A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer.

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.

Expanded polyglutamine peptides alone are intrinsically cytotoxic and cause neurodegeneration in Drosophila.

Several dominant, late-onset neurodegenerative diseases (e.g. Huntington's disease) are caused by expansion of polyglutamine (polyQ) repeats within specific proteins. The diverse, yet overlapping, pathology of these diseases could be due to novel deleterious functions unique to each protein or to a common pathophysiology mediated by the long polyQ chains themselves. By engineering Drosophila to express different polyQ peptides, we find that expanded polyQ chains alone are intrinsically cytotoxic and cause neuronal degeneration and early adult death. We further find that this intrinsic toxicity is dependent on cell type and polyQ length and that the inclusion of other amino acids modifies and reduces toxicity. This is the first in vivo evidence that polyQs, when removed from their disease gene context, cause neurotoxicity. These studies provide a basis for understanding the diverse clinical presentations in terms of the intrinsic cytotoxic effect of polyQ peptides being modulated by protein context. Parallel experiments in which cytotoxic polyQ expansions were engineered into Dishevelled, a Drosophila protein containing a naturally occurring polyQ tract, strongly suggest that the effect of a toxic polyQ peptide can be neutralized by protein context. This animal model provides a simple and effective means of screening for therapeutics that relieves the polyQ-induced lethality, independent of any particular disease gene. By quantifying the degree of lethality in several transgenic lines, we have identified a number of genetically modified strains that are suitable for eventual testing of compounds or genes that ameliorate the pathology of polyQ peptides.

Regeneration in insects.

@9cIntroduction@21T issues exhibit an impressive ability to respond to a myriad of insults by repairing and regenerating complex structures. The elegant and orderly process of regeneration provides clues to the mechanisms of pattern formation but also offers the hope that the process might one day be manipulated to replace damaged body parts. To manipulate the process, it will be necessary to understand the genetic basis of the process. In the case of the insect leg, we are coming close to such a level of understanding and many of the lessons learned are relevant to vertebrate systems. A dynamic web of gene regulatory networks appears to create a robust self-organizing system that is at once extremely intricate but also perhaps simple in its reliance on a few key signaling pathways and a few simple processes, e.g. autoactivation and lateral inhibition. Here we will summarize what has been learned about the networks of gene regulation present in the Drosophila leg discs and then we will explore how the regenerative responses to different insults can be understood as predictable responses to these networks. Each of the regulatory networks could themselves serve as the subject of a detailed review and that is beyond the scope of this discussion. Here we will focus on the interplay between the regulatory networks in patterning the tissue.

Shaggy and dishevelled exert opposite effects on Wingless and Decapentaplegic expression and on positional identity in imaginal discs.

The finding that Wingless (WG) and Decapentaplegic (DPP) suppress each others transcription provides a mechanism for creating developmental territories in fields of cells. Here, we address the mechanism of that antagonism. The dishevelled (dsh) and shaggy (sgg) genes encode intracellular proteins generally thought of as downstream of WG signaling. We have investigated the effects of changing either DSH or SGG activity on both cell fate and wg and dpp expression. At the level of cell fate in discs, DSH antagonizes SGG activity. At the level of gene expression, SGG positively regulates dpp expression and negatively regulates wg expression while DSH activity suppresses dpp expression and promotes wg expression. Sharp borders of gene expression correlating precisely with clone boundaries suggest that the effects of DSH and SGG on transcription of wg and dpp are not mediated by secreted factors but rather act through intracellular effectors. The interactions described here suggest a model for the antagonism between WG and DPP that is mediated via SGG. The model incorporates autoactivation and lateral inhibition, which are properties required for the production of stable patterns. The regulatory interactions described exhibit extensive ability to organize new pattern in response to manipulation or injury.

Developmental territories created by mutual antagonism between Wingless and Decapentaplegic.

Drosophila appendages develop from imaginal discs which become subdivided into distinct regions during normal patterning. At least 3 axes of asymmetry are required to produce a chiral appendage such as a leg. The A/P compartments provide one axis of asymmetry in all discs. In leg and antennal discs, the anterior compartment becomes asymmetric in the D/V axis with decapentaplegic (dpp) expression defining dorsal anterior leg, and wingless (wg) expression defining ventral anterior leg. However, unlike wing discs, no D/V compartment has been demonstrated in legs or antennae. How are the dorsal anterior and ventral anterior territories defined and maintained? Here we show that wg inhibits dpp expression and dpp inhibits wg expression in leg and eye/antennal discs. This mutual repression provides a mechanism for maintaining separate regions of wg and dpp expression in a developing field. We propose the term 'territory' to describe regions of cells that are under the domineering influence of a particular morphogen. Territories differ from compartments in that they are not defined by lineage but are dynamically maintained by continuous morphogen signaling. We propose that the anterior compartment of the leg disc is divided into dorsal and ventral territories by the mutual antagonism between WG and DPP signaling.

dishevelled is required during wingless signaling to establish both cell polarity and cell identity.

The dishevelled gene of Drosophila is required to establish coherent arrays of polarized cells and is also required to establish segments in the embryo. Here, we show that loss of dishevelled function in clones, in double heterozygotes with wingless mutants and in flies bearing a weak dishevelled transgene leads to patterning defects which phenocopy defects observed in wingless mutants alone. Further, polarized cells in all body segments require dishevelled function to establish planar cell polarity, and some wingless alleles and dishevelled; wingless double heterozygotes exhibit bristle polarity defects identical to those seen in dishevelled alone. The requirement for dishevelled in establishing polarity in cell autonomous. The dishevelled gene encodes a novel intracellular protein that shares an amino acid motif with several other proteins that are found associated with cell junctions. Clonal analysis of dishevelled in leg discs provides a unique opportunity to test the hypothesis that the wingless dishevelled interaction species at least one of the circumferential positional values predicted by the polar coordinate model. We propose that dishevelled encodes an intracellular protein required to respond to a wingless signal and that this interaction is essential for establishing both cell polarity and cell identity.

Effects of iodide on class II-MHC antigen expression in iodine deficient hyperplastic thyroid glands.

The expression of major histocompatibility complex class II molecules (Ia antigen) has been analyzed by immunoperoxidase staining in thyroids of normal C3H mice, of iodine-deficient mice with a hyperplastic goiter and of mice during goiter involution induced by administration of either a high iodide dose (HID, 10 micrograms/day) for 0.5 to 8 days or a moderate iodide dose (MID, 1 microgram/day) or triiodothyronine (T3, 1 micrograms/day) for 2 days. In normal and in hyperplastic thyroids, few interstitial cells were Ia positive (monoclonal antibodies, mAb, M5/114, ER-TR3). Their number was unchanged when goiter involution was induced by MID or by T3, but was significantly increased (p less than 0.05) after HID. It was maximal at days 1 and 2 of involution, decreased thereafter but remained higher (p less than 0.05) than in controls after 8 days. The Ia positive cells were mainly macrophages and, to a lesser extent, dendritic cells. Macrophages were identified by their heterogeneous content and their numerous lysosomes. They were stained with anti-Mac-1 (M1/70) and anti-Mac-2 (M3/38) mAb. Dendritic cells were characterized by their slender cytoplasmic processes, indented nucleus and pale cytoplasm. They were positive for NLDC-145 and MIDC-8 mAb whose specificity for dendritic cells has been demonstrated in lymphoid organs. During the whole period of involution analyzed, Ia antigens were not expressed on follicular cells. Since macrophages and dendritic cells are known to be involved in the pathogenesis of immune disorders, the inflammation induced by administration of HID to iodine-deficient mice could be considered as the early step of an immunological reaction.

Dosimetry by ESR spectroscopy following a radiation accident.

On 2 September, 1982, one of the employees of the gamma-irradiation facility at The Institute for Energy and Technology (Kjeller, Norway) entered the irradiation cell with a 65.7-kCi 60Co source in unshielded position. The victim received an unknown radiation dose and died after 13 days. Using electron-spin resonance spectroscopy (ESR), the radiation dose in this accident was subsequently determined based on the production of long-lived free radicals in nitroglycerol tablets carried by the operator during accident. He used nitroglycerol for heart problems and free radicals are easily formed and trapped in sugar which is the main component of the tablets. Calibration experiments were carried out and the dose given to the tablets during the accident was determined to be 39 Gy. Phantom experiments based on this result indicate an average whole-body dose in the accident of 22.5 Gy.