In insects, juvenile hormone (JH) is a key hormone that regulates development, metamorphosis, and reproduction. Nevertheless, little is known about the molecular mechanisms of JH action. This project will study the JH signaling pathways in the fruit fly, Drosophila melanogaster, and the red flour beetle, Tribolium castaneum. A Drosophila genetic screen was developed and conducted in the PI's laboratory in order to isolate potential JH signaling components. This work identified four genes that encode negative regulators of the Wnt signaling pathway, a pathway important in the development of all animals. This project will determine the functional conservation of these four genes in Tribolium using RNAi. Methoprene-tolerant (MET) is considered to be a candidate for the JH receptor, but in Drosophila Met null mutants are completely viable and fertile. Research in the PI's laboratory has demonstrated that MET is functionally redundant with another protein, germ cell-expressed (GCE) and mosaic analysis of Met-gce double mutants will be employed to determine the function of JH signaling in different tissues. Overall this research project will identify more players in JH action, clarify the interaction of Wnt and JH signaling pathways, and evaluate the function of JH in different tissues. Because JH action is an important target for a group of insecticides, this project could have important implications for new insecticide design, insecticide resistance management, and environmental safety. Results of this project will also be integrated into the undergraduate and graduate courses. The proposed experiments offer educational and training opportunities for graduate, undergraduate and high school students from diverse backgrounds. They will be exposed to the process of scientific discovery through active learning in developmental biology coursework and hands-on experience in the principal investigator's laboratory. The broader impacts of this project also include international collaboration and international training program.
Insect metamorphosis is a fascinating process whereby a larva, specialized to feed and grow, is transformed into a sexually mature adult. Juvenile hormone (JH) coordinates with 20-hydroxyecdysone (20E) in regulating insect molting and metamorphosis. The molting process is orchestrated by 20E, whereas the nature of the molt is determined by JH during critical JH-sensitive periods. Studies on gene expression during Drosophila metamorphosis have led to a detailed molecular understanding of the genetic regulatory hierarchies that underlie the 20E response. However, molecular action of JH remains an enigma. In this project, we designed and conducted a forward genetic screen to identify genes involved in JH biosynthesis and signaling pathway and performed reverse genetic studies to analyze the functional redundancy of Methoprene-tolerant (Met) and Germ-cell expressed (Gce) in transducing JH action. Functional analysis was also conducted for kr-h1 promoter to isolate JH response element. From 4,400 lethal lines, we isolated 55 mutations that cause JH-deficiency phenotypes. The genes associated with these mutations were found to encode proteins with various molecular functions, including enzymes, signal transduction molecules, and transcription factors. Some of the identified genes are known to be involved in JH biosynthesis or signaling, such as fpps and Kr-h1. Others are linked to JH action at the first stage. Among these genes, there are three Wnt signaling components, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), and two TGF-β signaling components, thick vein (tkv) and mothers against Dpp (mad). Our genetic screen also demonstrated that the Tkv and Mad mediated TGF-β signaling stimulates JH biosynthesis by upregulating the expression of JH acid methyltransferase (jhamt), a key regulatory enzyme in the JH biosynthesis pathway. On the other hand, Wnt signaling cross-talks with JH signaling by suppressing the transcription of Met and gce, genes that encode for putative JH receptors. We have generated a double mutation line that carries null mutant alleles for both the Met and gce genes. Both Met and gce null single mutants are fully viable, but the Met gce double mutant dies during the larva-pupa transition. The defective phenotypes and gene expression changes observed in the Met gce double mutants are similar to those found in JH-deficient flies. The difference is that exogenous application of JH analogs has the ability to rescue JH-deficient animals but not Met gce mutants. Our data suggest that Drosophila Met and Gce redundantly transduce JH action. Kr-h1 is a JH primary response gene. We dissected the regulatory regions of the Kr-h1 gene and identified a 25-bp DNA fragment upstream of the Kr-h1 promoter region that is required for JH-induced Kr-h1 expression. This project has significantly enhanced the PI’s consistent efforts to promote teaching and provided training opportunities for students at different levels, including one postdoctoral fellow, three graduate students, five undergraduate students, one high school student, and two high school teachers of underrepresented group. It also dramatically facilitates PI’s ongoing international collaboration with the scientists at the Shanghai Institute of Plant Physiology and Ecology, China. The results collected from this project were published in the high profile peer-reviewed journals, such as Development and Insect Biochemistry and Molecular Biology, and presented in the international conferences, such as the annual conference of the Entomological Association Society and the Drosophila Research Conference. The fly strains generated in this project have been freely shared with more than ten labs throughout the world.
