Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles ¹ and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies ². Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.     

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.     

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.     

Distant activation of Notch signaling induces stem cell niche assembly

Here we show that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, we studied the formation of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to our understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.     

Melatonin and alcohol-related disorders

ABSTRACT

This review concerns the current knowledge of melatonin and alcohol-related disorders. Chronobiological effects of ethanol are related to melatonin suppression and in relation to inflammation, stress, free radical scavenging, autophagy and cancer risk. It is postulated that both alcohol- and inflammation-induced production of reactive oxygen species (ROS) alters cell membrane properties leading to tissue dysfunction and, subsequent further ROS production. Lysosomal enzymes are often used to assess the relationships between intensified inflammation states caused by alcohol abuse and oxidative stress as well as level of tissue damage estimated by the increased release of cellular enzymes into the extracellular space. Studies have established a link between alcoholism and desynchronosis (circadian disruption). Desynchronosis results from the disorganization of the body’s circadian time structure and is an aspect of the pathology of chronic alcohol intoxication. The inflammatory conditions and the activity of lysosomal enzymes in acute alcohol poisoning or chronic alcohol-dependent diseases are in most cases interrelated. Inflammation can increase the activity of lysosomal enzymes, which can be regarded as a marker of lysosomal dysfunction and abnormal cellular integrity. Studies show alcohol toxicity is modulated by the melatonin (Mel) circadian rhythm. This hormone, produced by the pineal gland, is the main regulator of 24 h (sleep-wake cycle) and seasonal biorhythms. Mel exhibits antioxidant properties and may be useful in the prevention of oxidative stress reactions known to be responsible for alcohol-related diseases. Naturally produced Mel and exogenous sources in food can act in free radical reactions and activate the endogenous defense system. Mel plays an important role in the normalization of the post-stress state by its influence on neurotransmitter systems and the synchronization of circadian rhythms. Acting simultaneously on the neuroendocrine and immune systems, Mel optimizes homeostasis and provides protection against stress.

Abbreviations: ROS, reactive oxygen species; Mel, melatonin; SRV, resveratrol; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ANT, arylalkylamine-N-acetyltransferase; EC cells, gastrointestinal enterochromaffin cells; MT1, melatonin high-affinity nanomolecular receptor site; MT2, melatonin low-affinity nanomolecular receptor site; ROR/RZR, orphan nuclear retinoid receptors; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; TAC, total antioxidant capacity; ONOO?^–, peroxynitrite radical; NCAM, neural cell adhesion molecules; LPO, lipid peroxidation; α-KG, α-ketoglutarate, HIF-1α, Hypoxia-inducible factor 1-α, IL-2, interleukin-2; HPA axis, hypothalamic-pituitary-adrenal axis; Tph1, tryptophan hydroxylase 1; AA-NAT, arylalkylamine-N-acetyltransferase; AS-MT, acetylserotonin O-methyltransferase; NAG, N-acetyl-beta-D-glucosaminidase; HBA1c glycated hemoglobin; LPS, lipopolysaccharide; AAP, alanyl-aminopeptidase; β-GR, β-glucuronidase; β-GD, β-galactosidase; LAP, leucine aminopeptidase.