Precocious evagination of the embryonic chick thyroid in ATP-containing medium

Recently, the cytological changes during early organogenesis have been described for a number of epithelial organs that undergo either invagination or evagination. During this process, a relatively flat sheet of tightly joined cells is converted to a curved structure, forming either a tube, vesicle, or branched tree.

The search for the intracellular control mechanisms responsible for these organotypic shape changes has centered primarily on the microfilaments and their possible role in cytoplasmic contractility (for reviews, see Wessells et al. 1971 ; Schroeder, 1973a; Pollard & Weihing, 1974). Although morphological and biochemical data support microfilament involvement, direct evidence is still lacking that a process homologous to a muscle contraction is responsible for the cell-shape changes occurring during organogenesis. Also, there has been a recent tendency to use cells in culture as models to study the role of microfilaments. Such systems have shortcomings in attempts to explain organotypic shape changes because the cells are in an entirely different arrangement (individuals and spread flat) from the cells of a primordium (joined at their apices and columnar).

We have used the thyroid placode as a model system to study changes in several extracellular and intracellular parameters during evagination (Shain, Hilfer & Fonte, 1972; Hilfer, 1973). These include cell elongation and blebbing, placement of microfilaments and microtubules, and dimensions of cell apex and base, as well an analysis of population dynamics (Smuts, Hilfer & Searls, unpublished). If the roles of various forces, such as increased cell numbers, cell elongation, and cytoplasmic contraction, are to be tested individually, a method must be devised which allows organotypic shape changes to occur rapidly rather than in the hours that are required in ovo.

The use of model systems to study shape changes dates at least to the work of Hoffman-Berling & Weber (1953). By modifying this original ‘contraction medium’ we have been able to study shape changes in organ primordia. Upon treatment, the thyroid region undergoes a rapid evagination that mimics normal organogenesis both in shape and in cytological detail. This paper reports the phenomenological events that occur; the complex fine structural changes will be reported separately. Preliminary results with this system have been described (Hilfer, Young & Fithian, 1974).

Triton X-100, adenosine 5’-monophosphate (no. A1877) (AMP), adenosine 5′-diphosphate (no. A0127) (ADP), adenosine 5’-triphosphate (no. A3127) (ATP), guanosine 5′-triphosphate (no. G5631) (GTP), and ethyleneglycol-bis-(β-aminoethyl ether) N,N-tetraacetic acid (EGTA) were obtained from Sigma Chemical Co., St Louis, Mo., U.S.A. Nutrient Medium 199 and HEPES buffer were purchased from GIBCO, Grand Island, New York, U.S.A, and 2,4-dinitrophenol (DNP) from Mann, Inc., New York, U.S.A.

The standard ‘contraction medium’ consisted of Medium 199 with the addition of 1·0 mM ATP and 0·05 % Triton X-100. The glycerol extraction procedure of Hoffman-Berling & Weber (1953) completely solubilized the embryonic tissues. Therefore, the detergent Triton X-100 was tested at concentrations from 0·01 to 1·0 %. Consistent results were obtained at a concentration of 0·05 % without gross damage to the cells. ATP was tested over a concentration range of 0·1–10 mM. As with Hoffman-Berling & Weber’s (1953) study, the minimum concentration that gave consistent results was 1·0 mM, prepared at pH 7·2 and kept on ice. Since the tissues were sensitive to changes in pH, all solutions were maintained within the range of 7·2–7·5. In later experiments 25 mM HEPES buffer was added to the medium to help maintain this range. Control media consisted of Medium 199, ATP in Medium 199, Triton X-100 in Medium 199, and Triton X-100 and 1-0 mM pyrophosphate in Medium 199.

Nucleotide specificity was tested by adding either 1·0 to 2 MM AMP, 1·0 to 2·0 MM ADP, or 1·0 to 10·0 mM GTP to Medium 199 containing 0·05 % Triton X-100. Intracellular ATP was eliminated by treatment with 50 μm to 1 DIM DNP, which should have not only prevented ATP formation but also stimulated ATPase activity (Lardy & Wellman, 1952; Cooper & Lehninger, 1957). Sensitivity to Ca²⁺ was tested by treating the tissues with Ca²⁺ and Mg²⁺-free Hanks’ saline containing 25 mM HEPES buffer, 0·05 % Triton X-100, and 1-100 mw EGTA before the addition of ATP.

Embryos at stage 14 of development (Hamburger & Hamilton, 1951) were removed from Rhode Island Red chicken eggs (Hardy’s Hatchery, Essex, Mass., U.S.A.) and placed in Medium 199. The pharyngeal region was excised and the pharyngeal roof slit longitudinally. All experiments were done at room temperature (20–23 °C).

Two procedures were followed. In the first, pharynxes having thyroids of equivalent developmental stage were placed in the wells of a three-depression spot plate (Corning no. 7200). The thyroid region was photographed through a Wild M5 dissecting microscope. The nutrient medium was replaced with complete ‘contraction medium’ in one well and control media in the other two wells. Photographs were then taken at timed intervals. In the second procedure, Triton X-100 and ATP were added in sequence with photographs taken at intervals to assess the effects of each component of the ‘contraction medium’ on the appearance of the same primordium.

Living preparations were photographed through a Wild M5 dissecting microscope equipped with an automatic exposure device and a Leica camera back. Representative samples were fixed in 2·0 % glutaraldehyde buffered with phosphate and postfixed in osmium tetroxide as described previously (Hilfer, Searls & Fonte, 1973). Samples either were embedded in Araldite (Cargille, Cedar Grove, N.J., U.S.A.) for light microscopy or were critical-point dried (Sorvall, Norwalk, Conn., U.S.A.) and coated with gold-palladium for scanning electron microscopy. One-half to one micron sections were stained with azure Il-methylene blue (Richardson, Jarett & Finke, 1960) and photographed with a Zeiss Photomicroscope II. Scanning electron micrographs were taken with an Etec Autoscan.

The portion of the embryo that was used is illustrated in Fig. 1 A. With the lower jaw (mandible) left attached to the pharynx, the embryo was cut off behind the attachment point of the bulbus arteriosus and the bulbus arteriosus also removed. After removal of the neural tube and notochord, the roof of the pharynx was slit longitudinally and the pharynx spread flat in the depression dish. The floor of the pharynx formed the center of the resultant piece of tissue and the gill arches and slits lay to either side.

At stage 14 the thyroid primordium forms a small depression at the midline in the floor of the pharynx at the level of the second pair of pharyngeal arches, just behind the mandibular arch. In the living preparation (Fig. 1 A), this thyroid placode is detectable as a dense patch. By stage 16 (Fig. 3 A), approximately 7 h later, the thyroid primordium forms a deep depression that is suspended from the pharyngeal floor into the space at the base of the bulbus arteriosus. The primordium forms an eccentric cup with a cranial bias and is surrounded by a ridge that is raised above the surface of the pharyngeal floor (Shain et al. 1972; Hilfer, 1973).

When the pharyngeal regions of stage-14 embryos (46 cases) were placed in nutrient medium containing 1 mM ATP and Triton X-100 (‘contraction medium’) at room temperature, 91 % of the samples rapidly changed shape (Fig. 2 A) to resemble the thyroid of a stage-16 embryo (Fig. 3 A). The change is diagrammed in Figs. 1, 2 and 3B. In all cases the maximal change was reached in 20 min ; in some cases a change could be seen in the first 2 min and the maximal change occurred by 5 min, as in this illustration. In contrast, development in ovo from stage 14 to stage 16 at 37 °C requires approximately 7 h. The shape change is even more obvious when judged with the scanning electron microscope. The placode is barely discernible at stage 14 prior to treatment (Fig. 4); the pharyngeal floor is only slightly indented. After the brief exposure to ‘contraction medium’ (Fig. 5), this region became a deep indentation, similar to that found in a stage-16 embryo (Fig. 6). The thyroid region did not change shape when the pharynx was treated with any of the control media: just Triton X-100 (0/6 cases), just ATP (2/14 cases), or Triton X-100 followed by 1 mM pyrophosphate (1/8 cases).

Specificity of the nucleotide requirement was tested by substituting AMP, ADP, or GTP for ATP. Neither AMP (0/5 cases) nor ADP (0/12 cases) resulted in evagination; however, GTP at the same concentration as ATP caused the same shape change (8/8 cases). When ATP was added to samples that had not responded to ADP, they did evaginate (6/6 cases). Since the GTP may have been used to phosphorylate ADP intrinsic to the tissue, DNP was added at the same time as the detergent before the addition of GTP. Addition of DNP should have degraded intrinsic ATP and prevented transphosphorylation of ADP (Cooper & Lehninger, 1957; Lardy & Wellman, 1952). At a concentration of 1 mM DNP, 1 mM GTP did not cause evagination (1/4 cases). Pretreatment with DNP also prevented evagination with 1 mM ATP (0/7 cases). When the ATP concentration was raised to 5 mM, at least partial evagination occurred (9/9 cases) while the same concentration of GTP did not allow evagination (0/6 cases).

Attempts were made to test calcium dependency of the ATP-elicited evagination by presoaking the Triton-treated samples in Hanks’ saline minus Ca²⁺ and Mg²⁺ (CMF Hanks’) containing EGTA. Even at 0·1 M EGTA, the point at which the tissue started to dissociate, the subsequent addition of ATP resulted in evagination that did not differ significantly from preparations treated with detergent and ATP in either CMF Hanks’ or Medium 199.

Observation of intact primordia in ‘contraction medium’ indicated that the region surrounding the stage-14 placode was the site of evagination. Selected areas of the pharyngeal preparation were excised to test whether the evagination resulted from changes in this area or from pressures generated external to the thyroid region. Removal of all gill arches to leave just the floor of the pharynx and the mandible still allowed the evagination to occur. When the floor of the pharynx was slit longitudinally through the thyroid placode, a lateral ridge was formed in ‘contraction medium’. When the incision passed along one edge of the placode, a ridge formed which surrounded the placode except at the place where the cut was made. However, if the placode region was excised to leave a hole in the pharynx, but the surrounding cells not destroyed, folds did not form after the addition of ‘contraction medium’. The excised placode in the bottom of the same depression dish also underwent no measurable folding. Evagination, then, does not require the presence of gill arches but does seem to be dependent upon the presence of the placode. The ridge that becomes raised above the pharyngeal surface must be contributed by the ring of cells just external to the placode.

The organization of the thyroid primordium at successive stages of development has been described in detail elsewhere (Shain et al. 1972; Hilfer, 1973). A description of the thyroid stage-14 embryos after incubation in control medium (9 cases) will be given for comparison with pharynxes treated with ‘contraction medium’ (9 cases). The thyroid placode is recognizable in sections even at low magnification because its cells are more densely packed and taller than those of the adjacent pharynx (Figs. 7, 8). In the pharynx (under the conditions of preservation that were used) the lateral cell surfaces adhered primarily at the apical and basal limits of the epithelial sheet. In the central region of the thyroid primordium, the lateral plasmalemmae were in close apposition over their entire length. In cross-section (Fig. 8 A, B), a marginal zone can be distinguished between the pharyngeal cells and the central region of the thyroid which contained cells approximately the same height as those of the placode, but with the lateral borders more loosely joined. Longitudinal sections (Fig. 7) show that the depression in the floor of the pharynx was biased in the cranial direction as early as stage 14; the cranial wall had a steeper slope than the caudal wall. In cross-section, the pharyngeal floor was curved slightly with the thyroid region forming the deepest portion of the curve.

After exposure to ‘contraction medium’, the thyroid region of stage-14 embryos folded to resemble the primordium of a stage-16 embryo. A deep and asymmetrical cavity was formed, giving the primordium the shape of a pit that was skewed cranially (Fig. 9). The lateral surfaces lay almost parallel to each other (Fig. 10 A, B) as a result of sharp bends (arrows) at the deepest part of the pit. Control thyroids had a cross-sectional dimension of approximately 55 cell diameters, consisting of a central zone of compact cells of approximately 25 cell diameters and a peripheral ring of approximately 15 cell diameters on each side, with characteristics intermediate between those of the central zone and the pharynx. In contrast, the thyroid region after treatment with ‘contraction medium’ had a cross-sectional dimension of approximately 95 cell diameters, consisting of a central zone of approximately 55 cell diameters and a peripheral ring of approximately 15 cell diameters on each side, containing less tightly packed cells. Thus, the diameter of the thyroid region increased approximately by 1·75 times after treatment.

The cytological characteristics of the cells were changed in the region that responded to ‘contraction medium’. In the central thyroid region of control preparations, the cells were pseudostratified and elongate. The cytoplasm contained large droplets and a dense apical band. These have been shown by electron microscopy to be lipid droplets and microfilament bundles, respectively (Shain et al. 1972; Hilfer, 1973). In the peripheral zone of the thyroid (Fig. 11), the cells contained only a few, smaller droplets and a dense apical band was seen in only an occasional cell. The cells of the central zone tended to be taller than those of the marginal zone or of the pharyngeal epithelium, which were both of approximately the same height. The pharyngeal epithelial cells were loosely arranged, rarely contained lipid droplets, and apical bands were absent under the conditions of preservation that were used for this study. The cells in all regions varied considerably in shape, as is characteristic of pseudostratified epithelia.

After treatment with detergent and ATP, the cells at the base of the vesicle differed little from those in the same location in nutrient medium, although more of the nuclei may have been at the cell bases (Fig. 10A). The appearance of the cells in the zone surrounding the original placode, however, had changed significantly (Fig. 12). The cells from the edge of the original placode to the edge of the gill bar epithelium were indistinguishable in shape from those of the central part of the primordium, having become taller than pharyngeal cells in the corresponding location in control medium. The tight association of the lateral surfaces, presence of apical bands and even of large lipid droplets, gave the impression of a single population of cells from the midline to the edge of the evagination. In this newly added region of the thyroid, the cells appeared to be less contorted than they were in untreated preparations, and the nuclei were more elongated.

This study represents a preliminary attempt to discover if bending of the thyroid placode during organogenesis involves a cellular contraction. We have shown that one condition demanded for a contractile system is satisfied, a requirement for ATP. The ATP appears to act in a specific manner, for it cannot be replaced by pyrophosphate, AMP, or ADP, nor by GTP in the presence of DNP. The effect occurs only under conditions in which ATP can enter the cell, suggesting that the response is intracellular.

Many studies have shown a circumstantial relationship between apical and/or basal microfilaments and bending movements of cell sheets (see Wessells et al. 1971). It has been suggested that bending results from purse-string type pinching of cell apices of many cells joined in a sheet (Baker & Schroeder, 1967; Wessells et al. 1971). Measurements of volume occupied by the filament bundles before and after neurulation have been interpreted as consistent with sliding of filaments past each other (Burnside, 1973). Many non-muscle cell types have been shown to contain actin and myosin by biochemical criteria (reviewed in Pollard & Weihing, 1974). Furthermore, an ATP-dependent and reversible dissociation of purified actins and myosins has been shown in many of these studies. Binding of heavy meromyosin to the contractile ring of cells undergoing mitosis (Schroeder, 1973 b) and by microfilaments of neural plate cells (Schroeder, 1973 a) and salivary primordia (Spooner et al. 1973) undergoing morphogenesis is strong evidence that the filaments are actin.

The localization of myosin fibrils within non-muscle cells has been more difficult. Filaments having the thickness and cross bridges of myosin rods have not been found. The inability to recognize myosin rods may be related to a difference in their size relative to muscle myosin. Reassociated myosin isolated from granulocytes (Stossel & Pollard, 1973) and platelets (Niederman & Pollard, 1975) yields rods woth a diameter of only 6–12 nm, a size that overlaps the range of presumed actin microfilaments. Immunological techniques suggest an association of myosin with actin. In immunofluorescent studies antimyosin stains in a fibrillar pattern (i.e. Weber & Groeschel-Stewart, 1974) in the same location as fibrils identified as actin. Although immuno-electron-microscopic methods confirm the presence of myosin, these studies have not shown it to be in a fibrillar form (Painter, Sheetz & Singer, 1975; Shibata et al. 1975). Therefore, no direct evidence exists that a sliding filament system is involved in organotypic shape changes, although suggestions have been made as to how such a mechanism might work in non-muscle cells (Spooner, 1973; Pollard & Weihing, 1974). The possibility also exists that the apical filament bands are not the only actin-containing system within cell sheets. The cortical cytoplasm of the lateral cell surface may contain a system of filaments involved in cell elongation (Burnside, 1975).

Although whole organs have not heretofore been described as responding to a ‘contraction medium’, the relatively long history of studies on glycerinated cell models dates to the work of Hoffman-Berling on fibroblasts (i.e. Hoffman-Berling & Weber, 1953). More recently portions of cytoplasm isolated from amebae (Taylor, Condeelis, Moore & Allen, 1973) and fibroblasts (Izzard & Izzard, 1975) have been shown to protrude pseudopod-like extensions only in the presence of ATP and calcium ions. Also, isolated intestinal brush borders shorten in the presence of ATP (Rodewald, Newman & Karnovsky, 1976; Mooseker, 1976). One must be cautious in interpreting results from model systems, however, because microtubule-containing systems also respond to ATP. Examples are the continued movement of chromosomes on isolated mitotic spindles (Cande et al. 1974) and the beating of sperm tails (Summers & Gibbons, 1973) in ATP-containing solutions. Thus, it is not clear if only one component of whole cells exhibits the ATP effect.

We have speculated that the apical microfilaments in the thyroid primordium may act to maintain shape of the apical surface rather than to cause constriction of cell apices (Hilfer, 1973). The sequential events in the enlargement of the thyroid placode during evagination, including the placement of longitudinally oriented microfilament bundles suggests to us that the thyroid is formed by the incremental addition of rings of cells at the periphery of the pre-existing placode. This conclusion is supported by the results of the present study. The region of maximal shape change after the addition of ‘contraction medium’ corresponds to the zone of cells surrounding the original thyroid placode. Within 15 min after the addition of ‘contraction medium’, the cells of this region acquire the same characteristics as those of the original placode. This region of the pharyngeal floor is in readiness for thyroid formation. Not only is it set apart from the already established placode and the rest of the pharynx by longitudinal bands of microfilaments and the beginnings of basal indentations (Hilfer, 1973), but it also corresponds to a region in which DNA replication is decreased relative to the pharynx (Smuts, 1974; Smuts et al., unpublished). The latter studies have shown that the cessation of DNA synthesis (and presumably of mitosis) follows a definite pattern. The formation of the placode at stage 11 is marked by a sharp reduction in the number of dividing cells. At later stages, the zone of cells with a reduced labeling index enlarges, with a narrow band of cells remaining totally unlabeled. The unlabeled cells correspond in position to the cells containing longitudinal microfilament bands. Additional rings of cells with a lower labeling index, separated by a narrow zone of unlabeled cells, were added to the primordium during later development stages.

The most striking feature of the response to ‘contraction medium’ was the elongation of the cells in the region surrounding the original placode. The new region of elongated cells came to lie almost perpendicular to the original placode, with a marked inflexion between the two regions. The combination of shape changes suggests that different forces might be acting in different regions of the placode. Evagination might result from the combined efforts of cell elongation at the margin and apical constriction at the bend. Similarly the apical filament bundles might serve different functions in the two regions. Although involved in constriction at the bend, they may prevent spreading of cell apices in the marginal zone.

Preliminary studies by transmission and scanning electron microscopy of primordia treated with ‘contraction medium’ indicate that evagination is probably the result of several forces. It is clear that the changes in shape of the thyroid primordium which are brought about by ‘contraction medium’ mimic the changes that occur during normal evagination in ovo. However, it is not clear what causes the changes. This system provides an excellent opportunity to test the role of the several possible forces that have been suggested to cause evagination -cell contraction, elongation and division.

We wish to thank Debbie Kogan for her help with the nucleotide experiments and Eva Hilfer for the line drawings.

S. R. Hilfer is supported by Grant Numbers 70-00580 and BMS 75-16744 from the National Science Foundation. Ms Palmatier has received support from Temple University in the form of a Research Assistantship.