Cellular polarity in cultured animal pole cells of Xenopus embryos

The expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos (stages 6, 8, and 10) was examined morphologically and immunocytochemically. When control embryos reached stage 23, daughter cells derived from a single or a few animal pole cells formed aggregates. Outer cells of the aggregates displayed intracellular and surface polarity and expressed an epidermis-specific antigen (XEPI-1) on the apical surface circumference, while these characteristics had not yet been established in the animal pole cells at the time of isolation. However, inner cells of the aggregates did not display the cellular polarity along an outer-inner axis of the aggregates and displayed the antigen randomly within the aggregates. These results indicate that the expression of cellular polarity in epidermal differentiation of Xenopus embryos in vitro depends on the position within the aggregates formed by daughter cells derived from isolated animal pole cells.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

Phenotypic expression of marrow cells when grown on various substrata

Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.     

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.     

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.     

Induction of neural cell adhesion molecule (NCAM) in Xenopus embryos

Using a classical neural induction protocol (H. Spemann and H. Mangold (1924). Roux' Arch. Entwicklungsmech. Org. 123, 389-517), it has been demonstrated that the sustained presence of NCAM in Xenopus embryos, as detected by immunohistochemistry, was confined to the experimentally induced nervous system and the primary host nervous system. Furthermore, in vitro NCAM expression by dorsal blastopore lip and animal pole tissue was detected only when the two tissues were cultured in contact. These and other results show that readily detected and sustained levels of NCAM expression in Xenopus can be used as a marker for neural tissue and an early positive indicator that neural induction has occurred. They suggest that the observed levels of NCAM are a consequence of and not a prerequisite for induction. Using NCAM expression in vitro to determine the minimum time necessary for this induction to occur in vivo, it was found that NCAM was first detected in cultured animal pole that had been removed at stage 10.75 or later. Thus, an inductive step necessary and sufficient for stimulation of NCAM expression in animal pole tissues had not occurred or was reversible prior to the first 2 to 2.5 hr of gastrulation.     

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

Autonomous mesoderm formation in blastocoelic roof explants from inverted Xenopus embryos.

Xenopus eggs, artificially fertilized, were prevented from undergoing equilibrium rotation by incubation in medium containing ficoll. Three orientations were selected: normal, with animal pole uppermost; inverted, with vegetal pole away from gravity; and an off-axis orientation, with embryos tilted approximately 90 degrees from the animal-vegetal axis. At blastula stage 8, cells forming the blastocoelic roof were cultured in isolation as explants. These cells are normally fated to from epidermis ventrally and neural derivatives dorsally. Unexpectedly, in the fragments originating from inverted or 90 degrees-off-axis embryos, axial structures were found: notochord, somites, neural cells, cement glands, and sometimes sensory organs. Inverted eggs could be exploited in studies of mesodermal specification.     

Clonal analysis of mesoderm induction in Xenopus laevis

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.     

被子植物的早期胚胎形态建成

被子植物早期胚胎形态建成是其有性生殖过程中一个重要发育阶段。在这一阶段中,被子植物形体基本特征形成,包括顶-基轴极性建立、不同细胞层分化以及分生组织形成。合子极性直接与顶基细胞命运相关,但其极性产生机理仍然不明。研究表明,WOX家族转录因子、生长素定向运输以及生长素响应应答可能参与了早期顶-基模型建成;辐射对称模型的建立可能由细胞与细胞间相互作用来介导;生长素流可能参与胚胎顶端组织形成。该文对近年来被子植物早期胚胎形态建成过程中的合子极性建立与生长、合子分裂及其顶基细胞的形成、胚根原特化及根极的形成、辐射对称模式及表皮原特化、顶端分生组织特化及子叶起始等方面的研究进展进行了综述。     

CELL POSITION-DEPENDENT RECIPROCAL FEEDBACK REGULATION OF TYPE I COLLAGEN GENE EXPRESSION IN CULTURED HUMAN SKIN FIBROBLASTS

In order to investigate possible cell positional effects on the gene expression of human dermal fibroblasts, the authors cultured the cells on non-coated polystyrene culture dishes, type I collagen-coated dishes, or collagen gels formed by type I collagen, or suspended them in type I collagen gels and measured collagen synthesis by the cells. The production rate of type I collagen was similar whether cells were cultured on non-coated polystyrene or on type I collagen-coated dishes, but it was suppressed significantly when the cells were placed within the collagen gel matrix. Time-dependent expression of genes for α1(I) and α2(I) collagen chains was measured by Northern blot analysis. A significant increase in mRNA levels for these chains was observed when the cells were cultured for three days on type I collagen-coated dishes or on collagen gels. On the other hand, a significant decrease in the mRNA levels was observed after 2 days and later, when the cells were cultured within type I collagen gel matrix. These results indicate that human dermal fibroblasts recognize their position on or in type I collagen (extracellular matrix) and respond by changing their expression patterns of type I collagen chain genes. The results of the kinetics of gene expression also suggest that upregulation and downregulation of type I collagen genes are controlled by different mechanisms.     

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified Xenopus Epidermis

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.     

Increased attachment and confluence of skin epidermal cells in culture induced by ascorbic acid: detection by permeation of trypan blue across cultured cell layers

In culture epidermal cells from the skin of newborn rats became attached to Millipore filters coated with type IV collagen much better than to filters coated with type I collagen. Ascorbic acid markedly increased the attachment and viability of epidermal cells seeded on type I collagen, but had no significant effect on cells seeded on type IV collagen. It was also found to enhance the synthesis of type IV collagen by the cells, which, we concluded, enabled the cells to become well attached to type I collagen. This conclusion was supported by studies on the penetration of trypan blue through the cell layers. There was a lag in penetration through cell layers cultured both with and without ascorbic acid on Millipore filters coated with either type I or IV collagen, indicating that the cells were confluent over the whole surface of the filters. The lag was much longer in the cultures with ascorbic acid, indicating greater confluence and tighter attachment of cells due to production of type IV collagen. The penetration was found to be due to destruction of the confluent cell layers by its cytotoxic effect. The time lag before penetration of trypan blue is a good index of the confluence and attachment of cultured cells to collagen layers.     

Patterns of junctional communication during development of the early amphibian embryo

Cell-cell communication through gap junctions was examined in Xenopus laevis embryos between the 16-cell and early blastula stages using Lucifer Yellow, Fluorescein, lead EDTA and dicyanoargentate as probes of junctional permeability. Injections were made into cells whose position was identified with respect to the primary cleavage axis and the grey crescent. FITC dextrans revealed cytoplasmic bridges between the injected cell and its sister only. In the animal pole at the 16-cell stage at the future dorsal side of the embryo, Lucifer Yellow was frequently and extensively transferred between cells through gap junctions. At the future ventral side gap junctional transfer of Lucifer Yellow was significantly less frequent and less extensive. The asymmetry of transfer between future dorsal and ventral sides of the animal pole was more marked at the 32-cell stage. In the vegetal pole also at the 32-cell stage, a dorsoventral difference in junctional permeability to Lucifer Yellow was observed. At the 64-cell stage the transfer of Lucifer Yellow was relatively frequent between cells lying in the same radial segment in the animal pole; transfer into cells outside each segment was infrequent, except at the grey crescent. At the 128-cell stage, Lucifer transfer between future dorsal or future ventral cells in the equatorial region was infrequent. A high incidence of transfer was restored at the future dorsal side at the 256-cell stage. At the 32-cell stage, fluorescein was infrequently transferred between animal pole cells although lead EDTA moved from cell to cell with high, comparable frequency in future dorsal and ventral regions. Dicyanoargentate always transferred extensively, both at the 32- and 64-cell stages. Treatment of embryos with methylamine raised intracellular pH by 0.15 units, increased the electrical conductance of the gap junction and produced a 10-fold increase in the frequency of Lucifer Yellow transfer through gap junctions in future ventral regions of the animal pole at the 32-cell stage.     

Cell polarity in sea urchin embryos: reorientation of cells occurs quickly in aggregates

Four apical components were used as markers for the apical end of the cell in studies centering on cell polarity in the early blastula stage of sea urchin embryos and in aggregates of cleavage stage cells. Cells were observed to maintain their polarity for several hours if dissociated and cultured in suspension. Orientation of cells in aggregates initially is random; however, within 3 hr the cells have reoriented so that their apical-basal axis corresponds to the correct inside-outside position in the aggregate. This reorientation occurs before formation of a basal lamina or a new hyalin layer in the aggregate, and appears to take place by a rotation or other movement of individual cells. The polarity within each cell is maintained during reorientation. An apical surface antigen is colocalized with concentrations of filamentous actin. Treatment of isolated cells with cytochalasin B causes the antigen to lose its apical position and eventually become distributed around the outside of the cell. Microtubules are visible radiating from two foci closely associated with the nucleus in untreated cells. Treatment of isolated cells with nocodazole leaves the apical cell surface marker and its associated actin undisturbed, but causes the nucleus to lose its apical position. Cytochalasin B and colchicine both prevent reorientation of cells in aggregates. Thus polarity appears to be a constant for the cells, and their reorientation in aggregates occurs prior to the polarized release of extraembryonic matrix and basal lamina.     

Ambystoma maculatum gastrulae have an oriented, fibronectin-containing extracellular matrix.

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin-containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastula (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip-animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN-containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN-coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge.     

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.     

beta-Catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.     

Fates and states of determination of single vegetal pole blastomeres of X. laevis

Vegetal pole cells of Xenopus morulae contribute progeny to all three germ layers, but from the midblastula stage onward they contribute only to the endoderm. We have investigated whether this restriction in fate reflects cell determination by implanting labeled vegetal pole cells into the blastocoels of host embryos and asking which structures later include labeled progeny. Single vegetal pole cells from the morula and also from the midblastula stage can contribute progeny to all germ layers. At the early gastrula stage the cells can contribute only to the endoderm. Thus the restriction of fate in the midblastula does not reflect cell determination. However, the cells do become determined by the beginning of gastrulation.