ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress.

Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). We established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS RNAi ) and challenged them with H2O2. Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H2O2-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS RNAi fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. We thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress.

Rab11 activation by Ik2 kinase is required for dendrite pruning in Drosophila sensory neurons.

Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons.

Cell-Autonomous Regulation of Dendrite Self-Avoidance by the Wnt Secretory Factor MIG-14/Wntless.

Self-avoidance allows sister dendrites from the same neuron to form non-redundant coverage of the sensory territory and is important for neural circuitry functions. Here, we report an unexpected, cell-autonomous role of the Wnt-secretory factor MIG-14/Wntless in mediating dendrite self-avoidance in the C. elegans multidendritic PVD neurons. Similar findings in Drosophila suggest that this novel function of Wntless is conserved. The mig-14 mutant shows defects in dendrite self-avoidance, and ectopic MIG-14 expression triggers dendrite repulsion. Functions of dendrite self-avoidance and Wnt secretion could be mapped to distinct MIG-14 domains, indicating that these two functions of MIG-14 are genetically separable, consistent with lack of self-avoidance defects in the Wnt mutants. We further demonstrate that MIG-14 engages Wiskott-Aldrich syndrome protein (WASP)-dependent actin assembly to regulate dendrite self-avoidance. Our work expands the repertoire of self-avoidance molecules and uncovers a previously unknown, Wnt-independent function of MIG-14/Wntless.

dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations.

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.

Probing the Fractal Pattern of Heartbeats in Drosophila Pupae by Visible Optical Recording System.

Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. We developed a non-invasive visible optical heart rate recording system especially suitable for long-term recording by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals (Mean ± SD: 1009.7 ± 151.6 ms) as compared to the control group (545.5 ± 45.4 ms, p < 0.001). The multiscale correlation of SERCA group (scaling exponent: 0.77 ± 0.07), on the other hand, is weaker than that of the control Drosophila (scaling exponent: 0.85 ± 0.03) (p = 0.016).

Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons.

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons.

BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development.

Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability.

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing.

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.

Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis.

Pruning is a widely observed mechanism for developing nervous systems to refine their circuitry. During metamorphosis, certain Drosophila sensory neurons undergo large-scale dendrite pruning to remove their larval branches before regeneration of their adult dendrites. Dendrite pruning involves dendrite severing, followed with debris removal. Little is known about the molecular mechanisms underlying dendrite severing. Here, we show that both the Ik2 kinase and Katanin p60-like 1 (Kat-60L1) of the Katanin family of microtubule severing proteins are required for dendrite severing. Mutant neurons with disrupted Ik2 function have diminished ability in severing their larval dendrites in pupae. Conversely, premature activation of Ik2 triggers precocious dendrite severing in larvae, revealing a critical role of Ik2 in initiating dendrite severing. We found a role for Kat-60L1 in facilitating dendrite severing by breaking microtubule in proximal dendrites, where the dendrites subsequently separate from the soma. Our study thus implicates Ik2 and Kat-60L1 in dendrite severing that involves local microtubule disassembly.

Integrins control the positioning and proliferation of follicle stem cells in the Drosophila ovary.

Adult stem cells are maintained in specialized microenvironments called niches, which promote self-renewal and prevent differentiation. In this study, we show that follicle stem cells (FSCs) in the Drosophila melanogaster ovary rely on cues that are distinct from those of other ovarian stem cells to establish and maintain their unique niche. We demonstrate that integrins anchor FSCs to the basal lamina, enabling FSCs to maintain their characteristic morphology and position. Integrin-mediated FSC anchoring is also essential for proper development of differentiating prefollicle cells that arise from asymmetrical FSC divisions. Our results support a model in which FSCs contribute to the formation and maintenance of their own niche by producing the integrin ligand, laminin A (LanA). Together, LanA and integrins control FSC proliferation rates, a role that is separable from their function in FSC anchoring. Importantly, LanA-integrin function is not required to maintain other ovarian stem cell populations, demonstrating that distinct pathways regulate niche-stem cell communication within the same organ.

Nuclear integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors during Drosophila visceral mesoderm induction.

Tissue induction during embryonic development relies to a significant degree on the integration of combinatorial regulatory inputs at the enhancer level of target genes. During mesodermal tissue induction in Drosophila, various combinations of inductive signals and mesoderm-intrinsic transcription factors cooperate to induce the progenitors of different types of muscle and heart precursors at precisely defined positions within the mesoderm layer. Dpp signals are required in cooperation with the mesoderm-specific NK homeodomain transcription factor Tinman (Tin) to induce all dorsal mesodermal tissue derivatives, which include dorsal somatic muscles, the dorsal vessel and visceral muscles of the midgut. Wingless (Wg) signals modulate the responses to Dpp/Tin along anteroposterior positions by cooperating with Dpp/Tin during dorsal vessel and somatic muscle induction while antagonizing Dpp/Tin during visceral mesoderm induction. As a result, dorsal muscle and cardiac progenitors form in a pattern that is reciprocal to that of visceral muscle precursors along the anteroposterior axis. Our present study addresses how positive Dpp signals and antagonistic Wg inputs are integrated at the enhancer level of bagpipe (bap), a NK homeobox gene that serves as an early regulator of visceral mesoderm development. We show that an evolutionarily conserved bap enhancer element requires combinatorial binding sites for Tin and Dpp-activated Smad proteins for its activity. Adjacent binding sites for the FoxG transcription factors encoded by the Sloppy paired genes (slp1 and slp2), which are direct targets of the Wg signaling cascade, serve to block the synergistic activity of Tin and activated Smads during bap induction. In addition, we show that binding sites for yet unknown repressors are essential to prevent the induction of the bap enhancer by Dpp in the dorsal ectoderm. Our data illustrate how the same signal combinations can have opposite effects on different targets in the same cells during tissue induction.

Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns.

Genetic approaches in Drosophila led to the identification of Forkhead, the prototype of forkhead domain transcription factors that are now known to comprise an evolutionarily conserved family of proteins with essential roles in development and differentiation. Sequence analysis of the recently published genomic scaffold sequence from Drosophila melanogaster has allowed us to determine the presumably full complement of forkhead domain encoding genes in this species. We show herein that the Drosophila genome contains 17 forkhead domain encoding genes; 13 of these genes have orthologs in chordate species, and their products can be assigned to 10 of the 17 forkhead domain subclasses known from chordates. One Drosophila forkhead domain gene only has a Caenorhabditis elegans ortholog and may represent a subclass that is absent in chordates, while the remaining three cannot be classified. We present the mRNA expression patterns of seven previously uncharacterized members of this gene family and show that they are expressed in tissues from all three germ layers, including central and peripheral nervous system, epidermis, salivary gland primordia, endoderm, somatic mesoderm, and hemocyte progenitors. Furthermore, the expression patterns of two of these genes, fd19B and fd102C, suggest a role for them as gap genes during early embryonic head segmentation.

Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers.

The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. Here we show that the Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as org-1, a Drosophila homologue of mammalian TBX1, in these cells.

The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp.

Dpp signals are responsible for establishing a variety of cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although we have a reasonably clear picture of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. In the present study, we describe three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, which are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. We show that these genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, we demonstrate that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, we show that the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend our knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals.