New directions in craniofacial morphogenesis

The vertebrate head is an extremely complicated structure: development of the head requires tissue-tissue interactions between derivates of all the germ layers and coordinated morphogenetic movements in three dimensions. In this review, we highlight a number of recent embryological studies, using chicken, frog, zebrafish and mouse, which have identified crucial signaling centers in the embryonic face. These studies demonstrate how small variations in growth factor signaling can lead to a diversity of phenotypic outcomes. We also discuss novel genetic studies, in human, mouse and zebrafish, which describe cell biological mechanisms fundamental to the growth and morphogenesis of the craniofacial skeleton. Together, these findings underscore the complex interactions leading to species-specific morphology. These and future studies will improve our understanding of the genetic and environmental influences underlying human craniofacial anomalies.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

The pattern of proliferation of the neuroblasts in the wild-type embryo of Drosophila melanogaster

Summary The pattern of neuroblast divisions was studied in thoracic and abdominal neuromeres of wild-type Drosophila melanogaster embryos stained with a monoclonal antibody directed against a chromatin-associated antigen. Since fixed material was used, our conclusions are based upon the statistical evaluation of a large number of accurately staged embryos, covering the stages between the formation of the cephalic furrow up to shortened germ band. Our observations point to a rather stereotypic pattern of proliferation, consisting of several parasynchronous cycles of division. The data suggest that all SI neuroblasts divide at least eight times, all SII neuroblasts six or seven times and all SIII neuroblasts at least five times. This conclusion is based on the mapping of mitotic neuroblasts and is supported by the progressive reduction of the neuroblast volume and by the results of cell countings performed on embryos of increasing age. No conclusive evidence was obtained concerning the fate of the neuroblasts after their last mitosis, i.e. it cannot be decided whether the neuroblasts degenerate or become incorporated as inconspicuous cells in the larval ventral cord. The duration of the cycles of division of the neuroblasts was found to be 40–50 min each, while in the case of ganglion mother cells about 100 min are required to complete one cell cycle.     

Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern

Summary During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.     

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles’ medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, α-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was α-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. γ-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.     

Elektronenmikroskopische Zytomorphologie der männlichen Brustdrüse

Zusammenfassung Im Vergleich zu lichtmikroskopischen Untersuchungen an der Mamma virilis wird anhand von 2 operativ entfernten Brustdrüsen eines 57 und 63 Jahre alten Mannes die elektronenmikroskopisch erfaßbare Zytomorphologie beschrieben. Die Befunde werden den physiologischen Wachstumsimpulsen dieses Organs gegenübergestellt und Fragen der Zelldifferenzierung, der Desquamation und apokrinen Sekretion beantwortet. Elektronenmikroskopisch werden am Drüsenepithel Basalzellen, größere Zellen der oberflächlichen Zellreihen und Myoepithelzellen unterschieden. Diese Zellen entsprechen den Gangepithelien der weiblichen Brustdrüse und besitzen intracytoplasmatische Filamente. Diese stellen ein häufiges Differenzierungsprodukt des Zytoplasmas dar. Mechanismen einer Sekretion waren nicht nachweisbar. In die Drüsenlichtung werden pseudopodienartig vorgewölbte Zytoplasmateile abgeschnürt (Extrasionsvorgang). — Superfiziale Zellen werden desquamiert, wobei die Zytolyse in den marginalen Zytoplasmaschichten erfolgt. Kern und Teile des Zytoplasmas gelangen in die Drüsenlichtung. — Die Befunde zeigen die von Lebensalter und Proliferationsreiz abhängigen Vorgänge eines permanenten Zellersatzes in der männlichen Brustdrüse an.

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Ultrastructure of the mammary gland of the human male

Summary The ultrastructure of two mammary glands obtained operatively from a 57-year old and a 63-year old man was compared to the structure observed in the light microscope, and related to stimuli controlling growth of the gland, cellular differentiation and desquamation, and apocrine secretion. The glandular epithelium, which is analogous to that of the female mammary gland, is differentiated into basic cells, large superficial cells, and myoepithelial cells. The cells have intracytoplasmic filaments, that may be a sign of differentiation. Mechanisms for secretion were not observed, although pseudopodia-like parts of the cytoplasm are extruded into the glandular lumen. Superficial cells are desquamated, followed by cytolysis of their margins. These findings illustrate the replacement of cells due to age and altered stimuli.

Frl. St. Walter, lt. Assistentin des elektronenmikroskopischen Labors, danken wir für Präparationen und Photoarbeiten.     

建兰根状茎增殖条件的研究

研究了建兰两个栽培品种的根状茎在固体及液体两种培养方式,以及培养基中有无激素和不同激素浓度配比对增殖及分化的影响。根状茎在液体培养基上的生长量都大于固体培养基上的生长量。根状茎在无激素的培养基上也能增殖。加入激素有促进作用。合适的激素浓度及配比因品种而异。     

In search of mammary gland stem cells

Summary The evidence for mammary epithelial stem cells and their phenotypic characteristics in normal and neoplastic development is reviewed. The presence of stem cells in all parts of the mammary parenchyma at all stages of differentiation has been demonstrated by transplantation experiments. The phenotypic characterization of stem cells has been defined by a battery of monospecific antibodies. These studies suggest that a mammary epithelium stem cell compartment exists in the basal layer of the gland as well as in the end bud. Whether these same stem cells are expressed in mammary preneoplasias and neoplasias has not been answered conclusively. Phenotypic markers specific for stem cells and stably expressed in transformed cell populations are needed to follow the fate of stem cells.Dedicated to Professor Stuart Patton on the occassion of his 70th birthday.