Domains of differential cell proliferation and formation of amnion folds in chick embryo ectoderm.код для вставкиСкачать
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, opment and Evolution. Plenum Press, New York. esis, but we know little about overall patterns of pro- Hamburger, V., and H.L. Hamilton 1951 A series of normal stages in liferation in whole intact embryos. Differential cell the development of the chick embryo. J . Morphol. 88:49-92. 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. mains of proliferative differentials and waves of inBrowder, L.W., ed. Plenum, New York, pp. 103-143. 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. LITERATURE CITED Minkoff, R. 1980 Regional variation of cell proliferation within the facial processes of the chick embryo: A study of the role of “mergAdamstone, F.B. 1948 Experiments on the development of the amnion ing” during development. J . Embryol. Exp. Morphol., 57:37-49. in the chick. J . Morphol., 83r359-371. Alvarez, IS., and G.C. Schoenwolf 1992 Expansion of surface epithe- Newman, S.A., and W.D. 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