The origin of elementary units of multicellularity and development of a spatial organization of cell layers

The concept of the elementary unit of multicellularity, the histion, which also serves as a morphofunctional unit of cell layers, is introduced. Histions are cell groups formed as a result of division of labor between cells. Cell layers are regarded as regular cell networks (lattices) formed through polymerization of histions. The theory constructed based on this concept allows the composition and structure of a multitude of histions to be calculated; their development to be quantified; and families of topological and geometrical models for the histoarchitecture of cell layers to be constructed, visualized with computer programs, and experimentally verified. In addition, this model can predict previously unknown topological variants of the histoarchitecture of epithelial tissues, as well as their presence in normal development and pathology.     

Identification of the Ubiquitous Human Desmoglein, Dsg2, and the Expression Catalogue of the Desmoglein Subfamily of Desmosomal Cadherins

Desmosomes are junctions between epithelial, myocardiac, and certain other kinds of cells. They represent plasma membrane domains enriched in specific transmembrane glycoproteins, notably desmoglein (Dsg) and desmocollin (Dsc), both of which have recently been identified as members of the larger family of Ca²⁺-dependent cell adhesion molecules, the cadherins. Previously described forms of desmoglein have been isolated as proteins and cloned as cDNAs from epidermis and related stratified epithelia but have not been detected in the majority of other desmosome-containing tissues and cell culture lines. Here we present the complete cDNA-derived amino acid (aa) sequence of a different desmoglein polypeptide, termed Dsg2 (1069 aa, mol wt 116,760) and its precursor molecule (1117 aa, mol wt 122,384), which occurs in all human and bovine desmosome-producing tissues, tumors, and cell lines examined, epithelial as well as nonepithelial ones. We conclude that Dsg2, the largest molecule in this protein family, is the fundamental desmoglein common to all desmosome-possessing tissues, including simple epithelia and myocardium, and many cell cultures. Furthermore, in several tissues and cell lines Dsg2 is the only Dsg isoform detected so far. By contrast, the epidermal isoforms Dsg1 and Dsg3 are restricted to certain specialized epithelia, mostly stratified squamous ones. The importance of the junction-specific cadherin Dsg2 in tissue formation and carcinogenesis as well as in the development of autoimmune diseases of the Pemphigus type is discussed. In addition, we propose to use Dsg2 as a general marker common to all epithelial cells and tumors and to use the specific pattern of occurrence of Dsg and Dsc isoforms as an additional criterion for cell typing in tumor diagnosis.     

A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to a non-metastasizing rat pancreatic carcinoma cell line and to non- metastasizing sarcoma cells. Homologues of this variant as well as several other CD44 splice variants are also expressed at the RNA level in human carcinoma cell lines from lung, breast, and colon, and in immortalized keratinocytes. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we studied the expression of variant CD44 glycoproteins in normal human tissues and in colorectal neoplasia. Expression of CD44 variant proteins in normal human tissues was readily found on several epithelial tissues including the squamous epithelia of the epidermis, tonsils, and pharynx, and the glandular epithelium of the pancreatic ducts, but was largely absent from other epithelia and from most non-epithelial cells and tissues. In human colorectal neoplasia CD44 variant proteins, including homologues of those which confer metastatic ability to rat tumors, were found on all invasive carcinomas and carcinoma metastases. Interestingly, focal expression was also observed in adenomatous polyps, expression being related to areas of dysplasia. The distribution of the CD44 variants in human tissues suggests that they play a role in a few restricted differentiation pathways and that in colorectal tumors one of these pathways has been reactivated. The finding that metastasis-related variants are already expressed at a relatively early stage in colorectal carcinogenesis and tumor progression, i.e., in adenomatous polyps, suggests the existence of a yet unknown selective advantage linked to CD44 variant expression. The continued expression in metastases would be compatible with a role in the metastatic process.     

Localization of a novel integrin of the beta 1 subfamily in human tissues.

We have previously described a novel integrin composed of a beta 1-chain non-covalently linked to an alpha-chain which is biochemically different from those known so far (i.e., alpha 1-alpha 7 and alpha v). This molecule has been identified with a monoclonal antibody (MAb) termed 10.1.2 raised against long-term cultured human thymic epithelial cells (TEC). In this study we analyzed the immunohistochemical distribution of this new integrin in a variety of human tissues. MAb 10.1.2 stains several types of endothelial and epithelial cells. Among the endothelia, a strong reaction was detected in the HEV of lymphoid organs including thymus, lymph node, tonsil, and mucosa-associated lymphoid tissue. Epithelial localizations of note were those in the basal layer of the epidermis and of other stratified squamous epithelia, where the lateral and apical but not the deep surfaces of most cells were stained. A variety of water-electrolyte transporting cells in sweat glands, salivary glands, and kidney were also stained at their deep surface. The latter findings suggest that this molecule may subserve other functions in addition to those related to cell adhesion.     

Epithelial apoptosis

Apoptosis is an essential part of the normal cellular phenotype repertoire. In the absence of appropriate survival factors, apoptosis is activated through specific signalling cassettes. Epithelia form distinctive three-dimensional cohesive structures that depend on adhesive interactions in order for these tissues to carry out their specialised roles, such as secretion and reproduction. The cellular programme that triggers apoptosis in epithelial cells has not yet been shown to differ from that in other cell types, yet the unique characteristics of epithelia endow them with specific determinants for survival. In particular, cell-matrix and cell-cell interactions are required to prevent entry of epithelial cells into apoptosis, and soluble factors that have profound effects on epithelia, such as steroid hormones or hepatocyte growth factor, also influence their survival. The regenerative capacity of certain epithelia is controlled by intrinsic expression of survival genes within stem cell populations, and may regulate the susceptibility of different epithelial tissues to undergo carcinogenesis.     

Phenotypic and developmental analysis of mutations at thecrumbs locus,a gene required for the development of epithelia inDrosophila melanogaster

Summary The genecrumbs (crb) ofDrosophila melanogaster provides an essential function for the embryonic development of ectodermally derived epithelia. Complete loss of function alleles of thecrb gene are recessive embryonic lethals and lead to a disorganization of the primordia of these epithelia, followed by cell death in some tissues. Incrb mutant embryos, different organs are affected to a different extent. Some tissues die almost completely (as the epidermis, the atrium and the pharynx) while others partially survive and conserve their basic epithelial structure (as the tracheal system, the oesophagus, the proventriculus, the salivary glands, the hindgut and the Malpighian tubules). Degeneration is first visible at stage 11 and continues successively throughout development. There is evidence that the loss of epithelial cell polarity may be the cause for the degeneration of these tissues, suggesting that thecrb gene product is involved in stabilizing the apico-basal polarity of epithelial cells. As previously shown, thecrb protein is specifically expressed on the apical side of embryonic epithelia in a reticular pattern outlining the borders of the cells. Here we demonstrate that thecrb protein shows the same subcellular localization in epithelial cells of imaginal discs and in follicle cells, indicating a similar function ofcrb during the development of embryonic, imaginal and follicle epithelia. Clonal analysis experiments indicate that the genecrb is not cell-autonomous in its expression, suggesting that the gene product may act as a diffusible factor and may serve as a signal in a cell-cell communication process. This signal is thought to be required for the formation and/or maintenance of the cell and tissue structure of the respective epithelia.     

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.     

Immunomorphological study of the distribution of prekeratin with a molecular weight of 49 kilodaltons in various epithelial cells in rats

Cryostat sections of various tissues of rat were stained using an indirect immunofluorescent method with monoclonal antibody against individual prekeratin with the molecular mass of 49 kilodalton (PK-49). Connective tissue endothelial cells, neurons, glia, haematopoetic tissue and smooth muscles were completely negative in this test. 46 morphological variants of epithelial structures were investigated. PK-49 was absent from all the stratified epithelia (epidermis, hair folliculi, oesophagus) but was expressed in virtually all simple epithelia of endodermal origin (exceptions: squamous lung alveolar epithelium and germinative epithelium of testis). There were negative (kidney tubules) as well as positive (bladder, mammary, glands) cell elements among mesodermal and ectodermal simple epithelia. High specificity of individual PK in respect to morphological variants of epithelia points out to the important role played by prekeratin-type intermediate filaments in morphogenesis.     

Topological Progression in Proliferating Epithelia Is Driven by a Unique Variation in Polygon Distribution

Morphogenesis is consequence of lots of small coordinated variations that occur during development. In proliferating stages, tissue growth is coupled to changes in shape and organization. A number of studies have analyzed the topological properties of proliferating epithelia using the Drosophila wing disc as a model. These works are based in the existence of a fixed distribution of these epithelial cells according to their number of sides. Cell division, cell rearrangements or a combination of both mechanisms have been proposed to be responsible for this polygonal assembling. Here, we have used different system biology methods to compare images from two close proliferative stages that present high morphological similarity. This approach enables us to search for traces of epithelial organization. First, we show that geometrical and network characteristics of individual cells are mainly dependent on their number of sides. Second, we find a significant divergence between the distribution of polygons in epithelia from mid-third instar larva versus early prepupa. We show that this alteration propagates into changes in epithelial organization. Remarkably, only the variation in polygon distribution driven by morphogenesis leads to progression in epithelial organization. In addition, we identify the relevant features that characterize these rearrangements. Our results reveal signs of epithelial homogenization during the growing phase, before the planar cell polarity pathway leads to the hexagonal packing of the epithelium during pupal stages.     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na：1K：2C1（乌贼轴突中是2Na：1K：3C1）,其活性被布美他尼（bumetanide）和呋塞米（furosemide）所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体：NKCCl和NKCC2。NKCCl存在于多个组织中,合有NKCCl的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密斑的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用（尤其是NKCCl亚基）是通过特定的激酶使该转运蛋白自身发生氧化／硝化、磷酸化／去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

Changes in localization of human discs large (hDlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hDlg variants

Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.     

Dome formation by oral epithelia in vitro

Summary Multicellular, cystic structures, termed domes, have been described previously in epithelia cultured from various tissues that have a known transport or secretory function in vivo and in vitro. We report for the first time dome formation in cells cultured from “covering” and “rest” epithelia of oral tissues: porcine gingival and alveolar mucosa epithelium and epithelial rests of Malassez. As demonstrated by light- and electron microscopy, the morphology of the domes varied with the location of their lumen and the number of cells or cell layers involved in their structure. Sequential observations using phase contrast microscopy and time lapse cinematography of living cultures showed that the domes were dynamic structures with expansion-collapse cycles of between 30 min and 17 h duration. Dome formation in oral epithelia was stimulated by dibutyryl cyclic AMP (dbcAMP, 10⁻³ to 10⁻⁶ M) and abolished by ouabain (10⁻¹⁰ M), an inhibitor of sodium transport. The morphological features and the dynamic nature of domes found in oral epithelia, and their dbcAMP and ouabain responsiveness are similar to those demonstrated previously in several other epithelia that have a known transport function in vivo and in vitro. Such fluid transport is not thought to be a property of oral epithelia in vivo. Our data, however, suggest a similar function of these epithelia cultured in vitro, and perhaps in pathological cyst formation in vivo.     

Hyaluronan Disappears Intercellularly and Appears at the Basement Membrane Region during Formation of Embryonic Epithelia

The distribution of tissue hyaluronan has been assessed in the neuraxial region of 8.5 to 10.5 day mouse embryos using a fragment of bovine nasal cartilage proteoglycan that binds specifically to hyaluronan. Hyaluronan is abundant in all mesenchymal tissues, predominantly intercellularly, but markedly diminishes when mesenchymal cells organize into epithelia, as in the formation of somites. Hyaluronan reappears in abundance when epithelia (e.g. sclerotome) disperse into mesenchyme. Hyaluronan is present between cells of early epithelia (e.g. neural plate), but is lost during their subsequent development when it becomes abundant at their basement membrane regions. These results show for the first time changes in hyaluronan distribution during the development of embryonic epithelia. The hyaluronan distribution found is consistent with the functions proposed for hyaluronan in embryonic mesenchyme: intercellular hyaluronan would allow the epithelial cells to move and reduced hyaluronan would allow the cells to associate. The absence of intercellular hyaluronan in later epithelia would allow increased membrane contacts that lead to the formation of intercellular junctions. The restriction of hyaluronan to basement membrane regions in later epithelia further substantiates the suggestion that hyaluronan is a bona fide component of the basal lamina and that it is involved in maintaining epithelial morphology.     

Cloning and sequence of the cDNA encoding the β4 integrin subunit in rat peripheral nerve.

β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia.     

Analysis of corneal development with monoclonal antibodies. II. Tissue autonomy in cornea-skin recombinants

Developmental autonomy of corneal epithelial and stromal components was assessed by their subsequent differentiation after recombination with feather-forming thigh dermis and epidermis, respectively. Work by others has shown that feather-forming dermis exhibits strong inductive ability when used in such epithelial-mesenchymal recombinations. After culture of the recombinants on the chorioallantoic membrane (CAM) of host embryos, differentiation as "cornea" was assessed immunohistochemically using the anti-corneal stromal matrix and anti-corneal epithelial antibodies described previously (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983). Feather initiation and outgrowth and keratin synthesis served as markers for differentiation as skin. It has been found that corneal epithelia from 5-day embryos, when grown in association with feather-forming dermis from the thigh, will participate in feather formation. In such recombinants, when the corneal epithelium became incorporated into feathers it failed to express the corneal epithelial antigen, but in regions of the recombinant where feathers did not form, de novo expression of the antigen was sometimes detected. The limited liability of the epithelium is not present in corneal epithelia taken from embryos a day or two older. When such epithelia were used for making the recombinants, no feathers were formed and the corneal epithelial antigen was extensively produced. Thus epithelial determination occurs long before the epithelium would begin to overtly differentiate and express the epithelial antigen in vivo (about 12 days of development). In reciprocal recombinations of corneal stromas with feather-forming epidermis, the stromas proceeded to express the corneal stromal matrix specific antigen de novo after culture on the CAM. They did not, however, redirect differentiation of the epidermis which never expressed the corneal epithelial antigen and in some cases went on to keratinize. These results indicate that development of both the corneal epithelial and stromal components becomes autonomous at least several days before these tissues overtly differentiate. This suggests that the component tissues of the cornea may not interact in a manner typical of those of other organs which, in general, are thought to require continual interaction of their epithelial and mesenchymal components for normal development.     

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.     

The quest for the function of simple epithelial keratins

Simple epithelial keratins K8 and K18 are components of the intracellular cytoskeleton in the cells of the single-layered sheet tissues inside the body. As members of the intermediate filament family of proteins, their function has been a matter for debate since they were first discovered. Whilst there is an indisputable case for a structural cell-reinforcing function for keratins in the mutilayered squamous epithelia of external barrier tissues, some very different stress-protective features now seem to be emerging for the simple epithelial keratins. Even the emerging evidence of pathological mutations in K8/K18 looks very different from mutations in stratified epithelial keratins. K8/K18-like keratins were probably the first to evolve and, whilst stratified epithelial (keratinocyte) keratins have diversified into a large group of keratins highly specialised for providing mechanical stability, the simple epithelial keratins have retained early features that may protect the internal epithelia from a broader range of stresses, including osmotic stress and chemical toxicity.     

Mechanisms of regulating cell topology in proliferating epithelia: impact of division plane, mechanical forces, and cell memory

Regulation of cell growth and cell division has a fundamental role in tissue formation, organ development, and cancer progression. Remarkable similarities in the topological distributions were found in a variety of proliferating epithelia in both animals and plants. At the same time, there are species with significantly varied frequency of hexagonal cells. Moreover, local topology has been shown to be disturbed on the boundary between proliferating and quiescent cells, where cells have fewer sides than natural proliferating epithelia. The mechanisms of regulating these topological changes remain poorly understood. In this study, we use a mechanical model to examine the effects of orientation of division plane, differential proliferation, and mechanical forces on animal epithelial cells. We find that regardless of orientation of division plane, our model can reproduce the commonly observed topological distributions of cells in natural proliferating animal epithelia with the consideration of cell rearrangements. In addition, with different schemes of division plane, we are able to generate different frequency of hexagonal cells, which is consistent with experimental observations. In proliferating cells interfacing quiescent cells, our results show that differential proliferation alone is insufficient to reproduce the local changes in cell topology. Rather, increased tension on the boundary, in conjunction with differential proliferation, can reproduce the observed topological changes. We conclude that both division plane orientation and mechanical forces play important roles in cell topology in animal proliferating epithelia. Moreover, cell memory is also essential for generating specific topological distributions.