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Domains of differential cell proliferation and formation of amnion folds in chick embryo ectoderm.

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THE ANATOMICAL RECORD 238:225-236 (1994)
Domains of Differential Cell Proliferation and Formation of Amnion
Folds in Chick Embryo Ectoderm
SUE ANN MILLER, KIMBERLEY L. BRESEE, CHERYL L. MICHAELSON,
AND DEAN A. TYRELL
Department of Biology, Hamilton College, Clinton, New York
ABSTRACT
Patterns of cell proliferation in ectoderm epithelium that
will form avian amnion correlate with morphogenesis, but not in an obvious pattern with respect to large-scalefolding. At sites where the pre-axial
amnion folds will first appear in 4- to 8-somite embryos, patterns of proliferation do not separate into domains that presage location of the single
pre-axial fold that is commonly described in embryology texts. Instead,
increased cell proliferation occurs in a significant, bilateral pattern. In
stages with 13 to 27 somites, when lateral amnionic folds are prominent,
five paraxial domains of cell proliferation correlate with morphology and
show decreasing levels of cell proliferation with distance from the neural
axis. Slowly growing areas surround rapidly growing areas and could assist buckling of epithelium by providing constraint on expansion of faster
growing areas. Proliferation domains in ectoderm correlate with morphology and morphological events when localized changes in cell shape are
lacking and suggest a role for differential cell proliferation in formation of
large-scale epithelial folds in early chick embryos. o 1994 Wiey-Liss,Inc.
Key words: Chick embryo, Amnion, Ectoderm, Epithelium, Morphogenesis, Autoradiography, Growth, Cell cycle
Avian amnion forms by elevation and apposition of
epithelial folds. Amniogenesis in domestic mammals
such as pig, cat, dog, horse, cow, sheep, and goat also
occurs by a bilateral folding and subsequent fusion of
somatopleure (Noden and de Lahunta, 1985). The
avian process is generally described (e.g., Carlson,
1988, p. 257) as creation of a pre-axial, crescentic, amnion head fold. Arms of the crescent extend along the
developing axis and elevate as lateral amnion folds.
The process that drives elevation of folds is still unclear. Formation of amnion and body folds is a largescale event that occurs concomitantly with folding that
forms neural and gut tubes. Roughly 40% of the 5.5 mm
length of a 20-somite chick embryo is morphologically
involved in folding somatopleure, so it is entirely possible that the mechanism behind these large-scale folds
involves more than localized cell shape changes. More
diffuse forces, such as proliferation differentials, could
be important in generating large-scale folds. Davidson
provided a detailed description of appearance of cells
during formation and fusion of amnion folds (Davidson,
1977), but cell proliferation was not a part of that
study. The possible role of differential cell proliferation
in formation of amnionic folds has not been considered.
Study of lateral body folds suggested a role for proliferation differentials in that folding process (Miller,
1982) and a subsequent, preliminary study of proximal
amnion suggested possible differentials that could contribute to buckling of epithelium to define body folds
(Miller et al., 1982). Expanded surveys supported the
existence of differentials in ectoderm (Bresee, 1991;
0 1994 WILEY-LISS. INC
Miller and Bresee, 1992). Although filaments are
abundant as amnion cells begin to differentiate, no
evidence of organized arrays or specific areas of cell
wedging were seen (Miller and Campbell, 1979). This
current study set out to test the hypothesis that proliferation differentials might have a role in formation of
avian amnion and to provide a more extensive “map” of
general proliferation patterns in avian ectoderm during early morphogenesis.
We examined embryonic and extraembryonic ectodermal epithelium distal to the area of amnionic folding to determine whether lateral growth asymmetries
and craniocaudal differentials exist during the formation of body folds and lateral amnionic folds. This
report presents data that support the hypothesis that
differentials in cellular proliferation within the somatopleure contribute to formation of lateral amnionic
folds and suggests that differential growth in the form
of differing rates of cell proliferation can be a contributor to large-scale general morphogenesis such as the
generation of folds in epithelia.
MATERIALS AND METHODS
White Leghorn (SPAFAS, SPF-1) embryos were labeled continuously during the final hours of incubation
Received June 21, 1993; accepted September 8, 1993.
Address reprint requests to Dr. Sue Ann Miller, Department of
Biology, Hamilton College, 198 College Hill Road, Clinton, NY 13323.
226
S.A. MILLER ET AL.
Pre-axial Amnion Fold I
Fig. 1 . Schematic of 3 pre-axial levels of extraembryonicectoderm sampled in 4- to 10-somite embryos.
with 5 pCi 3H-thymidine (specific activity, 20 Ci/
mmole) in 0.05 ml saline, dropped onto each embryo a t
3-hour intervals according to the method of Smuts et
al. (1978) modified by Miller (1982). Embryos studied
in stages HH 8 to 10 (4-to 10-somitepairs) (Hamburger
and Hamilton, 1951) were labeled during the final 6
hours of incubation, and embryos studied in HH stages
11to 16 (13- to 27-somite pairs) were labeled over the
final 12 hours of incubation. Embryos were removed
with generous amounts of surrounding membranes to
provide extensive areas of epithelium for study.
Lengths of epithelium ranged from 13 to 18 mm across
transverse sections. Embryos were staged a t fixation in
buffered formalin. Transverse serial sections cut 4-10
pm thick were mounted on albumin-coated slides, deparaffinized, and dipped in Kodak NTBS emulsion.
Standard histological and autoradiographic techniques
were used in preparation of slides.
Proliferation was estimated using a proliferation index (PI, percentage of nuclei labeled with tritiated thymidine). We used thymidine labeling rather than mitotic figures to infer proliferation, because counts of
mitotic figures are not a reliable indicator of rate of
growth in embryonic tissues (Dalcq 1937, 1938; Woodard, 1948; Dondua, 1966). Mitotic figures were noted
when seen, but mitotic indices were not collected.
Data were collected in specifically defined sample
sites in epithelial ectoderm at specific morphological
levels. Each site within each level was sampled five
times (i.e., five sections) by placing an ocular grid that
defined an area of 12,343 pm2 a t magnification x 450
over the area of ectoderm to be counted and scoring all
nuclei as labeled or unlabeled. A nucleus was considered labeled when the number of autoradiographic
grains over the nucleus exceeded the number expected
from calculations of normal background for each preparation. Nine sample sites were specifically defined for
six morphological levels in embryos with 4- to 10somite pairs. Figure 1 represents three of these levels
to show placement in and around the area of epithelium that produces the pre-axial amnion fold. Three
subsequent sample levels (preoral gut, oral membrane
1,and oral membrane 2) are not pictured. Twenty-six
sample sites were specifically defined for six morphological levels in embryos with 13- to 27-somite-pairs.
Figure 2 represents general locations of sample sites in
a section with lateral amnion folds.
Counts were tallied on a digital denominator, and
those data were recorded directly into a Microsoft@Excel spreadsheet. Data were collected by four investigators working independently. Statistical analyses were
performed using J M P Statistical Visualization Software (SAS) in a Macintosh IIfx microcomputer. Analysis of Variance (ANOVA) and student’s t-test were
used to evaluate raw data for differences between proliferation indices between groupings of pooled data for
each level of amnion formation. Analyses were performed on individual embryos, and groups of embryos
to determine appropriate groupings for interpreting
data. Similarly, data from levels were examined
pooled, and by level or site, to determine patterns
within the large data base. Established groupings of
data were analyzed in both transverse and sagittal presentations.
227
ECTODERM CELL PROLIFERATION DOMAINS
Lateral Amnion Fold
Lateral Amnion Fold
Fig. 2. “racing of a representative section showing general location of sample sites in a n embryo with
lateral amnion and body folds.
RESULTS
Distinct domains of proliferation occur in ectoderm
epithelium during amniogenesis, but correlation with
the morphology of the process is different a t the site of
the initial pre-axial amnion fold than it is for later
lateral folds. Data are presented in graphs displaying
summary statistics by site in the two major morphological groups: youngest embryos that have only a preaxial amnion fold and older embryos that have fused
pre-axial folds into amnion membrane and have obvious lateral amnion folds. Mitotic figures were rare in
sites where folds develop.
Pre-Axial Amnion Fold
Eighteen embryos fixed at stages 8-10 (4- to 10somite pairs) provided data for the initial pre-axial amnion fold. Figure 3 presents data according to the three
sample levels illustrated in Figure 1. Although the visible pre-axial amnion head fold appears as a single
crescentic fold that rises over the emerging head process, proliferation is higher in a distinctly bilateral pattern that does not reflect the form of the pre-axial amnion fold.
Right distal PIS appear slightly lower than left, but
differences are not &gnifica& ( p = .3273, .0399, and
.3232, respectively). Interestingly, median PIS decrease
along a caudal gradient toward the emerging axis (40,
34, 31%), and these are significantly different groups
(P = .0003). This gradient suggests the appearance of
a pre-axial fold, despite the bilateral “elevations” of
proliferation that flank it in transverse sections.
Lateral Amnion Folds
Twenty-eight embryos fixed at stages 11-16 (13- to
27-somite pairs) (224,508 cells) provided data for lateral amnion folds. Domains of cell proliferation that
differ significantly from each other and that correlate
with morphology and morphogenesis are consistent
across 28 individuals of a large data base comprised of
embryos collected and counted by several investigators
working independently. These patterns are real and
are significant with respect to morphogenesis and the
correlated morphology at each site.
Figure 4 shows data from all levels in all embryos
with lateral amnion folds. Means represent domains
with highly significant differences (P < .00005). Five
domains are obvious in this analysis, and each domain
represents a grouping that differs statistically from the
others. Proportion of the cell population dividing decreases in a medial-to-lateral gradient in 13- to 27somite embryos. Proliferation at fold sites is different
from adjacent areas, but is not a peak flanked by relatively lower growth.
When data from embryos with lateral folds are
grouped by levels and arranged in a cephalo-caudal
sequence, more details emerge (Fig. 5). Four to five
domains are common between the levels, but the contents of the domains vary according to progress of fold
generation. Body (including body folds) is always one
domain with the highest PI. Distal epithelium is always a separate domain with lowest PI. Amnion sites
are a significant domain that overlaps comparison circles for the body domain in pre-fold epithelia, but amnion folds are a separate distinct domain.
Figure 6 arranges the same data for folds and adjacent sites in sagittal series. Sagittal arrangement
shows the general reduction in PI with time and makes
it easier to see the differential between chorion and
large amnion fold that may be contributing to elevation of the fold.
Figure 7 presents the data in three age groups that
correspond to Hamburger and Hamilton’s (1951) descriptions of amniogenesis and show differences between profiles early and late in the establishment of
lateral folds. Early lateral fold stages (13-19 somites)
have four proliferation domains, but amnion domains
overlap. The greatest range of proliferation and five
distinct domains occur a t the time lateral amnion folds
are elevating (20-23 somites). Amnion fold is a clearly
separate domain in the middle of the range of five domains. Chorion and distal epithelium are also more
distinctly separate during the stages of lateral fold elevation. After lateral amnion folds are elevated and
fusing (25-27 somites), increased cell division provides
general growth of amnion and chorion. Four domains
are less clear and overlap. Inclusion of proximal amnion cells in the body domain rather than the amnion
domain may reflect acceleration of growth in lateral
body folds.
In summary, significant increases precede appearance of the pre-axial fold, but the bilateral pattern does
not mirror the single fold that will appear. Proliferation domains around lateral amnion folds do not show
a peak corresponding to the site of incipient folds,
228
S.A. MILLER ET AL.
Pre-axial Fold I
80 -
70 -
__
~
60
Domains:
50
Laterals
40
Median
P < 0.00005
PI range = 40 53
30
lateral
median
I
I
lateral
-
’
Pre-axial Fold II
Domains:
L. Distal
R. Distal
Proxirnals
Median
P < 0.00005
PI range = 34 48
-
distal proximal
proximal distal
median
Proamnion
Domains:
Distals
Proximals
Median
P e 0.00005
PI range = 31 50
I
-
I
distal proximal
proximal’ distai
median
Sites in Pre-axial Extraembryonic Ectoderm
B
Means with 95% confidence interval
Quantiles: 90%,75%,50%,25%,10%
Means Comparison Circles, 95%
Fig. 3. Mean proliferation indices of sample sites in transverse series from pre-axial levels of embryos that had not yet formed pre-axial
amnion folds. Data are from 18 embryos fixed at HH stages 8-10 (4to 10-somite pairs). “Domains” are significantly different regions of
proliferation indicated by the statistical program. Dense stippling indicates the body domain in all subsequent figures. Less dense stippling indicates the domain containing amnion fold sites. The legend
included in this figure also applies to statistical summaries in subsequent figures. Size and shape of means indicators is also informative.
Flatter diamonds indicate greater confidence in a mean from a larger
sample. The smaller the comparison circle, the greater the sample size
and confidence in the mean it represents. Comparison circles that do
not overlap indicate significantly different sets of data.
229
ECTODERM CELL PROLIFERATION DOMAINS
Domains:
Body
- Prox. amnion
9 7 Yo
9 1 Yo
folds 8 4 Yo
Chorion
7 6 Yo
-Amnion
- Distal epithel. 6 7 Yo
’ 0
O
P < .00005
L
distal chorion amnion
body
EMBRYO
body
amnion chorion distal
Right
Left
Sites
Fig. 4. Composite of mean proliferation indices at sample sites in embryos with lateral amnion folds.
Data are from 224,508 cells in 28 embryos fixed at HH stages 11-16 (13-to 27-somite pairs). Fine
stippling indicates body sites; coarse stippling marks amnion domains.
but high proliferation precedes elevation of lateral
folds. There is a gradient of decreased proliferation
with increased distance from the body axis and with
increased elevation of lateral folds. Differentials in
proliferation at sites flanking largest folds are most
dramatic, and it is the differentials that appear to be
significant in morphogenesis. Once amnion folds have
fused, proliferation in ectoderm is again high (8689%).
DISCUSSION
Patterns of cell proliferation in ectoderm epithelium
anticipate amniogenesis and complement other phenomena that may contribute to the creation of largescale folds. Domains vary through the period of lateral
fold formation and the differences between domains is
greatest when the greatest amount of amnion fold elevation is occurring. Once amnion folds have fused, high
levels of proliferation suggest growth to generate new
structure and support the textbook description that
“growth in the somatopleure itself tends to extend the
amniotic fold caudad over the head of the embryo”
(Carlson, 1988, p. 257). This new information about
differential cell proliferation must be placed into context with an extensive body of data on amniogenesis
and morphogenesis in general.
Avian amniogenesis is large-scale folding morphogenesis, and complex events such as morphogenesis appear to be effected by several mechanisms acting concurrently or in sequence. Kolega (1986) suggests
epithelial morphogenesis of sheets, tubes, spheres, or
pockets is a result of varying combinations of a few
basic processes. It is reasonable to consider that regional differentials in cell proliferation might contribute to morphogenesis. Indeed, several investigations
have suggested a link between localized cell division
and morphogenesis.
Proliferative differentials play an important role in
the formation of the lateral body folds of 20-somite
chick embryos (Miller, 1981, 1982). Localized changes
in cell proliferation correlate with bending of neural
plate (Smith and Schoenwolf, 19871, and expansion of
surface epithelium has been suggested as a major extrinsic force in bending of chick neural plate (Alvarez
and Schoenwolf, 1992). Regional differences in cellular
proliferation rates have been noted in branching salivary rudiment (Bernfield et al., 1972), elongating tubular glands in chick oviduct (Wrenn, 19711, and during rupture of oral membrane (Miller and Olcott, 1989)
and rupture of pharyngeal closing plates (Miller et al.,
1993). Localized increases in proliferation of mesoderm
cells precede visible creation of mounds of tissue in
early limb buds (Searls and Janners, 1971) and correlate with formation and fusion of facial swellings
(Minkoff, 1980). Our data suggest that significant differences in cell proliferation precede a visible amnion
fold.
Development may occur through temporal waves of
mitosis in epithelia. Part of the temporal wave is visible in the sagittal presentations in Figure 6 . The high
proliferation rate that returns to cells proximal to the
axis included in the new amnion membrane (not included in figure) represents another wave. We have
also seen waves of proliferation in chick endodermal
230
S.A. MILLER ET AL.
Riaht
Left
C)omalns:
100
90
80
... ... ... ... ... ... .
70
60
20
10
~~~
.....
. 2.
. .....
. .Y. :f
. .. .. .. .. . . . .
.......
.....-.
... . ... ... ... ... ..
. . . . . . . .. .. .
. .. . ... ... ... ... ...
.. .. .. .. .. ..
. . . .. .. .. .. .
......
...
1:..1.. .......
.. .. '. . : ..
I . .
I
//I
-1
I
I
. . . . . .
. . . .. .. .. .. .
. .. .. .. .. .. ..
0 distal chorio
amhion
,Body
.Amnion
Chorion
Distal Epithelium
'body
'
I
EMBRYO
I
,
body '
I
1
amhion 'chorioi distal
YU
Left Chorion
...
80
...... ... ... ... ...
. .. .. .. .. .. .
. .. .. . . .- .. .
. . . .. .. .. .. .
.. .. .. .. .. ..
. . .. .. .. .. ..
.. .. .. .. .. ..
......
. . . .. .. .. .. .
. . . .. .. .. .. .
. . . . . . . . . . ..
. . .. .. .. .. ..
. . .. .. .. .. ..
......
.. .. . . . . . .. .
......
. .. ... ... ... ... ...
. . . . . . .. .. ..
. .. .. .. .. .. .
. . . . . . .. .. ..
. . . . . .
. . . .. .. .. .. .
Amnion Folds
distal chorion amnion
body
EMBRYO
Left
body
amnion chorion distal
Right
Sites
Fig. 5. Mean proliferation indices of sample sites pooled in Figure 4,arranged in transverse series, and
displayed according to morphological levels.
231
ECTODERM CELL PROLIFERATION DOMAINS
Cephalad
Caudad
Sianificantlv Different GrouDs:
100
- Medium and Small Folds
--$-
90
Large Fold and Pre-fold
Proximal Amnion Sites
P = 0.0437
PI range = 89 92
3 0 ~
20
10
+
I
Large
Fold
Medium
Fold
I
Small
Fold
I
-
Pre-fold '
Sianificantlv Different GrOUDS:
- Medium, Small, Pre-fold
- Large Fold
Amnion Fold Sites
0'
' Medium '
Large
Fold
Fold
Small
Fold
' Pre-fold I'
I
P < 0.00005
PI range = 78 88
-
Sianificantlv Different G r o u m
- Small Fold and Pre-fold
- Medium Fold
- Large Fold
:i
20
U
-
Chorion Sites
-
10
-
04
Large
Fold
'
Medium
Fold
Small
Fold
Pre-fold
'
P < 0.00005
PI range = 66 82
Level in Sagittal Plane
Fig. 6. Mean proliferation indices of proximal amnion, amnion fold, and chorion sites arranged in
sagittal series.
-
232
S.A. MILLER ET AL.
Right
Left
Domain5:
Body
Proximal Amnion
Amnion Fold
Chorion-Distal
,0°5----90
40
30
20
13-19 Somites
10
distal chorion
amnion
body
body
EMBRYO
amnion
chorion distal
Domains:
Body
Proximal Amnion
Amnion Folds
1
Chorion
Distal Epithelium
20-23 Somites
.
distal chorion' amnion
'body
EMBRYO
'
body
100
'
.
amnion
chorion distal
Domains:
Body-Proximal Amnion
Amnion Folds
Chorion
Distal Epithelium
~
90
. . .. .
. . . ..
.. .. .
. . . ..
. . .. .
. .. . .
. -. .
. .. ... .
. .. ..
. ... .
. . . ..
. . .
. .. . .
. . . ..
. . .
. .. . .. .
. ... .
.. . .. . ..
80
70
60
50
40
'"_
30
10
,
I
I_~~~_I
. . .
. . .
...
0
distal'chorion
an In
.
body
EMBRYO
body
25-27 Somites
I
,
am on
Left
Sites
Fig. 7. Mean proliferation indices of sample sites pooled in Figure 4, arranged in groups of increasing
age.
ECTODERM CELL PROLIFERATION DOMAINS
epithelium forming gut (Miller, 1984, 1986). Further
study of endoderm epithelium and correlation of waves
of proliferation with tube morphogenesis is in progress
in our laboratory. Asynchronous proliferative events
could be the result of timing proliferative waves such
that asymmetries occur in specific adjacent areas. The
resulting differential growth could contribute significantly to initiation of folds, grooves, and tubes.
Differential rates of cellular proliferation combined
with mechanical constraint have been used to explain
several developing systems. Localized and relatively
higher rates of proliferation in areas physically constrained would lead to changes in shape of crowded
cells (a placode) and ultimately to buckling of epithelium (invagination or evagination). Such explanations
have been suggested for evaginating chicken lens epithelium (Zwaan and Hendrix, 19731, evaginating thyroid gland (Hilfer, 1973; Smuts et al., 19781, and developing pancreas rudiment (Pictet et al., 1972). The idea
of regional differences in rates of cellular proliferation
contributing to mechanical constraints on the direction
in which growth can occur was also proposed for early
morphogenetic changes in implanting mouse embryos
(Copp, 1979).
Devices that contribute to small-scale folding, such
as localized changes in cell shape (Spooner and Wessells, 1973)may not be necessary to produce large-scale
folds. Large-scale folding could be effected by more diffuse forces such as localized differential growth in the
form of differentials in cell proliferation.
Several investigations have addressed the question
of what causes somatopleure to double upon itself to
form amnionic folds in chick embryos. Lillie (1903)performed a series of classic cauterization experiments
that demonstrated varying effects on folding. Dalcq
(1937, 1938) proposed that forces of epiblastic epiboly
were involved in elevation of amnionic folds. Adamstone (1948) expanded the work of Dalcq and Lillie
with additional cauterization experiments. Hamilton
(1965) incorporated that work into his expansion of Lillie’s treatise on chick embryo development. Davidson
(1977) explored several aspects of the microanatomy of
amniogenesis. Overton (1989) focused on forces associated with fusion of epithelial sheets. All these studies
focused on elevation and closure of established folds,
attributed amniotic folding to tension located along the
somatopleure and changes in cell shape, and suggested
that elevation is not an intrinsic property of the lateral
folds but that it may be related to fusion of established
folds. Differential cell proliferation was not a part of
any of these studies.
Our data provide new information about formation
of large-scale folds and support several earlier observations. Domains in our data (Fig. 4) correspond to
Davidson’s morphological regions in ectoderm during
amnion development. Our data also corroborate Davidson’s (1977) suggestion of medial constraint and add
a component of lateral constraint produced by significantly slower rates of growth in the ectoderm of the
extraembryonic somatopleure. Slow relative rates of
proliferation at distal sites, combined with the folding
body wall and constraint of a fast-growing axial
center, could lead to buckling of epithelium along
the amnionchorion sites. This effect could even
produce multiple folds similar to the secondary
233
amnion folds described in later stages by Lillie
(Hamilton, 1965).
Our data also support earlier observations about
growth of later amnion. Hamburger and Hamilton
(1951) describe amnion in stages occurring immediately after those in this study (HH17; 29-32 somites)
as having “considerable variability” concerning how
much coverage of the body exists and completeness of
closure of the raphe. Our data suggest this reflects increased and more uniform growth rates in embryonic
and amniotic sites. The beginnings of this can be seen
in our data from 25-27-somite embryos (HH stages
15-16). Proliferation indices are more uniform and
generally high once amnion is established and elaboration of the structure, rather than initial shaping, is
the emphasis.
Differential proliferation must be put into context
with other explanations that have been offered for the
elevation of chicken amniotic folds. Constraint of expanding epithelium, packing forces, differential adhesion, and the energy of fusion of existing folds have all
been considered.
Consfraint of Expanding Epithelium
The notion that epithelium will buckle when growth
is constrained was considered by His (1874) when he
suggested that amphibian neurulation might be caused
by growth pressure. Although the proposal that only
cell proliferation drives neurulation is now considered
too simple, growth pressure is still a likely candidate
for the force causing epithelium to form accordion folds
(Kolega, 1986). The first irregular and unevenly distributed folds of mouse embryo pancreatic diverticulum (Pictet et al., 1972) and convolutions of the human
cerebral cortex (Richman et al., 1975) have been explained by constricted growth pressure. Additional evidence that rapid proliferation in constrained epithelium leads to folding was provided by Desmond and
Jacobson (1977) with experiments in which extensive
atypical folding of neuroepithelium was found in embryonic chick brains a few hours after intubation removed normal hydrostatic support of rapidly expanding epithelium. Cell proliferation in constrained
epithelium would be expected to produce folds. Growth
alone, even highly localized growth, merely expands a
sheet unless there are structural limitations on that
expansion.
Medial constraint of amnion growth is provided by
body epithelium growing at a comparatively faster rate
while lower rates of growth distally provide lateral constraint. It is possible to view the flanking extraembryonic epithelium growing at significantly slower rates
as a constraint on expansion of body ectoderm that
forces buckling and concomitantly supports amnion
folding. Proliferation indices a t lateral distal sites are
much lower in folding levels. This observation supports
the hypothesis of lateral constraint (Miller et al., 1982).
Lateral amnion folding is most apparent in levels
where the proliferation index of distal sites is significantly lower than that in more medially located sites.
Proliferation occurs throughout extra-embryonic epiblast during epibolic expansion and is not restricted to
the blastoderm periphery in stage 2-26 chick embryos
(Downie, 1976).Our samples of blastoderm were extensive (13-18 mm wide), but did not include the entire
234
S.A. MILLER ET AL.
blastoderm between epibolic edges. Downie reports
that even after the yolk has been covered (4 days, later
than the period of our study), epiblast continues to
grow with proliferation restricted largely to a band just
distal to the advancing edge of the area vasculosa. Distal proliferation provides new membrane and eases
tension created by the early imbalance between expansion by migration and expansion by proliferation, but it
could also be another source of lateral constraint during the period of elevation of lateral amnion folds. A
circumferential belt of increased growth could serve as
a lateral constraint to the growth patterns seen in our
samples.
Packing Forces
Additional evidence of a n impact of cell proliferation
differentials may be seen in the elongated cells of amniochorionic ridge (ACR), a n arc of tightly packed cells
described by Davidson (1977) as the precursor to lateral amniotic folds. Pressure from surrounding tissues
(distal epithelium and body folds) combined with differential cell cohesive forces could produce a n ACR in a
manner similar to the packing forces contributing to
the maintenance of elongated shape of neuroepithelial
cells (Schoenwolf and Powers, 1987). Differential cell
proliferation could be the “unspecified process of selfdifferentiation” that Davidson mentioned a s being responsible for the formation of the ACR. Furthermore,
the ACR marks the site of amnion fold formation (Davidson, 1977) and could be viewed as a hinge combined
with forces of differential growth that assists folding
similar to the way thyroid placode assists evagination
(Smuts et al., 1978) and avian neural tube is shaped
(Schoenwolf and Smith, 1990). Our data support the
possibility of packing forces operating within extraembryonic epithelium.
Regionalized increased cell proliferation is a form of
pressure. Weiss (1955)observed that gross mechanical
factors do not create differentials manifested in caveins, outpocketing, fissures, or folds, but merely translate them into more conspicuous configurations. Goldin’s (1980) review of epithelial morphogenesis also
suggested that initiation of folding is not only the result of pressure from adjacent tissues and cell shape
changes, but localized differences in cell proliferation
as well.
Differential Adhesion
Differential adhesion could provide fixation of attachments and sites of constraint and must be considered as another force involved in folding epithelial
sheets. Extracellular matrix and desmosomes are two
sources of localized adhesion. A preliminary study correlating general matrix staining with body and gut
folds (unpublished observations of Miller and PinolRoma) suggested a role for matrix adhesion in fixing
areas of epithelia undergoing folding. Interfacial tension suggested by Newman and Comper (1990) could be
the result of adhesive differences between blocks of
cells. Differential effects provided by cell division,
along with cell movement and cell death, are ultimately influenced by cellular adhesions (Gustafson et
al., 1986). Cell adhesion molecules and/or desmosomes
within body fold ectoderm would increase cell adhesions and may contribute to folding. Cell adhesions
within the amniotic fold ectoderm need to be explored
in greater depth to determine their contribution to
buckling of somatopleure and the possible role of adhesion in the process of folding.
Energy of Fusion
Cell-cell fusion in the area of the raphe, may drive
the posterior movement of the amnion headfold (Davidson, 1977). Lillie (1903) called it the energy of fusion.
Adamstone (1948) suggested that formation of the amnion is based on mutual dependance of right and left
amniotic folds. Overton (1989) also suggested that fusion of opposite folds is enough to lift the somatopleure
up in the form of lateral folds around the embryo. The
notion that the developing lateral amniotic folds are
dependent upon fusion of the folds themselves is supported by a n experiment whereby cauterization of one
fold rendered folding on the opposite side insignificant
(Overton, 1989). Regarded in this way, amniogenesis is
seen a s a process of a “level-for-level” fusion of amniotic folds, driving the posterior movement of the amnion headfold like a zipper and coincidentally elevating
lateral folds. Davidson (1977) contends that cell-cell
fusion may contribute to the antero-posterior progression of the amniotic headfold, but that it falls short of
supporting amniotic headfold formation, tailfold formation, and closure of the orifice. All of these explanations assume amniotic folds in place in order to have
something to fuse. They do not address the question of
initial formation of those folds. Fusion does not act
alone in the formation of the amnion.
Other
Amniotic fold formation could be a passive process
resulting from mechanical events occurring elsewhere
in the blastoderm. Weight of the embryo could push the
body into the yolk and force the somatopleure to buckle
and fuse over the sinking axis. Cephalic flexure and
torsion (both occurring around the 14- to 15-somite
stage), may aid amniotic fold morphogenesis and headfold formation by forcing the head into the yolk (Goodrum and Jacobson, 1981). However, formation of amnion in acephalic embryos suggests that morphogenetic
movements in the cephalic axis are not necessary.
In conclusion, patterns of differential cell proliferation correlate with elevation of lateral folds and establishment of amnion. Patterns of proliferation correlate
with morphology and can be seen as assisting the
caudad progression of fusing folds. If differential
growth is not a primary cause of elevation of amnion
folds, it may be necessary to help fine-tune the morphogenesis and fill in areas of membrane as folds form.
Several questions remain unanswered. Although it
is plausible that growth expansion of a constricted
sheet of cells causes folding, much of the evidence remains correlative or circumstantial. It is difficult to
demonstrate experimentally that mitosis is necessary
for folding to occur. Creation of holes in a n epithelium
produces mixed results owing to the variable of a cocontribution from contractile filaments. Poisoning cells
to stop division is not precise and defeats the purpose of
the experiment.
Localized regulation of the cell cycle is a likely contributor to epithelial morphogenesis (Smithand Schoenwolf, 19911, but our experiments did not address ques-
ECTODERM CELL PROLIFERATION DOMAINS
235
tions about duration of the cell cycle or its control. The Downie, J.R. 1976 The mechanism of chick blastoderm expansion. J .
Embryol. Exp. Morphol., 35:559-575.
question of whether a decrease in proliferation index is Goldin,
G.V. 1980 Towards a mechanism for morphogenesis in epia result of a decline in the percentage of replicating
thelio-mesenchymal organs. Quart. Rev. Biol. 55:251-265.
cells, or a change in the length of the cell cycle, remains Goodrum, G.R., and A.G. Jacobson 1981 Cephalic flexure formation
in chick embryo. J. Exp. Zool., 216:399-408.
unanswered.
J.P., G.L. Stebbins, and F.J. Ayala 1986 Genetics, DevelCell division is a fundamental process of embryogen- Gustafson,
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proliferation is a form of differential growth that may Hamilton, H.L. 1965 Lillie’s Development of the Chick. Holt, Rinehart and Winston, New York.
be a significant contributor to large-scale morphogen- Hilfer,
S.R. 1973 Extracellular and intracellular correlates of organ
esis events, but until patterns of proliferation are
initiation in the embryonic chick thyroid. Am. Zool., 13:1023available, that contribution remains invisible to inves1038.
tigators. Knowledge of proliferation domains will as- His, W. 1874 Unsere Koperform und das physiologische Probleme
ihrere Entstehung. Vogel, Liebzig.
sist interpretation of specific studies of the control of Kolega,
J. 1986 The cellular basis of epithelial morphogenesis. In:
cell proliferation and variation of the cell cycle. DoDevelopmental Biology: A Comprehensive Synthesis. Vol. 2.
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creased proliferation along an axis may be seen as an Lillie, F.R. 1903 Experimental studies on the development of the organs in the embryo of the fowl (Gallus domesticus). Biol. Bull.,
expression of these controls, but we must know the
5:92-124.
distribution of proliferation patterns to interpret fully Miller, S.A. 1981 Closure of lateral body folds and axial rotation in
the effect of morphogens and growth factors. This study
chick embryos. Am. Zool., 21:976. (abstr.)
provided information about proliferation patterns in Miller, S.A. 1982 Differential proliferation in morphogenesis of lateral body folds. J. Exp. Zool., 221:205-211.
chick embryo ectoderm epithelia. Our analysis of pro- Miller,
S.A. 1984 Differential proliferation may be the major contribliferation in endoderm and the relationship of proliferutor to folding in formation of the chick embryo gut tube. Am.
ation to formation of chick gut folds and tube formation
Zool., 24:158A (abstr.).
Miller, S.A. 1986 Differential cell proliferation and formation of the
will be the subject of a subsequent report.
chick embryo gut tube. Anat. Rec., 224:87A (abstr.).
Miller, S.A., and K.L. Bresee 1992 Domains of differential cell proliferation in chick embryo ectoderm confirm that amnion constrains
body folding during folding morphogenesis. Anat. Rec., 232:62A
This report includes preliminary work done in par(abstr.).
tial fulfillment of requirements for Senior Thesis by
S.A., and R.B. Campbell 1979 Ultrastructure of avian soK.L.B., C.L.M., and D.A.T. Funding was provided by Miller,
matopleure in lateral body folds during axial morphogenesis. Am.
the Casstevens Family Fund, Hughes Medical InstiZool., 19:1002 (abstr.).
tute, Hamilton College Academic Fund for Seniors, and Miller, S.A., and C.W. Olcott 1989 Cell proliferation in chick oral
membrane lags behind that of adjacent epithelia at the time of
Hamilton College Faculty Research Funds. The data
rupture. Anat. Rec., 223,204-208.
base reported here includes embryos prepared by Joel Miller,
S.A., J.P. Amidon, and F.E. Price 1982 Amnion is a constrainAmidon and Megan Cavanaugh. Dr. Frank Price proing support for lateral body folding in chick embryos. Am. Zool.,
22:930 (abstr.).
vided valuable consultation on use of computer appliMiller, S.A., A.M. Favale, and S.J. Knohl 1993 A role for differential
cations in this analysis.
cell proliferation in perforation and rupture of chick pharyngeal
closing plates. Anat. Rec., 237~408-414.
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