Temperature-controlled microscopy for imaging living cells: apparatus, thermal analysis and temperature dependency of embryonic elongation in Caenorhabditis elegans.

A new experimental apparatus for temperature-controlled microscopy has been developed for the study of the temperature dependency of developmental processes in the nematode Caenorhabditis elegans. However, the application of this apparatus is rather general and can be used for a wide range of temperatures between - 10 and 90 degrees C. The new apparatus is easy to use, inexpensive, simple to construct, and is designed for precise temperature control of oil-immersion microscopy using epifluorescence. Thermal analysis of the experimental apparatus shows the effects of each of its components, as well as the effects of uncertainty in temperature measurements. Finally, results of this study indicate that: (i) embryos incubated and imaged at temperatures of 8 degrees C and below do not elongate; (ii) the initial elongation rate is strongly temperature-dependent between 9 and 25 degrees C.

Fusomorphogenesis: cell fusion in organ formation.

Cell fusion is a universal process that occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. Very little is known about the molecular and cellular mechanisms of cell fusion during pattern formation. Here we review the dynamic anatomy of all cell fusions during embryonic and postembryonic development in an organism. Nearly all the cell fates and cell lineages are invariant in the nematode C. elegans and one third of the cells that are born fuse to form 44 syncytia in a reproducible and stereotyped way. To explain the function of cell fusion in organ formation we propose the fusomorphogenetic model as a simple cellular mechanism to efficiently redistribute membranes using a combination of cell fusion and polarized membrane recycling during morphogenesis. Thus, regulated intercellular and intracellular membrane fusion processes may drive elongation of the embryo as well as postembryonic organ formation in C. elegans. Finally, we use the fusomorphogenetic hypothesis to explain the role of cell fusion in the formation of organs like muscles, bones, and placenta in mammals and other species and to speculate on how the intracellular machinery that drive fusomorphogenesis may have evolved.

Ring formation drives invagination of the vulva in Caenorhabditis elegans: Ras, cell fusion, and cell migration determine structural fates.

Directed cell rearrangements occur during gastrulation, neurulation, and organ formation. Despite the identification of developmental processes in which invagination is a critical component of pattern formation, little is known regarding the underlying cellular and molecular details. Caenorhabditis elegans vulval epithelial cells undergo morphological changes that generate an invagination through the formation of seven stacked rings. Here, we study the dynamics of ring formation during multivulva morphogenesis of a let-60/ras gain-of-function mutant as a model system to explore the cellular mechanisms that drive invagination. The behavior of individual cells was analyzed in a let-60/ras mutant by three-dimensional confocal microscopy. We showed that stereotyped cell fusion events occur within the rings that form functional and nonfunctional vulvae in a let-60/ras mutant. Expression of let-60/ras gain-of-function results in abnormal cell migration, ectopic cell fusion, and structural fate transformation. Within each developing vulva the anterior and posterior halves develop autonomously. Contrary to prevailing hypotheses which proposed three cell fates (1 degrees, 2 degrees, and 3 degrees), we found that each of the seven rings is a product of a discrete structural pathway that is derived from arrays of seven distinct cell fates (A, B, C, D, E, F, and H). We have also shown how autonomous ring formation is the morphogenetic force that drives invagination of the vulva.

Formation of the vulva in Caenorhabditis elegans: a paradigm for organogenesis.

The genes involved in the inductive interactions that specify cell fates in the vulva of Caenorhabditis elegans are known in some detail. However, little is known about the morphogenesis of this organ. Using a combination of cell biological and anatomical approaches, we have determined a complete morphogenetic pathway of cellular events that lead to the formation of the vulva. These events include reproducible cell divisions, migrations, remodeling of adherens junctions, cell fusions and muscle attachments. In the course of these events, an epithelial channel comprising a stack of 7 toroidal cells is formed that connects the internal epithelium of the uterus with the external body epithelium, forming the vulva. Vulval muscles attach to the epithelial channel and the whole structure everts during the final molt. The mature vulva has rotational, two-fold symmetry. Using laser microsurgery, we found that the two halves of the vulva develop autonomously.

ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions.

Cell fusions in the developing epithelial of C. elegans.

In this paper we characterize the order of hypodermal cell fusions in the Caenorhabditis elegans hermaphrodite. Somatic cell fusions are part of the developmental program of many tissues in a variety of organisms. The formation and remodeling of tissues and organs can be studied at the cellular level in C. elegans. Here we establish a system for studying cell fusion by characterizing somatic cell fusions during morphogenesis in C. elegans. Fusion is a common cell fate in this nematode; numerous epithelial fusions occur in the hypodermis, vulva, uterus, and excretory gland cells (Sulston et al., 1983. Dev. Biol. 100, 64-119). Some but not all pharyngeal muscles also fuse (Albertson and Thomson, 1976. Philos. Trans. R. Soc. London Ser. B 275, 299-325). We have studied the behavior of epithelial adherens junctions before and during cell-to-cell fusions in embryonic and postembryonic development. Our results define the timing and sequence of short-range migrations followed by fusions that generate syncytia. We have made use of an antibody that stains adherens junctions to study the behavior of hypodermal cells during development. Fusion of specific cells in the hypodermis causes rearrangements of the adherens junctions between cells. Fusion events usually start in the anterior part of embryos or larvae. There is some variation in the specific order in which cells fuse, but the final positions, boundaries, and sizes of syncytia are the same. In some cases fusion causes isolation of a mononucleate cell or group of cells by a surrounding, growing syncytium. Our characterization of the order of cell fusions will provide a basis for the identification of molecular events required for regulated membrane fusion during development.

ATP and cytosol requirements for transferrin recycling in intact and disrupted MDCK cells.

We have developed an in vitro system for studying membrane transport during receptor-mediated endocytosis. Using nitrocellulose disruption to permeabilize selectively the apical domain of filter-grown MDCK cells, the recycling of receptor-bound transferrin (Tfn) from an intracellular pool was reconstituted in vitro with a rate and efficiency similar to that of intact cells. Tfn and Tfn receptor recycling from endosomes back to the cell surface was dependent on added ATP and cytosol-derived proteins. Thus, incubation of intact cells under conditions of ATP depletion resulted in the clearance of Tfn receptors from the basolateral membrane, this was reversible upon removal of the energy poisons. Reappearance of previously internalized receptors could also be obtained in disrupted cells but required the addition of both ATP and cytosol to the assay mixture. Similarly, when intact cells were allowed to internalize labeled Tfn prior to disruption, efficient and rapid release of ligand back into the medium was markedly stimulated by ATP and cytosol. Recycling was judged to be both selective and vectorial since only the expected small fraction of a previously internalized horseradish peroxidase was released after addition of ATP and cytosol, and release was primarily into the basal medium. While the cytosol contributed one or more protein factors, none was sensitive to N-ethylmaleimide. Alkylation of the disrupted cells, however, did inactivate recycling.