Divalent metal content in diet affects severity of manganese toxicity in Drosophila.

Dysregulation of manganese (Mn) homeostasis is a contributing factor in many neuro-degenerative diseases. Adult Drosophila are sensitive to excessive levels of dietary Mn, dying relatively early, and exhibiting biochemical and mobility changes reminiscent of Parkinsonian conditions. To further study Mn homeostasis in Drosophila, we sought to test lower levels of dietary Mn (5 mM) and noted a striking difference in Canton-S adult survivorship on different food. On a cornmeal diet, Mn-treated flies live only about half as long as untreated siblings. Yet, with the same Mn concentration in a molasses diet, adults survive about 80% as long as untreated siblings, and adults raised on a sucrose-yeast diet are completely insensitive to this low dose of dietary Mn. By manipulating metal ion content in the cornmeal diet, and measuring the metal content in each diet, we traced the difference in lifespan to the levels of calcium and magnesium in the food, suggesting that these ions are involved in Mn uptake and/or use. Based on these findings, it is recommended that the total dietary load of metal ions be considered when assessing Mn toxicity.

Long disordered regions of the C-terminal domain of Abelson tyrosine kinase have specific and additive functions in regulation and axon localization.

Abelson tyrosine kinase (Abl) is a key regulator of actin-related morphogenetic processes including axon guidance, where it functions downstream of several guidance receptors. While the long C-terminal domain (CTD) of Abl is required for function, its role is poorly understood. Here, a battery of mutants of Drosophila Abl was created that systematically deleted large segments of the CTD from Abl or added them back to the N-terminus alone. The functionality of these Abl transgenes was assessed through rescue of axon guidance defects and adult lethality in Abl loss-of-function, as well as through gain-of-function effects in sensitized slit or frazzled backgrounds that perturb midline guidance in the Drosophila embryonic nerve cord. Two regions of the CTD play important and distinct roles, but additive effects for other regions were also detected. The first quarter of the CTD, including a conserved PxxP motif and its surrounding sequence, regulates Abl function while the third quarter localizes Abl to axons. These regions feature long stretches of intrinsically disordered sequence typically found in hub proteins and are associated with diverse protein-protein interactions. Thus, the CTD of Abl appears to use these disordered regions to establish a variety of different signaling complexes required during formation of axon tracts.

Gia/Mthl5 is an aorta specific GPCR required for Drosophila heart tube morphology and normal pericardial cell positioning.

G-protein signaling is known to be required for cell-cell contacts during the development of the Drosophila dorsal vessel. However, the identity of the G protein-coupled receptor (GPCR) that regulates this signaling pathway activity is unknown. Here we describe the identification of a novel cardiac specific GPCR, called Gia, for "GPCR in aorta". Gia is the only heart-specific GPCR identified in Drosophila to date and it is specifically expressed in cardioblasts that fuse at the dorsal midline to become the aorta. Gia is the only Drosophila gene so far identified for which expression is entirely restricted to cells of the aorta. Deletion of Gia led to a broken-hearted phenotype, characterized by pericardial cells dissociated from cardioblasts and abnormal distribution of cell junction proteins. Both phenotypes were similar to those observed in mutants of the heterotrimeric cardiac G proteins. Lack of Gia also led to defects in the alignment and fusion of cardioblasts in the aorta. Gia forms a protein complex with G-αo47A, the alpha subunit of the heterotrimeric cardiac G proteins and interacts genetically with G-αo47A during cardiac morphogenesis. Our study identified Gia as an essential aorta-specific GPCR that functions upstream of cardiac heterotrimeric G proteins and is required for morphological integrity of the aorta during heart tube formation. These studies lead to a redefinition of the bro phenotype, to encompass morphological integrity of the heart tube as well as cardioblast-pericardial cell spatial interactions.

Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution.

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family. Using in situ hybridization techniques, we detect expression of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large "superclade" that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl "superclade" paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family.

In the absence of frazzled over-expression of Abelson tyrosine kinase disrupts commissure formation and causes axons to leave the embryonic CNS.

BACKGROUND: In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. METHODOLOGY/PRINCIPAL FINDINGS: The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. CONCLUSION/SIGNIFICANCE: Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.

Life span extension and neuronal cell protection by Drosophila nicotinamidase.

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

Frazzled cytoplasmic P-motifs are differentially required for axon pathway formation in the Drosophila embryonic CNS.

Frazzled is a Netrin-dependent chemoattractive receptor required for axon pathway formation in the developing Drosophila embryonic CNS. The cytoplasmic domain is important and contains three conserved P-motifs (P1, P2, and P3) thought to initiate intracellular signaling cascades and to crosstalk with other receptors during axon pathway formation. Here, we rescue homozygous frazzled embryos by pan-neurally expressing a series of mutants lacking either the cytoplasmic domain or one of the conserved P-motifs and assess the ability of these mutants to rescue frazzled defects in commissural, longitudinal and motor axon pathways. Surprisingly, while the cytoplasmic domain is required, removal of an individual P-motif does not prevent gross formation of commissures. However, removal of P3 from Fra does prevent eagle-expressing commissural axons from crossing the midline in the posterior commissure suggesting that some neurons have a stronger requirement for P3-dependent signaling. Indeed, axons within the longitudinal connective as well as a small subset of motor neurons within the ISNb pathway also specifically require P3 to project to their targets correctly. In these latter axon projections, deleting the P1-motif appears to de-regulate the receptor's activity, actually increasing the frequency of motor neuron projection errors and inducing ectopic midline crossing errors. Collectively, these data demonstrate the critical nature of both the P1 and the P3-motifs to Frazzled function in vivo during axon pathway formation.

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS.

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

Frazzled regulation of myosin II activity in the Drosophila embryonic CNS.

Frazzled (Fra) is a chemoattractive guidance receptor regulating the cytoskeletal dynamics underlying growth cone steering at the Drosophila embryonic midline. Here, by genetically evaluating the role of Rho GTPases in Fra signaling in vivo, we uncover a Rho-dependent pathway apparently regulating conventional myosin II activity. Midline crossing errors induced by expressing activated Cdc42(v12) or Rac(v12) are suppressed by a heterozygous loss of fra(4) signaling but, in a Fra(wt) gain-of-function condition, no interaction is detected. In contrast, the frequency of crossovers is enhanced approximately 5-fold when Fra(wt) is co-expressed with activated Rho(v14) and this interaction specifically requires the cytoplasmic P3 motif of Fra. Expression of Rho(v14) and activated MLCK (ctMLCK) synergistically increase ectopic crossovers and both require phosphorylation of the regulatory light chain (Sqh) of myosin II. Abelson tyrosine kinase may also help regulate myosin II activity. Heterozygous abl(4) abolishes the midline crossing errors induced by ctMLCK alone or in combination with Fra(wt); suppression of Rho(v14) crossovers is not observed. Interestingly, an interaction between Fra and an activated Abl (Bcr-Abl) also specifically requires the P3 motif. Therefore, the P3 motif of Frazzled appears to initiate Rho and Abl dependent signals to directly or indirectly regulate myosin II activity in growth cones.

Abelson tyrosine kinase is required to transduce midline repulsive cues.

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. Here, a genetic approach is used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. Our data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but we suggest that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues.

Regulation of rho family GTPases is required to prevent axons from crossing the midline.

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. Our data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho.

Activation of the repulsive receptor Roundabout inhibits N-cadherin-mediated cell adhesion.

The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of beta-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos.

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.