Signaling networks involved in patterning dorsal chorion structures in Drosophila

In Drosophila oogenesis, patterning of the follicle cells covering the developing oocyte is achieved by inductive signaling. Two major signaling pathways converge to induce a subpopulation of dorsal anterior follicle cells to adopt cell fates which give rise to operculum and dorsal appendages. One of the signals is initiated by the TGF-ß/BMP signaling pathway. Decapentaplegic (Dpp), one of the BMP like ligands in Drosophila, forms a morphogenetic gradient along the AP axis in the follicular epithelium and promotes operculum fate at highest level while moderate levels promote dorsal appendage fate. The second signal is provided by EGF/TGF-alpha like ligand Gurken (Grk) which is locally secreted by from the developing oocyte and forms a gradient along the dorsoventral axis. High concentrations of Grk induce the operculum fate, moderate concentrations the dorsal appendage fate. Clonal analysis shows that in absence of Dpp activity Grk cannot induce any of the dorsal cell fates in the follicular epithelium indicating that Dpp acts as a competence factor for Grk signaling. Moreover, Dpp also restricts the range of Grk signaling. The combined misexpression of Grk and Dpp leads to an expansion of dorsal fates along both the axes. A phenotype could be generated in which all main body follicle cells except those at the termini of the egg chamber were tansformed into operculum fate. The Dpp gradient and its action on target genes is modulated by several inhibitors which themselves are targets of the Dpp and EGF pathways. This results in a complex network of feedback control. Based on its intriguing expression pattern within the follicular epithelium I investigated the function of Drosophila snoN, a member of the Ski family proteins which are known as transcriptional co-factors of TGF-ß signaling in vertebrates. snoN mutant females lay eggs with enlarged operculum while misexpression of snoN in the whole follicular epithelium reduces the operculum size. Thus, SnoN acts as a transcriptional repressor of operculum fate genes. The intracellular Dpp inhibitors, brinker (brk) and daughters against dpp (dad) act together with snoN to regulate the Dpp readout in the follicular epithelium. Loss of function clones for brk, show that brk acts as a transcriptional repressor of operculum fate genes. Interestingly, loss of function clones of the extracellular Dpp inhibitor short gastrulation (sog) lead to a posterior expansion of the dorsal appendage fate indicating that in contrast to its role in the embryo Sog limits the diffusion of Dpp within the follicular epithelium. Like the Dpp pathway, the EGF pathway induced by Grk is modulated by several genes which themselves are targets of both pathways. In particular, the activation of rhomboid (rho) leads to a secondary amplification of the EGF signal which is thought to play an important role in follicle cell patterning. By performing clonal analysis for rho, we disprove this hypothesis and show that that a graded activity of Grk itself is sufficient to induce different dorsal fates and that the amplification is not essential for defining the midline fate

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-kappaB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFkappaB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes

Uba1 functions in Atg7- and Atg3-independent autophagy

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 used in ubiquitination, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy

Larval midgut destruction in Drosophila: Not dependent on caspases but suppressed by the loss of autophagy

Orientador: Demian CastroMonografia(Especializaçao) - Universidade Federal do Paraná,Setor de Ciencias Sociais Aplicadas, Curso de Especializaçao em Desenvolvimento Economic

Autophagy as a trigger for cell death : autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor of apoptosis is a novel mechanism of triggering cell deat

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field