/. Embryol exp. Morph. Vol. 42, 163-175 pp. 1977
163
Printed in Great Britain © Company of Biologists Limited 1977
Precocious evagination of the embryonic chick
thyroid in ATP-containing medium
By S. ROBERT HILFER, 1 BERNICE Y. PALMATIER AND
EILEEN M. FITHIAN
From the Department of Biology, Temple University, Philadelphia
SUMMARY
The identification of actin and myosin in many cell types other than muscle has given
support to the hypothesis that contractile proteins are involved in cell shape changes. However, there is no direct evidence that a contractile process participates in morphogenetic
movements during organogenesis. As a first step in testing this possibility, thyroid placodes
of chick embryos were treated with an incubation medium containing Triton X-100 and ATP.
Pharyngeal regions, isolated in Medium 199, were photographed at timed intervals. At
a concentration of 10~3 M ATP, the thyroid region formed a deep pit within minutes after
addition of this 'contraction medium', whereas evagination requires approximately 7 h in ovo.
Treatment with Medium 199 containing either Triton X alone, ATP alone, or Triton X and
pyrophosphate did not result in evagination of the thyroid. Substitution of other nucleotides
for ATP suggested a specific requirement for ATP. Surgical removal of selected portions of the
pharyngeal floor and examination of sectioned material by light microscopy indicated that
the cells involved in the shape change were located at the periphery of the thyroid placode
before treatment. The sharp bends that were formed in 'contraction medium' cannot be
explained entirely by pinching of cell apices at the point of folding; rather the effect of several
forces is indicated in the evagination.
INTRODUCTION
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 WessellseJtf/. 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
1
Author's address: Department of Biology, Temple University, Philadelphia, Pa. 19122,
U.S.A.
164
S. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
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).
MATERIALS AND METHODS
Reagents. 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-(/?-aminoethyl ether) A^JV-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.
Incubation medium. The standard 'contraction medium' consisted of Medium
199 with the addition of 1-0 HIM 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 HIM. 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.
In vitro thyroid eyagination
165
Nucleotide specificity was tested by adding either 1-0 to 2 mM AMP, 1-0 to
2-0mM ADP, or 1-0 to 100mM GTP to Medium 199 containing 0-05 % Triton X-100. Intracellular ATP was eliminated by treatment with 50 jam to 1 mM
DNP, which should have not only prevented ATP formation but also stimulated ATPase activity (Lardy & Wellman, 1952; Cooper & Lehninger, 1957).
Sensitivity to Ca 2+ was tested by treating the tissues with Ca 2+ and Mg2+-free
Hanks' saline containing 25 mM HEPES buffer, 0-05 % Triton X-100, and
1-100 mM EGTA before the addition of ATP.
Procedure. 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.
Microscopy. 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 goldpalladium for scanning electron microscopy. One-half to one micron sections
were stained with azure II-methylene blue (Richardson, Jarett & Finke, 1960)
and photographed with a Zeiss Photomicroscope II. Scanning electron micrographs were taken with an Etec Autoscan.
RESULTS
Living preparations
Normal development. 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.
166 l"3fs. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
In vitro thyroid evagination
167
46
8B
JOB
12
FIGURES
1-10
Fig. 1 A. Living pharynx of a stage-14 embryo in nutrient medium. The left half of
the mandibular arch (M) has been removed. Arches II and III lie to either side of
the pharyngeal floor. The thyroid is visible as a dense patch (arrow) between the
second pair of arches, x 55.
Fig. 2 A. The same pharyngeal preparation as in Fig. 1,10 min after the addition of
Triton X and 5 min after the addition of ATP. A ridge is visible in the thyroid region
(arrow) and the tissue has become a bit more transparent. The mandible has rotated
downward, bringing a remnant of the bulbus arteriosus into focus behind the
thyroid (x). x55.
Fig. 3 A. Ventral portion of the pharynx of a stage-16 embryo in nutrient medium.
The thyroid (arrow) has formed a pouch in the floor of the pharynx. The base of the
pouch is skewed caudally. x 55.
Fig. 4. Scanning electron micrograph of a stage-14 embryonic pharynx fixed after
20 min in nutrient medium containing Triton X. Thefloorof the pharynx between the
second arches (II) is slightly curved. The slight indentation at the midpoint (arrow)
is the thyroid placode. x 120.
Fig. 5. Scanning electron micrograph of another stage-14 pharynx, fixed after a
20-min exposure to contraction medium. The region between the second arches (II)
has become deeply indented, x 120.
[For continuation of legends see overleaf
168
S. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
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).
Fig. 6. Scanning electron micrograph of a stage-16 pharynx,fixedafter removal from
the embryo. The photograph was taken at a greater tilt angle than in the previous
illustrations. The thyroid evagination forms a pouch surrounded by a ridge that
projects above the surface of the pharyngeal floor, between the second pharyngeal
arches (II). x 120.
Fig. 7. Sagittal section of a thyroid placode at stage 14 after incubation in nutrient
medium for 20 min. The cells within the placode are tightly packed and contain
lipid droplets (L) and apical blebs (Bl). Basal indentations are seen towards the
cranial (Cr) and caudal (Cd) boundaries of the placode. The placode is skewed
cranially, producing a more gradual sloping of the caudal wall. A portion of the
bulbus arteriosus (H) remains attached ventral to the primordium. Bright field,
x260.
Fig. 8 A. Cross-section near the deepest point of the thyroid placode in a stage-14
embryo, after 20 min in nutrient medium containing Triton X. The section passes
through the aortic arches (aa) slightly cranial to the bulbus arteriosus. Arrows
demarcate the limits of the central and marginal zones of the primordium. A bracket
marks the region enlarged in Fig. 11. Bright field, x 350.
Fig. 9. Sagittal section of a thyroid placode at stage 14 after 20 min in contraction
medium. The cranial (Cr) and caudal (Cd) walls of the placode are bent at sharp
angles. Bright field, x 260.
Fig. 10 A. Cross-section through the deepest point of a thyroid placode at stage 14,
after 20 min in contraction medium. Near the midline the nuclei are seen toward the
cell bases. In contrast, the cells of the lateral walls have randomly distributed nuclei.
The lateral surfaces of the primordium are almost perpendicular to the floor.
A bracket marks the region enlarged in Fig. 12. Arrows demarcate the distances
equivalent to the limits of the central and marginal zones of untreated primordia.
Bright field, x 350.
Figs. 1B, 2B and 3B. Idealized three-dimensional drawings of the living preparations
shown in Figs. 1 A, 2 A and 3 A. Note the rounded shape of the pit that forms in
contraction medium (2B) in comparison to the elongate depression surrounded by
a ridge which forms in ovo at stage 16.
Figs. 8B and 10B. Tracings of the sections in Figs. 8 A (control) and 10 A (treated).
The floor of the pharynx has been divided into a series of zones based upon the shapes
of the cells in each region. These are: C = central region of the thyroid placode,
M = marginal zone, and P = ventral pharynx. The average number of cell
diameters from the midline to the edges of each region is indicated. After treatment
with 'contraction medium', cells that were peripheral to the marginal zone have
become part of the thyroid and form a new marginal region (M'). The broken line
through zone P of Fig. 8B corresponds to the outer limit of zone M' of Fig. 10B.
In vitro thyroid evagination
M
169
M
Fig. 11. Portion of a stage-14 thyroid in nutrient medium containing Triton X-100,
enlargement of the region bracketed in Fig. 8. Intertwined cells of the marginal zone
(M) are on the left and the looser pharyngeal cells (P) on the right side of the figure.
The transition from one type to the other is relatively abrupt (A). Most of the lipid
droplets (L) are small. B corresponds to the position of B in Fig. 12. A few cells
contain an indistinct apical band. Bright field, x 1400.
Fig. 12. Portion of a stage-14 thyroid in contraction medium at approximately the
same level as Fig. 11. Enlargement of the region bracketed in Fig. 10, but from an
adjacent section, rotated counterclockwise. The cells to the left are near the edge of
the evagination (in zone M'); the cells to the right are towards the midline (in
zone M). Lipid droplets (L) are larger and more numerous than in control preparations. A dense band forms an almost continuous line across the cell apices. The
narrow cleft (A) is equivalent in position to the outer edge of the untreated placode,
and to A in Fig. 11. Thus, the indentation at B is in an equivalent position to B in
Fig. 11. Bright field, x 1400.
Treatment with 'contraction medium'. When the pharyngeal regions of
stage-14 embryos (46 cases) were placed in nutrient medium containing 1 HIM
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).
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S. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
Specificity of the response. 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 HIM 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 Mg 2+ (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.
Localization of evaginating cells. 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.
Histological preparations
Analysis of the shape change. The organization of the thyroid primordium at
successive stages of development has been described in detail elsewhere (Shain
etal. 1972; Hilfer, 1973). A description of the thyroid stage-14 embryos after
incubation in control medium (9 cases) will be given for comparison with
In vitro thyroid evagination
171
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.
Cytological changes. 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 ah 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.
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S. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
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. 10 A). 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.
DISCUSSION
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
In vitro thyroid eyagination
173
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 irnmuno-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 etal. 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. HoffmanBerling & 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
12
EMB 42
174
S. R. HILFER, B. Y. PALMATIER AND E. M. FITHIAN
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.
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(Received 28 March 1977, revised 27 May 1977)