Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.
Upon dissociation, metazoan cells exert an intrinsic potential of sorting out by themselves. It has been repeatedly studied to date as a model to elucidate the principle of morphogenesis. For example, dissociated Hydra cells reassemble and restore the normal whole body [1,2]. Similarly, dissociated chick limb bud cells sort out [
In amphibian early embryos, it is also widely known that dissociated cells derived from different germ layers first unite indiscriminately and reconstruct normal tissue structures under relatively simple culture conditions [
In this paper, we have focused on the following two kinds of macromolecules: 1) C-cadherin and E-cadherin, both of which are major forms of Xenopus cell adhesion molecules at the embryonic stage [13-16], and 2) the cortex actin, cytoskeleton protein known to cooperate with cadherin during the regulation of the cell shape and cell motility [
Sexually mature Xenopus laevis colonies were purchased from Sato Yosyoku (Chiba, Japan) and embryos were obtained by artificial fertilization. Oocytes were stripped from females injected 10 hours earlier with 800 units of human chorionic gonadotrophin (Gonatropin, Asuka Pharmaceutical Co., Tokyo, Japan), and fertilized with minced testis in De Boer’s solution (110 mM NaCl, 1.3 mM KCl, 0.45 mM CaCl2, 3 mM HEPES, pH 7.3 at 23˚C). The embryos were maintained in 10% Steinberg’s solution at 23˚C (1× Steinberg’s solution; 58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3)2·4H2O, 0.83 mM MgSO4·7H2O, 10 mM HEPES, pH 7.3 at 23˚C). The development stage was determined according to Nieuwkoop and Faber [
At stage 10 (the early stage of gastrulation), the animal caps were collected from five dejellied and devitellined embryos using an eyebrow knife and tungsten needle. Likewise, parts of the vegetal hemisphere were carefully collected to be free of not only the animal caps but also the marginal zones. They were individually transferred into Ca2+- and Mg2+-free Modified Barth Saline (CMFMBS: 88 mM NaCl, 1 mM KCl, 5 mM HEPES, 2.5 mM NaHCO3, pH 7.8 at 23˚C) containing 50 μg/ml gentamicin statically for one hour. The outer layer of the animal cap was discarded because it was difficult to dissociate, and only the inner layer was dissociated by gentle pipetting. AC (animal cells) were mixed with an equal volume of VC (vegetal cells), then transferred to an agar-coated 4-well Nunc dish (Thermo Fisher Scientific, Roskilde, Denmark) filled with Ca2+, Mg2+-containing MBS. The mixtures were rotated at 70 rpm and 23˚C for one hour, and then they were subjected to a stationary culture at 23˚C. Aggregates were incubated for 2.5, 5, 7.5, 12, 24, 48, 72, 96, 120 and 148 h, observed by a streomicroscope, and fixed with MEMFA [21,22]. For the long culture, culture media were refreshed every 12 hours. They were dehydrated through an ethanol series and embedded in paraffin wax (Shandon Histoplast, Thermo Scientific, Cheshire, UK).
At the two-cell stage, dejellied embryos were transferred into 4% Ficoll in 10% Steinberg’s solution, and both blastomeres of them were injected with a total volume of 20nl containing 1% Dextran rhodamine (DR, D3312, Molecular Plobes, Eugene, USA) at their animal pole side. At different times in culture, the aggregates consisting of the DR-labeled and unlabeled cells were fixed with 4% paraformaldehyde (PFA) in amphibian CMFPBS (6.4 g NaCl, 0.2 g KCl, 2.9 g NaHPO3·12H2O, 0.2 g KH2PO4, and 200 ml water), then embedded in paraffin, and sectioned into 4 μm slices. The slices were deparaffinized, mounted using Fluoromount (Diagnostic Biosystems, Pleasantom, USA), and observed with a fluorescent microscope (OLYMPUS BX50, Tokyo, Japan). The nuclei had been stained with DAPI (4, 6-diamidino-2-phenylindole dihydrochloride, 0.5μg/ml).
The paraffin sections were deparaffinized and stained with Alcian blue.
The paraffin sections were deparaffinized, rinsed with CMF-PBS, blocked with 1% normal horse serum in CMF-PBS for 20 minutes at room temperature, incubated with the primary antibody overnight at 4˚C, then extensively washed with CMF-PBS. The secondary antibody was then added and incubation was carried out for 2 hours at room temperature. The samples were stained for nuclei with DAPI, mounted using Fluoromount, and observed by fluorescent microscope. The following primary antibodies were used: the anti-E-cadherin monoclonal antibody (supernatant 5D3, Developmental Study Hybridoma Bank, Iowa, USA, 1/3 dilution), anti-Ccadherin monoclonal antibody (supernatant 6B6, Developmental Study Hybridoma Bank, 1/2 dilution), and anti-actin monoclonal antibody (MAB1501, Millipore, Billerica. USA, 1:100 dilution). An Alexa 488 conjugated goat anti mouse IgG2b (A21141; Molecular Probes, Eugene, USA, 1:500 dilution) and an Alexa 594 conjugated goat anti mouse IgG1 (A21125; Molecular Probes, Eugene, USA, 1:1000 dilution) were used as the secondary antibodies. The fluorescence intensities specific to the actin filament at the edge of the AC clusters were determined using the software Image J (National Institutes of Health, USA).
Xenopus embryos at stages 10.5, 12.5, 15, 19, and 21 and aggregates were lysed in extraction buffer (1 × CMFPBS, 1% triton X-100, 20 units/ml aprotinin, 1 mM EDTA (pH 8.0)) containing 1 mM diisopropyl fluorophosphates. The samples were quantified by the BCA protein assay kit (Thermo Fisher Scientific, Illiois, USA). They were combined with an equal amount of Leammli’s 2× sample buffer containing 5% 2-mercaptoethanol, boiled for 2 minutes at 90˚C, and separated in 8% SDSPAGE gel by running for 60 minutes at 60 mA. Gels were transferred to an immobilon-P membrane (Millipore, Billerica, USA) using a semidry apparatus (Nihon Eido Co., Ltd., Tokyo, Japan) for 3 hours at 100 mA. All membranes were blocked with 1% NHS in PBT for 40 minutes at room temperature, and incubated with the primary antibody overnight at 4˚C. The following primary antibodies were used: anti-E-cadherin mAb (supernatant 5D3, Developmental Study Hybridoma Bank, Iowa, 1/10 dilution), anti C-cadherin mAb (supernatant 6B6, Developmental Study Hybridoma Bank, Iowa, 1/10 dilution), and anti β-tubulin mAb (T4026; Sigma, St. Louis, USA, 1:1000 dilution). The HRP-conjugated mouse immunoglobulin (Dako, Glostrup, Denmark, 1:3000 dilution) was used as the secondary antibody for 2 hours at room temperature. The membranes conjugated with HRP were detected by DAB solutions (3 mg of 3-3’ diaminobezidine (Dojindo Laboratories, Kumamoto, Japan), 10 ml of 50 mM TrisHCl (pH 7.6), and 8 μl of 30% H2O2). The intensity of bands was quantitatively estimated by image J (National Institutes of Health, USA).
The thick paraffin sections (20 μm) were reacted with the anti-actin antibody, followed by incubation with Alexa 488 conjugated goat anti-mouse IgG1 (A21121; Molecular Probes, 1:500 dilution). The samples were observed using a Leica TCS SP5 conforcal microscope (Leica Microsystem, Wetzlar, Germany) equipped with immersion lens. All images were taken with the size of 1024 × 1024 pixels, and individual 1.5 μm optical slices were assembled into stacks. Some of them ware axially tilted in the X and Y directions.
The phenomenon called “sorting out of cells”, including those found in the pioneering studies by Townes and Holtfreter [
To clarify whether or how the dissociated cells move to their own positions in the present aggregate culture system, we visualized the process of cell sorting using dextran rhodamine labeled cells (
two kinds of cells could individually recognize themselves. The shape of the AC aggregates became nearly spherical with time (Figures 1 (A)-(C)), whereas the VC aggregates remained flat and rugged (Figures 1(D)-(F)). A temporal and spatial evaluation of the cell arrangement within the AC+VC aggregate was made by preparing the horizontal (Figures 1(G)-(I)) and vertical (Figures 1(J)-(M)) sections against the area facing the agar at different times during the stationary culture. Each of the AC and VC formed clusters and was arranged at random (
and gradually self-assembled into large clusters, then they concentrically arranged, that is, the AC cluster was enveloped by the VC cluster (Figures 1(G) and (I)). This concentrification of clusters suggested that cell sorting progressed. Interestingly, as shown in the vertical sections, the AC and VC clusters started to be rearranged relatively up and down from about 7.5 hC (Figures 1(J) and (L)), and such rearrangement was completed by 12 hC (
The expression of the cell-cell adhesion molecule, cadherin, during the present in vitro culture was immunohistochemically examined (
deed, this tendency of upregulation might explain the fact that the aggregates became more spherical with time. The persistence and up-regulation of cadherin further suggested that the expression of cadherin was also essential for maintaining the morphology of the aggregates during the polarization step, as will be described in Discussion.
The process of sorting out is generally thought to result in an architecture with some concentric arrangements composed of different cell elements, as shown by the preceding theoretical studies (for example, [
brane, but extremely low in the VC (Figures 4(A)-(D)). At 2.5 hC, actin bundles were observed at the edge of AC clusters (
which the polarization was completed, cells closely contacted with each other, so that the space between the clusters was almost lost, and moreover, newly formed actin bundles were seen in the outermost margin of the aggregates (
To assess how these actin bundles were localized in the AC clusters, we also analyzed the expression of the actin filament in thick sections (20 μm) using confocal laser microscopy (
artifacts after fixation would be minimized. When 15 to 20 optical slices (1.5 μm distance) were assembled into stacks (Figures 5(A), (D) and (G)), and were X axially tilted at 40 degrees (Figures 5(B), (E) and (H)), and Y axially tilted at 35 degrees (Figures 5(C), (F) and (I)), the presence of the cortex actin, namely, the accumulation of the actin along the margin of the AC cluster was disclosed. At 2.5 hC, there were remarkable gaps between the AC and VC clusters (
The dissociated AC and VC were intermingled and underwent a long-term stationary culture. At first, they indiscriminately united and formed a flat aggregate. They then gradually rounded up (Figures 5(A)-(D)). After 12 hC, the pigmented AC began to occupy the upper part of the aggregate as if it reproduced the arrangement of the ectoderm and endoderm just like in vivo (Figures 5(E)- (G)). The AC and VC clusters were mutually apposed up and down, as shown by using the Dextran rhodamine labeled cells (
Our experimental results (
the whole process of cell sorting of the AC and VC dissociated during the early gastrula stage can be roughly divided into two steps, i.e., concentrification and polarization. The cells seem to be devoted to “cell sorting in a conventional meaning” for the initial several hours to form a concentric-layered structure, and then the concentrificated aggregates started to restore or mimic the in vivo animal-to-vegetal polarity by rearrangement of the AC and VC clusters. Our finding that these two steps sequentially proceeded but being temporally separated has never been argued by the past studies until now. It took about half a day for the aggregates to establish a polarized embryoid structure. The subsequent embryogenesis in the present in vitro culture system also proceeded very slowly, so that, for example, 96-hour cultured embryos (
Homophilic binding molecule cadherins are responsible for cell sorting, which produces segregation of the cell populations and formation of a tissue boundary [25,26]. In Xenopus laevis embryos, the regional identity starts to be created before midblastula and the cell sorting behavior gradually becomes remarkable as the development advances [9,10]. Accordingly, cadherins are thought to play active roles in the specification of the germ layers. Indeed, C-cadherin is known to ubiquitously express as a maternal cadherin in Xenopus laevis, while E-cadherin starts to exclusively express in the ectoderm from the gastrula stage [13-16]. The ectoderm-derived AC would express both cadherins, and the endoderm-derived VC express only C-cadherin.
Although the homophilic binding dependence of the behavior of the cadherin-expressing cells has not been verified in this study, homotypic reassembly (AC-AC or VC-VC recombination) took place and the cell-typespecific expression patterns of these cadherins in vivo was reproduced in the present in vitro system (
We have withheld the consideration of the role of cadherins, and tried to examine the expression and localization of the cytoskeletal actins, another major intracellular element in the cell motility, in order to find a clue to the question of what is the definitive change within the cells for the concentrified-to-polarized rearrangement of the cell clusters.
Morphogenetic movements linked to the active rearrangement of the cell populations progress during the gastrula stage in Xenopus laevis [
We also examined the possible participation of fibronectin (FN) in the polarization step, as this extracellular matrix component serves as a substrate for mesoderm migration during gastrulation [
Although the immunohistochmical analyses did not show any definitive involvement of the actin filaments and fibronectins in the motility of individual cells such as lamellipodal movement (though not yet experimentally ruled out), another finding to be considered is that a significant amount of actin bundles at the edge of the AC cluster was accumulated at least until completion of the concentrification (
We should now pay attention to the fact that the tangential accumulation of actin at the edge of the AC clusters showed a tendency to decrease as polarization of the clusters proceeded, as observed by an image analysis of these actin bundles (
DAH explains that the tissue surface tension is simply proportional to the cell adhesion energy, while the differential interfacial tension hypothesis, DITH, was proposed in which the interfacial tension in individual cells is responsible for regulation of the tissue surface tension [
Apart from the polarization, we also observed a series of interesting phenomena with the entire aggregate. By the completion of the polarization, the aggregate itself became compacted, and the space between the AC and VC clusters was almost lost. More interestingly, the actin bundles at the outermost surface of the aggregate became remarkable (
The cell sorting properties of the dissociated cells from Xenopus laevis embryos at the blastula and gastrula stages have been studied to date, mainly on their involvement in determination of the germ layer identities in vivo and/or influence of the properties by exogenous inducing substances [9,39]. In this study, we employed the sorting out assay using the same staged Xenopus embryos as those in preceding studies. As a series of detailed temporal analyses of cell sorting, we tried a long term stationary culture up to 7 days. The AC clusters, which had established a polarized arrangement by 12 hC, were found to exhibit the differentiation of cartilage tissues though slowly but normally (
AC is derived from the blastocoelic roof (animal cap) possessing a multi-differentiatiation potencies from the blastula to early gastrula stage. The animal cap is known to be competent to respond to a member of TGF-β, activin, and differentiate into the mesoderm and endoderm in a dose-dependent manner [
In conclusion, our study showed that the cell sorting process of AC and VC from Xenopus early gastrulae could be divided into two steps, concentrification and polarization, and that, in this in vitro system, the expression of cadherin like in vivo and a reduction of actin accumulation at the edge of AC cluster occurred. The actin downregulation was considered to account for the rearrangement of the clusters. Moreover, by extending the period of the cell sorting culture, the AC cluster was differentiated into mature cartilage tissues probably via inductive interactions between the AC and VC. Thus, the aggregate culture system of Xenopus AC and VC allows progress of a certain number of principal events involved in the embryogenesis in vivo.
We would like to thank the members of our Morphogenesis Laboratories for their supports, including helpful discussions in weekly seminars, during this study.