Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.     

Cells that emerge from embryonic explants produce fibers of type IV collagen

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro

A number of adult and embryonic epithelia, when suspended within native type I collagen gels, give rise to elongate bipolar cells that migrate freely within the three-dimensional matrix. The morphology of these newly formed mesenchyme-like cells is indistinguishable from "true" mesenchymal cells at the light and ultrastructural level. In this report, we extend previous observations on the transformation of embryonic avian lens epithelium to mesenchyme-like cells. Lens epithelia, dissected from 12-day chick embryos, were cultured either within a collagen matrix or on a two-dimensional surface. Cells derived from explants on the surface of type I collagen express the epithelial phenotype. The cells form new basal lamina, continue to express delta-crystallin protein and secrete both type IV collagen and laminin. In contrast, epithelia suspended within collagen gels lose epithelial morphology, phenotype, and cytodifferentiation. The newly formed mesenchyme-like cells lack the ability to synthesize lens-specific delta-crystallin protein, type IV collagen, and laminin. They do, however, express type I collagen de novo, a characteristic of mesenchymal cells. The changes in cytodifferentiation and tissue phenotype which occur during the transformation are stable under the conditions studied here. When mesenchyme-like cells are removed from the gel and replated onto two-dimensional surfaces, they remain bipolar, will invade collagen matrices, and are unable to synthesize delta-crystallin protein.     

Collagen IV in the developing lens capsule.

The lens capsule is a specialized thickened basement membrane that completely surrounds the lens and provides anchoring sites for zonules, the filamentous bodies that suspend the lens. Like other basement membranes, the lens capsule contains collagen IV, which is a family of six polypeptides, subunits alpha1(IV)-alpha6(IV), each of which is encoded by a distinct gene. We have investigated the presence of collagen IV subunits in the developing lens capsule by using confocal immunohistochemistry and antibodies against each of the six collagen IV subunits. In murine embryos, subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) were detected in the basement membrane surrounding the lens vesicle, and they persisted in the capsule until adulthood. In contrast, neither collagen alpha3(IV) nor alpha4(IV) was detected in the lens capsule until 2 weeks postnatal. Similarly, we detected no collagen alpha3(IV) or alpha4(IV) in lens capsules of 54-day human embryos, while collagen alpha3(IV) and alpha4(IV) were detected in adult humans. Thus, in the lens capsule, there is a developmental shift in detectable collagen IV subunits; early in development we observed subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV), which is consistent with the presence of fibrillar [alpha1alpha1alpha2] and elastic [alpha5alpha5alpha6] protomers, but later in development components of the more cross-linked [alpha3alpha4alpha5] protomer appear. An elastic lens capsule may be necessary in order to accommodate rapid lens growth in early development, whereas later in development a stronger, more cross-linked capsule may be necessary in order to tolerate the stress caused by postnatal accommodation and disaccommodation of the lens.     

Steady-state levels of mRNAs coding for the type IV collagen and laminin polypeptide chains of basement membranes exhibit marked tissue-specific stoichiometric variations in the rat

Rat retina, lens, and kidney from 8-week-old animals were assayed for the steady-state levels of mRNAs for four basement membrane components: The alpha 1 chain of type IV collagen, the alpha 2 chain of type IV collagen, the B1 chain of laminin, and the B2 chain of laminin. Each tissue exhibited markedly different ratios of the four mRNAs. The mRNA ratio for the alpha 1 chain of type IV collagen to the B1 chain of laminin varied from a value of 0.7 in retina to a value of 17 in lens. Also, the mRNA ratio for the alpha 1 chain to the alpha 2 chain of type IV collagen varied from 1.6 in retina to 17 in lens, and the mRNA ratio for the B1 chain to the B2 chain of laminin varied from 0.6 in lens to 2.9 in kidney. The mRNA coding for the alpha 1 chain of type IV collagen decreased in all three tissues as the animals increased in age from 8 to 16 weeks, with the rate of decline being greater in retina than in lens of kidney. The levels of mRNA coding for the B1 and the B2 chains of laminin decreased in the kidney between 8 and 16 weeks but at different rates. Comparison of mRNAs from kidney of rats over this time period showed that the ratio of alpha 1 to B1 remained relatively constant with age, whereas the ratio of B1 to B2 increased. One possible explanation for the results is that each tissue has elaborate, tissue-specific controls for translation that provide synthesis of basement membrane components in the same proportion, in spite of the varying steady-state levels of the mRNAs. A more likely explanation is that different tissues synthesize type IV collagen and laminin at different rates, and that even the subunit compositions of the type IV collagen and laminin molecules vary from tissue to tissue and in an age-dependent manner.     

Development and characterization of the lens capsule of mouse embryos (day 12 to day 19 of gestation)

The development of the lens capsule (LC) of mouse embryos was investigated between days 12 and 19 of gestation using immunomorphological (collagen type I, II, III or IV, laminin, BL-heparan sulfate, fibronection) and electron microscopic techniques. The lens capsule contains the typical components (collagen type IV, laminin and BL-heparan sulfate) of the basal lamina (BL) and can therefore be considered as thickened BL. Tannic acid fixation is especially suited for an electron microscopic demonstration of the lens capsule. The development of the lens capsule starts on day 12 of gestation. Its thickening is due to BL accumulation from the outside. This mode of thickening can be explained by the tendency to two-dimensional self assembly of collagen type IV. Electron-dense granules occur in the basal cytoplasm of lens epithelial cells. These granules can be considered as secretion granules. Their increased occurrence towards the end of gestation is attributed to a delayed secretion rather than to an increased synthesis.     

Biochemical, ultrastructural and immunological study of in vitro production of collagen by bovine lens epithelial cells in culture.

Lens epithelial cells isolated from adult bovine were maintained in long-term culture. They synthesised important extracellular fibrils which had a similar pattern to capsule-like material in electron microscopic pictures. These fibrils were sensitive to a highly purified collagenase. After addition of labelled proline in the culture medium, the solubilized material obtained from culture preparation contained radioactive hydroxyproline. When the culture was maintained at confluency for several months, extracellular fibres with a pattern similar to fibrous long spacing collagen were observed. The collagen newly synthesised by epithelial cells has been isolated from the cells and from the medium. It has been chromatographed on agarose A5m column and analysed on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Confluent lens cells were stained with anti-serum to lens capsule collagen type IV, the anti-serum stained the fibrils which are shed between the cells. Thus the collagen newly synthesised by epithelial cells has been isolated and tentatively identified as basement membrane collagen.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.     

Type II collagen mRNA containing an alternatively spliced exon predominates in the chick limb prior to chondrogenesis.

A series of cDNA clones corresponding to the 5' end of the chicken type II collagen mRNA were generated using a single-sided polymerase chain reaction technique. Analysis of these cDNAs showed that the second exon of the gene is alternatively spliced such that it is either present or absent in the mRNA. This exon encodes a 70-amino acid cysteine-rich globular domain which is present in the amino-terminal propeptides of alpha 1(I), alpha 1(III), and alpha 2(V) procollagen chains but which was previously thought to be absent from type II procollagen. Analysis of the expression of the two alternatively spliced forms of the chicken type II collagen mRNA showed that the mRNA without the second exon was the predominant form (approximately 90%) in sternal cartilage from 14-day embryos, but in precartilage limb mesenchyme only the form including the second exon was detected. This later form was also present in a number of non-cartilage tissues including embryonic calvaria, skin, heart, skeletal muscle, and brain; no type II collagen mRNA was detected in liver. Studies of developing limbs from progressive embryonic stages suggest that the appearance of the mRNA lacking the second exon is a relatively late event during chondrogenesis.     

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.     

Extracellular matrix production by embryonic epithelium cultured on type IV collagen Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts whcih, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens.We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils.When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter.In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.