Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Over the duration of this project I have participated in dissemination of scientific knowledge to target audiences through efforts including participation in scientific meetings, formal classroom instruction, laboratory instruction, participation in workshops, and outreach programs. My lab has participated in the Annual Drosophila Research Conference (2011, 2013) which attracts Drosophila researchers doing both basic and applied research. This provided an opportunity to share research results and confer with other labs interested in Drosophila immunity, and steroid/neurohormone signaling as a potential tool for pest control. My efforts also included teaching undergraduate courses that draw a student base from both the College of Biological Sciences and the College of Agriculture and Environmental Sciences. The instruction provided allows many of these students to pursue internships in local biotechnology and agriculture-oriented businesses in the area, as well as to pursue further postgraduate training and education. Each year I teach an upper division laboratory course in Genetics (MCB 160L) which provides students with hands-on experiences using genetic model systems relevant to agriculture and biotechnology to address genetic problems, including Drosophila melanogaster (insects), yeast, Arabidopsis thaliana (plants), and Caenorhabditis elegans (nematode). From 2010-2013 I also taught an upper division undergraduate course in Developmental Genetics (MCB 163) that instructs students in central concepts in development, molecular biology and genetics using insect (Drosophila melanogaster), nematode (C. elegans) and mammalian (mouse) model systems. From 2013-2015 I additionally taught an upper division undergraduate course in Advanced Molecular Biology (MCB 121) that instructs students in principles of DNA structure, chromatin packaging, DNA repair and recombination, transcription, RNA processing and translation. These are concepts central to basic and applied biology. For the past three years I have been a trainer of the Research Scholars Program in Insect Biology, administered by Joanna Chiu and Jay Rosenheim in the UC Davis Entomology Department. This program provides research and training opportunities in insect biology labs for talented undergraduate students. In 2013-14 I provided materials and academic support to a high school biology instructor in Modesto (Delgel Pabalan) to enable her to set up and teach a Drosophila genetics project to two classes of biology students. For the past three years I have served as a panelist for the UC Davis STEM Transfer Day Career Discovery Workshop to provide advice and information regarding academic success, career and UCD research opportunities to under-represented and economically disadvantaged prospective transfer students in the biological sciences. Each year I have also participated in outreach/recruitment days for potential new freshman and transfer students with academic interests in the biological sciences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the duration of this project, training and professional development opportunities for eight undergraduate students have been provided ( Biyan Feng, Thanh Du, Victoria Nieciecki, Han Nguyen, Maryam Zaman, Manreet Randhawa, Payal Pratap, Amy Du). The training received helped these students meet their goals of acceptance into professional schools, graduate training, or working in biotech/pharmaceutical research. Prof. John A. Kiger (Emeritus) has jointly worked on experiments concerning post-eclosion regulation of development by Bursicon and that project also includes a collaborator, Dr. Ben White, an insect neurobiologist/physiologist at the National Institute of Mental Health (NIMH). Dr. Deborah Kimbrell, an emeritus Associate Research Geneticist with expertise in the insect immune response, has collaborated on the insect immunity studies. We have also collaborated with Dr. Walter Leal ( Dept. Entomology and more recently a member of Dept of Molecular and Cellular Biology) and provided technical support and advice for some of his studies related to insect olfaction, including training post-doctoral fellows from his lab in applications of genetic approaches to their work. These collaborations have provided rewarding intellectual opportunities for scientific interaction for all members involved. How have the results been disseminated to communities of interest?Results have been disseminated via participation in scientific meetings and publication of research results. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goals of our work focus broadly on the molecular mechanisms of developmental communication systems that control the physiology and development of insects. Within this framework, our objectives included (1) analysis of the role of the insect steroid hormone 20-hydroxyecdysone in controlling development and function of the insect immune system at metamorphosis (2) analysis of other targets of the steroid response that control events during molting and metamorphosis (3) identification of neuronal pathways that control insect behavior and physiology at and immediately following adult eclosion. These research objectives encompass critical periods in insect development that could be exploited as targets for pest management strategies. Progress in these areas over the duration of the project is briefly summarized below. (1) Analysis of the role of the insect steroid hormone 20-hydroxyecdysone in controlling development and function of the insect immune system at metamorphosis. Our work in this area was initiated by an observation that reducing the function of the Ecdysone Receptor (EcR) by targeted dsRNA interference specifically in immune tissues led to pupal lethality and the production of melanotic masses in larvae and pupae. A heterozygous loss of function mutation (normal phenotype by itself) for an independent locus (dappled) that produces melanotic masses as a homozygote can act as a genetic enhancer of the pupal lethal phenotype resulting from steroid response suppression in the immune tissues. Our studies of insect immune function and its regulation by steroid hormones have revealed that pupal lethality in response to depletion of steroid hormone signaling in immune cells requires cell autonomous steroid response alterations in the immune cells themselves, as well as abnormal persistence of larval tissues into the pupal phase, which may act as a trigger for hyperactivation of the immune response. Consistent with these results, we have determined that the lethality is not likely to be simply due to loss of function of the immune cells leading to the inability to fight septic conditions during the tissue reorganizations at metamorphosis. Rather, our current hypothesis is that hyperactivation of the immune response at metamorphosis in the absence of moderating steroid regulation can be toxic for the organism. Our research into basic mechanisms of insect immunity and their regulation by steroid hormones has provided useful insights that will facilitate development of new approaches for manipulation of insect life cycles and physiology, and new or improved methods for improving crop production by enhancing the effectiveness of fungal and bacterial entomopathogens. (2) Analysis of other targets of the steroid response that control events during molting and metamorphosis. Our investigations of a steroid dependent gene (IMP-E1) that affects epithelial development in Drosophila have shown that reduction in activity of this gene affects development of adult epithelia (such as wings, abdominal epidermis), with loss of adult abdominal epithelia (and resulting absence of the adult abdominal cuticle) as well as production of smaller, aberrantly shaped pupal wings. Studies of cell proliferation in the abdominal epithelial precursors show that the initial establishment of these precursors is normal, and the initial rounds of cell division in early pupal development occur but are delayed in time. We extended these investigations using heat-shock RNAi-induced loss of function studies in a time course analysis to define critical periods for gene function during development. This work defined a period in early pupae that is dependent on IMP-E1 for normal production of the abdominal epidermis and cuticle, perhaps due to a defect in the second wave of histoblast proliferation and/or their spreading and displacement of the larval epidermis. We are currently investigating whether the later cell divisions and tissue movements that close and form a continuous abdominal epidermis occur normally. Analysis of the wing defects reveals that the wing area is reduced but spacing and shape of the cells is unchanged, suggesting a defect in cell proliferation and/or regulation of cell death. We have also uncovered a role for the gene product in regulating larval cell growth, as more severe depletion results in larval lethality, producing tiny larvae that fail to grow normally although they continue molting to the third instar. (3) Identification of neuronal pathways that control insect behavior and physiology at and immediately following adult eclosion. We have identified the disco-related locus as the location of the dominant negative mutation batone (bae) which blocks normal wing expansion after eclosion and delays cuticle tanning. We used the CRISPR-cas9 gene editing approach to create 8 different targeted loss of function alleles of the disco-related locus in a batone mutant background. The disco-related loss of function alleles were verified by DNA sequencing to contain frameshift mutations in that locus, and we observed that all of these loss of function alleles suppressed the batone phenotype allowing normal wing expansion, adding strong evidence for identification of disco-r as the locus affected by the batone mutation, and the causal agent of the post-eclosion phenotype. We have determined that a pair of neuronal transcription factor genes, disco-related and its very similar adjacent gene disco, are likely regulators of the neuropeptide cascade that results in post-eclosion wing maturation and cuticle tanning in insects. This classic pathway involves neuronal release of the peptide neurohormone signal Bursicon. Our results suggest that these transcription factors may act as partially redundant modulators to directly regulate production and/or release of the hormone. Our collaborator Ben White had shown that patterns of Bursicon release into circulation following eclosion are perturbed in the bae mutant background. Synthesis of one subunit of Bursicon is not detected in key regulatory neurons in the subesophageal ganglion (Bseg), suggesting that one effect of the mutation is to block these critical neural circuits. Our hypothesis is that these Zn-finger transcription factors could affect the fate and/or behavior of neurons that orchestrate release of Bursicon and integration of the behavioral and physiological network. Our work has therefore identified new upstream regulatory steps in this critical pathway in insect development.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: During the past year I participated in dissemination of scientific knowledge to a broad range of students at UC Davis. These activities included teaching an upper division laboratory course in Genetics (MCB 160L) which provides students with hands-on experiences using genetic model systems relevant to agriculture and biotechnology to address genetic problems, including Drosophila melanogaster (insects), yeast, Arabidopsis thaliana (plants), and Caenorhabditis elegans (nematode). I also taught an upper division undergraduate course in Advanced Molecular Biology that instructs students in principles of DNA structure, chromatin packaging, DNA repair and recombination, transcription, RNA processing and translation. Students in these courses come from both the College of Biological Sciences and the College of Agriculture and Environmental Sciences. The instruction provided allows many of these students to pursue internships in local biotechnology and agriculture-oriented businesses in the area, as well as for further postgraduate training and education. I also mentored one undergraduate student doing research in my lab: Payal Pratap (graduated June 2014). I was a trainer on the Research Scholars Program in Insect Biology, administered by Joanna Chiu and Jay Rosenheim in the UC Davis Entomology Department. This program provides research and training opportunities in insect biology labs for talented undergraduate students. I provided materials and academic support to a high school biology instructor in Modesto (Delgel Pabalan) to enable her to set up and teach a Drosophila genetics project to two classes of biology students. I served as a panelist for the UC Davis STEM Transfer Day Career Discovery Workshop to provide advice and information regarding academic success and research opportunities as an undergraduate at UC Davis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training opportunities for an undergraduate researcher, Payal Pratap, who is currently pursuing research job opportunities in the Bay Area following her graduation. In addition, my undergraduate teaching efforts, particularly with regard to the upper division genetics lab course, provide valuable introductory research skills and training to the students in the course. In addition to the technical and scientific laboratory training, students also develop their skills in scientific communication via writing a 10 page lab report formatted as a scientific journal publication. How have the results been disseminated to communities of interest? I am active in scientific and educational outreach efforts for prospective undergraduate freshmen and transfer students as a regular participant in campus information events such as Decision Day and UCD STEM Transfer Day Career workshops. Our research on the Bursicon neurohormone pathway was submitted for publication and is in the process of being revised for resubmission. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period we plan to complete the remaining work confirming the identity of the batone mutation and the involvement of the disco-related gene in regulation of post-eclosion insect maturation and to successfully publish this work. We also plan to apply similar CRISPR-cas9 technologies to engineer IMP-E1 knockout mutations specific for mRNA transcript isoforms to clarify the production of mutant phenotypes, and to complete analysis of the cellular basis of these phenotypes.
Impacts
What was accomplished under these goals?
We have continued to investigate signal transduction pathways that regulate insect development, maturation, and physiology. (1) One research area focuses on identification of new regulatory steps in the Bursicon pathway, a neuropeptide hormone signaling cascade that regulates insect maturation following adult eclosion from the pupal stage (behavior, wing expansion, cuticle tanning). Our previous work on the dominant negative batone mutation, which blocks normal wing expansion, had identified the disco-related locus, encoding a zinc finger transcription factor, as a likely candidate gene. Our hypothesis is that this transcription factor affects the fate or function of neurons that are required for normal production of one subunit of the Bursicon neuropeptide hormone. During the current reporting period we conducted experiments needed to revise a submitted manuscript by obtaining more definitive genetic evidence to identify the disco-related gene as the site of the mutation. We used the CRISPR-cas9 gene editing approach to create 8 different targeted loss of function alleles of the disco-related locus in a batone mutant background. The disco-related loss of function alleles were verified by DNA sequencing to contain frameshift mutations in that locus, and we observed that all of these loss of function alleles suppressed the batone phenotype allowing normal wing expansion, adding strong evidence for identification of disco-r as the locus affected by the batone mutation, and the causal agent of the post-eclosion phenotype. This work has been conducted in collaboration with the lab of Ben White at NIH. (2) We are also continuing investigation of steroid responsive genes that play a role in tissue morphogenesis during embryogenesis and pupal development in Drosophila. Previous work with hypomorphic loss of function constructs had demonstrated a role for one of these genes, IMP-E1, in regulation of formation of imaginal disc and abdominal histoblast derived structures (eg. the abdominal epidermal segments, head capsule, and wings), perhaps via defects in cell proliferation and/or cell death. By using RNAi constructs that can be expressed at higher levels, we found that a strong, widespread reduction of expression of this gene produces an unusual "tiny larva" phenotype, with delayed development of small larvae that nonetheless appear to undergo normal molts but fail to produce pupae. We are currently investigating the developmental basis of this phenotype to test the hypothesis that this is due to an early defect in division or polytenization and growth of larval cells.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
The batone mutation of the disco-related gene targets command neurons in the Bursicon signaling network of Drosophila. Jeanette E. Natzle, Fengqui Diao, Benjamin White, and John A. Kiger Jr. (submitted) G3: Genes|Genomes|Genetics.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: During the past year I participated in dissemination of scientific knowledge to a broad range of students at UC Davis. These activities included teaching an upper division laboratory course in Genetics (MCB 160L) which provides students with hands-on experiences using genetic model systems relevant to agriculture and biotechnology to address genetic problems, including Drosophila melanogaster (insects), yeast, Arabidopsis thaliana (plants), and Caenorhabditis elegans (nematode). I also taught an upper division undergraduate course in Developmental Genetics (MCB 163) that instructs students in central concepts in development, molecular biology and genetics using insect (Drosophila melanogaster), nematode (C. elegans) and mammalian (mouse) model systems. Students in these courses come from both the College of Biological Sciences and the College of Agriculture and Environmental Sciences. The instruction provided allows many of these students to pursue internships in local biotechnology and agriculture-oriented businesses in the area, as well as for further postgraduate training and education. I also mentored two undergraduate students doing research in my lab: Thanh Du (graduated June 2013) and Payal Pratap (currently enrolled). In addition I became a trainer on the Research Scholars Program in Insect Biology, administered by Joanna Chiu and Jay Rosenheim in the UC Davis Entomology Department. This program provides research and training opportunities in insect biology labs for talented undergraduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training and professional development opportunities for several undergraduate researchers, who learned research skills under the supervision of the PI. How have the results been disseminated to communities of interest? Our research in the area of the neurohormonal control of insect development following eclosion has been submitted for publication and is currently under review. I have been active in dissemination of knowledge about current research technologies in the areas of genetics, molecular biology and animal development through teaching undergraduate lab courses and lecture courses in these areas. This provides the student community at UC Davis with hands-on training and theoretical instruction that allows them to pursue careers in biotechnology, health care delivery, and bio-agricultural fields. I am also active in outreach activities to prospective freshman and community college transfer students as a regular participant in campus information events such as Decision Day. I have provided information to students and their families about research and educational opportunities on campus and options for pursuing training in areas of biology (applied and basic), biotechnology, genetics, etc. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we hope to complete work on the immunity project to further define the cellular populations that are primarily affected by diminished steroid responsiveness by creating additional genetic mosaics that will better characterize the major site of action and cause of the observed phenotypes. This will allow us to publish the body of work that we have completed in this area. We also plan to complete work in the area of steroid regulation of IMP-E1 expression and its role in pupal morphogenesis to define the common cellular mechanism that results in the abdominal, appendage and larval phenotypes.
Impacts
What was accomplished under these goals?
During the past year we have continued our efforts to understand the hormonal regulation of insect development and physiology with the goal of producing knowledge that can be applied to minimize negative impacts of insects on agricultural productivity. We completed a set of studies focused on regulation of the Bursicon signaling pathway in insects and this work has been submitted in a publication that is currently under review. We found that the batone mutation blocks normal pathways critical for cuticle tanning and maturation and expansion of insect wings following pupal eclosion. The mutation blocks normal post-eclosion behaviors as well as release of the neuropeptide Bursicon into the circulation. Synthesis of one subunit of Bursicon is not detected in key regulatory neurons in the subesophageal ganglion (Bseg) in this background, suggesting that one effect of the mutation is to block these critical neural circuits. Genetic analysis identified the disco-related gene as the site of the bae mutation. Further genetic analyses established that bae behaves as a dominant negative allele and that disco-r and the related disco gene function in a redundant manner with respect to regulating this Bursicon-production pathway. These genes encode Zn-finger transcription factors that could affect the fate and/or behavior of neurons that orchestrate release of Bursicon and integration of the behavioral and physiological network. Our work has therefore identified new upstream regulatory steps in this critical pathway in insect development. We have also made progress in a second area, investigating the role of the steroid hormone ecdysone in regulating a target gene (IMP-E1) important for maturation and production of the adult abdominal epidermis and imaginal disc derivatives. We extended our previous investigations using heat-shock RNAi-induced loss of function studies in a time course analysis to define critical periods for gene function during development. This work defined a period in early pupae that is dependent on IMP-E1 for normal production of the abdominal epidermis and cuticle, perhaps due to a defect in the second wave of histoblast proliferation and/or their spreading and displacement of the larval epidermis. Function of this gene is also required for normal development of imaginal disc derived structures such as the wing and the head capsule during pupal. Analysis of the wing defects reveals that the wing area is reduced but spacing of the cells is unchanged, suggesting a defect in cell proliferation and/or regulation of cell death. A third research area focuses on clarification of the role of steroid hormones in regulation of function and development of the Drosophila immune system, based on our initial observations that reducing function of the steroid response pathway in immune cells results in pupal lethality. In this area we have continued experiments that established that the steroid response deficit is not equivalent to loss of function of the immune cells, and that aberrant persistence of larval tissues into pupal development may enhance the abnormal immune responsiveness. These results suggest the hypothesis that loss of steroid responsiveness triggers a hyperactive immune response.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
The batone mutation of the disco-related gene targets command neurons in the Bursicon signaling network of Drosophila. Jeanette E. Natzle, Fengqui Diao, Benjamin White, and John A. Kiger Jr. (submitted 2013) G3: Genes|Genomes|Genetics. (Support by AES/Hatch was acknowledged)
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The goal of our work is to understand insect physiology and development with respect to regulation by steroid and other neurosecretory hormones, particularly as related to insect metamorphosis and insect immunity, including response to pathogens. Our project outputs consisted mainly of experiments conducted in several areas related to our project goals: (1) we investigated possible mechanisms for transcriptional regulation of release of the neuropeptide hormone bursicon, which controls events at insect eclosion (2) we conducted experiments to identify mechanisms associated with pupal lethality due to depression of steroid signaling in immune tissues in insects and (3) we analyzed the role of a steroid inducible gene, IMP-E1, in regulating epithelial development and insect growth. Over the course of this work, we trained 3 undergraduate students, who carried out research in support of these projects and employed/trained a recent UC Davis graduate, who also assisted part-time with this work. Part of this work was presented at a poster presentation at the 53rd Annual Drosophila Research Conference in Chicago, Illinois in March 2012, attended by Drosophila researchers from the US and other countries. PARTICIPANTS: The PI for the project, Jeanette Natzle, planned and supervised work on the project as well as carrying out much of the experimental work and analysis. Three undergraduate researchers have worked part-time on the projects, providing laboratory support and conducting experiments under the supervision of the PI: (1) Thanh Du, Genetics major, planned graduation June 2013 (2) Maryam Zaman, Biochemistry & Molecular Biology major, graduated December 2012 (3) Manreet Randhawa, Biochemistry & Molecular Biology major, graduated December 2012. In addition, a recent UC Davis graduate, Victoria Nieciecki (Genetics major) was employed part-time for about 6 months to help with the work. Prof. John A. Kiger (emeritus) has jointly worked on experiments concerning post-eclosion regulation of development by Bursicon and that project includes a collaborator, Dr. Ben White, and insect neurobiologist/physiologist at the National Institute of Mental Health (NIMH). Dr. Deborah Kimbrell, an emeritus Associate Research Geneticist with expertise in the insect immune response, has collaborated on the insect immunity studies. During this time period I had the opportunity to attend the 53rd Annual Drosophila Research conference in Chicago Illinois, which was an excellent opportunity for professional development and updates on current research and technology in fields related to my work. TARGET AUDIENCES: Presentation of our work at the 53rd Annual Drosophila research conference provided the opportunity to disseminate our results to a community of scientists with similar interests in insect physiology, development and immunity. In addition, I participate in teaching a lab course in genetics (MCB 160L) at UC Davis, which provides instruction to students in the College of Biological Sciences as well as in the College of Agriculture and Environmental Science. Students are taught to use a number of genetic model systems (Drosophila, Arabidopsis, yeast, C. elegans, bacteria) that are relevant to many aspects of biotechnology and agricultural research. This training is directly useful to many of these students that use the experience to obtain internships in local biotechnology and agriculture-oriented businesses in the area, as well as for further postgraduate education and training programs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our research this year produced a number of new insights into basic mechanisms of insect development and immunity that will have applications in manipulation of the insect life cycle to reduce negative impacts on crop production. Our studies of insect immune function and its regulation by steroid hormones have revealed that pupal lethality in response to depletion of steroid hormone signaling in immune cells requires alterations in the immune cells themselves, as well as abnormal persistence of larval tissues into the pupal phase, which may trigger hyperactivation of the immune response. Consistent with these results, we have determined that the lethality is not likely to be due to inactivity of the immune cells leading to the inability to fight septic conditions during the tissue reorganizations at metamorphosis. We have also established that co-expression of the Dicer RNAse component of the dsRNA inhibition pathway can produce a more complete removal of steroid receptor function in our RNAi experiments, leading to a stronger, more reproducible phenotype. In other experiments investigating control of post-eclosion behaviors by the neuropeptide hormone Bursicon, we have obtained genetic evidence for a complex interdependence of Bursicon function on two related transcription factors, disco and disco-related. Our results suggest that these hormones may directly regulate production and/or release of the hormone rather than affecting the fate or connectivity of neurons that produce the hormone. Finally, our investigations of a steroid dependent gene that affects epithelial development in Drosophila have shown that this gene affects development of adult epithelia (such as wings, abdominal epidermis), with loss of adult abdominal epithelia as well as production of smaller, aberrantly shaped pupal wings. Studies of cell proliferation in the abdominal epithelial precursors show that the initial establishment of these precursors is normal, and the initial rounds of cell division in early pupal development occur but are delayed in time. We are currently investigating whether the later cell divisions and tissue movements to close the epidermis occur normally. We have also uncovered a role for the gene product in regulating larval growth, as more severe depletion results in larval lethality, producing tiny larvae that fail to grow normally although they continue molting to the third instar. We plan to publish these results during the ensuing year.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The overall goal of this project is to apply the tools of Drosophila genetics to understand and potentially manipulate critical aspects of insect physiology, in particular related to post-eclosion behaviors and insect immunity and response to pathogens. Our project outputs during this period consisted mainly of experiments conducted in several areas to meet our project goals: (1) identification of neuronal pathways that control insect behavior and physiology post eclosion (2) analysis of the effects of steroid depletion on function and development of different parts of the insect immune system (3) modulation of the Drosophila steroid response using double-stranded RNA inhibition technology. In addition, an undergraduate research student (Biological Sciences major) was trained and assisted in this research project. Our recent results were disseminated to the broader community through a poster presentation at the 53rd Annual Drosophila Research Conference in Chicago, Illinois. PARTICIPANTS: The PI for this project, Jeanette Natzle, planned and supervised work on the project as well as carrying out some of the experimental work and analysis. Two undergraduate researchers have worked part-time on the project, providing laboratory support and conducting some experiments under supervision by the PI: (1) Thanh Du, Biological Sciences major, planned graduation June 2013 (2) Victoria Nieciecki, Genetics major, graduated June 2011. Prof. John Kiger, has jointly worked on the experiments looking at post-eclosion development and the project has expanded to include an additional collaborator, Dr. Ben White, an insect neurobiology/physiologist at the National Institute of Mental Health (NIMH). Dr. Deborah Kimbrell, an emeritus Associate Research Geneticist with expertise in the area of the insect immune response, has collaborated on the insect immunity studies. TARGET AUDIENCES: Our research was presented at the 53rd Annual Drosophila Research Conference held in Chicago Illinois. This conference attracts Drosophila researchers doing both basic and applied research. We were able to share results and confer with other labs interested in Drosophila immunity as a potential tool for pest control. In addition, I participate in teaching a genetics laboratory course (MCB 160L) at UC Davis, which provides instruction to students in the College of Biological Science and the College of Agriculture and Environmental Sciences. Students are taught to use a number of genetic model systems (Drosophila, Arabidopsis, C. elegans, yeast, bacteria) that are relevant to many aspects of biotechnology and agriculture. This training is directly useful to many of these students that use the experience to obtain internships in local biotechnology and agriculture-oriented businesses in the area, as well as for further postgraduate education and training programs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our research into basic mechanisms of insect immunity and their regulation by steroid hormones has provided useful insights that will facilitate development of new approaches for manipulation of insect life cycles and physiology, and new or improved methods for improving crop production by enhancing the effectiveness of fungal and bacterial entomopathogens. We have recently shown that interference with steroid signaling in several immune cell types results in lethality during pupal development, blocking eclosion of the adult insect. We are currently investigating whether the lethality results from increased susceptibility to natural infection during tissue reorganization at metamorphosis, or from an enhanced, unregulated immune response that may damage normal tissue. Along with these studies, we have also identified a pair of neuronal transcription factor genes, disco and disco-related, as likely regulators of the neuropeptide cascade that results in post-eclosion wing maturation and cuticle tanning in insects. In addition, we have identified a Drosophila steroid-responsive gene that is required for production of an intact abdominal epidermis, and for wing and head capsule morphogenesis during insect pupal development.
Publications
- J.E. Natzle, P. Finnegan, D. Kuo, P. Nguyen, D. Kimbrell. 2012. Steroid modulation of immune function in Drosophila. 53rd Annual Drosophila Research Conference, Program and Abstract Book. Chicago, Illinois. p. 66 (Abstract # 603C) (work completed and submitted in 2011, published in 2012)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The overall goal of this project is to produce, characterize and apply tools for manipulating in vivo levels of the insect steroid, 20-hydroxyecdysone, in Drosophila melanogaster in order to understand and potentially manipulate critical aspects of insect physiology and development, such as immune responses to pathogens and post-eclosion wing maturation. Drosophila is an important model for other pest insects, as it offers the opportunity to apply powerful genetic and molecular approaches to investigate conserved mechanisms. Our main activities during this period consisted of conducting and analyzing experiments designed to further our research goals. Our effort was directed in three main areas: (1) identification of the batone gene and its mechanism of action in controlling the expansion of adult insect wings following eclosion (2) characterizing the immune response in Drosophila pupae (3) preparing reagents that can be used for assaying molecular endpoints of the immune response in pupae. I initiated a collaboration with the lab of Prof Frank Zalom (UCD, Entomology) to share some of our knowledge and expertise in insect molecular biology and immune response with the goal of developing novel control mechanisms for the recent California invasion of the agricultural pest Spotted Wing Drosophila. In addition,we participated in training a post-doc from the lab of Prof. Walter Leal in the UCD Entomology Department in the application of genetic techniques in Drosophila. We also trained an undergraduate student (B.S. Microbiology, Fall 2010) in application of Drosophila research tools for analysis of insect immunity and hormone response. PARTICIPANTS: The PI for the project, Jeanette Natzle, planned and supervised work on the project as well as carrying out some of the experimental work and analysis. Two undergraduate researchers have worked on the project during this period. An undergraduate student, Biyan Feng (B.S. Microbiology, Fall 2010), worked part time on the project from January 2010- August 2010, prior to her graduation. From October 2010 through December 2010 (and continuing into the current year) another undergraduate, Thanh Du, has helped with the project part-time. This work hs provided valuable training and experience in laboratory techniques and conduct of scientific research for these undergraduates and helps them pursue their career goals. A collaborator in the Department of Molecular and Cellular Biology, Prof. John Kiger, has jointly worked on the wing maturation project, and the project has expanded to include an additional collaborator, Dr. Ben White, an insect neurobiologist/physiologist at the National Institute of Mental Health (NIMH). Prof. Frank Zalom, UC Davis Dept of Entomology has provided consultation and advise on studies using the Spotted Wing Drosophila and pest management approaches. TARGET AUDIENCES: I participate in teaching an undergraduate Genetics laboratory course, MCB 160L, which provides instruction in several genetic model systems that are useful in many aspects of biotechnology and agriculture (Drosophila, Arabidopsis, C. elegans, yeast, bacteria), as well as basic genetic and molecular biology techniques. Research similar to that undertaken in this Experiment Station project forms part of the basis for instruction in the class. Students in the class come from both the College of Biological Sciences and the College of Agriculture and Environmental Sciences. Many of the undergraduates are working, or have worked, in agriculture-related laboratories or professional internships. This training prepares them for entry into the workforce in agricultural or biotechnology-oriented fields, as well as for further postgraduate education and training programs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The tools that we are applying in our research will provide useful insights for development of new approaches for management of pest insect life cycles, new mechanisms to enhance crop production and control disease vectors. For instance we are specifically working toward understanding how the effectiveness of natural fungal and bacterial entomopathogens can be enhanced by possible manipulation of the insect immune system. Our preliminary studies indicate that depressing the insect steroid response in immune cells can produce some enhancement in susceptibility to fungal infection in adults. We are currently extending these studies to the pupal developmental period. We have collected and are in the process of testing PCR reagents that will allow us to include an analysis of molecular endpoints of the immune response in our studies. We have also studied the mechanism of wing maturation after insect eclosion, as this represents a vulnerable window during insect development. We are seeking to identify the molecular signaling pathways responsible for this physiological event, with the goal of possible intervention to disrupt maturation of adults. We are close to identifying a previously unknown regulator of this pathway, the product of the batone gene. Genetic analysis has narrowed the set of potential transcription units and we are currently testing these genes for identity with the mutant locus.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The overall goal of this project is to develop and utilize tools for manipulating in vivo levels of the insect steroid hormone, 20-hydroxyecdysone, in Drosophila melanogaster in order to better understand how the hormone controls critical aspects of insect physiology and development, such as immune response to pathogens, and post-eclosion wing maturation. Drosophila serves as an important model system for other pest insects, as it offers the opportunity to apply genetic and molecular approaches that are not widely available in other insects. Understanding the hormonal control mechanisms for these developmental events may open the way for potential new strategies in pest management. One objective of our efforts during this reporting period has been to determine the role of ecdysteroids in regulating function and development of Drosophila hemocytes (circulating blood cells). We have completed or are in the process of conducting the following studies to support this objective: (1) Analysis of hormone-dependent changes in hemocyte subpopulations using in vivo and in vitro dye labeling studies during larval and pupal development (2) Construction and analysis of stocks to monitor antibacterial peptide induction during the hypothesized inflammatory response that may occur after steroid receptor depletion (3) Quantitative PCR analysis of immune activation targets following steroid receptor depletion in immune tissues. PARTICIPANTS: The PI for this project, Jeanette E. Natzle, planned and supervised work on the project as well as carrying out some of the experimental work and analysis. Two undergraduate researchers have worked on this project during the reporting period. An undergraduate student, Jenifer Kim, worked part-time on the projects from January 2009 - June 2009 while completing her Bachelor's degree. Ms. Kim subsequently obtained employment in the biotechnology industry. From October 2009 through December 2009 (and continuing into the current year) another current senior-level undergraduate student, Biyan Feng, has helped with the project. This work has provided valuable training and experience in laboratory techniques and conduct of scientific research for these talented undergraduates and has helped them to pursue their goals of careers in research science. TARGET AUDIENCES: I participate in teaching an undergraduate Genetics laboratory course, MCB 160L, which provides instruction in several genetic model systems that are useful in many aspects of biotechnology and agriculture (Drosophila, Arabidopsis, C. elegans, yeast, bacteria). Research similar to that undertaken in this experiment station project forms part of the basis for instruction in the course. Undergraduates in this course receive instruction in molecular biology and genetic techniques that prepare them for further post-graduate work in research as well as for entering jobs after graduation. Many students in majors in the College of Agriculture (eg. Biotechnology) as well as in the College of Biological Sciences are trained in this laboratory course. In addition, when the next segment of this work is completed it will be prepared for publication and will be available to the larger scientific community in the scientific literature and through presentation at scientific meetings. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Insects rely extensively on modulation of steroid hormone levels to regulate initiation and completion of critical developmental events. The tools that we are applying in our research will provide useful insights for development of new approaches for management of pest insect life cycles, providing new mechanisms to enhance crop production and control disease vectors. In particular our research into hormonal regulation of the insect immune response will enhance effectiveness of pest control strategies that use natural pathogens such as bacteria, fungus and parasites. Potential strategies for manipulating the immune response could increase susceptibility to these biological control agents. With respect to our first project output (analysis of hormone-dependent changes in hemocyte subpopulations during larval and pupal development) we found that diminishing functional ecdysone receptor in circulating hemocytes (insect blood cells) was associated with an increase in levels of some populations of blood cells and decreases in others relative to controls. Our results suggest that it is the population of hemocytes released from the lymph glands during early pupal development that may be primarily affected by the depletion of steroid receptor function. We postulate that steroid receptor depletion in a subset of lymph gland cells results in aberrant signaling to other lymph gland cells to activate an inflammatory response, perturbing their function and resulting in melanotic nodule formation and pupal lethality. Our second project output (monitoring antibacterial peptide induction after steroid receptor depletion) was approached via construction of stocks that would allow steroid receptor depletion to be followed by changes in fluorescent intensity of GFP-tagged antibacterial peptides. This is an important endpoint to follow since antibacterial peptides form a major mechanism in insects for overcoming pathogen invasion. While some results were promising, suggesting that steroid depletion may be able to ectopically activate the antimicrobial peptide response, fluctuating fluorescent background in the stocks limited the sensitivity and reliability of the approach. We were however able to demonstrate that introducing a bacterial infection in pupae triggers antimicrobial peptide induction, a fact that hadn't been previously established. This led us to initiate the third approach (quantitative PCR analysis of immune activation targets following steroid receptor depletion in immune tissues). We are identifying appropriate molecular targets to monitor targets of several arms of the inflammation/pathogen response pathway so that we can assay the effect of steroid receptor depletion on the response. We will be able to detect ectopic activation of the inflammation/pathogen response in the absence of pathogen challenge, as well as depression of responses following introduced bacterial infection in pupae. This work will provide the basis for further studies (eg microarray analysis) to define the full extent of the effect of the steroid response on insect immunity.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The overall goal of this project is to develop and utilize tools for manipulating in vivo levels of the insect steroid hormone, 20-hydroxyecdysone, in Drosophila melanogaster in order to better understand how the hormone controls critical aspects of insect physiology and development, such as immune response to pathogens, and post-eclosion wing maturation. Drosophila serves as an important model system for other pest insects, as it offers the opportunity to apply genetic and molecular approaches that are not widely available in other insects. Understanding the hormonal control mechanisms for these developmental events may open the way for potential new strategies in pest management. During this reporting period our efforts have been directed toward experiments that address project objectives focused on two areas. One of these objectives is to determine the role of ecdysteroids in regulating function and development of Drosophila hemocytes (circulating blood cells). In support of this objective, we have carried out an analysis of hormone dependent changes in circulating hemocyte populations before and after pupariation, using hemocyte-specific dyes and in vivo GFP labeling. In addition, we have constructed genetic stocks that will allow us to test the hypothesis that a functional ecdysone receptor is necessary to prevent aberrant hemocyte signaling to trigger an abnormal inflammatory state. We also identified post-eclosion wing maturation as another critical insect developmental milepost that could be sensitive to perturbation of the normal hormonal response machinery. We initiated investigations of the role of ecdysone signaling in regulating the epithelial mesenchymal transition and programmed cell death that are essential for removing the wing epithelium from the maturing wing blade following eclosion of the insect from the pupal case. We were able to disseminate our results by discussion of our work with other researchers in the field at the annual Drosophila Research conference in San Diego, April 2008. PARTICIPANTS: The PI for this project, Jeanette E. Natzle, planned and supervised work on the project as well as carrying out some of the experimental work and analysis. The PI wrote the paper listed as a publication, with the assistance of the co-authors (Kiger and Green) who are faculty and emeritus colleagues respectively in the Department of Molecular & Cellular Biology at UC Davis. Two undergraduate researchers have worked on this project during the reporting period. Steve Swanson worked as a Junior Specialist for seven months following his graduation before beginning a Masters program in Biotechnology at Columbia University. A current undergraduate, Jenifer Kim, continued work part-time on the projects for the three remaining months during the reporting period, and has continued working while completing her Bachelor's degree this year. This work has provided valuable training and experience in laboratory techniques and conduct of scientific research for these talented undergraduates and has helped them to pursue their goals of careers in research. In addition, a Genetics Graduate Group rotation student worked for 5 weeks on the project, providing an opportunity for training and professional development, allowing her to learn to work with the Drosophila genetic model system and to apply genetic and developmental biology approaches. TARGET AUDIENCES: I participate in teaching an undergraduate Genetics laboratory course, MCB 160L, which provides instruction in several genetic model systems that are useful in many aspects of biotechnology and agriculture (Drosophila, Arabidopsis, C. elegans, yeast, bacteria). Undergraduates in this course receive instruction in molecular biology and genetic techniques that prepare them for further post-graduate work in research as well as for entering jobs after graduation. Many students in majors in the College of Agriculture (eg. Biotechnology) as well as in the College of Biological Sciences are trained in this laboratory course. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Insects rely extensively on modulation of steroid hormone levels to regulate initiation and completion of critical developmental events. The tools that we are applying in our research will provide useful insights for development of new approaches for management of pest insect life cycles, providing new mechanisms to enhance crop production and control disease vectors. In particular our research into hormonal regulation of the insect immune response will enhance effectiveness of pest control strategies that use natural pathogens such as bacteria, fungus and parasites. Potential strategies for manipulating the immune response could increase susceptibility to these biological control agents. During this reporting period we found that diminishing functional ecdysone receptor in circulating hemocytes (insect blood cells) was associated with an increase in levels of blood cells relative to controls, a characteristic that may be related to the observed production of aberrant melanotic nodules that indicate abnormal blood cell function. We are currently investigating whether the changes reflect increased proliferation of larval blood cells or early/increased release of cells from the lymph glands. We have also generated genetic stocks that will allow us to investigate the potential role of ecdysone in regulating signaling by circulating hemocytes to stimulate antibacterial peptide synthesis and regulation of the inflammatory response. Production of antibacterial peptides by immune-responsive tissues is a major mechanism in insects for overcoming pathogen invasion. These efforts will form the groundwork for further experiments to monitor the effect of ecdysone receptor depletion on survival after pathogen introduction. Another critical event that contributes to insect survival is the hormonally controlled maturation of the wing into a functional flight organ following eclosion from the pupa case. By manipulating ecdysone receptor levels in the wing epithelium at this time we found that decreasing ecdysone receptor function in the wing epidermis can block epithelial apoptosis and therefore normal wing maturation, but the effect is most likely on establishment of permissive conditions for response to other later signals. We established that the epithelial mesenchymal transition (which is essential for formation of a functional wing) depends on the peptide hormone bursicon and its receptor, rickets, and this is regulated independently of the simultaneous apoptosis of the epithelium.
Publications
- Natzle, J.E., Kiger, J. A., Jr., Green, M.M. (2008) Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation. Genetics 180 (2): 885-893.
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Progress 01/01/07 to 12/31/07
Outputs
Steroid hormones provide signals to modulate development and physiology of many multicellular organisms. The life cycle and maturation of insects are particularly sensitive to fluctuations in the level of the insect steroid hormone 20-hydroxyecdysone (20-HE), with steroid-dependent developmental events occurring during embryogenesis, larval molts, metamorphosis and germline/oocyte maturation. As a result of our past research efforts, and in fulfillment of Experiment Station research objectives, we generated a tool for in vivo regulation of circulating 20-HE levels through controlled expression of the baculoviral ecdysteroid glucosyl transferase gene (EGT). In recent years we have documented the inhibitory effects of EGT expression on 20-HE dependent gene expression and developmental transitions. We have applied this tool, as well as gene cassettes capable of reducing Ecdysone Receptor levels via dsRNA inhibition, to analyze the role of 20HE in development and function
of hemocytes, the cellular mediators of the insect immune response. As the insect immune system is the first line of defense against natural pathogens that can be used as biocontrol agents, it is important to search for control points that could be used to modulate the immune response in vivo, in order to improve the efficacy of these biocontrol agents. During the past year we have established assays to approach our objective of determining how 20-HE regulates hemocyte development and function. We have successfully adapted and developed assays that can be used to analyze the following parameters in hemocytes obtained from single larvae: (1) phagocytosis of fluorescently labeled bacteria (2) microaggregation as a measure of changes in cell surface properties (3) proportions of different hemocyte subtypes in the circulating population using selective differential staining (4) numbers of hemocytes in the circulation. We are currently using these assays to compare hemocyte properties in
Ecdysone Receptor-depleted hemocytes and matched controls. We have also used immunocytochemical assays for Ecdysone Receptor (EcR) levels in hemocyte nuclei to show that the level of EcR reduction achieved with different dsRNA expression constructs correlates with pupal lethality. We expect that our continued efforts to understand the mechanisms by which steroids regulate the insect immune system will lead to understanding of critical control points and allow manipulation of host hormones to improve efficacy of biocontrol efforts.
Impacts
In recent years our work, and that of other labs, has helped to define several key genes that encode enzymes in the biosynthetic pathway for the insect steroid hormone 20-hydroxyecdysone (20HE), but the regulatory relationships and the mechanisms that regulate expression of the gene products are still not well understood. Because insects rely extensively on this steroid system to regulate timing and initiation of key developmental events, the tools that we have developed to regulate 20HE levels in vivo with the EGT enzyme will provide critical insight for development of new approaches for management of pest insect life cycles. We will also use the genetic tools that we have developed to study the role of 20HE in programming the development and/or function of the Drosophila immune system. Our experiments in this area are directly relevant to biological control of insect pests because several strategies for natural control of insect pests rely on infection by fungus,
bacteria and other insect parasites or pathogens. The insect immune response is the first line of defense against pathogen attack. To improve the effectiveness of these pest control strategies, it is important to understand how development of the capacity for an immune response is regulated in vivo. As we learn more about this point, we plan to directly test whether alteration of host ecdysteroid levels by infection with baculovirus capable of reducing host ecdysteroid levels might act synergistically to enhance the effectiveness of introduced insect pathogens by weakening the host insect immune response.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Steroid hormones act as humoral signals to regulate gene expression in many eukaryotes. They play a particularly important role in insects where the single major ecdysteroid, 20-hydroxyecdysone, coordinates events during embryogenesis, larval molts, metamorphosis, and female fertility. Several of the objectives of our research rely on application of a regulatable system that we have developed to manipulate ecdysteroid levels in vivo through controlled expression of the baculoviral ecdysteroid glucosyl transferase gene (EGT). During this reporting period we have confirmed by quantitative real-time PCR that induction of EGT expression at the beginning of pupal development results in a long-lasting reduction in transcription from steroid-regulated genes. The development of these qPCR assays will also provide us with a sensitive way to monitor outcomes of changes in hormone levels with respect to gene expression in target tissues such as the Drosophila immune system, one
of the objectives of our research. We have also made progress in our objective to determine the role of ecdysteroids in regulation of development and function of hemocytes, the major cells that mediate the innate immune response in Drosophila. We had previously shown that reduction of the steroid response capability specifically in the hematopoietic system using Ecdysone Receptor specific RNAi controlled by a hemocyte/pericardial cell specific promoter (DotGAL4) leads to pupal lethality and the production of melanotic masses that are hallmarks of a defective immune response. We have confirmed that a second independent hematopoietic promoter (HemeseGAL4) expressed in hemocytes but not pericardial cells gives a very similar phenotype. To test the potential efficacy of the mutagenesis screen that we proposed as a way to genetically identify loci that may be responsible for or targets of the steroid response in hemocytes, we have tested the ability of known mutations that affect hemocyte
function to act as dominant enhancers of this pupal lethality. We have identified the dappled locus, which produces melanotic tumors when homozygous, as a dominant enhancer of pupal lethality resulting from Ecdsyone Receptor depletion. This result suggests that our genetic screen should be successful in identifying additional unknown loci that are important for steroid regulation of hemocyte development and function. As the insect immune system is the first line of defense against natural pathogens that can be used as biocontrol agents, it is important to search for control points that could be used to modulate the immune response in vivo, in order to improve the efficacy of these biocontrol agents. We expect that our continued efforts to understand the mechanisms by which steroids regulate the insect immune system will lead to understanding of these control points.
Impacts
In recent years our work, and that of other labs, has helped to define several key genes that encode enzymes in the biosynthetic pathway for the insect steroid hormone 20-hydroxyecdysone (20HE), but the regulatory relationships and the mechanisms that regulate expression of the gene products are still not well understood. Because insects rely extensively on this steroid system to regulate timing and initiation of key developmental events, the tools that we have developed to regulate 20HE levels in vivo with the EGT enzyme will provide critical insight for development of new approaches for management of pest insect life cycles. We will also use the genetic tools that we have developed to study the role of 20HE in programming the development and/or function of the Drosophila immune system. Our experiments in this area are directly relevant to biological control of insect pests because several strategies for natural control of insect pests rely on infection by fungus,
bacteria and other insect parasites or pathogens. The insect immune response is the first line of defense against pathogen attack. To improve the effectiveness of these pest control strategies, it is important to understand how development of the capacity for an immune response is regulated in vivo. As we learn more about this point, we plan to directly test whether alteration of host ecdysteroid levels by infection with baculovirus capable of reducing host ecdysteroid levels might act synergistically to enhance the effectiveness of introduced insect pathogens by weakening the host insect immune response.
Publications
- Kiger, J.A., Natzle, J.E., Kimbrell, D.A., Paddy, M.R., Kleinhesselink, K., Green, M. 2006. Tissue remodeling during maturation of the Drosophila wing. Developmental Biology, in press.
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Progress 01/01/05 to 12/31/05
Outputs
Steroid hormones play a critical role in the development of arthropods and many other organisms. The insect steroid, 20-hydroxyecdysone (20HE), initiates the larval molts and regulates many of the developmental events that occur during insect maturation and metamorphosis. During the past year we made progress on our research objective to develop a regulatable system for reducing ecdysteroid levels in vivo by ectopic expression of baculovirus ecdysteroid glucosyl transferase (EGT). During previous project years we had placed the baculoviral EGT gene under the control of the yeast UAS (Upstream Activating Sequence) and successfully produced several transgenic Drosophila lines with stable insertions of UAS:EGT. This gene can be temporally regulated by expression of the yeast GAL4 transcription factor controlled by specific Drosophila promoters. Using this system we have shown that EGT expression driven by GAL4 during embryogenesis or during late larval periods results in
a developmental block with a phenotype reminiscent of that of reduced 20HE. In situ hybridization during embryogenesis and RNA-blot analysis verified that the induction of steroid-inducible transcripts at these time periods is specifically blocked when EGT activity is induced. We have shown that induction of EGT activity at different points during pupal development can be used to manipulate 20HE responses at these times, providing previously unavailable versatility in altering hormone dependent events. We are in the process of determining the time course and duration of the reduction in 20HE-dependent responses upon expression of the EGT enzyme. In conjunction with our earlier work, these analyses provide confirmation that these genetic lines will be useful tools for manipulating the ecdysone response in vivo. In addition, we have carried out preliminary experiments to investigate the usefulness of the inducible EGT lines, as well as dsRNA-interference lines that reduce 20HE receptor
activity (produced in the lab of C. Antoniewski), to study the role of steroid hormones in regulation of development and function of the insect innate immune system. As the insect immune system is the first line of defense against natural pathogens that can be used as biocontrol agents, it is important to search for control points that could be used to modulate the immune response in vivo. We have found that reduction of the steroid response capability specifically in the hematopoietic system (which produces circulating hemocytes that mediate the immune response) leads to pupal lethality and the production of melanotic masses that are hallmarks of a defective immune response. We will use the new tools that we have developed to manipulate the steroid response in vivo to gain a deeper understanding of how steroid hormones control the host insect response to pathogens.
Impacts
In recent years our work, and that of other labs, has helped to define several key genes that encode enzymes in the biosynthetic pathway for the insect steroid hormone 20-hydroxyecdysone (20HE), but the regulatory relationships and the mechanisms that regulate expression of the gene products are still not well understood. Because insects rely extensively on this steroid system to regulate timing and initiation of key developmental events, the tools that we have developed to regulate 20HE levels in vivo with the EGT enzyme will provide critical insight for development of new approaches for management of pest insect life cycles. We will also use the genetic tools that we have developed to study the role of 20HE in programming the development and/or function of the Drosophila immune system. Our experiments in this area are directly relevant to biological control of insect pests because several strategies for natural control of insect pests rely on infection by fungus,
bacteria and other insect parasites or pathogens. The insect immune response is the first line of defense against pathogen attack. To improve the effectiveness of these pest control strategies, it is important to understand how development of the capacity for an immune response is regulated in vivo. As we learn more about this point, we plan to directly test whether alteration of host ecdysteroid levels by infection with baculovirus capable of reducing host ecdysteroid levels might act synergistically to enhance the effectiveness of introduced insect pathogens by weakening the host insect immune response.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The insect steroid hormone 20-hydroxyecdysone (20HE) coordinates key aspects of the growth and development of holometabolous insects, including embryonic development, larval molts and transformation of the larva into the adult during metamorphosis. Experimental manipulation of hormone levels would allow investigation of hormone dependent events at the molecular and cellular level, and would provide opportunities for new avenues for regulating the life cycle of agricultural pests as well. During the initial years of this project we have succeeded in creating transgenic Drosophila lines that express the baculovirus EGT gene (ecdysteroid glucosyl transferase) under regulation of a yeast UAS (upstream activating sequence). The EGT enzyme inactivates endogenous steroids by sugar conjugation. In our system, it can be regulated according to the expression pattern of the yeast GAL4 transcription factor coupled to various Drosophila promoters. We previously showed that
embryonic and late larval expression of EGT causes a developmental arrest similar to that produced by reduction in 20HE receptor levels. Analysis of steroids following EGT expression (in collaboration with L. Gilbert, Univ. North Carolina) shows a reduction in active steroid levels. During the past year we have investigated the use of heat shock GAL4 induction to activate EGT expression at precise, experimentally controllable times during larval and pupal development. We have established that heat shock induction in late third instar blocks initiation of metamorphosis as expected. We have verified by Northern blot analysis that heat shock induced EGT expression during pupal development blocks subsequent hormone-dependent gene transcription. We have also adapted and optimized a biochemical assay for EGT activity to establish that expression of our EGT transgene generates an enzymatic activity in our larval extracts that is capable of conjugating radiolabeled sugars onto ecdysone. We
are currently using this biochemical assay to monitor the kinetics of EGT induction, and the persistence of enzymatic activity following heat shock. We will then be able to use this tool to analyze in more detail than previously possible the role of the steroid in regulating specific events during pupal development. We are also working on modifying the structure of the EGT gene to remove the coding region for the signal peptide that leads to enzyme secretion. This would allow us to generate a tissue-localized EGT that would be very useful for analyzing ecdysone requirements for specific tissue function without perturbing the overall physiology of the insect. We will also now be able to manipulate hormone levels in vivo in order to study the effect on mechanisms that control normal hormone biosynthesis and degradation.
Impacts
Insects are an agriculturally important pest for food crops as well as important vectors for diseases of humans and animals. The insect steroid hormone that controls key aspects of insect development is potentially an important intervention point for insect control, since this particular hormone is not found in vertebrates or humans. We have developed a system that allows experimental manipulation of insect hormone levels in vivo with a precision not previously possible. This will be a valuable tool for analysis of the mechanisms that regulate hormone biosynthesis and homeostasis and for furthering our understanding of hormone dependent events during the insect life cycle. This knowledge may facilitate the design of effective strategies for insect pest regulation.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The growth and development of holometabolous insects, those that undergo a complete metamorphosis, is coordinated by changes in the concentration of the insect steroid hormone, 20-hydroxyecdysone. We are using the genetically tractable insect model system, Drosophila melanogaster, as a tool to learn more about the molecular and developmental mechanisms that control steroid-dependent events in insect development. The goal of this project is to identify genes that regulate insect steroid levels during development and to test experimental strategies for modulating ecdysteroid levels in vivo. During the initial years of this project we created transgenic Drosophila lines that express the baculovirus EGT gene (ecdysteroid glucosyl transferase) under regulation of a yeast UAS (upstream activating sequence). This enzyme, which inactivates endogenous steroids by sugar conjugation, can thus be regulated by controlled expression of the yeast GAL4 transcription factor. During
this report period we have established that activation of EGT during early embryogenesis with several different promoter/GAL4 fusions leads to a developmental arrest similar to that produced by decreased levels of ecdysteroid receptors. In addition, transcription of known ecdysteroid-dependent genes in the embryo is abolished. Promoter/GAL4 fusions that direct expression at the close of larval development result in arrest at or just after pupariation, the predicted result for reduction of ecdysteroid levels at the initial phases of metamorphosis. We have measured the levels of circulating steroids at these two periods in EGT-expressing and control animals to establish that EGT expression reduces active steroid concentrations. We are currently carrying out an analysis of the effect of heat-shock regulated EGT expression at defined points during pupal development to clarify the nature of hormone fluctuations during metamorphosis and to define the pupal developmental events that are
dependent on elevated steroid titers. Our work this year has established the EGT system as a useful and reliable tool for in vivo modulation of hormone titers. During the remaining years of the project we plan to initiate efforts to identify the endogenous genes that inactivate ecdysteroids during the normal course of development. We will also investigate the feasibility of removing the coding region for the signal peptide from the secreted enzyme to produce a tissue-localized EGT that would be useful for analyzing the requirements for ecdysteroids in specific tissues without perturbing the overall hormone concentrations.
Impacts
Regulation of insect steroid hormone biosynthesis and degradation is poorly understood. Our progress in experimental manipulation of ecdysteroid levels in via EGT expression provides a valuable new tool to elucidate mechanisms that regulate hormone levels and to analyze steroid-dependent steps in the insect life cycle. This knowledge can be used to design new strategies for insect pest regulation.
Publications
- Harris, D., Orme, C., Kramer, J., Namba, L., Champion, M., Palladino, M.J., Natzle, J., Hawley, R.S. (2003. A deficiency screen of the major autosomes identifies a gene (matrimony) that is haplo-insufficient for achiasmate segregation in Drosophila oocytes. ) Genetics 165(2): 637-52.
- Natzle, J.E., Miller, R., Losev, Y. (2003). Ecdysteroid UDP-Glucosyl Transferase: A new tool to manipulate ecdysone levels in vivo. A. Dros. Res. Conf. 44: a177.
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Progress 01/01/02 to 12/31/02
Outputs
Insect growth and development is regulated by the circulating concentration of the steroid hormone 20-hydroxyecdysone, which is dependent on a finely tuned balance between biosynthesis and degradation. Regulation of hormone turnover is poorly understood at the molecular level but provides a potentially useful new control point for modifying the insect life cycle. The goal of this project is to identify genes that regulate the levels of insect ecdysteroids during development and to investigate experimental strategies for manipulating ecdysteroid levels using Drosophila melanogaster as a genetic model system. To that end, we have developed a transgenic Drosophila line that expresses a baculoviral UDP-ecdysteroid glucosyl transferase gene (EGT) in an experimentally controllable manner. We used PCR to amplify the EGT gene from the baculovirus genome and then placed it in a Drosophila P-element transformation vector, under the control of a yeast UAS (upstream activating
sequence). We recovered several independent transformant lines that are viable in the absence of EGT activation. Lethality results when transcription of the UAS-EGT gene is activated by expression of the yeast GAL4 transcription factor under the regulation of a large number of different Drosophila promoters. We are currently confirming that lethality is associated with reduction in circulating ecdysteroid titers and absence of ecdysteroid-dependent gene transcription. We are using a heat-shock inducible GAL4 line to investigate the developmental consequences of ecdysteroid depletion at early (embryonic) and later (larval and pupal) developmental periods. During the next year we plan to modify the EGT sequence to remove the signal peptide that targets the enzyme for secretion into the hemolymph. We will produce transgenic lines with this modified EGT that will inactivate steroids within specific tissues without resulting in a general systemic depletion. This will allow us to
investigate more precisely the developmental and physiological requirements for ecdysteroids in specific tissues. We will also initiate approaches to identify the endogenous gene(s) that inactivate ecdysteroids to control hormone levels during the normal course of development.
Impacts
Regulation of insect steroid biosynthesis and degradation is poorly understood. A genetic approach allows identification of genes essential for steroid metabolism. EGT lines that allow experimental regulation of ecdysteroid levels at specific times and tissues will provide an important new tool for insect endocrine research. This work will ultimately suggest new strategies for insect pest control.
Publications
- Natzle, J.E., Clark, K.E., Vesenka, G.D., Robertson, J.P. (2002). Independent regulation of the IMP-E1 gene by ecdysone and the Jun Kinase pathway. A. Dros. Res.Conf. 43: 364A.
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Progress 01/01/01 to 12/31/01
Outputs
Steroid hormones regulate the development of insects and many other eukaryotes by binding to and activating nuclear receptors to influence gene transcription patterns. Hormone titers are maintained by a balance between biosynthesis and degradation within the organism. The goal of this project is to identify genes that regulate the levels of insect ecdysteroids during development and to investigate experimental strategies for manipulating ecdysteroid levels using Drosophila melanogaster as a model system. During the past year we have focused on development of a transgenic Drosophila line that will express a baculoviral UDP-ecdysteroid glucosyl transferase gene (EGT) in an experimentally controllable manner. The EGT enzyme adds a sugar to ecdysteroid hormones, leading to their inactivation and clearance from the hemolymph. We have used PCR to amplify the EGT gene from a baculovirus genome and have inserted it into a Drosophila P-element transformation vector, under the
control of a yeast UAS (upstream activating sequence). We are currently analyzing potential transgenic lines. We will activate transcription of the UAS-EGT by genetically introducing a chromosome carrying the yeast GAL4 transcription factor, which activates transcription through the UAS, under the regulation of various Drosophila promoters. We will monitor ecdysteroid levels and developmental outcomes following activation of EGT transcription in our transgenic lines. We are also modifying the EGT sequence to remove the signal peptide that targets the enzyme for secretion into the hemolymph. This will allow us to inactivate ecdysteroids in specific tissues by directing EGT synthesis there without producing a general depletion of the hormone. This should provide an important tool for understanding tissue specific responses to the steroid. We will test this modified EGT construct by expressing it in a well-characterized ecdysteroid target tissue such as the larval salivary gland. We will
demonstrate its utility by expression in hemocytes, which are important mediators of insect immune responses, phagocytosis and tissue remodeling during metamorphosis, but are poorly characterized with respect to regulation by steroids.
Impacts
Regulation of the insect steroid biosynthesis pathway is poorly understood. A genetic approach allows identification of genes essential for steroid metabolism. EGT lines that allow experimental regulation of ecdysteroid levels at specific times and tissues will provide an important new tool for insect endocrine research. This work will ultimately suggest new strategies for insect pest control.
Publications
- Kiger, J.A., Natzle, J.E., Green, M.M (2001). Hemocytes are essential for wing matuation in Drosophila. Proc. Natl. Acad. Sci. USA 98, 10190-10195.
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Progress 01/01/00 to 12/31/00
Outputs
Steroid hormones play a critical role in coordinating development in insects as well as in other eukaryotic organisms. The goal of this project is to identify new genes involved in the maintenance of insect ecdysteroid hormone levels and to investigate strategies for experimental manipulation of hormone levels during development. We recently completed and published the initial characterization of the disembodied locus (dib), a p450 steroid hydroxylase that is essential for 20-hydroxyecdysone synthesis and proper embryonic development in Drosophila. During the past year we have initiated work on identification of the genes associated with three other mutations that perturb ecdysteroid production or response: shroud (sro), spook (spo) and phantom (phm). This work is facilitated by the recently completed Drosophila genome sequence. We have also initiated work on production of a transgenic fly line containing a regulatable EGT gene. At the time of activation, this
ecdysteroid glucosyl transferase activity would reduce endogenous levels of steroid and should allow experimental perturbation of the normal ecdysteroid response. We have PCR-amplified the EGT gene from a baculovirus genome and have inserted it into a vector from which we can construct a P-element insertion construct. We have also characterized a number of deletions near an endogenous Drosophila EGT gene to identify those that remove all or part of the locus. We are currently analyzing the phenotypes of these mutants to gain insight into the possible functions of members of the EGT gene family in Drosophila.
Impacts
Little is known about the production and role of steroid hormones in insect embryogenesis. We are among the first to identify specific genetic loci involved in embryonic ecdysteroid production in insects. This information should ultimately provide new opportunities for insect pest control with biorational methods.
Publications
- Chavez, V.M., Marques, G., Delbecque, J.P., Kobayashi, K., Hollingsworth, M., Burr, J., Natzle, J.E. and O'Connor, M.B. (2000) The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127, 4115-4126.
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Progress 01/01/99 to 12/31/99
Outputs
Periodic release of the insect steroid hormone 20-hydroxyecdysone (20-HE) coordinates a number of important events during insect development via transcriptional activation of specific sets of target genes. The objectives of this research project include (1) identification of genetic loci that are important for the production of the active steroid hormone during embryogenesis of the model insect Drosophila (2) utilization of mutations in these loci to determine which steps in embryonic development are 20-HE-dependent. In collaboration with the O'Connor lab, we recently identified the disembodied locus (dib) as a steroid hydroxylase whose activity is required for normal embryonic development and for expression of ecdysone-dependent genes during embryogenesis. Radioimmunoassays to determine the levels of total ecdysteroids and 20-HE (in collaboration with J.-P. Delbecque) in a set of six mutants that share the inability to produce a first instar cuticle, revealed that
both steroid classes are severely depressed in dib and spo (spook) homozygotes, confirming a role for dib and identifying spo as another potential locus required for embryonic production of ecdysteroid hormones. We are also constructing germline transformation vectors carrying an inducible copy of the baculovirus ecdysteroid UDP-glucosyltransferase gene, which should allow us to deplete endogenous 20-HE levels in a controlled manner.
Impacts
Little is known about the production and role of ecdysteroids in insect embryogenesis. We are among the first to identify specific genetic loci involved in embryonic ecdysteroid production in insects. This information should ultimately provide new tools for analysing pathway biochemistry and potentially identifying new regulation points for biorational insect pest control efforts.
Publications
- Chavez, M., Delbecque, J.P., Marques, G., Kobayashi, K., Burr, J., Hollingsworth, M., Natzle, J. and O'Connor, M.B. 2000. The Drosophila diembodied gene controls epidermal and midgut morphogenesis and codes for a cytochrome P450 enzyme required for ecdysone biosynthesis. In preparation.
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Progress 01/01/98 to 12/01/98
Outputs
The insect steroid hormone, 20-hydroxyecdysone (20-HE), regulates developmental events during the life cycle of holometabolous insects. The role of the hormone during embryonic development and the mechanism of its production and release during mid-embryogenesis are poorly understood. The objective of this research project is to identify the targets of hormone action during embryogenesis in Drosophila by analyzing mutations that block the normal release of ecdysteroids in embryos. We recently identified the disembodied (dib) locus as a candidate for a gene involved in regulating embryonic ecdysone release based on its cuticle-deficient phenotype and the absence of normal expression of known ecdysone-target genes in homozygous mutant animals. In collaboration with the laboratory of M. O'Connor, the dib gene has been isolated and is predicted to encode a steroid hydroxylase. Homozygous mutant animals produce reduced levels of 20-HE during embryogenesis. We are currently
testing whether injecting 20-HE into mutant embryos partially or fully rescues the developmental defects. Because many developmental mechanisms are conserved among holometabolous insects, the information we learn about the production and function of steroid hormones during Drosophila embryogenesis should be applicable to other insects.
Impacts
(N/A)
Publications
- , M., KOBAYASHI, K., NATZLE, J., O'CONNOR, M. 1999. The disembodied locus encodes a steroid hydroxylase necessary for normal gene expression and embryonic development in Drosophila melanogaster. 40th Annual Drosophila Research Conference.
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Progress 01/01/95 to 12/30/95
Outputs
Critical developmental events in the life cycle of holometabolous insects are controlled by circulating hormones such as the steroid hormone, 20- hydroxycdysone, and the isoprenoid hormone, juvenile hormone. Our research objective was to utilize molecular responses to ecdysone in imaginal discs (IMP-E1 gene transcription) as an assay system to determine the mechanisms by which these hormones interact to regulate gene expression during development in insects. We established that the hormone-dependent induction of imaginal disc ecdysone-responsive mRNAs is regulated at the level of transcription and includes both primary responses (including IMP-E1) and secondary hormone responses. PCR-based cloning of the 5' end of the IMP-E1 cDNA identified a single transcriptional start site that is utilized during metamorphosis and embryogenesis. While transcriptional regulation plays the primary role in induction of the IMP-E1 mRNA, message instability may contribute to the dynamic
changes in spatial expression patterns in imaginal discs during morphogenesis. Based on the expression profile and a dissociated embryonic cell assay system we conclude that the IMP-1 gene is ecdysone-responsive in embryogenesis. Unexpectedly, IMP-E1 is not inducible in an imaginal disc cell line (W8), although some other ecdysone-responsive genes can be induced. IMP-E1 induction is significantly reduced to about 20% normal levels in imaginal discs removed from the overgrowth mutant, 1(1)d.1g.1.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/94 to 12/30/94
Outputs
Embryonic development, molting and metamorphosis in holometabolous insects are controlled by circulating hormones such as the steroid hormone 20-hydroxyecdysone (20-HE) and the isoprenoid juvenile hormone (JH). Our objective is to understand how insect development is regulated by transcriptional gene activation resulting from the interactions of these hormones with the genome. The Drosophila melanogaster 20-HE responsive locus IMP-E1 is being studied as a model system. During this reporting period the nucleotide sequence of the IMP-E1 cDNA has been completed and reveals that the product is expected to be a secreted protein of at least 163 kD, containing three amino terminal EGF-like repeats followed by a short mucin-like domain. Seven mutations in the IMP-E1 locus have been isolated including severe alleles that cause embryonic lethality due to failure to complete the morphogenetic movements required for dorsal closure, head involution and segment formation and less
severe pupal lethal alleles that produce a few adults, exhibiting deformed imaginal disc derivatives. These phenotypes suggest that the IMP-E1 product is involved in epithelial morphogenesis. The expression of this locus in embryos is apparently dependent on embryonic pattern formation products, based on the spatial expression domains in embryos and on alterations in IMP-E1 expression in pattern mutant backgrounds.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/93 to 12/30/93
Outputs
Circulating hormones such as the steroid 20-hydroxecdysone (20-HE) and the isoprenoid juvenile hormone (JH) regulate embryonic development, molting and metamorphosis in holometabolous insects such as Drosophila melanogaster. The objective of this proposal is to understand how these biochemical signals interact to regulate the expression of specific genes during insect development. During the reporting period the 5' end of the 20-HE responsive locus IMP-E1 was cloned using reverse transcription-PCR methods. This 5' terminus has been mapped on genomic DNA containing the gene, allowing initiation of efforts to identify the important regulatory regions that control gene expression. Transcription apparently initiates at the same location during the embryonic and the imaginal expression periods. This work will allow a comparison of function of these regions in the presence and absence of JH. Two imaginal disc cell lines were tested to determine the induction characteristics
of 20-HE responsive genes in these cells in culture. Preliminary results suggest that specific 20-HE responsive promoters that are hormone-inducible in cultured intact imaginal discs are differentially responsive to 20-HE in these continuously proliferating cell lines. We will test the hypothesis that a proliferative state is antagonistic to steroid-induced differentiation in insect tissues, as has been suggested for some vertebrate systems.
Impacts
(N/A)
Publications
- VESENKA, G.D. 1993. An Analysis Of The Expression And Regulation Of The Ecdysone-Inducible Genes, IMP-E1 and IMP-L1, During Drosophila Embryogenesis And Pupariation. Ph.D. Thesis. University of California, Davis, 159p.
- NATZLE, J.E. and VESENKA, G.D. 1994. Isolation and organ culture of imaginal tissues. Methods Cells Biology (In press).
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Progress 01/01/92 to 12/30/92
Outputs
Circulating hormonal signals control important developmental events during the life cycle of holometabolous insects, including initiation of each molt and metamorphosis. The nature of the response to one of these hormones, 20-hydroxyecydysone, appears to be regulated by the presence or absence of a second hormone, juvenile hormone. The objective of this project is to use molecular markers for the ecdysone response in DROSOPHILA to understand how juvenile hormone modulates this signaling mechanism. During the reporting period, the imaginal disc and embryonic spatial expression patterns of two ecdysone-inducible test genes (IMP-E1 and IMP-L1) have been determined. The transcription patterns indicate that regulatory input must be provided by a spatial pattern regulation system in addition to the hormonal signal because expression is not uniform throughout the imaginal disc or the embryonic epidermis. Seven mutant alleles that may map within the IMP-E1 locus have been
isolated. Analysis of these mutations is in progress to determine whether any of them affect regulatory regions of the gene. Two cell lines derived from imaginal discs (kindly provided by Dr. M. Milner) are being tested to determine the culture conditions required for ecdysone-responsive expression of the test genes.
Impacts
(N/A)
Publications
- NATZLE, J.E. 1993. Temporal regulation of DROSOPHILA imaginal disc morphogenesis: A hierarchy of primary and secondary 20-hydroxyecdysone-responsive loci. Developmental Biology 155:in-press.
- NATZLE, J.E., ROBERTSON, J.P., MAJUMDAR, A., VESENKA, G., ENLOW, B. and CLARK, K. 1992. Sequence and expression of IMP-L1, an ecdysone-inducible gene expressed during DROSOPHILA imaginal disc morphogenesis. Developmental Genet. 13:331-344.
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Progress 01/01/91 to 12/30/91
Outputs
Metamorphosis of holometabolous insects is triggered by an increase in the concentration of the steroid hormone, 20-hydroxyecdysone. The nature of the response to ecdysone depends on the presence or absence of a second hormone, juvenile hormone, with the former case leading to a larval molt and the latter case leading to a pupal molt. The objective is to use the molecular response to ecdysone in DROSOPHILA imaginal discs as an assay system to determine the mechanisms by which juvenile hormone and ecdysone interact to regulate gene expression. During the reporting period the regulatory mechanism by which the model genes are controlled by ecdysone have been determined, in order to appropriately plan experiments designed to monitor the effect of juvenile hormone. It has been established that the test genes, IMP-E1 and IMP-L1, fall into two regulatory classes with respect to induction by ecdysone (IMP-E1, primary response; IMP-L1, secondary response). It has also been
established that induction of these genes is regulated by ecdysone at the level of transcription, rather than by post-transcriptional mechanisms. Experiments designed to determine the location of the 5-prime regulatory regions that initiate transcription from these loci have been initiated.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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