulate material in the dense vesicles shows no apparent order. x 82,000. Fig. 5. Junctional region of two cells from an intact stage 25 thyroid after 24 hr in culture. The section passed through only a few microvilli, all cut longitudinally. Centrioles and vesicles with pale contents are in close proximity to the microvilli. x 33,000. Fig. 6. Junctional region of two cells from a stage 25 thyroid after 24 hr in culture medium containing 50 pg/ml LACA. The mixture of tangentially and longitudinally sectioned microvilli seems to occupy a depression in only one cell surface. A cilium (C) in a cytoplasmic pocket is visible in cross-section. x 22,500. Fig. 7. The junctional region of three cells in a stage 25 thyroid which was cultured with its capsule for 18 hr. Most of the microvilli were cut at an angle, but microfilaments are visible in their cores. The involvement of three cells in this incipient follicle is recognized on the basis of three regions of adherent junctions. x 26,000. Fig. 8. Apical region of three cells from a normal Rhode Island Red thyroid at stage 35 (8) days). Most of the microvilli were cut in cross-section with portions of two cilia (C) lying among the microvilli. Two centrioles (arrows) are present in the cell at the lower right. Many small vesicles with contents of different densities surround the Golgi region at the lower left. x 31,000. Fig. 9. Light micrograph of a small portion of the central region of a stage 32 Rhode Island Red thyroid (7+ days). This represents the earliest time at which follicles (F) can be positively identified by light microscopy. Cilia are visible as line lines within the follicular spaces. x 1260. Fig. 10. Small follicle from a stage 35 Rhode cell on the right appears to loop out of and back villi or flaps of cytoplasm are associated with the contains many vesicles with both electron-dense
Island Red thyroid. The cilium of the into the plane of section. Long microjunctional zones. The apical cytoplasm and lucid contents. x 17,000.
Fig. 1I. A small follicle in a stage 25 thyroid that was fixed after 3 days in nutrient medium containing 50pg/ml LACA. Although the microvilli may seem to be within the cell, the participation of two cell apices is evident from the interruption of the adherent junction (arrow). Portions of two cilia are visible, one in the follicular space and the other within a cytoplasmic pocket. The cytoplasm contains a large number of vesicles with dense contents. x 20,000.
THYROID
FOLLlCLE
FORMATION
the orientation of the microvilli changes as the extracellular space expands. The microvilli adjacent to the junctional region frequently have a shape and length reminiscent of the marginal cytoplasmic flaps in mature follicles (Fig. 10). Follicles in this size range could be sectioned in the plane of the junctional zone, thus giving the impression that the follicular space is intracellular (Fig. I I), similar to those of the initial stages of follicle formation described above. Microvilli become a less prominent feature of the follicular space in later stages of development (Figs. 12, 14). Cilia A common characteristic of the earliest detectable follicles and a means of identifying incipient follicles is the presence of centrioles near the apical cell surface or of a cilium lying between the interdigitated microvilli. Sections frequently pass through a centriole or cilium in one cell of the incipient follicle and often the second cell also is sectioned through a part of either a centriole or cilium (Figs. 5, I I, 13). Usually a single centriole is visible but in a few sections a second centriole lies in the plane of section. Cilia commonly are included within pockets in the cytoplasm or extend into the follicular space (Figs. 8, 10, I I). The central pair of tubular subunits frequently is absent (Figs. 6, 10). The preponderance of cell apices that possess either centrioles or cilia for each cell participating in an early stage of follicle formation indicates that these structures are a constant feature of the follicles. Centrioles and cilia are seen as frequently at later stages in larger follicles (Figs. 12, 14). The sampling problems with the larger structures make it difficult to ascertain how many cells comprising each follicle possess a cilium, however. Cytoplasmic
Polarizution
organization
qf‘organelles
Polarization of cytoplasmic organelles is not as distinct at early developmental stages as it is at later stages because the endoplasmic reticulum is sparse (Fig. 4). The cells tend to be polygonal, with small portions of the cytoplasm of one cell inserting between the bulk of adjacent cells. Thus, it is difficult to trace one cell from base to apex in thin sections. Many of the apical cell regions appear trun-
733
cated, with two faces meshing with another row of cells. The nuclei tend to be basal or to one side. A few short channels of endoplasmic reticulum lie in the basal cytoplasm near the nucleus. An extensive network of rough endoplasmic reticulum is not seen until much later stages (10 days, Hilfer, 1964). Golgi lamellae and vesicles invariably are supranuclear and sometimes form more than one stack of channels. The apical cytoplasm contains vesicles of different sizes and densities, microtubules, and microfilaments. Apical vesicles During the initial stages of follicle formation, the apical cytoplasm contains large numbers of vesicles with contents that preserve differently. Basically, they fall into two main groups; those with dense, particulate contents and those with little electron density. The vesicles with dense contents tend to have a greater size range than the others. For reasons that are not obvious, the contents stain more intensely in cells from organ culture than in thyroids from embryos i/z ovo but the cells contain comparable numbers of vesicles under all conditions that were examined. The particulate contents do not appear to be crystalline (Fig. 4~). Material of the same particle size and density is found in vesicles of various sizes and shapes (Figs. 4, 6, II, 12, 17). A few vesicles with material of similar density, primarily in cells in culture, also contain membrane whorls or smaller vesicles (Figs. 4, 6). It is possible that these vesicles represent dense bodies (primary lysosomes) and autophagic vacuoles (secondary lysosomes). However, without appropriate identification, the possibility cannot be excluded that the contents represent a secretory product destined to enter the follicular space. Dense vesicles are less numerous in the cells of larger follicles and tend to be smaller (Figs. 14, 15). The vesicles with pale contents exhibit less variation in size and are present in larger numbers between the Golgi lamellae and the cell surface than the dense vesicles. In contrast to the dense vesicles, some pale vesicles lie close to the plasmalemma and appear continuous with the plasma!emma from the earliest stages of follicle formation (Figs. 5. 8, 10, 12). The pale vesicles contain fine material that stains much less intensely than the particulate material of the dense vesicles
734
HILFER
(Figs. 14c, 1%). It is possible that some of the contents are lost during tissue preparation, especially since there is some variation in the amount of material found in different vesicles. The number of vesicles in follicle cells at different stages of development shows no apparent change.
common along lateral surfaces of follicle cells at later stages. Other specialized junctions are associated with follicles having larger extracellular spaces. Incipient desmosomes are first seen in follicles with small lumina, sometimes as plaques without tonofilaments (Fig. 16).
Cell junctions
Microfilaments
Extensive adherent junctions are a prominent feature of the early stages in follicle formation. The junctions surrounding the region of microvillar interdigitation consist ofrelatively straight, parallel membranes separated by a gap of approximately 200 A and with increased density along the inner membrane leaflet (e.g. Figs. 4-7). Irregular interdigitations characteristic of prefollicular stages (Fig. 3) often exist peripheral to the adherent junction-microvillar complex (Fig. 4a). lnterdigitations of this sort also are
Microfilaments (SO-70 A in diameter) form a core of parallel filaments within the microvilli from the time the microvilli first appear (Figs. 4-7). The bundle is oriented parallel to the longitudinal axis of the microvillus. Additional microfilaments are found in the apical cytoplasm apparently continuous with the core filaments. In larger follicles these apical filaments tend to be visible as bundles that penetrate somedistance into thecell body (Fig. 17). Additional filaments in follicle cells at all stages lie in the cortical cytoplasm in no
and microtubules
Fig. 12. (a) Another follicle which appears superficially to be within a single cell, found in a normal stage 35 White Leghorn embryo. Adjacent sections show that this is a tangential section of a follicular space between at least two cells. A small amount of fine, filamentous material is present within the follicular space and the cytoplasm contains vesicles with lucent and dense contents. x 19,750. (b) Enlargement of the upper right corner of (a). The membrane of a dense vesicle is continuous with the plasmalemma (arrow) and several vesicles with finer material (L) lie close to the surface. The figure also contains a profile of a microtubule (M) and microfilament (MF). x 59,000. Fig. 13. A small follicle in a thyroid from a normal embryo. Microtubules are associated with the centrioles
stage 30+ Rhode Island Red (C) in both cells. x 14,100.
Fig. 14. A larger follicle from a normal stage 35 White Leghorn embryo. (a) The microvilli are a less prominent component of the follicular space than in smaller follicles and the space contains more of the fibrillar and granular components. A large membranous whorl with filamentous contents is also visible. The junctional regions possess cytoplasmic ‘flaps’ extending into the follicular space but no new specialized attachment site is visible along the lateral cell surfaces. The section passed close to the surface of a cilium (C). x 13,600. The lumen contains clumps of dense granules with radiating filaments (G), enlarged in (b). The apical cytoplasm contains pale vesicles (L) with fine filaments that are enlarged in (c). (b) and (c) x 57,000. Fig. 15. Part of a follicle in the thyroid of a White Leghorn embryo that was decapitated at stage 14 and fixed at stage 36 (10 days). (a) Although the follicular space is smaller than in the previous illustration, the space contains a greater concentration of dense material (arrows, enlarged in (b)). The apical cytoplasm contains a few small vesicles with dense contents (D) and paler vesicles with filamentous contents (L), enlarged in (c). The base of a cilium is included in the section. (a) x 19,000; (b) and (c) x 66,500. Fig. 16. Junctional region of a larger follicle at stage 35. (a) The density on the cytoplasmic side at the desmosome is not clearly organized. The follicular space contains fine strands and clumps of dense granules. x 65,500. (b) Enlargement of several clumps of dense granules within the same follicular lumen. x 80,000.
736
HILFER
Fig. 17. A smaller follicle from a stage 25 thyroid primordium after culture for 24 hr without its mesenchymal component. Microfilaments of the microvilli and of the cortical cytoplasm are especially prominent, perhaps as a result of the plane of section. Microtubules are associated with the centrioles in the cell to the left. Some vesicles with dense contents lie close to the plasmalemma. x 36,600.
apparent orientation with respect to the surface. The identification of microfilaments depends to a large extent upon the plane of section since they cannot be recognized in cross-section. Since sufficient landmarks do not exist for determining the orientation of follicle cells in thin sections at early stages, the question of the distribution of microfilaments cannot be answered from the present study. Microtubules can be found at all levels within follicle cells, from the earliest developmental stage. Frequently tubules are seen close to the apical cell surface (Figs. 6, 12-15, 17). In only some of these is a centriole in the
vicinity (Figs. 13, 17), with the microtubules radiating from it. In the other sections, the orientation ranges from perpendicular to the apical surface to parallel with it. The follicular space In incipient follicles, the extracellular space is limited to a few hundred angstrom between the microvillar surfaces (Figs. 4-7). The extracellular space contains fine material that probably is a continuation of the plasmalemma1 surface coat (Fig. 4b). In follicles with a slightly larger extracellular space, the expanded lumen has a density equivalent to
THYROID
FOLLICLE
FORMATTON
the contents of the paler apical vesicles (Figs. 1l-15). Almost all of the material that is electron dense is associated with microvillar surfaces and consists of fine strands (Fig. 12b). Follicles with larger lumina than these contain similar fine strands and packets of dense particles (Figs. 14b, 16). In these larger lumina the particulate material is not confined to the surface of microvilli but forms loose strands throughout the extracellular space (Figs. 1417). The amount of extracellular material varies more or less with the size of the follicular lumen, from these loose meshworks (Figs. 1Sb, 16b) in smaller follicles to the dense accumulations that fill the lumina of mature follicles. A small number of follicles with larger lumina are seen as early as stage 35 (S$ days) in both normal and decapitated embryos. Cultures of stage 25 (4f5 days) thyroids, whether treated with drugs or incubated with or without the mesenchymal capsule, form a few larger follicles as early as 24 hr after explantation. Substantial numbers of these larger follicles do not appear before IO days in ova (Stage 36) or 48 hr in vitro. Even in these older thyroids many immature stages of follicle formation are found among the larger follicles. The follicular lumina change in shape from flattened spaces between two cell apices through cavities with scalloped margins to the rounded lumina of mature follicles. The intermediate sized follicles tend to have the irregularly shaped lumina with contents of intermediate packing of the particulate material. These follicular lumina also contain accumulations of membranes either as single layers (Fig. 15) or as multiple layers (Fig. 14). It is not known if these membrane profiles are formed during expansion of the follicular space or result from inadequate fixation of the follicular contents (Hasty and Hay, 1978).
Discussion A difference of opinion has arisen over the way in which thyroid follicle formation is initiated. Two basically different mechanisms have been proposed to describe the formation of the extracellular space. A possible reason for the different points of view is interpretation of electron microscope images. The obvious dangers in interpreting isolated
737
images of thin sections in terms of threedimensional structure, when the plane of section cannot be determined, has been pointed out in previous studies. Thus, a section that appears to show an intracellular droplet may in reality be a section cut parallel to an apical surface that contained a depression. Furthermore, there are dangers in using an older embryonic stage and selecting images to construct a developmental sequence without checking the earlier stages, as was done by Cordier et al. (1976). Calvert and Pusterla (1973) ‘intracellular canaliculi’ are associated with plasmalemmae and may actually be tangential sections of extracellular spaces. Apparently, serial sections did not reveal any droplets large enough to correspond with the cytoplasmic droplets of light microscopy. Light microscopic images that have been interpreted as fusion of apical colloid droplets (Bradway, 1929; Waterman and Gorbman, 1935) probably represent the later period of expanding follicular size during which the small follicular space takes on a scalloped appearance (Fujita and Tanizawa, 1966; Hilfer, 1964, 1969; Hilfer and Hilfer, 1966). In this study thyroids were investigated under several conditions that allowed images of the first stages of follicle formation to be obtained in the absence of later stages. Under these conditions the largest number of profiles consisted of interdigitated coarse microvilli that were continuous with the cell surface. The few profiles that gave the superficial appearance of being intracellular on closer inspection either contained a tangentially sectioned adherent junction or such junctions appeared at the margin of the extracellular space when such ‘follicles’ were traced through adjacent sections. Thus, in the chick embryo, interdigitations of the apical plasmalemma associated with specialized junctions preceded the formation of any sizeable droplets in the apical cytoplasm. The absence of large droplets from the cell apex at early stages appears to be characteristic of the rat thyroid also (Tice et al., 1977). There is one welldocumented case of follicle formation proceeding from intracellular lumina (Remy et al., 1977). In porcine thyroid cells large, intracellular vesicles containing colloid can be demonstrated before multicellular follicles are formed. It is possible that the mechanism of follicle formation differs in different species. An alternate possibility is that
738
isolated cells from mature follicles in culture differ in their morphogenetic pattern when reforming follicles as compared with the process in the early embryo. Processes similar to the opening of the follicular lumen occur in other developing organs. Some organs begin as solid primordia which secondarily hollow out. Examples include cavitation of the caudal portion of the chick neural tube (Criley, 1969) formation of bile canaliculi in the liver (Wood, 1965), and hollowing of nephric tubules (Saxen and Wartiovaara, 1966). In the chick embryo the esophagus at first is an open tube but becomes occluded only to open again before hatching commences (Allenspach, 1964, 1966). These processes of cavity formation have been suggested to occur either by erosion of the cell apices or by secretion of material into the extracellular space at the bases of coarse microvilli. In all cases the cell apices are joined by tight junctions, the apical membranes interdigitate, and the apical cytoplasm contains numerous small vesicles. In some cases, the cells have been reported to contain increased numbers of lysosomes (Saxen and Wartiovaara, 1966; Wood, 1965). Opening of the esophagus before hatching also corresponds to a time of increased activity of lysosomal enzymes (Wilson and Allenspach, 1974). At present experimental evidence is lacking on the method by which the extracellular spaces expand. The images of early thyroid follicles, showing the complete range from no extracellular space between interdigitated membranes to round follicular spaces, give the impression that expansion is gradual. It is possible that two types of material are secreted during follicle formation, based upon the difference in appearance of the extracellular lumen at early stages and at later stages of development. The electron-lucent contents of the small follicles that are found at early developmental stages slowly changes to a particulate component in the larger follicular spaces. Vesicles with two types of contents are seen in the apical cytoplasm at these stages. Many of the small vesicle have electron-lucent contents. Larger, irregularly shaped vesicles with a dense, particulate content are present in the apical cytoplasm of small follicles but are present in much reduced numbers in the apical cytoplasm of cells in larger follicles. We suggested pre-
HILFER
viously that these dense bodies might be lysosomes (Hilfer, 1964; Hilfer et al., 1968) but the appropriate cytochemistry to demonstrate acid hydrolases has not been done. The increased number of dense bodies and concomitant increase in acid phosphatase activity during reopening of the developing esophagus (Wilson and Allenspach, 1974) is suggestive of a similar process during expansion of the follicular space and resulting in the scalloped cell surfaces. It seems odd, however, for lysosomal enzymes to be secreted into the follicular space. Perhaps some or all of the dense material in the larger vesicles of the smaller follicles is an early form of colloid. This question will not be resolved until the pathway of synthesis and secretion is followed by labelling techniques. The types of junctions that form between early follicle cells may differ in chicken and rat. In the rat (Tice et al., 1977) it has been shown by freeze-fracture and tracer studies that occluded junctions form around the earliest interdigitated membranes. The earliest junctions consist of only a few furrows constructed of incompletely fused particles. Yet they exclude the tracer, horseradish peroxidase, and must be functional in preventing at least some loss of follicular contents. In the chick embryo, the simplest interdigitations and smallest follicles are associated only with adherent junctions. Whether or not these junctions are sufficient to contain the material in the extracellular space has not been tested so far. Only the larger follicles have a junctional complex consisting of occluded zones and desmosomes in addition to adherent zones. The question of what event causes initiation of follicle formation remains unanswered. Follicles are not found until mesenchyme invasion occurs at stage 30 (Shain et al., 1972). In the absence of mesenthyme or in the presence of inhibitors of collagen synthesis that prevent formation of the fibrillar material within the epithelial component of the gland, follicle initiation occurs normally (Hilfer and Pakstis, 1977). Thus, neither the mesenchyme nor the presumed epithelial collagen that precedes its invasion can be responsible for initiation of follicle formation. Similarly, TSH need not be present since follicle formation proceeds in the absence of the pituitary. It is not clear, however, if some pituitary hormones might
THYROID
FOLLICLE
739
FORMATION
be provided by the yolk in ovo in decapitated embryos or by the serum in organ culture. At present it appears that the control is intrinsic to the epithelial component of the primordium, itself.
Acknowledgements This work was supported by grant no. PCM 77 17686 and its predecessors from the National Science Foundation.
References ALLENSPACH,A. L. 1964. Experimental analysis of closure and reopening of the esophagus in the developing chick. J. Morph., 114, 287-302. ALLENSPACH, A. L. 1966. The reopening process of the esophagus in the normal chick and the crooked neck dwarf mutant. J. Embryol. exp. Morph., 15, 67-76. BRADWAY,W. 1929. The morphogenesis of the thyroid follicles of the chick. Ann?. Rec., 42, 157-167. CALVERT, R. 1972. Electron microscopic observations on the contribution of the ultimobranchial bodies to thyroid histogenesis in the rat. J. Amt., 133, 269-288. CALVERT, R. and PUSTERLA,A. 1973. Formation of thyroid follicular lumina in rat embryos studied with serial fine sections. Gen. camp. Endow.. 20, 584-597. CAU. P., BECHET-MICHEL, M. and FAYET, G. 1976. Morphogenesis of thyroid follicles in virro. Adv. Amt.. 52, l-66.
CORUIER, A. C., DENEF, J.-F. and HAUMONT, S. M. 1976. Ultrastructure
of the thyroid
in dwarf mice. J. Annt.,
146. 339-357.
CRILEY, B. B. 1969. Analysis of the embryonic sources and mechanisms of development of posterior levels of chick neural tubes. J. Morph., 128, 465-502. Fucjo, N. W. 1940. Effects of hypophysectomy in the chick embryo. J. exp. Zool., 85, 271-297. FWITA. H. and TANIZAWA, Y. 1966. Electron microscopic studies on the development of the thyroid gland of chick embryo. Amt. Entwickl.-Gesch.. 125. 132-151. HAMBURGER,V. and HAMILTON, H. 1951. A series of normal stages in the development of the chick embryo. J. Morph.,
88, 49-92.
HASTY, D. L. and HAY, E. D. 1978. Freeze-fracture studies of the developing cell surface. IT. Particle-free membrane blisters on glutaraldehyde-fixed cornea] fibroblasts are artefacts. J. Cell Biol., 78, 756-768. HILFER, S. R. 1964. Follicle formation in the embryonic chick thyroid. I. Early morphogenesis. J. Morph,. 115. 135-152.
HILFER. S. R. and HILFER, E. K. 1966. Effects of dissociating agents on the fine structure of embryonic chick thyroid cells. J. Morph., 119. 217-231. HII.FER, S. R., TSZARDL. B. and HILFER, E. K. 1968. Follicle formation in the embryonic chick thyroid. Il. Reorganization after dissociation. Z. Zellforsch. mikrosk. Amt., 92, 256-269. HILFER, S. R. and PAKSTIS, G. L. 1977. Interference with thyroid histogenesis by inhibitors of collagen synthesis. J. Cell Biol., 75, 446-463. HILFER, S. R. and SEARLS,R. L. 1979. Differentiation of the thyroid in the hypophysectomized chick embryo, De~a. Biol. (submitted). ISHIKAWA, K. 1965. Electron microscopic studies of the thyroid gland of the rat in embryonic life. Okajimas Fol. Amt. Jap.. 41, 295-31 I. OLIN. P.. EKHOLM, R. and ALMQVIST,S. 1970. Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology, 87, 1000-1014. MICHEL-BECHET, M., CAU, P., FAYET, G. and COURRIER, R. M. 1973. Etude ultrastructurale de la morphogenese du follicule thyroiden: reorganisation des cellules thyroidiennes de port en culture. C. Y. h&d SPanc. Acad. Sci. Paris., 277, 1029-1032. R~MY, L., MICHEL-BECHET, M., CATALDO, C., BOTTINI, J., HOVSEPIAN, S. and FAYET, G. 1977. The role of intracellular lumina in thyroid cell for follicle morphogenesis itr vitro. J. UNrtrtrsttrccf. Res., 61, 243-253. RICHARDSON,K. C., JARETT, L. and FINKE, E. M. 1960. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Tech&., 35, 313-323. R~Q~J~s, M. J., TORRESANI, M., BEQUET, M. and LISSITSKY, S. 1972. Relationship between thyroglobulin synthesis, iodine metabolism and hisrogenesis in the developing rabbit fetal thyroid gland. ~ornp. Endowi%,
19, 457-472.
SAXON,L. and WARTIOVAARA,J. 1966. Cell contact 271 290.
and cell adhesion
during tissue organization.
J. Cancc,r. I,
740
HILFER
SHAIN, W. C., HILFER, S. R. and FONTE, V. G. 1972. Early organogenesis of the embryonic chick thyroid. I. Morphology and biochemistry. DA. Biol., 28, 202-218. SHEPARD, T. H. 1967. Onset of function in the human fetal thyroid: biochemical and radioautographic studies from organ culture. J. clin. Endocrin. Metab., 21, 945-958. SHEPARD, T. H. 1968. Development of the human fetal thyroid. Gen. camp. Endocrin., 10, 174-184. TICE, L. W., CARTER, R. L. and CAHILL, M. C. 1977. Tracer and freeze-fracture observations on developing tight junctions in fetal rat thyroid. Tissue & Cell, 3, 395-417. WATERMAN,A. and GORBMAN, A. 1935. Development of the thyroid gland of the rabbit. J. exp. Zoo/., 132, 509-53s. WILSON, J. R. and ALLENSPACH, A. .L 1974. The role of acid phosphatase and fi-glucuronidase in reopening and remoulding of the developing chick esophagus. Devl. Biol., 41, 288-300. WOOD, R. L. 1965. An electron microscope study of developing bile canaliculi in the rat. Anat. Rec., 151, 507-530.