Lynn M. Riddiford, PhD - US grants

Our long-term goal is to understand how ecdysteroids and juvenile hormone (JH) act to coordinate insect growth and development. Ecdysteroids cause molting, both suppressing on-going intermolt gene expression and activating the cascade of genes necessary for the molt. Moreover, ecdysteroids are important for maintenance of tissue synchrony during the molt. JH determines whether the molt will be progressive or not; in its presence metamorphosis cannot occur. Thus, JH prevents ecdysteroid's activation of previously unexpressed genes that allow the cell to change its differentiated state. Our model for studying this action is the insect epidermis which makes the cuticle, i.e. the exoskeleton, and which is a single cell layer thick and can readily be cultured. Our specific aims for the next 5 years are: 1) To characterize the regulatory regions of the several larval cuticular genes and dopa decarboxylase and to study their possible interactions with ecdysteroid and JH receptors and/or the transcription factors that these hormones induce. 2) To clone, characterize, and study the hormonal control of a pupal-specific cuticle gene to complete our arsenal of epidermal genes that are hormonally regulated at metamorphosis. 3) To determine the role in molting and metamorphosis of the ecdysteroid-induced transcription factor ("RAR") that shows significant similarity to the mammalian retinoic acid receptor. 4) To examine possible changes in EcR subtypes in the epidermis through larval molting and metamorphosis by in situ hybridization and immunocytochemistry and how they are regulated by the hormonal milieu. These studies will allow us to define at the molecular level how ecdysteroids and JH regulate insect growth, molting, and metamorphosis. This information can lead to design of new types of insect growth regulators essential to the control of disease vectors. Also, emerging similarities between the actions of JH and of retinoic acid in the regulation of cell differentiation, particularly in mammalian epidermis, make the insect epidermis a possible nonvertebrate model for study of common mechanisms involved.

This project will examine the molecular structure and the regulation of synthesis of the neuropeptide, eclosion hormone (EH), in the tobacco hornworm, Manduca sexta. EH is synthesized both in the brain and in the ventral CNS and released before each molt to elicit a preprogrammed pattern of neural activity culminating in ecdysis. The 62 amino acid peptide has recently been sequenced and we intend to isolate the EH gene using synthetic oligonucleotide probes designed based on the protein sequence. The isolation of the EH gene will offer the opportunity to study the synthesis and action of a neuropeptide in the relatively simple nervous system of an insect with it's identifiable neurons. We will examine the structure of the EH gene by a combination of nucleic acid sequencing and transcript mapping studies and will identify other potential peptides derived from the prohormone. We will determine the developmental regulation of EH mRNA synthesis and will analyze the endocrine factors that regulate its expression. The cellular localization of the EH transcript will be determined by in situ hybridization both in the brain and in the cells associated with the ventral CNS, the two sources of EH. Finally, the presence of EH-like sequences in other insects will be examined by hybridization to the EH gene.

The proposed research is a continuation of an interdisciplinary collaboration on the structure, dynamics, and physiology of proteins which bind and degrade juvenile hormone (JH) and JH mimics (JHM). The original goals of this collaborative research were to develop chemical and biochemical techniques to approach the molecular biology of juvenile hormone (JH) reception and catabolism. These initial objectives have been acheived, and in this renewal request, further goals are proposed: First, to study the structure of the JH and JHM binding proteins (JHBP an RP) in Manduca larval epidermal cell cytosol and nuclei, and of JH esterases (JHE) and epoxide hydrolases (JHEH) through (a) synthesis of radioligands, (b) JHBP/RP and JHE/EH purification and sequencing, (c) active site labeling and sequencing, (d) antibody production, (e) gene isolation and sequencing, and (f) overproduction of proteins; second, to examine the dynamics of the ligands and macromolecules, including (a) timing and location of JHBP/RP, JHE/EH, (b) movement of hormones and proteins between cell compartments, and (c) protein-ligand, protein-protein, and protein-DNA interactions. A key to the structure and dynamics goals will be the development of a baculovirus expression system in Bombyx for protein overproduction. The third goal is to correlate structure and dynamics with physiology by characterizing, (a) the appearance of JHBP/RP and JHE/EH in the life cycle, and (b) comparative biochemistry of JHBP/RP and JHE/EH in insects, with respect to ligand recognition and protein similarities. The characterization of receptor proteins for juvenile hormone (JH) and JH mimics (JHM) and knowledge of the pathways of their degradation are major keys to understanding the molecular basis for hormonal regulation of gene expression in insects. This research is an interdisciplinary collaboration which combines expertise in molecular biology and physiological manifestations of gene expression in insects (L. M. Riddiford), biochemical and molecular studies of JH metabolism (B. D. Hammock), and synthesis and use of high specific activity labeled JHs and JHMs (G. D. Prestwich). This proposal was submitted under the NSF initiative in the Chemistry of Life Processes.

The proposed research, jointly supported by the Division of Cellular Biosciences and the Division of Chemistry, is the continuation of an interdisciplinary project to understand the action of juvenile hormone at the molecular level combining the disciplines of chemistry, molecular biology, and physiology. Major keys include understanding the molecular basis for hormonal regulation of gene expression in insects (L.M. Riddiford), biochemical and molecular studies of JH metabolism and synthesis (B.D. Hammock), and use of high specific activity labeled JHs and JHs (G.D. Prestwich). Three benefits result from this project. First, the scientific work is directed at a fundamental problem in regulation of gene expression, and the metamorphosis of insects in an easily accessible and fascinating model system for this study. Second, the practical implications of this work may lead to the development of new biologically specific and environmentally safer insect control strategies for crop pests. Third, the interdisciplinary training required for the students, postdoctoral fellow and faculty researchers involved enhances the quality and breadth of scientific inquiry in each laboratory.

Lynn Riddiford #IBN 9419957 Proposed are studies to elucidate the role of the nuclear 29kDa protein that specifically binds juvenile hormone (jp29) in the regulation of larval molting and metamorphosis. Studies on the tobacco hornworm, Manduca sexta, will concentrate on three aspects: 1) isolation of other nuclear protein(s) with which jp29 may interact and their subsequent cloning and sequencing to elucidate their function, 2) immunocytochemcial localization within the nucleus, and 3) developmental and endocrine regulation of jp29. We also plan to isolate the homologous gene from Drosophila containing the Manduca jp29 cDNA under the control of a heat shock promoter will be used to determine the effects of over-expression of this protein on development, reproduction, and JH esterase production in the presence and absence of JH. Also, immunocytochemical studies will concentrate on its developmental and endocrine regulation in various tissues. This study in the two insects should help elucidate the molecular mode of action of JH in directing metamorphosis and whether jp29 has a functional role in adult responses to JH. ***

9514187 Riddiford Ecdysteroids cause insect molting and metamorphosis, and juvenile hormone is present during larval life to allow growth and molting and to prevent metamorphosis. Interference with the action of these hormones is one way to control insect pests. Dr. Riddiford has found that there is a dynamic and complex pattern of changing ecdysteroid receptor isoforms and its partner USP during a molt and at metamorphosis in the epidermis of the tobacco hornworm, Manduca sexta. She now plans to study both the hormonal and the spatiotemporal regulation of these receptors and the initial transcription factors that they induce at the cellular level using primarily immunocytochemical and in situ hybridization analysis of tissue exposed to these hormones both in vivo and in vitro. These studies will provide deeper insight into how the ecdysteroids initiate and coordinate the cellular responses that lead to the formation of a new cuticle during a molt and during the switch in cell fate that is essential for metamorphosis. By studying the influence of juvenile hormone on these cellular dynamics, she will shed new light on its mode of action in the prevention of the switch of cell fates. ***

The project examines the role of juvenile hormone (JH) in the evolution of
metamorphosis and of the larval stage in insects. A key finding is that
in embryos of basal insect groups, JH suppresses the growth of embryonic
primordia and induces their precocious maturation. In insects with
complete metamorphosis, by contrast, JH treatment has little effect on
embryonic development but later application can suppress the resumption of
"embryonic" growth that is the prelude to metamorphosis. An advancement
in JH secretion during embryogenesis of the latter insects may have
provided a mechanism by which a late embryonic instar, the pronymph, was
modified to become an active, feeding larval stage characteristic of
insects with complete metamorphosis.
The proposal examines the effects of JH on the expression of three genes
involved in proximal-distal (P-D) patterning of the leg: Distal-less (Dll),
dachshund (dac), and rotund (rn). The objectives are: 1) to determine if
the spatial and temporal expression of P-D patterning genes seen in
developing leg imaginal discs in Drosophila also occurs during the
patterning of the embryonic leg in a more basal insect such as the
grasshoppers, Schistocerca americana; 2) test whether JH treatment to
these embryos during limb formation results in a truncation or alteration
in P-D gene expression? 3) examine P-D gene expression in embryos of the
moth, Manduca sexta, to determine if the novel leg of the caterpiller is
associated with a novel (truncated?) program of P-D gene expression; 4)
examine the transition in P-D gene expression during metamorphosis and its
sensitivity to juvenile hormone treatment.

Requested is partial support for a new Gordon Conference on Metamorphosis to be held July 10-15 1999 at Connecticut College, New London, CT. Its purpose is to bring together people working on metamorphosis in insects and amphibians and a variety of other organisms to discuss the common cellular and molecular events that occur in metamorphosis and the questions that it poses about coordination and control systems in development. The 8 scientific sessions will deal with hormonal regulation of metamorphosis at both cellular and organismal levels, mechanisms of gene activation and repression, intracellular receptors and gene regulation, transcription factor cascades in metamorphosis, the evolution of metamorphosis, cellular specification and differentiation during insect metamorphosis, cellular alterations during amphibian metamorphosis, and cell death during metamorphosis. One keynote speaker, 27 session speakers, and 8 discussion leaders are invited. The remainder of the 135 participants will be selected from among applicants to the Conference and will be encouraged to present posters on their work. Eight poster presenters will be selected to give 15 minute talks in the sessions. Partial support for travel and conference fees is requested for the invited speakers and discussion leaders and for 10 selected graduate student and postdoctoral researchers from among the conference applicants.

The long-term goal of the parent grant is to understand how ecdysteroids and juvenile hormone (JH) act at the cellular and molecular level to coordinate insect molting and metamorphosis using the polymorphic epidermis of the tobacco hornworm, Manduca sexta, as a model system. We have characterized some of the major members of the ecdysteroid-induced transcription factor cascade that is activated during the molt including a switch in isoforms of the ecdysone receptor (EcR) and its partner USP (a RXR homolog), and how they are regulated by 20-hydroxyecdysone and JH. Our main specific aim in this FIRCA proposal is to determine whether the new technique of double stranded RNA interference will work in Lepidoptera to provide us with a means of studying the effects of loss-of-function of these various factors to ascertain their specific roles in molting and metamorphosis. We will also determine the role of the natural antisense RNA encoding a putative cuticle gene found in the 3' untranslated region of the ecdysone receptor gene. If the RNAi technique does not work, we will begin initial explorations of the use of transgenic Lepidoptera mediated by the piggyBac vector for ectopic expression of candidate genes or dominant negative forms at critical times in development. These studies should provide insight into the key roles of the switching of nuclear receptor isoforms during a well-defined developmental process and into the importance of the action of JH in regulating both the types and quantity of transcription factors induced by ecdysteroids for the prevention of metamorphosis. Since the effects of JH on metamorphosis in Lepidoptera is typical of most insects, the information gained also can provide a basis for the rational design of new insect growth regulators to control insect vectors of human disease and avoid possible side effects of these regulators or their environmental degradation products interfering with the endocrine and developmental roles of the RXR in vertebrates.

In insects ecdysone causes molting and metamorphosis and juvenile hormone (JH) allows molting but prevents the metamorphic action of ecdysone. Ongoing immunocytochemical studies of how JH modulates the ecdysone-induced cascade of transcription factors in the tobacco hornworm, Manduca sexta, pupal wing at the outset of adult development will be completed. The technique of RNA interference to eliminate specific mRNAs in this pathway will be used to determine the roles of key transcription factors of this cascade in pattern formation and cuticle deposition. This same transcription factor cascade will be analyzed immunocytochemically during pupal commitment of the wing imaginal disc and of the eye primordium during the molt to the final larval stage and at the initiation of pupal development. The role of JH in the maintenance of the larval state of these two adult precursors will be assessed by experimental manipulations, both in the animal and in tissue culture
Moling, an epidermal protein that appears only in the last larval stage of Manduca, belongs to the Takeout family of proteins that includes some JH-binding proteins. Recombinant Moling will be produced by a baculovirus system, then used to test whether it binds JH and, if so, the binding will be characterized. Also, the pattern of its disappearance in the epidermis relative to the time of pupal commitment and the appearance of Broad expression will be studied immunocytochemically. Putative homologs of Moling in Drosophila that are regulated by JH will be analyzed for their presence and possible role in larval epidermal development and metamorphosis.
The role of USP as a possible JH receptor in Drosophila is being studied in collaboration with Dr. Grace Jones. Bioassays of JH in the prevention of abdominal metamorphosis will be done with animals bearing mutated forms of USP hypothesized to have little or no JH binding activity.

[unreadable] DESCRIPTION (provided by applicant): Proposed is a continuing study of the hormonal regulation of growth, molting and metamorphosis in Drosophila melanogaster, concentrating on the key role of the endocrine gland (prothoracic gland) that produces the steroid molting hormone ecdysone in size assessment and thus the timing of the onset of metamorphosis. Juvenile hormone (JH) plays an important role in allowing larval growth and molting by preventing metamorphosis. The presence of JH at the onset of the molt prevents the ecdysone-induced switch from a larval program to the metamorphic program during the molt by regulation of the ecdysone-induced transcription factor Broad. The loss of JH after the attainment of the critical size for metamorphosis then determines the final size of the larva and subsequent adult. Growth itself is dependent on the nutrient-dependent insulin signaling system. Therefore, size control depends on an interaction between the classical endocrine system and this insulin signaling system. Our specific aims in the next 4 years are: 1) To complete the study of the regulation of second pupal cuticle formation by JH, concentrating on the regulation of the Broad and Kruppel-homolog transcription factors. 2) To determine how the prothoracic gland interacts with the insulin signaling pathway and other growth factors to assess the size of the animal and signal the onset of metamorphosis. 3) To determine how JH regulates the final size at pupariation. 4) To determine what are the critical tissues for size assessment. 5) To determine how insulin signaling affects growth differentially before and after the critical weight stage. 6) To determine the role of E75A in the prothoracic gland and/or target tissues to ensure sufficient ecdysteroid for larval molting. These studies should lead to a better understanding of the ways in which JH prevents the ecdysone- induced gene switching that is necessary for metamorphosis and how the nutrition-dependent insulin signaling pathway interacts with the JH regulatory pathway so that metamorphosis occurs at a time optimal for reproduction. The control of growth and the tinning of its cessation leading to reproductive maturation is a problem that all animals including humans face. These studies in insects will provide models for the types of cellular and molecular mechanisms underlying the cessation of growth at puberty in humans. These studies also should provide new insights into how developmental switches common to all developing systems are controlled by endocrine signals. Finally, the studies may provide the basis for novel strategies of insect control that will provide for new strategies for control of the vectors of human disease such as mosquitoes and biting flies. [unreadable] [unreadable] [unreadable]