Collagen involvement in branching morphogenesis of embryonic lung and salivary gland

The possibility that extracellular collagen is involved in branching morphogenesis of mouse embryo lung and salivary glands has been explored duringin vitro organ culture. Control cultures of both rudiment types contain abundant collagen in extracellular spaces between mesenchymal cells and in the epithelial-mesenchymal interface. Branching morphogenesis of lungs and salivary glands is not perturbed by the presence of β-aminopropionitrile, implying that extracellular collagen cross-linking is not required, but is perturbed by α,α′-dipyridyl orl-azetidine-2-car?ylic acid (LACA), agents reported to interfere with collagen synthesis and secretion. Analysis of the structural and biosynthetic effects of LACA revealed a severe inhibition of collagen synthesis, as monitored by hydroxyproline synthesis, and extracellular collagen accumulation. Cell and tissue integrity was not affected, but a slight inhibition of general protein synthesis, protein accumulation, and epithelial expansion was observed. The strong correlations between collagen biosynthesis, extracellular collagen presence, and branching morphogenesis are consistent with an integral role for collagen in embryonic lung and salivary gland morphogenesis.     

Invagination of the otic placode: normal development and experimental manipulation

The inner ear forms from paired ectodermal primordia that lie to either side of the developing hindbrain. Initially each primordium forms a shallow depression in the ectodermal surface. Invagination to form an otic pit coincides with the formation of several deep folds in the epithelial surface. An initial fold appears parallel to the embryonic axis and at the junction of the rhombencephalon with somitomeric mesoderm. This is followed by formation of cranial and caudal folds perpendicular to the axis and minor folds that are within the pit formed by earlier folding. The central region of the otic primordium remains in close apposition to the lateral surface of the neural tube during the process of fold formation, until the otic pit becomes quite deep. At that time, mesenchymal cells penetrate between the two layers. Experimental analysis of invagination supports the conclusion that otic invagination is controlled differently from that of similar organ primordia, such as the eye and thyroid. Whereas these other primordia can be stimulated to undergo normal morphogenetic shape changes precociously by treatments that presumably activate motile processes in the cytoskeleton, the same conditions have little effect on the otic placode. Similarly, neither inhibitors of calcium transport nor inactivators of calmodulin activity prevent otic pit formation, while these drugs block invagination of other primordia. These results suggest that otic invagination may be caused by changes in the surrounding tissues rather than by an activation of motility within the primordium.     

Biosynthesis of the basal lamina as a result of interaction between thyroid and mesenchymal cells in culture

Porcine thyroid cells were cultured alone or in mixed cultures with mesenchymal cells. The formation of a basal lamina in vitro was investigated ultrastructurally. Follicular reassociation of thyroid cells occurred in both types of culture; however, it was followed by formation of the basal lamina only when mesenchymal cells were present. The present findings suggest an epithelial origin of the basal lamina resulting from an interaction with mesenchymal cells.     

Collagen synthesis by cultured skin fibroblasts from siblings with hydroxylysine-deficient collagen.

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both alpha 1(I) and alpha 2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by alpha, alpha'-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.     

Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells : Induction of ‘Assembly’ on fibrillar type I collagen substrata

Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable [3H]hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the all over rates of collagen synthesis and degradation. The proportion of the [3H]collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibres. Immunoprecipitation of the labelled extracts, electrophoresis, indirect immunofluorescence and immunoperoxidase techniques reveal the presence of type IV collagen, along with laminin and heparan sulfate proteoglycan in this layer, in excess over the amounts detectable on cells cultured on plastic. Transformed cells on collagen produce and accumulate more [3H]collagen, yet are less effective in basement membrane formation than normal cells, indicating that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface.     

Identification and partial characterization of mesenchyme-derived growth factor that stimulates proliferation and inhibits functional differentiation of mouse mammary epithelium in culture

The effect of mesenchyme on both proliferation and differentiation of mammary epithelial cells was investigated in a primary cell culture system. Mammary cells cultured on collagen gel for 4 days produced casein in response to the synergistic action of insulin, cortisol, and prolactin. When mammary epithelial cells were co-cultured with fibroblasts derived from three different kinds of fetal mesenchymal tissues, casein production was suppressed. The addition of conditioned media obtained from cultures of these mesenchymal cells stimulated DNA synthesis and reduced casein synthesis in a dose-dependent fashion in the cultured mammary cells. Although such biological actions are similar to those of epidermal growth factor (EGF), the capability to compete with EGF for EGF receptor was not found in this conditioned medium. Sephadex G-200 column chromatography revealed that molecular weight of the peak which has these biological activities was around 100,000. These results indicate that fetal mesenchymal cells secrete a substance(s) which has a stimulatory effect on proliferation and an inhibitory effect on differentiation of mammary epithelial cells.     

The histology of tooth formation in the Australian lungfish, Neoceratodus forsteri Krefft

The histology of developing toothplates of Neoceratodusforsteri from the time of first appearance of the tooth primordia to the adult condition has been investigated. The dentition develops by the formation of a shell of primary epithelial and mesenchymal matrices. Within the shell, secondary mesenchymal matrix and central material, both containing columns of tertiary matrix, are laid down. Primary epidielial matrix appears to contain collagen and is closely associated with the epithelium of the mouth. All other tooth tissues as well as the supporting bone develop in association with mesenchyme. Primary, secondary and tertiary mesenchymal matrices appear to contain collagen. Central dentine contains some fibres, possibly of reticulin or collagen, within a matrix of unknown composition.
The tooth is attached to the underlying bone by a pedestal of bone and this grows with the tooth material.
New tooth tissues are formed in the pulp cavity in layers below the older material, causing the toothplate to grow in every dimension as the animal grows.
An evolutionary pathway is suggested for lungfish with a dentition of cusps arranged in radiating ridges.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

Effect of an analog of proline (L-azetidine-2-carboxylic acid) on in vitro differentiation of the rat fetal testis

The initial stages of the development of the seminiferous cords involve the differentiation and the aggregation of primordial Sertoli cells opposite to cells which acquire a mesenchymal-like aspect. The hypothesis that the development of the seminiferous cords depends on epithelial-mesenchymal relations between the two cell types was submitted to experimental test. Male gonadal primordia of rat fetuses were cultured in vitro in a synthetic medium containing the proline competitor, L-Azetidine-2-Carboxylic Acid. This drug is known to disturb the synthesis and secretion of collagen and proline-containing proteins. It prevents testicular organogenesis or destroys it if it has begun. It suppresses the expression of laminin and fibronectin in the gonadal primordium. These observations are taken as evidence that cellular correlations of the epithelial-mesenchymal type play a role in the development of the testis as they do in that of other organs.     

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.     

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.     

Identification and partial purification of a large, variant form of type XII collagen.

A large, alternate form of type XII collagen has been identified in cultures of the human epidermoid cell line WISH. This form, designated XIIA, is comprised of alpha chains that are approximately 90 kDa larger than the 220-kDa alpha chain previously characterized in extracts of fetal chicken and bovine tissues. Results from both collagenase digestion and rotary shadow analysis of partially purified material show that the increase is due to a larger NC3 domain. While both the large (XIIA) and the small (XIIB) forms of type XII collagen are identified in pulse-chase radiolabeling of fetal bovine skin explant culture, they are not related in a precursor-product fashion. Inhibition studies with alpha, alpha'-dipyridyl indicate that proper folding of the collagen helix is required for complete assembly and secretion of type XIIA in WISH cell culture. The 310-kDa alpha 1A chain is likely to represent the bovine equivalent of a second translation product, estimated to be 340 kDa, predicted from analysis of one complete chick cDNA sequence. Additionally, the amino-terminal amino acid sequence of the 220-kDa bovine alpha 1B chain was determined. This sequence is very near a potential alternate splice site predicted from analysis of chicken type XII cDNA.     

Alternative pathways of tobacco placental development: time of commitment and analysis of a mutant

Previous work from our lab identified mutants, Mgr3 and Mgr9, of tobacco (Nicotiana tabacum) that produced unusual elongated green outgrowths from placentae in vivo. Similarly appearing stigmatoid growths were described developing from some in vitro cultures of excised placentae of tobacco (Hicks and McHughen, 1974, 1977). Here we report a developmental analysis and comparison of the unusual stigmatoid outgrowths seen in in vitro cultures of wild-type and mutant placentae, as well as the green outgrowths seen in vivo in the mutants. The growths produced by wild-type and mutant placental cultures in vitro, and the growths produced by the mutants in vivo, are identified as abnormal stigmas and styles. Wild-type in vitro placental cultures also produce outgrowths identified as homologs of whole carpels. Carpel fusion is not required for differentiation of stigma, style, transmitting tract, vascular traces, ovary, and ovules in these structures. The type and extent of stigmatoid growth production depends upon the age of the explant at excision and culture initiation. Before ovule primordium initiation, few growths are seen in culture; for a short window of time afterward, the primordia are competent to give rise to stigmatoid and carpelloid growths when cultured. After commitment to ovule development occurs, the primordia produce only ovules when cultured. The behavior of the mutant placental cultures is dimorphic. Explants from early stages behave similarly to wild-type when cultured, but differences between wild-type and the mutant behaviors in culture arise at the time when the stigmatoid growths begin to appear in vivo in the mutants. These results imply that ovule primordia pass through stages of distinct sequential restrictions of fate, first to growth as gynoecia, and then second to growth as ovules. The mutant strains described here perturb the commitment to differentiation as ovules.     

Hemidesmosome formation by embryonic chick corneal epithelium in vitro

This study was undertaken in order to determine whether 15-day embryonic chick corneal epithelial cells can form hemidesmosomes when cultured on a variety of substrata. It was found that hemidesmosomes were formed on gelatin films, hydrated collagen gels, lens capsule, scraped corneal stroma, matrix produced by corneal endothelial cells and untreated tissue culture plastic. Hemidesmosomes were found after 5 days in cultures produced from either dissociated epithelial cells or whole epithelial explants. Hemidesmosomes occurred both singly and in groups and their morphology varied between well-defined structures with attachment plaques, sub-basal dense plates and connections to intracellular filamentous networks, and more rudimentary forms. The presence of extracellular material was often associated with the hemidesmosomes, although it was also possible to find hemidesmosomes where this material was absent. This work suggests that, in the embryonic chick cornea, extracellular structures such as anchoring filaments and anchoring fibres often associated with mature hemidesmosomes are not essential for hemidesmosome formation.     

Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III

This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. Northern hybridization showed the bone, cartilage, intestine, skin, and striated muscle to contain mRNAs for type XIII collagen. An intense in situ hybridization signal was obtained with the type XIII collagen cDNAs in the epidermis, hair follicles, and nail root cells of the skin, whereas the fibrillar collagen mRNAs were detected in the dermis. Cells in the intestinal mucosal layer also appeared to contain high levels of alpha 1(XIII) collagen mRNAs, but contained none of the fibrillar collagen mRNAs. In the bone and striated muscle, alpha 1(XIII) collagen mRNAs were detected in the mesenchymal cells forming the reticulin fibers of the bone marrow and endomycium. The hybridization signal obtained with the alpha 1(XIII) collagen cDNA probe in cartilaginous areas of the growth plates was similar, but less intense, to that obtained with the type II collagen probe. A clear hybridization signal was also detected at the (pre)articular surfaces and at the margins of the epiphyses, whereas it was weaker in the resting chondrocytes in the middle of the epiphyses. The brain, heart, kidney, liver, lung, placenta, spleen, testis, tendon, and thymus did not appear to contain alpha 1(XIII) collagen mRNAs.     

Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

Interaction of embryonic corneal epithelium with exogenous collagen,laminin, and fibronectin: Role of endogenous protein synthesis

Previously, we have shown that the embryonic corneal epithelium is capable of interacting with exogenous collagen, laminin, and fibronectin in soluble form, each of which causes isolated epithelium cultured on Millipore filter to stop blebbing, reorganize the basal cytoskeleton, and flatten. Here we examine the involvement of endogenously derived extracellular matrix (ECM) molecules in the interaction of the basal epithelial cell surface with the added ECM molecules. We demonstrate here that the isolated avian corneal epithelium cultured on Millipore filter is capable of synthesizing collagens and laminin, but not fibronectin. To examine whether the epithelium is capable of interacting directly with exogenous ECM components or if there is the necessity for production of a linker molecule, epithelial protein synthesis was inhibited with cycloheximide (CHX). The blebbing epithelium in the presence of CHX was then confronted with soluble ECM molecules added to the medium under the filter; such epithelia are able to interact with, and flatten in response to, both collagen and laminin. However, such inhibited epithelia continue to bled in the presence of fibronectin. We next used l-azetidine-4-carboxylic acid (LACA) to interfere with collagen secretion. Epithelia exposed to LACA are still capable of interacting with collagen and laminin, but not fibronectin, indicating a dependence on collagen secretion. These results suggest that fibronectin requires a linker protein, probably collagen, to interact with the basal epithelial surface, whereas both collagen and laminin may interact directly with the cell surface to transform the basal cytoskeleton into the cortical mat typical of differentiating corneal epithelium in situ.     

Development of fetal rat intestine in organ and monolayer culture

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.