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Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells.

Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induce ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed when the MFC were cultured either on plastic or within a layer of RBM. Although most MFC maintained on plastic died after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.

Animals↗

The use of retinoids as probes for analyzing morphogenesis of glands from epithelial tissues.

Thirty-five years ago Honor Fell and Edward Mellanby were studying effects of high doses of vitamin A on skeletal development in chick embryos when they noticed that a piece of epidermis, accidentally included in an organ culture, had undergone mucous metaplasia. Further studies by Fell and others eventually led to an understanding of the important role of vitamin A in modulating epithelia in vivo. Fifteen years later another organ culture experiment showed me that excess vitamin A could also initiate the morphogenesis of branching and mucus-secreting glands from developing vibrissa follicles in upper lip skin of embryonic mice. Since then our group has shown that induction of this novel structure by naturally occurring retinoids resembles a normal embryonic induction in that it is stage-dependent, time-dependent, and irreversible. Tissue separation and recombination studies showed that isolated upper lip epidermis can form these glands when combined with retinoid-treated upper lip dermis. Untreated mouse epidermis can form similar glands after combination with chick dermis containing higher retinoid levels. The hamster cheek pouch, normally devoid of glandular structures, can also form mucous glands when treated with a retinoid, either in vivo or in vitro. Recombination studies in organ culture have now shown that mesenchyme exposed to retinoid is essential for gland morphogenesis from pouch epithelium. Evidence is accumulating that retinoic acid may even be the active morphogen in some normally developing systems.

Animals↗

Can morphogenesis be understood in terms of physical rules?

Because the morphogenesis of biological systems is not fully understood, researches from various points of view are necessary. The present author has recently made computer simulations with his colleagues to construct branching systems of human organs, such as the lung airway and the liver blood vessels. In the simulations certain rules are assumed to govern bifurcating processes of the systems. These rules are expressed in terms of physical and geometrical concepts, such as minimum energy consumption and uniform filling of branches in the space of organs. Results of computer simulation are quite similar to real structures. However, actual mechanisms of morphogenesis, i.e. effects of genes or proteins, are not considered in these studies. In this article, the present work is discussed in relation to the concept of biological pattern formation by Meinhardt and a recent study by Miura and Shiota on lung growth.

Computer Simulation↗

The use of antibiotics for studies of morphogenesis and differentiation in microorganisms.

Numerous antibodies with a known mechanism of action are utilized as possible means for studying morphogenesis and differentiation. Inhibitors of biosynthesis of nucleic acids and proteins, compounds intervening with the synthesis and/or function of cell walls and membranes or compounds influencing the energy metabolism are particularly useful. The use of antibiotics for studies of the life cycle of viruses, bacteria, fungi, myxomycetes, protozoa and algae is analyzed in the present communication. Certain aspects of morphogenesis and functions of mitochondria and plastids were clarified with the aid of antibiotics. Relationships between production of antibiotics and differentiation of their producers are discussed in the final part of the paper.

Anti-Bacterial Agents↗

Hepatocyte growth factor promotes epithelial morphogenesis and occludin linkage to the cytoskeleton in cultured retinal pigment epithelial cells.

BACKGROUND: Although hepatocyte growth factor (HGF) is also known as scatter factor, it induces epithelial morphogenesis in cultured bovine retinal pigment epithelial (RPE) cells. To elucidate the mechanism of epithelial morphogenesis, we investigated the influence of HGF on occludin, a major component of tight junctions. METHODS: RPE cells were plated on collagen type 1-coated chamber slides or dishes, 20 ng/ml HGF was added and the cells were incubated for 1 week. Cells were harvested at several time-points, and occludin expression was examined by immunohistochemistry. Detergent extraction protocols to identify the intensity of occludin linkage to the cytoskeleton were also used. Occludin expression was determined semiquantitatively by Western blotting. RESULTS: Fluorescence microscopy revealed that HGF treatment increased the levels of insoluble occludin at the cell borders after detergent extraction. These level of insoluble occludin and the associated epithelial morphology were maintained for more than 3 weeks after withdrawal of HGF, whereas cells not treated with HGF had a fibroblastic appearance. Western blotting also showed that insoluble occludin was more abundant in HGF-treated cells. Furthermore, immunoreactive bands of insoluble occludin were somewhat larger than those of soluble occludin, suggesting that insoluble occludin may be modified in comparison to soluble occludin. CONCLUSION: Our results suggest that HGF promotes linkage of occludin to the cytoskeleton. HGF may become a therapeutic candidate in physiological recovery of RPE cells and in preparation of RPE monolayers for transplantation.

Animals↗

Auxin-cytokinin and auxin-gibberellin interactions during morphogenesis of the compound leaves of pea (Pisum sativum).

A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression.

Benzyl Compounds↗

The genetic control of arista lateral morphogenesis in Drosophila.

The sensory bristles and epidermal hairs of Drosophila have proven to be valuable model cell types for studying the role of the cytoskeleton in cellular morphogenesis. We have recently begun to use the arista laterals as a third model cell type. The laterals display a combination of bristle and hair characteristics and provide a system where we can compare the relative importance of specific genes and subcellular structures for the morphogenesis of different polarized cellular extensions. We have characterized the lateral phenotype of a collection of mutations selected because of their phenotypes in hairs and bristles. In many but not all ways the lateral phenotypes are similar to the hair and bristle phenotypes. We provide compelling genetic evidence for the importance of the actin cytoskeleton in lateral elongation, shaping and integrity. Our observations provide evidence that defects in actin bundling can destabilize laterals so that they split during growth. Temperature shift experiments suggest that a defect in lateral initiation can lead to subsequent splitting. These observations provide a link between multiple hair and lateral cells forming by both multiple initiation events and by the splitting of individual cellular extensions. We also found that mutations that lead to lateral splitting typically alter the stereotypic arrangement of actin filament bundles and microtubules in laterals.

Actins↗

Left and right contributions to the Xenopus heart: implications for asymmetric morphogenesis.

The left-right asymmetry of the vertebrate heart is evident in the topology of the heart loop, and in the dissimilar morphology of the left and right chambers. How left-right asymmetric gene expression patterns influence the development of these features is not understood, since the individual roles of the left and right sides of the embryo in heart looping or chamber morphogenesis have not been specifically defined. To this end, we have constructed a bilateral heart-specific fate map of the left and right contributions to the developing heart in the Xenopus embryo. Both the left and right sides contribute to the conoventricular segment of the heart loop; however, the left side contributes to the inner curvature and ventral face of the loop while the right side contributes to the outer curvature and dorsal aspect. In contrast, the left atrium is derived mainly from the original left side of the embryo, while the right atrium is derived primarily from the right side. A comparison of our fate map with the domain of expression of the left-right gene, Pitx2, in the left lateral plate mesoderm, reveals that this Pitx2-expressing region is fated to form the inner curvature of the heart loop, the left atrioventricular canal, and the dorsal aspect of the left atrium. We discuss the implications of these results for the role of left-right asymmetric gene expression in heart looping and chamber morphogenesis.

Animals↗

Clonal analysis of Distal-less and engrailed expression patterns during early morphogenesis of uniramous and biramous crustacean limbs.

In order to investigate the correlation of cell lineage, gene expression, and morphogenesis of uniramous and biramous limbs we studied limb formation in the thorax and pleon of the amphipod Orchestia cavimana and the isopod Porcellio scaber. We took advantage of the fact that in amphipod and isopod crustaceans--both Malacostraca--uniramous limbs evolved independently in the thorax whereas ancestral biramous limbs are formed in the pleon (abdomen). The gene Distal-less is expressed in the early limb buds as in other arthropods. Accordingly, it is likely to be responsible for the development of the proximodistal axis of the appendages. Double staining of Distal-less and Engrailed proteins suggests that Distal-less in the pleon of the amphipod Orchestia might not be under the control of the Wingless protein. Additionally, we studied axis formation of the uniramous and biramous limbs. In both species investigated, biramous limbs originate exclusively by the subdivision of the original limb bud. Both distal elements continuously express Distal-less. There is flexibility in the suppression of the development of additional branches in the crustacean limb. In the amphipod O. cavimana, uniramous thoracopods are formed by downregulation of Distal-less in the area where, in biramous limbs, the exopodites would occur. In contrast, this region never expresses Distal-less in the uniramous thoracopods of the isopod P. scaber. Our results suggest that the gene expression pattern is independent of the cell division pattern. Gene expression domains and morphogenesis of limbs and segments, on the other hand, show a good correlation.

Amphipoda↗

From head to toe: conservation of molecular signals regulating limb and craniofacial morphogenesis.

Recent evidence indicates that many molecules involved in generating and patterning the limbs also play a role during craniofacial morphogenesis. On the surface, this is an unexpected finding given that these regions of the body have separate evolutionary origins, are composed of different embryonic tissues, and are quite dissimilar in their anatomy. Results from several experiments involving Sonic hedgehog and retinoic acid point to a remarkable conservation of the signaling pathways mediated by these morphogens across multiple organ systems. Moreover, mutants such as the extra-toes and doublefoot mouse, and the talpid chicken also provide insights on common developmental processes that underlie the formation of the limbs and face. The identification of highly conserved aspects of morphogenesis is important for understanding fundamental mechanisms of development, as well as for revealing the common denominator of countless birth defects and providing new strategies for their prevention and cure.

Animals↗

Developmental regulation and ultrastructure of glycogen deposits during murine tooth morphogenesis.

The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5-17 (E11.5-E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14-E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum.

Animals↗

Morphogenesis of an anomalous ligamentum venosum terminating in the superior left hepatic vein in a human liver.

BACKGROUND/PURPOSE: We aimed to clarify the morphogenesis of an anomalous ligamentum venosum terminating in the trunk of the superior left hepatic vein, because the ligamentum venosum ordinarily terminates into the root of the left hepatic vein or directly into the inferior vena cava. METHODS: We examined an anomalous ligamentum venosum found in the cadaveric liver of an 84-year-old Japanese woman. RESULTS: The ligamentum venosum in this liver was not found in the usual course, the fissure for the ligamentum venosum. It lay on the posterior surface of the liver, connecting the left branch of the portal vein and the trunk of a small left hepatic vein. The small left hepatic vein draining the cranio-dorsal part of the lateral segment of the liver was revealed to be a superior left hepatic vein. This type of anomaly was found only in this 1 liver, among 125 cadaveric livers that were dissected. CONCLUSIONS: Taking previous reports into consideration, the morphogenesis of the anomalous ligamentum venosum in the present case may be explained as being due to the persistence of the right half of the subdiaphragmatic anastomosis, which receives the blood from the ductus venosus in the embryonal period.

Aged, 80 and over↗

Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis.

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt's lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogenesis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not-9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.

Animals↗

Ultrastructural study of myxoma virus morphogenesis.

Poxviruses are among the largest and most complex viruses known. Vaccinia virus, the prototype of the family Poxviridae, has been studied much more than myxoma virus. The aim of this work was to have a better knowledge about myxoma virus morphogenesis. The characterization of the main stages of MV morphogenesis was achieved by ultrastructural and immunological analysis. Specific antibodies were raised against M022L and M071L, two envelope proteins of extracellular enveloped virus and intracellular mature virus, respectively. The main stages of assembly were similar to those seen with other poxviruses, and the duration of the whole replication cycle was estimated to be around 16 h, longer than what was described for vaccinia virus. Morphological changes of infected cells were associated with the development of long cellular projections and enlarged microvilli. Intracellular enveloped viruses are associated with the cytoskeleton to move through the cell. Unlike earlier studies, as many cell-associated enveloped viruses as intracellular enveloped viruses were observed in relation with specialized microvilli, although these structures were rarely noticed. Finally, an unusual spreading process was observed, which uses cytoplasmic corridors.

Animals↗

Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia.

The human breast epithelium is a branching ductal system composed of an inner layer of polarized luminal epithelial cells and an outer layer of myoepithelial cells that terminate in distally located terminal duct lobular units (TDLUs). While the luminal epithelial cell has received the most attention as the functionally active milk-producing cell and as the most likely target cell for carcinogenesis, attention on myoepithelial cells has begun to evolve with the recognition that these cells play an active part in branching morphogenesis and tumor suppression. A major question that has been the subject of investigation pertains to how the luminal epithelial and myoepithelial lineages are related and precisely how they arise from a common putative stem cell population within the breast. Equally important is the question of how heterotypic signaling occurs between luminal epithelial and surrounding myoepithelial cells in normal breast morphogenesis and neoplasia. In this review we discuss data from our laboratories and from others regarding the cellular origin of human myoepithelial cells, their function in maintaining tissue polarity in the normal breast, and their role during neoplasia.

Animals↗

Research on control of craniofacial morphogenesis: an NIDR State-of-the-Art Workshop.

To assess the wide clinical ramifications of control of craniofacial morphogenesis, a State-of-the-Art Workshop was conducted by the National Institute of Dental Research at the initiative of Richard L. Christiansen, Chief of the Craniofacial Anomalies Program. In conjunction with the authors listed above, the format for the workshop was developed and participants were selected. The workshop was designed to provide an in-depth review of present knowledge and to identify future goals and directions for research on guiding, altering, and thus controlling growth and development of the cranofacial skeleton. The agenda for discussion ranged from molecular biology to clinical arts such as orthopedics and surgery. It was evident during the workshop that the mechanisms and procedures for controlling craniofacial morphogenesis must be derived from many biologic, physical, and clinical fields of knowledge. It is hoped that there will evolve an interdisciplinary clinical art which is aimed at preventing and correcting craniofacial deformities. Substantial biologic information has already been accumulated on the craniofacial skeleton. The clinical art of correcting malocclusion through mechanical forces is now applicable to the entire skull. The outstanding technical accomplishments of radical surgery in the correction of congenital craniofacial anomalies show that the needed surgical skills are now available. When these resources are combined, an area of knowledge and a clinical discipline which might be called "orthocephalics" is already identifiable. The workshop was held at the National Institutes of Health in Bethesda, Maryland, on Feb. 12 and 13, 1974. The ideas exchanged were integrated and summarized by the planning committee to produce this report.

Animals↗

The extracellular matrix between the optic vesicle and presumptive lens during lens morphogenesis in an anophthalmic strain of mice.

Extracellular matrix material present during early lens morphogenesis in anophthalmic strain ZRDCT-Ch mice was studied histochemically by the Alcian blue 8GX pH 2.5, Alcian blue 8GX pH 2.5/periodic acid-Schiff combined, high iron diamine, and Van Gieson methods. Observed staining patterns were compared with results from an analysis of a normal strain of mice (E.H. Webster, Jr., A.F. Silver, and N.I. Gonsalves, 1983, Develop. Biol. 100, 147-157). No differences in constituents were found between the strains in staining patterns of the ectodermal basal lamina. However, the optic vesicle basal lamina in the anophthalmic strain was found to have a relatively lower staining intensity for sulfated glycosaminoglycan associated with it than was observed in the normal strain, although these mutant optic vesicles were morphologically normal. Results from this and the earlier study on normal mice indicate that one function of sulfated glycosaminoglycan in early lens morphogenesis may be to serve as a cementing medium between the optic and lens rudiments. This sulfated glycosaminoglycan deficiency on the anophthalmic optic vesicle basal lamina is temporally correlated with and may be causally related to precocious lens cup formation and frequently observed separation of the normally adherent eye rudiments. Conclusions drawn from this study are consistent with the speculation of H.B. Chase and E.B. Chase (1941, J. Morphol. 68, 279-301) that there may be abnormal contact between the optic vesicle and presumptive lens ectoderm in the mutant strain, although there is a differing view on the cause of the abnormal contact.

Animals↗

Altered keratin biosynthesis follows inhibition of scale morphogenesis by hydrocortisone.

Hydrocortisone, administered onto the chorioallantoic membrane (CAM) of 7- to 10-day-old chick embryos, inhibits scale development, in a dose- and stage-dependent manner. The response is also region specific in that hydrocortisone treatment, at a specific dose and time, will completely block scutellate and interstitial scale development while leaving other scale types unaffected. Using histological, biochemical, and immunofluorescence techniques, we have shown that inhibition of scutellate scale morphogenesis prevents the subsequent formation of a beta stratum and alters expression of the alpha keratins. These data support the hypotheses that each avian scale type has its own distinctive temporal, morphological, and biochemical pattern of development; and in the case of scutellate scale development, hydrocortisone treatment alters keratin biosynthesis by interfering with earlier steps in morphogenesis.

Allantois↗