Characterization of cytokeratin patterns in the developing human tongue.

The characterization of cytokeratin (CK) in adult oral mucosa and developing teeth have been well documented in human. Cytokeratin distribution in developing oral mucosa has not yet been described. The aim of this study was to identify the expression of CK in human fetal tongue (week 10 to week 23) and to correlate the results with morphological maturation. Simple epithelial CK are expressed in all cell layers during the early stages, essentially in peridermal cells. From the 14th week, CK 18 is present only in the taste buds, making this polypeptide a reliable marker for this sensory organ. CK 4 and 13 are expressed from the 10th to the 23rd week by both ventral and dorsal lingual epithelia. Terminal differentiation keratins (CK 1, 2 and 10-11) can only be detected immunohistochemically at the 14th week in some cells on the external surface of some papillae. The number of these papillae and positive cells increase at the 19th and 23rd weeks. The terminal differentiation markers are expressed several weeks earlier than the formation of a well-distinguished keratinized layer.     

Innervation of developing human taste buds. An immunohistochemical study

 Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud’s progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7–18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds’ primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds. Accepted: 31 July 1997     

Intermediate filament typing of the human embryonic and fetal notochord

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.     

An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 Å; electron-dense band 20–40 Å), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 Å in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 Å separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.     

Comparative study on the appearance of various bioactive peptides in foregut derivates during the ontogenesis.

Bioactive peptides have an important multifunctional role in the gastrointestinal tract. In the present study we have investigated the dynamism of the appearance of PACAP (pituitary adenylate cyclase activating polypeptide), VIP (vasoactive intestinal polypeptide), gastrin, and secretin immunoreactivities in human foregut derivates during the ontogenesis using an immunohistochemical approach. None of these peptides were observed in the foregut derivates of an 8-week-old embryo. VIP immunoreactive nerve fibers appeared by the 11th week in the smooth muscle layers of the stomach. No other peptide immunoreactivities were observed of this stage. In 18- and 20-week old fetuses PACAP, secretin, and gastrin immunoreactive cells appeared in the developing glands of the stomach. In the duodenum gastrin immunoreactivity was present in the Lieberkühn's glands and secretin immunoreactive cells were seen between the surface epithelial cells. In the pancreas secretin immunoreactivity was found in the Langerhans islets; however, PACAP immunreactivity was observed in the exocrine portion. The distribution of VIP fibers did not change during the fetal life and it was similar to the adult pattern. According to our results the appearance of PACAP, secretin, and gastrin in the developing glands suggests their role in the proliferation and differentiation of the epithelial derivates.     

Embryogenesis of the human tongue]

Developmental peculiarities of the tongue as a whole and its main structural elements (muscles, membranes, glands, lingual tonsil) have been studied in 120 human embryos and fetuses 5-week-old--9-month-old. Transmissive electron microscopy, electron histochemistry (for estimation of 5'-nucleotidase activity), staining of semithin slices with toluidine blue and aniline++ pink have been applied. Problems on migration of myogenic elements in the developing tongue have been discussed. Ultrastructure of gustatory bulbs of the human fetus tongues has been investigated. A complete formation of the tongue takes place by the 8th-9th months of the prenatal development.     

Scanning Electron Microscopical Studies of Developing Gustatory Papillae in Humans

Development and morphological changes of human gustatory papillaeduring postovulatory weeks 6–15 have been studied usingscanning and transmission electron microscopy. The first papillaof the tongue appears around postovulatory week 6 in its caudalmidline near the foramen caecum. In contrast, the dorsal epitheliumof the anterior part of the tongue shows only small hillock-or papilla-like elevations from week 6 on, which comprise anaggregation of 5–20 epithelial cells. From week 7 on,most prominent fungiform papillae develop near the median sulcusand at the margins of the anterior part of the tongue. At theirtops, the first primitive taste pores are found around week10; these are often covered with processes of adjacent epithelialcells. Most pores, however, develop around weeks 14–15.The maturation of taste buds does not coincide with the appearanceof taste pores, since taste bud cells are not fully differentiatedin the observed period of time. Fungiform papillae are developedbefore filiform papillae, which do not occur within the first15 weeks of gestation. Fungiform papillae tend to grow betweenweeks 8 and 15 of gestation, whereas the size of vallate papillaeseems to be constant during this period. Chem. Senses 22: 601–612,1997.     

Urethral seam formation and hypospadias

Knowledge of the formation of the normal male urethra may elucidate the etiology of hypospadias. We describe urethral formation in the mouse, show the similarities and relevance to human urethral development, and introduce the concept of the epithelial seam formation and remodeling during urethral formation. Three mechanisms may account for epithelial seam formation: (1) epithelial-mesenchymal transformation similar to that described in the fusion of the palatal shelves, (2) apoptosis, and/or (3) tissue remodeling via cellular migration. Urethral development in the embryonic mouse (14-21 days of gestation) was compared with urethral formation in embryonic human specimens (8-16 weeks of gestation) by using histology, immunohistochemistry, and three-dimensional reconstruction. The urethra forms by fusion of the epithelial edges of the urethral folds, giving a midline epithelial seam. The epithelial seam is remodeled via cellular migration into a centrally located urethra and ventrally displaced remnant of epithelial cells. The epithelial seam is remodeled by narrowing approximately at its midpoint, with subsequent epithelial migration into the urethra or penile skin. The epithelial cells are replaced by mesenchymal cells. This remodeling seam displays a narrow band (approximately 30 microns wide) of apoptotic activity corresponding to the mesenchymal cells and not to epithelial cells. No evidence was seen of the co-expression of cytokeratin and mesenchymal markers (actin or vimentin). Urethral seam formation occurs in both the mouse and the human. Our data in the mouse support the hypothesis that seam transformation occurs via cellular migration and not by epithelial mesenchymal transformation or epithelial apoptosis. We postulate that disruption of epithelial fusion remodeling, and cellular migration leads to hypospadias.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

Invasiveness-triggered state transition in malignant melanoma cells

Cancer cells are considered to have high morphological heterogeneity in human melanoma tissue. Here, we report that epithelial cancer cells are dominant in different development stages of human melanoma tissues. The cellular and molecular mechanisms that maintain melanoma cells in the epithelial state are further investigated in the A2058 cell line. We find that micropore (8 µm) transwell invasion, but not superficial migration in the scratch assay, can induce remarkable morphological changes between epithelial and mesenchymal melanoma cells within 4 days. The morphological switch is associated with dynamic changes of epithelial–mesenchymal transition (EMT) hallmarks E-cadherin and vimentin. Further immunoflurencent staining and co-immunoprecipitation assay showed the uncoupling of the M3 muscarinic acetylcholine receptor (mAChR) and the p75 neurotrophin receptor (p75NTR) in epithelial melanoma cells. Specific knockdown of M3 mAChR by small interfering RNA (siRNA) significantly abrogates the transition of spindle-shaped mesenchymal cells to epithelial cells. Collectively, we report a cellular model of invasiveness-triggered state transition (ITST) in which melanoma cell invasion can induce morphological changes between epithelial and mesenchymal cells. ITST is one of the biological basis for maintaining metastatic melanoma cells in the epithelial state. Furthermore, M3 mAChR receptor-mediated ITST provides a novel therapeutic strategy to inhibit the development of malignant melanoma.     

The gastric mucosa of the human fetus. I. The surface epithelium

The ontogenesis of the surface epithelium in the gastric mucosa was studied by means of light and electron microscopy in 41 human foetuses ranging from 7th to 12th week of gestational age. The results obtained can be summarized as follows: 1) At the 7th week the gastric mucosa shows a simple pseudostratified epithelium; the epithelial cells are undifferentiated and filled, with glycogen clusters. 2) From the 8th week the epithelial surface shows small depressions that become deeper in the mesenchyme making the first bud of the gastric foveolae. 3) At the 9-10th week the gastric foveolae are more developed. The cells of the gastric epithelium can be therefore separated in two populations: a) the cells of the foveolae; b) the cell of the mucosal surfaces. 4) At the 12th week the cells of the mucosal surface become, on the basis of their histochemical and ultrastructural characteristics, surface mucous cells. The morphological differentiation is testified mainly by the transposition of the nuclei in the basal parts of the cells and by the gradual substitution of the cytoplasmic glycogen by mucous granules.     

Differentiation of cells of the bronchial wall in the early period of embryonal development of the lungs]

Electron microscopical analysis of cellular elements of the developing lung has been performed during early stages of the prenatal development. The mesenchymal anlage of the organ is characterized with an essential cell polymorphism. The precursors of the smooth myocytes are formed on the 13th day of embryogenesis and they develop in a close contact with epithelial tubules. Synthesis and accumulation of contractile elements are determining signs of myoblasts differentiation. Already at first stages of differentiation they interact by means of specialized junctions of adhesive type and from clusters, situating on periphery of the epithelial tubules. Increase in amount of contractile filaments in cytoplasm of myoblasts is accompanied with formation of the basement membrane of these cells. Together with myoblasts in the mesenchyme, cells of fibroblastic differon at various levels of development are described.     

Tetranectin immunoreactivity in normal human tissues. An immunohistochemical study of exocrine epithelia and mesenchyme

A recently discovered human plasma protein, tetranectin (TN), which has previously been demonstrated immunohistochemically within various endocrine tissues, was in this study identified in an additional number of epithelial and mesenchymal cells by two polyclonal antibodies and one monoclonal using the conventional immunoperoxidase staining technique and a modification of the CLONO-GLAD procedure. TN was found in endothelial and epithelial tissues, particularly in cells with a high turn-over or storage function such as gastric parietal and zymogenic cells, absorptive surface epithelium of the small intestine, ducts of exocrine glands and pseudostratified respiratory epithelium. Also mesenchymal cells produced a TN positive staining reaction, which was most conspicuous in mast cells, but also present in some lymphocytes, plasma cells, macrophages, granulocytes, striated and smooth muscle cells and fibroblasts. SDS-PAGE and Western blotting analysis of cultured human embryonal fibroblasts (WI-38) showed that the cells besides TN contain another protein with a molecular weight of 82,000. As this protein, however, reacted with our affinity purified antibodies it probably represents a precursor of TN or a protein with a molecular weight of approximately 60,000, which is covalently linked to TN. This and the fact that TN shows amino acid sequence homologies to the carboxyterminal part of the asialo-glycoprotein receptor and a cartilage proteoglycan core protein as well a binding affinity to plasminogen points to TN as being part of a larger molecule, which possibly has been cleaved by proteolysis at the cellular site and then passed into the blood, where it polymerizes into a tetramer.     

Development of the thoracic duct in the prenatal period of human ontogeny

In 40 series of histological sections performed in human embryos and prefetuses from 4 up to 20 weeks of development, as well as in 20 corpses of fetuses and stillborns, it has been stated that the anlage of the thoracic duct appear in 6-7-week-old fetuses as lymphatic clefts surrounded with mesenchymal cells that are situated near large veins in the areas of the most active morphogenesis. Connecting with each other, the clefts form the jugular and retroperitoneal lymph sacs and a well branching network of canals. From the latter, on the 7th-8th week of development a plexus of lymph vessels appear, and later on (on the 8th-9th week)--bilaterally situating trunks of the thoracic duct. Further development of the thoracic duct is connected with the lymph nodes formation, their germs appear on the 9th-10th week along the course of the left trunk, as well as along the ductal branches and anastomoses. The formation of the lymph nodes results in reduction of some trunks and plexuses of the thoracic duct. Owing to this, its form in 14-15-week-old prefetuses resembles the one in newborns. Disturbances in the formation processes of the lymph nodes along the course of the reducing ductal areas, as well as their formation along the course of its main trunk can result in various structural variants of the thoracic duct in children and grown-up persons. Histogenesis of the thoracic duct wall and formation of the lymph nodes are not completed by birth.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.     

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.