The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila.

PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.

Pro-apoptotic and pro-proliferation functions of the JNK pathway of Drosophila: roles in cell competition, tumorigenesis and regeneration.

The Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It appears to be conserved in all animal species where it regulates important physiological functions involved in apoptosis, cell migration, cell proliferation and regeneration. In this review, we focus on the functions of JNK in Drosophila imaginal discs, where it has been reported that it can induce both cell death and cell proliferation. We discuss this apparent paradox in the light of recent findings and propose that the pro-apoptotic and the pro-proliferative functions are intrinsic properties of JNK activity. Whether one function or another is predominant depends on the cellular context.

Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling.

Bone morphogenetic proteins (BMPs) are growth factors that provide essential signals for normal embryonic development and adult tissue homeostasis. A key step in initiating BMP signaling is ligand induced phosphorylation of receptor Smads (R-Smads) by type I receptor kinases, while linker phosphorylation of R-Smads has been shown to cause BMP signal termination. Here we present data demonstrating that the phosphatase Dullard is involved in dephosphorylating the Drosophila R-Smad, Mad, and is integral in controlling BMP signal duration. We show that a hypomorphic Dullard allele or Dullard knockdown leads to increased Mad phosphorylation levels, while Dullard overexpression resulted in reduced Mad phosphorylations. Co-immunoprecipitation binding assays demonstrate phosphorylated Mad and Dullard physically interact, while mutation of Dullard's phosphatase domain still allowed Mad-Dullard interactions but abolished its ability to regulate Mad phosphorylations. Finally, we demonstrate that linker and C-terminally phosphorylated Mad can be regulated by one of two terminating mechanisms, degradation by proteasomes or dephosphorylation by the phosphatase Dullard.

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.

N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development.

Ubiquitination and the reverse process deubiquitination regulate protein stability and function during animal development. The Drosophila USP5 homolog Leon functions as other family members of unconventional deubiquitinases, disassembling free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin, and maintaining cellular ubiquitin homeostasis. However, the significance of Leon/USP5 in animal development is still unexplored. In this study, we generated leon mutants to show that Leon is essential for animal viability and tissue integrity during development. Both free and substrate-conjugated polyubiquitin chains accumulate in leon mutants, suggesting that abnormal ubiquitin homeostasis caused tissue disorder and lethality in leon mutants. Further analysis of protein expression profiles in leon mutants shows that the levels of all proteasomal subunits were elevated. Also, proteasomal enzymatic activities were elevated in leon mutants. However, proteasomal degradation of ubiquitinated substrates was impaired. Thus, aberrant ubiquitin homeostasis in leon mutants disrupts normal proteasomal degradation, which is compensated by elevating the levels of proteasomal subunits and activities. Ultimately, the failure to fully compensate the dysfunctional proteasome in leon mutants leads to animal lethality and tissue disorder.

Detecting caspase activity in Drosophila larval imaginal discs.

Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.

Existence of prophenoloxidase in wing discs: a source of plasma prophenoloxidase in the silkworm, Bombyx mori.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster.

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster.

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function.