Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development.

Background: The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. Materials and Methods: To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. Results: We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Conclusion: Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Mechanisms Underlying Influence of Bioelectricity in Development.

To execute the intricate process of development, cells coordinate across tissues and organs to determine where each cell divides and differentiates. This coordination requires complex communication between cells. Growing evidence suggests that bioelectrical signals controlled via ion channels contribute to cell communication during development. Ion channels collectively regulate the transmembrane potential of cells, and their function plays a conserved role in the development of organisms from flies to humans. Spontaneous calcium oscillations can be found in nearly every cell type and tissue, and disruption of these oscillations leads to defects in development. However, the mechanism by which bioelectricity regulates development is still unclear. Ion channels play essential roles in the processes of cell death, proliferation, migration, and in each of the major canonical developmental signaling pathways. Previous reviews focus on evidence for one potential mechanism by which bioelectricity affects morphogenesis, but there is evidence that supports multiple different mechanisms which are not mutually exclusive. Evidence supports bioelectricity contributing to development through multiple different mechanisms. Here, we review evidence for the importance of bioelectricity in morphogenesis and provide a comprehensive review of the evidence for several potential mechanisms by which ion channels may act in developmental processes.

Imaging Dpp Release from a Drosophila Wing Disc.

The transforming Growth Factor-beta (TGF-ß) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-ß superfamily includes TGF-ß, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-ß superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Ion Channel Contributions to Wing Development in Drosophila melanogaster.

During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development.

Ion Channels in Bone Morphogenetic Protein Signaling.

How a single fertilized egg develops into a complex multicellular organism is one of the great mysteries of life. Developmental biology textbooks describe cascades of ligands, receptors, kinases, and transcription factors that designate proliferation, migration, and ultimately fate of cells organized into a multicellular organism. Recently, it has become apparent that ion channels are integral to the process of developmental signaling. Ion channels provide bioelectric signals that must intersect with the known developmental signaling pathways. We review some evidence that bioelectric signaling contributes to bone morphogenetic protein signaling.