ABSTRACT
The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Ovary , Reactive Oxygen Species , Superoxide Dismutase , Animals , Drosophila melanogaster/genetics , Female , Superoxide Dismutase/metabolism , Superoxide Dismutase/genetics , Reactive Oxygen Species/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Ovary/metabolism , NADPH Oxidases/metabolism , NADPH Oxidases/genetics , Reproduction , NADPH Oxidase 5/metabolism , NADPH Oxidase 5/genetics , Oviposition , Chorion/metabolismABSTRACT
The 57B region of Drosophila melanogaster includes a cluster of the three homeobox genes orthopedia (otp), Drosophila Retinal homeobox (DRx), and homeobrain (hbn). In an attempt to isolate mutants for these genes, we performed an EMS mutagenesis and isolated lethal mutants from the 57B region, among them mutants for otp, DRx, and hbn. With the help of two newly generated deletions from the 57B region, we mapped additional mutants to specific chromosomal intervals and identified several of these mutants from the 57B region molecularly. In addition, we generated mutants for CG15651 and RIC-3 by gene targeting and mutants for the genes CG9344, CG15649, CG15650, and ND-B14.7 using the CRISPR/Cas9 system. We determined the lethality period during development for most isolated mutants. In total, we analysed alleles from nine different genes from the 57B region of Drosophila, which could now be used to further explore the functions of the corresponding genes in the future.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila/genetics , Drosophila Proteins/genetics , DNA-Binding Proteins/genetics , Genes, Homeobox , ras Proteins/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolismABSTRACT
Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.
Subject(s)
Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Trans-Activators/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Phosphorylation/genetics , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Trans-Activators/genetics , YAP-Signaling ProteinsABSTRACT
The Drosophila homeodomain-interacting protein kinase (Hipk) is the fly representative of the well-conserved group of HIPKs in vertebrates. It was initially found through its characteristic interactions with homeodomain proteins. Hipk is involved in a variety of important developmental processes, such as the development of the eye or the nervous system. In the present study, we set Hipk and the Drosophila homeodomain proteins Homeobrain (Hbn), Empty spiracles (Ems), and Muscle segment homeobox (Msh) in an enzyme-substrate relationship. These homeoproteins are transcription factors that function during Drosophila neurogenesis and are, at least in part, conserved in vertebrates. We reveal a physical interaction between Hipk and the three homeodomain proteins in vivo using bimolecular fluorescence complementation (BiFC). In the course of in vitro phosphorylation analysis and subsequent mutational analysis we mapped several Hipk phosphorylation sites of Hbn, Ems, and Msh. The phosphorylation of Hbn, Ems, and Msh may provide further insight into the function of Hipk during development of the Drosophila nervous system.
