Showing papers on "Transdifferentiation published in 1996"

Xylogenesis: initiation, progression, and cell death

Abstract: Xylem cells develop from procambial or cambial initials in situ, and they can also be induced from parenchyma cells by wound stress and/or a combination of phytohormones in vitro. Recent molecular and biochemical studies have identified some of the genes and proteins involved in xylem differentiation, which have led to an understanding of xylem differentiation based on comparisons of events in situ and in vitro. As a result, differentiation into tracheary elements (TEs) has been divided into two processes. The "early" process involves the origination and development of procambial initials in situ. In vitro, the early process of transdifferentiation involves the dedifferentiation of cells and subsequent differentiation of dedifferentiated cells into TE precursor cells. The "late" process, observed both in situ and in vitro, involves a variety of events specific to TE formation, most of which have been observed in association with secondary wall thickenings and programmed cell death. In this review, I summarize these events, including coordinated expression of genes that are involved in secondary wall formation.

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

Abstract: When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.

Glucocorticoids, tumor necrosis factor‐α, and epidermal growth factor regulation of pulmonary morphogenesis: a multivariate in vitro analysis of their related actions

Abstract: The mouse lung commences development on embryonic day 11 as an epithelial evagination from the posterior pharyngeal wall into undifferentiated mesenchyme, this epithelium bifurcating to form the lung primordium. Branching morphogenesis, as well as terminal differentiation, requires epithelial-mesenchymal interactions utilizing precise regulatory controls. Not surprisingly, specific hormones and growth factors appear to play a key role in this regulation. We report here a series of experiments designed to investigate morphodifferentiation (epithelial branching number, generation number, and fractal dimension) and histodifferentiation (cell morphology and SP-A immunolocalization), as they relate to glucocorticoid (CORT)-regulation of growth factor function and expression (Northern analysis). These experiments were conducted in embryonic lung primordia (E11.5-E12) cultured under defined conditions in the presence of single or combined CORT, TNF-alpha, and EGF supplementation. EGF supplementation enhances branching morphogenesis, but not immunodetectable SP-A expression, in embryonic lung primordia cultured for 4 or 7 days. TNF-alpha supplementation also enhances branching morphogenesis on days 4 or 7 in vitro; on day 7, SP-A expression is also enhanced. By contrast, the introduction of exogenous CORT to embryonic explants cultured 4 or 7 days markedly alters morphodifferentiation and histodifferentiation. Early on it would appear to enhance morphodifferentiation by changing the process of branching, while contemporaneously initiating precocious SP-A expression; later on, it alters morphogenesis by continued terminal differentiation of normal lung epithelium and a singular transdifferentiation of lung mesenchyme into an epithelioid morphotype expressing SP-A. This is correlated with a CORT-induced, highly significant, down-regulation of TGF-beta 2 and TGF-beta 3 transcripts. Explants supplemented with CORT + TNF-alpha or CORT + EGF.demonstrate a microanatomy and SP-A expression pattern identical to that seen with CORT supplementation alone. EGF inhibits the accelerated lung maturation normally seen in the presence of exogenous TNF-alpha alone, suggesting a relationship between these two seemingly disparate regulatory pathways.

High-density culture of mouse Meckel's cartilage cells stimulates phenotypic conversion to osteocyte-like cells

Abstract: We examined the influence of cell density on the phenotypic conversion of Meckel's cartilage cells. The cells were isolated by enzymatic digestion and plated at a density of 0.5 × 104 or 2 × 104 cells/penicylinder and cultured under 5% CO2 in air for up to 4 weeks. The cultures were analyzed histologically by electron microscopy, histochemistry, and immunostaining. At the early stage of high-density culture, metachromatic chondrocytes appeared concomitantly with cartilage-specific type II collagen synthesis. The cells gradually transformed from large polygonal cells to multilayered small round cells, and formed nodules. During the later stage of culture, the cells exhibiting alkaline phosphatase (ALPase) activity, type I collagen, and osteocalcin as bone-type marker proteins consisted of spindle-shaped cells showing phenotypic conversion into osteocyte-like cells in the calcified nodules. In contrast, during low-density culture, most of the cells changed from fibroblastic cells to large flattened cells without cellular nodule formation, but these cells lacked chondrocytic features. Only solitary cells exhibited chondrocyte-specific features such as metachromasia, proteoglycan synthesis, and matrix calcification, but they did not undergo osteocytic transdifferentiation. These results indicate that well-organized nodule-forming cellular and extracellular components in high-density culture stimulate transdifferentiation into osteocyte-like cells.

Light and electron microscopy of stage-specific features of the transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro

Abstract: The changes in the morphological characteristics of Meckekl’s cartilage cells in culture can be divided into five stages during their transdifferentiation to cells with an osteocyte-like phenotype, as

Suppression by 5-Bromo-2'-deoxyuridine of Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

Abstract: 5-Bromo-2'-deoxyuridine (BrdU), a thymidine analog, suppressed the transdifferentiation into tracheary elements (TE) of isolated mesophyll cells of Zinnia elegans without affecting cell division. Tracer experiments with [3H]BrdU indicated that 76% and 24% of the incorporated radioactivity was located in the DNA and the RNA, respectively. Both thymidine and uridine counteracted the inhibitory effect of BrdU on transdifferen tiation but thymidine was much more effective than uridine. These results suggest that BrdU might interfere with transdifferentiation via its incorporation into DNA. The timing of effective suppression by BrdU was examined by monitoring the effects of the sequential addition of BrdU and thymidine with an interval of 12 h at various times in culture. Transdifferentiation was most sensitive to BrdU between the 24th and the 36th hour of culture. This result suggests that this window of time is critical for DNA synthesis during the transdifferentiation of isolated mesophyll cells of Zinnia elegans into TE.

[Cell sources, regulatory factors and gene expression in the regeneration of the crystalline lens and retina in vertebrate animals]

Abstract: Over the past century extensive experimental materials have been accumulated concerning cell sources of lens and retina regeneration, successive transformations of the cells, regulatory factors, and gene expression during restitution of these eye structures. The use of nuclear and cytoplasmic markers provided convincing evidence that the removed lens is restituted from the dorsal iris cells in vivo or from embryonic cells of the pigment epithelium and retina in vitro. The removed or destroyed retina is restituted as a result of transdifferentiation of the pigment epithelium cells in amphibians, fish, birds, and mammals during embryogenesis, in larvae of some anuran amphibians, and in adult newts. Cell precursors of rods are a cell source of retina regeneration in adult fish. A subpopulation of randomly distributed cells, which are a cell source of rod formation during the normal development of the eye was found in the external nuclear layer with the use of electron microscopy and nuclear and cytoplasmic markers. These cells are not only a source of regeneration of rods, but also of cones and cells of the internal nuclear layer after destruction of the corresponding retina layers. There is a peripheral growth area in the retina of vertebrates, where multi- and unipolar cells are localized, which provide for the retina growth during ontogenesis. A paradox of retina regeneration consists in that these little differentiated cells are not a source of complete restitution of the removed or destroyed retina. They make only a small contribution to its regeneration corresponding to the growth potential of cells of this eye region, while restitution of the retina proceeds only at the expense of cells of another type of differentiation. A factor controlling the differentiated state of the cell was found in the dorsal iris during studies of lens regeneration. Removal of this factor in the early stages of cell transformations leads to the initiation of lens regeneration. The factor is not specific and was identified in many cells of vertebrates, including the pigment epithelium and limb tissues, which, as is known, may be fully restituted. Studies of gene expression during lens and retina regeneration are now at the initial stage. The greatest advances were achieved on the model of transdifferentiation of the pigment epithelium cells of chick embryos into lentoids. Expression of genes MMP115 and pP344 was established in the pigment epithelium cells, which characterize the pigmented phenotype of the initial cells. Expression of the alpha-, beta-, and delta-crystallin genes was found in the lentoids, which characterize the phenotype of regenerating structures. The gene activity appears to be switched at an intermediate stage during cell dedifferentiation. Expression of the gamma-crystallin genes during lens regeneration in adult newts is initiated after completion of dedifferentiation and cell proliferation in the dorsal iris. The genes specifically expressed in the dorsal and ventral iris and in the retina rudiment have been identified by the method of gene subtraction. Expression of homeobox-containing genes from the family of PAX genes was found during lens regeneration in adult newts and retina regeneration in adult fish. The role of growth factors (FGF) as morphogenetic factors was proved, which are involved in a yet unknown way of altering the differentiation pathway of the initial cells during formation of the neuroepithelium rudiment in chick embryos, adult newts, and fish.

Neuronal Properties of Thyroid C-Cell Tumor Lines

Abstract: Thyroid C-cells present a paradoxical phenotype of neuronal properties in multiple endocrine neoplasia type 2 (MEN 2). The neuronal phenotype is in contrast to the primarily endocrine nature of the parental C-cells. What can account for this transdifferentiation? In this chapter, we will try to answer that question by comparing the neuronal features of C-cell lines with those of normal C-cells treated with nerve growth factor. From that perspective, we will then present a model proposing that ret tyrosine kinase activity is responsible for the neuronal properties in C-cell tumors. Interestingly, the neuronal phenotype is partly reversible. Overexpression of the ras oncogene or treatment with glucocorticoids inhibits both cell growth and induces a more endocrine state. An attractive hypothesis is that these agents might be decreasing ret activity, although this remains to be tested. The utility of thyroid C-cell lines for identification of differentiation and growth genes will be exemplified by description of a novel splice product of the Gsα transcript that is selectively expressed in a subset of neuroendocrine and neuronal cells. Finally, the applicability of C-cell lines for studying fundamental transcriptional control mechanisms will be described using the calcitonin/CGRP gene as a model. In summary, the induction and repression of neuronal properties in C-cell tumors provide a useful system for studying the genetic mechanisms underlying gene expression and differentiation in MEN 2.

Distribution of beta1 integrin during development of chick tarsometatarsal skin in vivo and in vitro.

Abstract: To determine the role of beta1 integrin in chick tarsometatarsal skin development, we examined the localization of the beta1 integrin immunohistochemically in vivo and in vitro by light and electron microscopy. Beta1 integrin was present over the entire cell surface of undifferentiated epidermis at early stages (Days 5, 9, 13). Marked changes in the localization of beta1 integrin occurred during epidermal keratinization and stratification, i.e., expression of beta1 integrin decreased in the superficial and intermediate cell layers from Day 13 to Day 17. After 17 days in vivo, when keratinization of the epidermis was completed, the distribution of beta1 integrin was confined to the basal layer of the epidermis in a pericellular distribution. During all stages examined, fibroblasts in the dermis were also stained. Immunoelectron microscopic study revealed that beta1 integrin was located on the plasma membrane of keratinocytes and dermal fibroblasts. The change in beta1 integrin localization that occurred in vivo could be reproduced in cultures of developing skin in which keratinization (differentiation) or mucous metaplasia (transdifferentiation) had been induced in vitro by hydrocortisone or retinol treatment, respectively. A monoclonal antibody against beta1 integrin caused striking changes in the epidermal keratinization process and in the basement membrane structure in vitro, i.e., inhibition of keratinization and detachment of the basement membrane from the basal surface of the epidermis. These results indicate that beta1 integrin plays an important role in cell-cell and cell-extracellular matrix interactions, which are important for epidermal development of the tarsometatarsal skin.