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The prototypical M13 peptidase, human Neprilysin, functions as a transmembrane “ectoenzyme” that cleaves neuropeptides that regulate e.g. glucose metabolism, and has been linked to type 2 diabetes. The M13 family has undergone a remarkable, and conserved, expansion in the Drosophila genus. Here, we describe the function of Drosophila melanogaster Neprilysin-like 15 (Nepl15). Nepl15 is likely to be a secreted protein, rather than a transmembrane protein. Nepl15 has changes in critical catalytic residues that are conserved across the Drosophila genus and likely renders the Nepl15 protein catalytically inactive. Nevertheless, a knockout of the Nepl15 gene reveals a reduction in triglyceride and glycogen storage, with the effects likely occurring during the larval feeding period. Conversely, flies overexpressing Nepl15 store more triglycerides and glycogen. Protein modeling suggests that Nepl15 is able to bind and sequester peptide targets of catalytically active Drosophila M13 family members, peptides that are conserved in humans and Drosophila , potentially providing a novel mechanism for regulating the activity of neuropeptides in the context of lipid and carbohydrate homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A 2 (iPLA 2 ) VIA gene CG6718 , the Drosophila melanogaster ortholog of human PLA2G6/PARK14 , mutations in which cause a suite of NDs collectively called PLA2G6 -associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA 2 -VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA 2 -VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA 2 -VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA 2 -VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA 2 -VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

Abstract Since the dawn of the 20th century, the fruit fly Drosophila melanogaster has been used as a model organism to understand the nature of genes and how they control development, behavior, and physiology. One of the most powerful experimental approaches employed in Drosophila is the forward genetic screen. In the 21st century, genome-wide screens have become popular tools for identifying evolutionarily conserved genes involved in complex human diseases. In the accompanying article “Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the  Phospholipase D   Pathway as a Potential Therapeutic Target,” Kankel and colleagues describe a forward genetic modifier screen to discover factors that contribute to the severe neurodegenerative disease amyotrophic lateral sclerosis (ALS). This primer briefly traces the history of genetic screens in Drosophila and introduces students to ALS. We then provide a set of guided reading questions to help students work through the data presented in the research article. Finally, several ideas for literature-based research projects are offered as opportunities for students to expand their appreciation of the potential scope of genetic screens. The primer is intended to help students and instructors thoroughly examine a current study that uses forward genetics in Drosophila to identify human disease genes.

Background and Objectives: The Drosophila retinal determination network comprises the transcription factor Teashirt (Tsh) and the transcription co-regulator C-terminal Binding Protein (CtBP), both of which are essential for normal adult eye development. Both Tsh and CtBP show a pattern of co-expression in the proliferating cells anterior to the morphogenetic furrow that demarcates the boundary between the anteriorly placed proliferating eye precursor cells and the posteriorly placed differentiating photoreceptor cells in the larval eye-precursor tissue, the eye–antennal disc. The disc ultimately develops into the adult compound eyes, antenna, and other head structures. Both Tsh and CtBP were found to interact genetically during ectopic eye formation in Drosophila, and both were present in molecular complexes purified from gut and cultured cells. However, it remained unknown whether Tsh and CtBP molecules could interact in the eye–antennal discs and elicit an effect on eye development. The present study answers these questions. Methods: 5′ GFP-tagging of the tsh gene in the Drosophila genome and 5′ FLAG-tagging of the ctbp gene were accomplished by the CRISPR-Cas9 and BAC recombineering methods, respectively, to produce GFP-Tsh- and FLAG-CtBP-fused proteins in specific transgenic Drosophila strains. Verification of these proteins’ expression in the larval eye–antennal discs was performed by immunohistological staining and confocal microscopy. Genetic screening was performed to establish functional interaction between Tsh and CtBP during eye development. Scanning Electron Microscopy was performed to image the adult eye structure. Co-immunoprecipitation and GST pulldown assays were performed to show that Tsh and CtBP interact in the cells of the third instar eye–antennal discs. Results: This study reveals that Tsh and CtBP interact genetically and physically in the Drosophila third instar larval eye–antennal disc to regulate adult eye development. This interaction is likely to limit the population of the eye precursor cells in the larval eye disc of Drosophila. Conclusions: The relative abundance of Tsh and CtBP in the third instar larval eye–antennal disc can dictate the outcome of their interaction on the Drosophila eye formation.

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A.sub.2 (iPLA.sub.2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA.sub.2 -VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA.sub.2 -VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA.sub.2 -VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA.sub.2 -VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA.sub.2 -VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

Since the dawn of the 20th century, the fruit fly Drosophila melanogaster has been used as a model organism to understand the nature of genes and how they control development, behavior, and physiology. One of the most powerful experimental approaches employed in Drosophila is the forward genetic screen. In the 21st century, genome-wide screens have become popular tools for identifying evolutionarily conserved genes involved in complex human diseases. In the accompanying article \"Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target,\" Kankel and colleagues describe a forward genetic modifier screen to discover factors that contribute to the severe neurodegenerative disease amyotrophic lateral sclerosis (ALS). This primer briefly traces the history of genetic screens in Drosophila and introduces students to ALS. We then provide a set of guided reading questions to help students work through the data presented in the research article. Finally, several ideas for literature-based research projects are offered as opportunities for students to expand their appreciation of the potential scope of genetic screens. The primer is intended to help students and instructors thoroughly examine a current study that uses forward genetics in Drosophila to identify human disease genes.

The Drosophila melanogaster Neprilysin like 15 (Nepl15) gene encodes a member of the M13 family of metalloendopeptidases, some of which have been linked to diabetes. My data reveals that Nepl15 transcript levels are significantly higher in larval fat body and in the adult male abdomen compared to the female abdomen. Adult age-matched flies in which the Nepl15 gene has been knocked out (w1118; Nepl15ko) feed normally relative to controls. As compared to the controls, adult w1118; Nepl15ko males have reduced glycerolipids and glycogen, but w1118; Nepl15ko females show elevated glycogen levels and no change in glycerolipids. However, both w1118; Nepl15ko adult male and female flies die sooner when starved and they both have a shortened lifespan when cultured on regular food compared to controls. Consistent with the reduced nutrient levels in w1118; Nepl15ko, adult male flies overexpressing Nepl15 in all tissues have elevated glycerolipids and glycogen compared to controls. Altogether, my results suggest that Nepl15 is necessary for both lipid and carbohydrate storage and thereby impacts longevity. Future studies using fruit fly M13 family members may help elucidate the mechanisms by which the activity of human Neprilysins are related with metabolic diseases.The D. melanogaster eye is a good model system to study development because most underlying processes are conserved in humans. The tissue containing the developing eye comprises both proliferating precursors and differentiating eye cells. The transition between proliferation and differentiation involves changes in gene expression. Regulation of chromatin structures is one of the mechanisms to control transcription of target genes. The D. melanogaster sequence-specific transcription factor Eyeless (Ey) (human ortholog of Pax6) is an eye specification factor. Studies from other groups suggest that Ey interacts with the sequence-specific transcription factor Teashirt (Tsh) to promote proliferation of eye precursors. Conversely, the sequence specific transcription factor Dachshund (Dac) promotes differentiation. D. melanogaster eye development is also controlled by C-terminal Binding Protein (CtBP). It regulates gene transcription by binding with sequence specific transcription factors and recruiting chromatin remodeling molecules. Previous studies from our lab revealed that the Ey and Dac proteins and the Ey and CtBP proteins are part of a complex or complexes in eye precursors. Genetic evidence showed that Ey and Dac interact with CtBP. However, there was no evidence of a direct biochemical interaction between Ey and CtBP. Therefore, we hypothesized that Dac and Tsh link Ey with CtBP in proliferating precursors and differentiating eye cells, respectively. In this work, I reveal that the CtBP and Dac, and the CtBP and Teashirt (Tsh) proteins can bind directly in vitro. Moreover, Tsh and CtBP interact in vivo in eye precursors. In future, we want to determine the mechanisms by which these protein complexes regulate the transition from proliferation to differentiation during eye development.

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is important for germline mitochondrial integrity in Drosophila, which may be relevant for understanding how PLAN develops. Competing Interest Statement The authors have declared no competing interest.

Drought is one of the biggest concerns in agriculture due to the projected reduction of global freshwater supply with a concurrent increase in global food demand. Roots can significantly contribute to improving drought adaptation and productivity. Plants increase water uptake by adjusting root architecture and cooperating with symbiotic soil microbes. Thus, emphasis has been given to root architectural responses and root–microbe relationships in drought-resilient crop development. However, root responses to drought adaptation are continuous and complex processes and involve additional root traits and interactions among themselves. This review comprehensively compiles and discusses several of these root traits such as structural, physiological, molecular, hydraulic, anatomical, and plasticity, which are important to consider together, with architectural changes, when developing drought resilient crop varieties. In addition, it describes the significance of root contribution in improving soil structure and water holding capacity and its implication on long-term resilience to drought. In addition, various drought adaptive root ideotypes of monocot and dicot crops are compared and proposed for given agroclimatic conditions. Overall, this review provides a broader perspective of understanding root structural, physiological, and molecular regulators, and describes the considerations for simultaneously integrating multiple traits for drought tolerance and crop improvement, under specific growing environments.