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Cellular differentiation in prostatic explant cultures: assessed by electron microscopy and X-ray microanalysis.

A method developed for X-ray microanalysis (XRMA) of cell monolayers cultured on Formvar film has been shown to be well suited for the study of explant cultures of human prostate. Adherence and epithelial cell outgrowth occur as readily on the formvar as on ordinary tissue culture plastic, giving rise to cultures with the same morphological characteristics. Conventional transmission electron microscopy revealed intracytoplasmic granules with the appearance of secretory vesicles. XRMA of electron-dense intracytoplasmic granules in freeze-dried cryosections showed significant local sequestration of calcium, but not of magnesium or zinc, elements that have previously been shown to colocalize with calcium in secretory granules in vivo. It is concluded that some aspects of the secretory phenotype are supported in this in vitro model. The factors regulating the expression of a differentiated phenotype in prostatic epithelium await further elucidation. XRMA may be useful in assessing the effects on secretory differentiation induced by variations in the culture conditions.

Biopsy↗

Ectoderm-mesenchyme and mesenchyme-mesenchyme interactions regulate Msx-1 expression and cellular differentiation in the murine limb bud.

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb mesenchyme that has been demonstrated to support limb outgrowth and proper limb development. The homeobox gene, Msx-1, is associated with the distal limb mesenchyme (progress zone) and its expression depends upon the presence of the AER in chick limbs. We demonstrate here that the expression of Msx-1 is dependent upon the limb ectoderm in the mouse, but that the inductive capacity of murine limb ectoderm is not restricted to the AER. Msx-1 can also be maintained in limb mesenchyme by the substitution of FGF 4 for the ectoderm; however, we see that local cell-cell interactions are required for high levels of expression. Disruption of cell-cell interactions in the limb mesenchyme results in a dramatic decrease in Msx-1 levels and a precocious expression of MyoD1, suggesting that the limb environment represses differentiation and promotes cell proliferation during early development. BMP 4 and FGF 2 can also maintain Msx-1 expression in limb mesenchyme as well as retinoic acid which is usually associated with polarizing activity in the early limb. Msx-2 expression does not appear to be dependent upon cell-cell interactions as measured in these experiments. Taken together, our data suggest that the expression of Msx-1, but not Msx-2, not only requires factors from the limb ectoderm, but also relies upon cues from local cell interactions and that the spatial distribution of inductive capacities in limb ectoderm differs between the avian and murine systems.

Animals↗

Development of the avian iris and ciliary body: mechanisms of cellular differentiation during the smooth-to-striated muscle transition.

The avian iris and ciliary body undergoes a transition from smooth-to-striated muscle during embryonic development. Using antibodies specific for smooth muscle-specific alpha-actin and myosin heavy chain, we confirm that a smooth-to-striated muscle transition occurs between E8 and E17 in both iris and ciliary body of the chick. To study the mechanisms regulating the transition in muscle type, we analyzed the fate of quail clones derived from E7 iris cells. When cells were cloned alone, 45/71 colonies differentiated into smooth muscle and 10/71 became striated muscle. None of the colonies were mixed with respect to muscle phenotype, indicating a lack of pluripotent stem cells. Furthermore, clones giving rise to nonstriated muscle could not be forced to incorporate into myotubes when cocultured with chick myocytes. Clones grown in coculture with chick embryo fibroblasts or E11 iris cells had very high cloning efficiencies (>98%). Significantly more clones differentiated into striated muscle when cocultured with E11 cells (60/156) than when cocultured with fibroblasts (29/108). This was due to an increased recruitment of undifferentiated cells into striated muscle, rather than a change in the percentage of cells differentiating into smooth muscle. In vivo and in vitro, various smooth and striated muscle-specific markers including contractile proteins, acetylcholine receptor subtypes, and transcription factors were colocalized in cells. Although our data argue against a multipotent stem cell for smooth and striated muscle cells, they cannot exclude a role for transdifferentiation. Cumulatively these results suggest that both smooth muscle and migratory myoblasts contribute to the development of myotubes in the avian iris and that this process is regulated in a non-cell-autonomous fashion by locally generated signals.

Acetylcholine↗

Nutrient signaling through TOR kinases controls gene expression and cellular differentiation in fungi.

The TOR kinases were first identified in Saccharomyces cerevisiae as the targets of the immunosuppressive drug rapamycin. Subsequent studies employing rapamycin as a tool in yeast have given us insight into the structure and function of the TOR kinases, as well as the biological role of the TOR signaling program in transmitting nutrient signals to promote cell growth. One of the major advances from this area has been in defining an unexpected role for TOR signaling in the regulation of transcription. The identification of target genes subject to regulation by TOR has provided a platform for the dissection of the signaling events downstream of the TOR kinases. Studies aimed at understanding TOR-regulated transcription have begun to shed light on how TOR signaling cooperates with other signaling programs. In addition, the TOR pathway regulates the developmental program of pseudohyphal differentiation in concert with highly conserved MAP kinase and PKA signaling programs. Remarkably, rapamycin also blocks filamentation in a number of important human and plant pathogens and the mechanism of rapamycin action is conserved in Candida albicans and Cryptococcus neoformans. The antimicrobial properties of less immunosuppressive analogs of rapamycin hold promise for the development of an effective antifungal therapy.

Antifungal Agents↗

Prenatal expression of growth hormone receptor/binding protein and insulin-like growth factor-I (IGF-I) in the enamel organ. Role for growth hormone and IGF-I in cellular differentiation during early tooth formation?

Immunohistochemistry was used to study the ontogeny of GH receptor/binding protein (GHR/BP) and IGF-I from the 13-day-old embryo (E13) to the E19 rat fetus in the developing incisor and molar. Analysis of serial sections revealed diffuse staining of GHR/BP and IGF-I at the bud and early cap stages within both the mesenchyme of the dental papilla and the ectodermal-derived enamel organ. Just before transition to the cap stage, immunoreactivity of GHR/BP and IGF-I increased in the epithelial bud and extended to the condensed dental mesenchyme. At the cap stage, the dental epithelium showed an intense expression of GHR/BP and IGF-I, whereas the dental mesenchymal cells showed very weak staining. The inner enamel epithelium and the outer enamel epithelium were positive for both GHR/BP and IGF-I in the bell stage. Differentiating ameloblasts, odontoblasts and the secretory ameloblasts and odontoblasts continued to express GHR/BP and IGF-I in incisors. These findings support the premise that growth hormone and IGF-I may play a role in embryonic tooth development by regulating the epithelial-mesenchymal interactions that influence events in growth and cytodifferentiation.

Animals↗

Human breast epithelium in vitro: the re-expression of structural and functional cellular differentiation in long-term culture.

Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/cell contact or topological organisation of cells prior to embedding within the collagen matrix.

Breast↗

Differential cellular distribution of two sulphur-containing amino acids in rat cerebellum. An immunocytochemical investigation using antisera to taurine and homocysteic acid.

An antiserum to homocysteic acid was raised in rabbits. Immunogens were prepared by coupling this amino acid to bovine serum albumin by means of glutaraldehyde and paraformaldehyde. When applied to semithin or ultrathin sections of rat cerebellum, the antiserum produced selective labelling of glial cells and processes, including the Bergmann fibers. No enrichment of immunoreactivity was detected in nerve terminals of the major excitatory fiber systems. The distribution of homocysteic acid-like immunoreactivity was very different from that of taurine (another sulphur-containing amino acid), as judged from consecutive semithin sections labelled with a postembedding immunoperoxidase procedure and from ultrathin sections labelled with a postembedding double immunogold procedure. Taurine-like immunoreactivity was concentrated in Purkinje cells and was low in glial elements. Our data suggest that the cerebellum contains a glial pool of homocysteic acid (and/or precursors that may undergo spontaneous oxidation to homocysteic acid) and that this amino acid is unlikely to act as a cerebellar transmitter.

Animals↗

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus.

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Differences between adrenomedullary adrenaline and noradrenaline cells: quantitative electron-microscopic evaluation of their differential cellular association with supporting cells.

Quantitative differences in cellular association of adrenomedullary chromaffin cells with other types of cells, mainly supporting cells, were studied. Adrenaline (A) and noradrenaline (NA) cells were compared. Electron micrographs (12000 x) of profiles of A and NA cells, bordering against other types of cells, were used for quantitative evaluation. Supporting cells constituted the majority of the non-chromaffin cell types. Occurrence frequencies of chromaffin cells contiguous with other types of cells were: (1) higher for A cells (68.9%, 199/289) than for NA cells (11.0%, 34/309) in case of small contact regions (chi 2-test: P < 0.001), and (2) higher for NA cells (68.3%, 211/309) than for A cells (9.7%, 28/289) in case of extended contact regions (P < 0.001). In conclusion, the extent of cellular association with supporting cells was remarkably lower in A cells than in NA cells. Such an arrangement is likely to be appropriate for the extensive, homogeneous control and amplified response characteristic of A cells, and for the close range, complex control and more diverse responses characteristic of NA cells.

Adrenal Medulla↗

Changes of membrane fluidity and Na+,K+-ATPase activity during cellular differentiation in the guinea pig epidermis.

The dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+,K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be associated with a decrease in plasma membrane fluidity and with a decline of Na+,K+-ATPase activity.

Animals↗

Cellular differentiations and structural characteristics in nasopharyngeal angiofibromas. An electron-microscopic study.

An electron-microscopic study of 9 nasopharyngeal angiofibromas was performed in order to elucidate the ultrastructural characteristics. Stromal fibroblasts and proliferating cells of the microvasculature were found. The stromal fibroblasts were subdivided into 3 different groups: (1) "classical" fibroblasts, (2) fibroblasts with histiocytelike features, and (3) fibroblasts with myoid features. By proliferation the cells of the capillary vessels change into stromal cells. A particular pattern of nuclei and dense intranuclear granules is only found in stromal fibroblasts. Consequently fibroblasts as well as cells of the microvasculature contribute to the pool of tumor cells.

Adolescent↗