Michael W. Young - US grants

The Notch locus of D. melanogaster is required early in embryogenesis for proper differentiation of the ectoderm. A complex complementation pattern is also found for mutations defining the gene. In an attempt to understnd the molecular basis of Notch function, DNA from the locus has been cloned and characterized in considerable detail. The Notch locus corresponds to a 37-kb transcription unit that generates polyA+ RNA about 11.7 kb in length. Most mutations at the Notch locus appear to alter mRNA coding sequences and therefore should produce aberrant Notch protein. In this proposal we will extend this analysis as follows: 1) Notch locus DNA has been used to generate E. coli hybrid plasmids that synthesize Notch peptides. These will be used to generate antibody to the in vivo Notch protein so that its structure and tissue distribution in mutant and wild-type flies can be assessed. 2) The structure and tissue distribution of Notch RNA in mutant and wild-type flies will be determined in order to decide how several DNA insertions near intron-exon boundaries generate mutant phenotypes. 3) Certain copia-like transposable elements produce tissue-specific, mutant phenotypes by interrupting intervening sequences. The molecular basis for their modulation of Notch locus expression will be explored by determining if the expression of each element corresponds temporally and spatially with suppression of Notch locus activity. We will also attempt to generate revertants of these insertion mutants. 4) The chromosomal intervals adjoining the Notch locus are heavily transcribed, but the functions of these DNA sequences are not apparent from previous genetic analyses. The structures of these transcription units and their precise boundaries with respect to the Notch locus will be mapped. 5) An attempt will be made to transform mutant flies with cloned Notch DNA and its derivatives. 6) A physical analysis of the Notch locus in a distantly related species of Drosophila will be undertaken.

A biological clock is housed within the brain of the fruit fly. In insects, reptiles and mammals these brain-based clocks govern temporal organization at many levels ranging from the regulation of gene activity in highly specialized tissues to overt periodic behaviors such as 24-hour cycles of activity and rest. Few clues exist with regard to the biochemical composition of such biological pacemakers, but clock mutants are available in Drosophila melanogaster, and these offer a means of probing the mechanism in great detail. We will be analyzing the molecular composition and function of the per locus. This gene appears to be expressed in the brain and establishes temporal organization within the fly through the action of a diffusible substance. Our approach will be as follows. 1) DNA corresponding to the per locus will be isolated and mutations defining the gene will be placed on the physical map. 2) Transcribed sequences at the per locus will be identified and related to the genetic and physical maps. 3) Protein coding sequences will be identified at the per locus and will be used to generate per-peptides in Escherichia coli. These peptides will be used to generate antibody which will in turn facilitate the characterization per protein with respect to size and distribution. 4) The possibility that the per locus encodes the humoral factor coupling the pacemaker to the rest of the organism will be tested. 5) An attempt to identify the biochemical form of the pacemaker will start with a search for temporal regulation in the synthesis of per RNA and protein.

The per locus plays a central role in the organization and function of the Drosophila biological clock. This gene has been mapped to a 7-kb DNA segment that codes for a 4.5 kb polyA+ RNA. The complete DNA sequence of the per locus has been determined, and this has allowed us to predict the primary structure of the encoded protein. In this proposal, we will extend our analysis as follows: 1) per RNA and protein titres will be assessed in transformed strains producing rhythms with different periodicities. 2) Certain per locus mutations will be physically mapped, and new mutations will be constructed in vitrok and tested for biological activity following transformation. 3) Antibody raised to per peptides will be used to test the hypothesis that per protein is a proteoglycan associated with cell surfaces. 4) The functions of several per-homologous Drosophila genes will be analyzed to determine if they have a role in the Drosophila clock. 5) The per locus is homologous to DNA from vertebrates. Cloned cDNAs from mouse brain that are homologous to per will be examined to see if they encode per-homologous proteins. 6) We will determine how large a fraction of the Drosophila genome is expressed according to a circadian rhythm, and how the per locus influences these rhythms.

For several decades it has been known that the Drosophila neurogenic genes are required early in development for proper differentiation of the ectoderm. In mutant flies, cells that should form hypoderm (skin) instead differentiate as nervous tissue. Recently we have discovered a similar role for the neurogenic genes in mesodermal differentiation: Loss of any of the genes causes hypertrophy of certain cell groups in mesoderm. From sequence analysis and biochemical and cellular studies, the best characterized of the neurogenic genes, Notch, produces a large, unprocessed, transmembrane protein that is similar to mammalian clotting and growth factors. The structure of the protein suggests action in cell-cell communication in ectoderm and mesoderm. In this proposal we will extend an analysis of the neurogenic genes and their products as follows: 1) Embryos genetically mosaic for Notch will be examined to determine whether mesodermal development is linked to autonomous action of the gene in that germ layer, or inductive interactions between mesoderm and ectoderm. 2) We will search for hypotrophied cell groups in mesoderm of neurogenic mutants using new mesodermal cell markers. We will also search for a possible role for the neurogenic genes in endoderm development. 3) The effects of amino acid substitutions in the different EGF-elements of Notch will be assessed in transgenic Drosophila. 4) A map of Notch and Delta protein segments governing interaction of these molecules will be produced in vivo and in vitro. We will search for additional molecular targets for interaction with the Notch and Delta proteins. 5) A Drosophila homologue of the neuromodulin/neurogranin gene family will be characterized at the genetic, molecular and cellular levels.

We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Homologues of genes initially characterized in the fly, have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. A central component of the fly clock is a feedback circuit in which two clock proteins, PERIOD (PER) and TIMELESS (TIM), repress their own transcription. Temporal delays in this feedback promote oscillatory gene expression. We have recently discovered novel cellular features controlling one such delay. Additional studies have identifed new genes and proteins affecting periodicity of the circadian clock. In this proposal we will examine the following: (1) PER and TIM appear to be physically modified in response to their interaction in the cytoplasm, allowing their subsequent, independent nuclear accumulation. We will identify and characterized modifications of these proteins that are associated with this regulation. (2) We will determine whether a newly discovered, PER/TIM cytoplasmic interval timer contributes to temperature compensation of the circadian clock. (3) We will conduct a high-throughput screen for new genes and proteins regulating the timed nuclear accumulation of PER and TIM in cultured cells. (4) A locomotor activity screen involving several hundred transgenic RNAi stocks has shown that reduction of a specific karyopherin substantially lengthens the period of the fly clock. The molecular pathway underlying this protein's contribution to rhythmicity will be explored in flies and S2 cells. (5) Cryptochrome (CRY) has a key role in the light-dependent degradation of TIM. We have produced new mutations that affect physical interactions of the CRY C-terminal tail with the CRY photolyase homology domain (PHD). Preliminary studies indicate that some of these mutations alter CRY stability only on exposure to light. We will determine whether light induces dissociation of the CRY C-terminal tail and the PHD. RELEVANCE (See instructions): Candidate gene approaches, originating in the forward genetic screens of Drosophila, allowed mutant orthologs of human PERIOD protein and casein kinase 1 to be connected to inborn errors of sleep. The early functional studies of these genes and proteins in Drosophila have also been used as the basis for exploring specific mechanisms underlying aberrant patterns of human sleep. We believe our proposed genetic, biophysical, and biochemical studies of Drosophila's circadian clock will continue to reveal new principles of organization and function that promote an understanding of human circadian rhythms.

DESCRIPTION (Applicant's Abstract): Impressed by the results of a particularly successful and oversubscribed international meeting on Molecular Clocks held at the Juan March Foundation in May of 1998, a Keystone meeting on the subject is proposed for 2001. The sub-areas to be represented include (1) Molecular and genetics studies of circadian behavioral rhythms in animal systems, with emphasis on newly identified genes, the strong homologies among clock genes in the animal kingdom, and their mechanisms of action. (2) The molecular pathways that afford resetting of clocks and the sleep/wake cycle in response to light. Our understanding of the molecular mechanisms associated with phototransduction and clock gene responses has grown significantly in just the past year. The role of cryptochromes, which act as circadian photoreceptors in some systems, is now widely studied. (3) Regulation of wake/sleep behavior. Biochemical strategies for conveying signals from molecular clocks to timed behavior have been clarified in the past year. (4) Genetic control of human wake/sleep cycles. The first hereditary variations in human circadian rhythms were reported last year as well as a partial molecular description of narcolepsy in mammalian models. (5) Plant, fungal, and bacterial clocks, and modeling molecular oscillators. Non-animal model systems continue to provide some of our greatest insights into biochemical mechanisms underlying circadian [clocks].

We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Orthologs of genes initially characterized in the fly,have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. Here we propose three classes of interdisciplinary investigations of the Drosophila clock. (1) We will conduct collaborative structural studies that can help us determine how specific regulatory actions, previously recognized genetically and biochemically, are performed by certain domains of the PER protein. Does the structure of PER indicate how it interacts with TIM and DBT? Do such data suggest how TIM suppresses PER's phosphorylation by DBT? Does a putative LOV domain near the N-terminus of PER possess a flavin binding domain? (2) We will generate new mutations for the analysis of vital clock genes by Conditional Protein Splicing. Our studies of hyper- and hypo-morphic mutations of GSK-3, dbt, vri, and Pdp-1 indicate that each is a key component of the Drosophila clock. However, because null mutations of these genes are lethal, it has been difficult to determine their full effects on the clock. We are developing a form of chemical genetics in which small diffusible molecules will reversibly control the presence of each vital protein in living flies. (3) We will produce new microarray and statistical approaches to clarify features of rhythmic genome activity and to determine if flies use a single molecular mechanism to generate all circadian rhythms. To begin to link the clock to specific behavioral and physiological outputs, we will investigate a novel set of genes that are regulated by a paired action of light and the circadian clock.

Our prior work has focused on the molecular mechanisms underlying Drosophila[unreadable]s circadian rhythms. Orthologs of genes initially characterized in Drosophila have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. Here we prose four classes of interdisciplinary investigations of the fly[unreadable]s rhythmic behavior. (1) We will extend a classical genetic screen (chemical mutagenesis) for mutations conferring aberrant activity/rest patterns. An X-chromosomal screen produced several mutant lines with effects on either sleep duration or phase. In future work, we will determine the identity of the affected genes, the cellular and molecular patterns of their expression, and will start a comparable screen on chromosomes 2 and 3. (2) We will characterize a novel, reduced- sleep mutant insomniac. These consistently rest for ~400 min/day, nearly 9 hours less than wild type. We will determine where insomniac is expressed within the brain, wheterh cells expressing insomniac have other roles in organizing sleep, and when insomniac function is required to determine sleep duration. (3) We have discovered that when Drosophila are maintained in a photocycle, period mRNA undergoes daily cycles of polyadenylation. The time of polyadenylation can be delayed by prolonged exposure to light. We will determine whether these responses provide a novel mechanism for day- length (seasonal) measurement.

DESCRIPTION (provided by applicant): Our prior work has focused on the molecular mechanisms underlying Drosophila's circadian rhythms. Orthologs of genes initially characterized in Drosophila have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. Here we propose three classes of interdisciplinary investigations of the fly's rhythmic activity/rest behavior. (1) We wil use elav-Gal4 to drive individual, UAS-RNAi transgenes from two extensive libraries in a search for novel genetic pathways regulating sleep. Our objective is to test the full complement of Drosophila's protein-coding genes using ~19,000 RNAi lines. (2) We will extend our studies of a novel, reduced-sleep mutant, insomniac. We will determine whether insomniac regulates sleep in an active/dynamic manner, or whether it regulates a developmental pathway that is essential for wild type levels of sleep. We will test the hypothesis that Insomniac functions as a substrate-specific adapter for the Cul3 ubiquitin ligase complex, and will use a variety of biochemical and molecular approaches to identify a target substrate(s). (3) We will further define the role of a small group of cell cycle genes, CycA, its regulator (Rca1), cdk2 and cdc42, in the regulation of sleep. As constitutive activation of a cluster of neurons expressing cell cycle genes induces excess sleep, we will isolate these cells by flow cytometry to discover patterns of gene expression that may underlie the contribution of these cells to sleep.

Project Summary/Abstract Our genetic screens in Drosophila previously identified several mutations with strong effects on patterns of sleep. Recently we found that three of these genes, insomniac, cullin3 and nedd8, are expressed in the blood-brain-barrier-forming subperineurial glia of the fly, and that the morphological and biophysical properties of the barrier are altered in inc, cullin3, and nedd8 mutants. In our proposed studies we will test the effects of classical barrier mutants on sleep, evaluate barrier function across the entire range of available sleep mutants and in aged vs young flies, and explore evidence for regulatory interactions among sleep and classical barrier-regulating genes. Most Drosophila sleep mutants severely reduce longevity. We will determine whether mutations that increase longevity also improve barrier function and sleep duration in sleep mutants. In a second branch of our proposed research we will examine the role of circadian clock genes in commonly encountered disorders of human sleep. We recently discovered a mutation of the circadian clock gene CRY1 that is associated with a form of delayed sleep phase disorder (DSPD) that affects ~1 in 100 individuals worldwide. The results of our study suggest a novel approach for exploring the heritability of similarly common sleep disorders: Predictive algorithms will be applied to several large human exome databases to select candidate circadian variants for cellular phenotyping. Prevalent alleles that are associated with altered circadian rhythmicity in cell culture assays also will be studied by behavioral phenotyping of carrier subjects identified by collaborators at Bilkent University in Ankara, Turkey. Disordered sleep is often accompanied by chronic diseases including diabetes, obesity, or certain mood and anxiety disorders. Although causality in such instances has been difficult to establish by traditional approaches, we will employ deep physiological and behavioral phenotyping of individuals sharing specific genetic variations affecting sleep in tests for linkage to specific co-morbidities. These studies may significantly enrich our understanding of biological pathways regulated by circadian clocks in humans as well as fundamental disease etiologies.

Project Summary/Abstract Genetic screens in Drosophila have identified mutations that significantly reduce both night- time and daytime sleep. Genes affected by these mutations are expressed in the blood-brain- barrier-forming subperineurial glia of the fly and alter the morphological and biophysical properties of the barrier. We have discovered novel genetic interactions among some of these mutations: surprisingly, certain mutant combinations restore both sleep and blood-brain-barrier function. We propose further studies that could reveal molecular pathways connecting their gene products and clarify their contributions to sleep and barrier function. We discovered that in wild type Drosophila the blood-brain barrier opens and closes with a rhythm that requires a circadian clock. We have also found that barrier permeability is closely connected to sleep need: sleep deprivation opens the barrier, but rebound sleep closes it. What is being exchanged across the barrier in such a dynamic fashion? Nervous system function is protected by a steep concentration gradient of K+ separating the haemolymph and brain. In our proposed studies we will develop tools to quantify K+ flux across the blood brain barrier with high temporal resolution in living flies. Are episodes of sleep and wakefulness correlated with these ion exchanges? Do such measurements reveal features of wake/sleep behavior that are not evident using standard locomotor activity monitoring? Our studies have also shown that sleep mutants reduce lifespan, but in a fashion that can be reversed by time-controlled access to food. These effects require a circadian clock and we will determine which tissues are responsible for this response and whether lifespan restoration depends on sleep recovery. Chronic exposure to psychogenic stressors can have profound, long-lasting effects on both physical and mental health and is often accompanied by a profound loss of sleep. Chronic social isolation provides a means by which a psychogenic stressor can be easily applied for an extended period, and we observe significant reductions in total sleep, day-time sleep, and night-time sleep in isolated flies when compared to sleep in siblings that are group reared. To search for genetic pathways that might respond to isolation-induced stress and depress sleep, comparative RNAseq assays were performed using Drosophila heads collected from group- reared flies, or from flies stressed through chronic isolation. Among the most highly responding genes are those thought to regulate appetite. These map to a small neuronal circuit which we will further characterize to determine its possible role in isolation-induced stress responses affecting sleep and hunger.