Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.

Developmental dissociation of thymic dendritic cell and thymocyte lineages revealed in growth factor receptor mutant mice

Thymocytes and thymic dendritic cell (DC) lineages develop simultaneously and may originate from a common intrathymic progenitor. Mice deficient for two growth factor receptor molecules [c-kit and the common cytokine receptor γ chain (γc)] lack all thymocytes including T cell progenitors. Despite this lack of pro-T cells, thymic DC compartments were identified in c-kit−γc− mice. Thus, c-kit- and γc-mediated signals are not essential to generate thymic DCs. In addition, pro-T cells do not appear to be obligatory progenitors of thymic DCs, because DC development is dissociated from the generation of thymocytes in these mice. Thymic DCs in c-kit−γc− mice are phenotypically and functionally normal. In contrast to wild-type mice, however, thymic DCs in c-kit−γc− and, notably, in RAG-2-deficient mice are CD8αneg/low, indicating that CD8α expression on thymic DCs is not independent of thymocytes developing beyond the “RAG-block.”

The new dysmorphology: application of insights from basic developmental biology to the understanding of human birth defects.

Information obtained from studies of developmental and cellular processes in lower organisms is beginning to make significant contributions to the understanding of the pathogenesis of human birth defects, and it is now becoming possible to treat birth defects as inborn errors of development. Mutations in genes for transcription factors, receptors, cell adhesion molecules, intercellular junctions, molecules involved in signal transduction, growth factors, structural proteins, enzymes, and transporters have been identified in genetically caused human malformations and dysplasias. The identification of these mutations and the analysis of their developmental effects have been greatly facilitated by the existence of natural or engineered models in the mouse and even of related mutations in Drosophila, and in some instances a remarkable conservation of function in development has been observed, even between widely separated species.

The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research

The Zebrafish Information Network, ZFIN, is a WWW community resource of zebrafish genetic, genomic and developmental research information (http://zfin.org). ZFIN provides an anatomical atlas and dictionary, developmental staging criteria, research methods, pathology information and a link to the ZFIN relational database (http://zfin.org/ZFIN/). The database, built on a relational, object-oriented model, provides integrated information about mutants, genes, genetic markers, mapping panels, publications and contact information for the zebrafish research community. The database is populated with curated published data, user submitted data and large dataset uploads. A broad range of data types including text, images, graphical representations and genetic maps supports the data. ZFIN incorporates links to other genomic resources that provide sequence and ortholog data. Zebrafish nomenclature guidelines and an automated registration mechanism for new names are provided. Extensive usability testing has resulted in an easy to learn and use forms interface with complex searching capabilities.

Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a novel paracrine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.

Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin

Three sublines of NIH 3T3 cells had the properties of non-neoplastic, preneoplastic, and neoplastic cells, respectively. The closer the cells were to neoplastic behavior, characterized by continuing growth at high density, the slower they multiplied at lower density. Under the conditions of high population density and low calf serum concentration used in the assay for transformed focus formation, the transformed or neoplastic cells were much more sensitive to killing by methotrexate (MTX) than were non-neoplastic cells in the same culture. This differential sensitivity of neoplastic cells was far more pronounced in molecular, cellular, and developmental biology medium 402 (MCDB 402) than in DMEM. It is associated with the presence in MCDB 402 of folinic acid, known clinically as leucovorin, which is a reduced form of the folic acid present in DMEM. Although leucovorin had been shown to selectively spare normal bone marrow and intestine in animals from the killing effect of MTX on tumor cells, we demonstrate the preferential killing of neoplastic over non-neoplastic cells of the same derivation. Neither neoplastic nor non-neoplastic cells were killed once they had stopped multiplying at their respective saturation densities. The development of the light foci characteristic of the preneoplastic cells was less sensitive to MTX than the formation of the dense foci produced by the fully neoplastic cells. The system should serve as a valuable model to establish basic principles and optimal conditions for selective killing of neoplastic cells by chemotherapeutic drugs.

Quantitative modeling of stochastic systems in molecular biology by using stochastic Petri nets

An integrated understanding of molecular and developmental biology must consider the large number of molecular species involved and the low concentrations of many species in vivo. Quantitative stochastic models of molecular interaction networks can be expressed as stochastic Petri nets (SPNs), a mathematical formalism developed in computer science. Existing software can be used to define molecular interaction networks as SPNs and solve such models for the probability distributions of molecular species. This approach allows biologists to focus on the content of models and their interpretation, rather than their implementation. The standardized format of SPNs also facilitates the replication, extension, and transfer of models between researchers. A simple chemical system is presented to demonstrate the link between stochastic models of molecular interactions and SPNs. The approach is illustrated with examples of models of genetic and biochemical phenomena where the UltraSAN package is used to present results from numerical analysis and the outcome of simulations.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Antigen-driven CD4+ T cell and HIV-1 dynamics: Residual viral replication under highly active antiretroviral therapy

Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.

Characterization and developmental patterns of telomerase expression in plants

Telomerase activity is developmentally regulated in mammals. Here we examine telomerase activity in plants, whose development differs in fundamental ways from that of animals. Using a modified version of the telomere repeat amplification protocol (TRAP) assay, we detected an activity in extracts from carrots, cauliflower, soybean, Arabidopsis, and rice with all the characteristics expected for a telomerase synthesizing the plant telomere repeat sequence TTTAGGG. The activity was dependent on RNA and protein components, required dGTP, dATP, and dTTP, but not dCTP, and generated products with a seven nucleotide periodicity. Telomerase activity was abundant in cauliflower meristematic tissue and undifferentiated cells from Arabidopsis, soybean, and carrot suspension cultures, but was low or not detectable in a sampling of differentiated tissues from mature plants. Telomerase from cauliflower meristematic tissues exhibited relaxed DNA sequence requirements, which might reflect the capacity to form telomeres on broken chromosomes in vivo. The dramatic differences in telomerase expression and their correlation with cellular proliferation capacity mirror changes in human telomerase levels during differentiation and immortalization. Hence, telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity.

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.