Histological assessment of developmental cell death in Drosophila pupae

This protocol describes the embedding and processing of Drosophila pupae in paraffin to monitor tissue changes during development. Although multiple methods are available to evaluate developmental changes in Drosophila embryos, imaging detailed changes during metamorphosis is challenging as the animal is enclosed in the cuticle, rendering it inaccessible to whole mount imaging. Here, we present a protocol that focuses on developmental clearance of the larval salivary glands in Drosophila pupae that can be extended to examine other tissues/stages for similar purposes. For complete details on the use and execution of this protocol, please refer to Velentzas et al. (2018)

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch

Data of sperm-entry inability in Drosophila melanogaster ovarian follicles that are depleted of s36 chorionic protein

This paper presents data associated with the research article entitled “Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis” [1]. Drosophila chorion is produced by epithelial follicle cells and one of its functional serving role is egg fertilization through the micropyle, a specialized narrow channel at the anterior tip of the egg [2]. Sperm entry during fertilization is necessary for the egg to complete meiosis [3]. D. melanogaster flies being characterized by severe downregulation of the s36 chorionic protein, specifically in the follicle-cell compartment of their ovary, appear with impaired fly fertility (Velentzas et al., 2016) [1]. In an effort to further investigate whether the observed infertility in the s36-targeted flies derives from a fertilization failure, such as the inability of sperm to pass through egg׳s micropyle, we mated females carrying s36-depleted ovaries with males expressing the GFP protein either in their sperm tails, or in both their sperm tails and sperm heads

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy

Summary: Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-κB pathway regulates autophagy during developmentally programmed cell death. : Nandy et al. show that Drosophila peptidoglycan (PGRP) receptors and NF-κB factor Relish drive salivary gland degradation by controlling the expression of Atg1, a key component of the autophagy pathway. Keywords: Drosophila, NF-κB, cell death, autophag

The β2 and β6 20S proteasome core subunits play essential roles in fly eye development.

<p>Stereo-microscopical (a, e and i), Optical (d, h and l {semi-thin sections}) and Scanning Electron Microscopy (SEM) (b, c, f, g, j and k) images, demonstrating the critical contribution of β2 and β6 20S proteasome core subunits in fly eye morphogenesis. (a–d) GAL4-ninaE.GMR/UAS-Prosbeta21 double transgenic fly eyes, overexpressing the conditionally (temperature sensitive {ts}) mutant β2^ts proteasome core protein form. (e–h) GAL4-ninaE.GMR/UAS-Pros261 double transgenic fly eyes, characterized by upregulated mutant β6^ts expression levels. (i–l) GAL4-ninaE.GMR/UAS-Prosbeta21 - UAS-Pros261 triple transgenic flies, bearing increased eye-specific contents of mutant (defective) β2^ts and β6^ts proteasome subunit forms. Dashed circle: ommatidium. Arrow: lesion area(s). Scale Bars: 50 µm.</p

The structural integrity of 20S proteasome core particle is critically implicated in Drosophila eye morphogenesis.

<p>Stereo-microscopical (a, e and i), Optical (d, h and l {semi-thin sections}) and Scanning Electron Microscopy (SEM) (b, c, f, g, j and k) images, revealing the damaged architectural pattern of fly eye in 20S proteasome deficient environments (arrow). (a–d) GAL4-ninaE.GMR/UAS-alpha5_RNAi double transgenic fly eyes, carrying reduced α5 protein levels. (e–h) GAL4-ninaE.GMR/UAS-dbeta5_RNAi double transgenic fly eyes, characterized by downregulated β5 proteasome core protein expression levels. (i–l) GAL4-ninaE.GMR/UAS-beta6_RNAi double transgenic flies, producing eyes with decreased β6 cellular contents. Dashed circle: ommatidium. Scale Bars: 50 µm.</p

Downregulation of autophagy is associated with mild Drosophila wing lesions.

<p>Stereo-microscopical (a, c, e, g and i) and Optical (b, d, f, h and j) images, demonstrating that, attenuation of autophagy can induce only marginally pathological phenotypes of fly wing formation (a–j) (arrows). (a and b) P{GawB}Bx^MS1096/UAS-Atg1_RNAi double transgenic fly wings, characterized by reduced Atg1 protein contents. (c and d) P{GawB}Bx^MS1096/UAS-Atg4_RNAi double transgenic fly wings, produced in Atg4 deficient cellular environments. (e and f) P{GawB}Bx^MS1096/UAS-Atg7_RNAi double transgenic fly wings, carrying downregulated Atg7 expression levels. (g and h) P{GawB}Bx^MS1096/UAS-Atg9_RNAi double transgenic fly wings, developed under cellular conditions of attenuated Atg9 autophagic protein synthesis. (i and j) P{GawB}Bx^MS1096/UAS-Atg18_RNAi double transgenic fly wings, bearing decreased amount of Atg18 protein. Asterisks (b {Atg1}, d {Atg4}, f {Atg7} and j {Atg18}): double (heterozygote) transgenic fly populations, containing both wild-type and mutant (at a low frequency) wing phenotypes. Scale Bars: 200 µm.</p

Disintegration of 20S proteasome core particle leads to fly wing dysmoprhia.

<p>Stereo-microscopical (a, c, e, g, i and k) and Optical (b, d, f, h, j and l) images, illustrating the critical roles of α- and β-based rings of the 20S proteasome catalytic core in wing morphogenesis. (a and b) P{GawB}Bx^MS1096/UAS-alpha5_RNAi double transgenic fly wings, carrying reduced cellular contents of α5 proteasome subunit. (c and d) P{GawB}Bx^MS1096/UAS-dbeta5_RNAi double transgenic fly wings, characterized by downregulated β5 expression levels. (e and f) P{GawB}Bx^MS1096/UAS-beta6_RNAi double transgenic flies, producing wings with diminished amount of β6 20S proteasome protein. (g and h) P{GawB}Bx^MS1096/UAS-Prosbeta21 double transgenic fly wings, overexpressing the conditionally (temperature sensitive {ts}) mutant β2^ts proteasome subunit. (i and j) P{GawB}Bx^MS1096/UAS-Pros261 double transgenic fly wings, carrying increased levels of conditionally mutant β6^ts protein specifically in the wing. (k and l) P{GawB}Bx^MS1096/UAS-Prosbeta21 - UAS-Pros261 triple transgenic flies, bearing dysmorphic wings due to organ-specific overexpression of defective (conditionally mutant) β2^ts and β6^ts 20S proteasome components. Scale Bars: 200 µm.</p