Neurons that make flies sleep

Sleep is known to be regulated by both intrinsic (what time is it?) and environmental factors (is it hot today?). How exactly these factors are integrated at the cellular level is a hot topic for investigation, given the prevalence of sleep disorders. Researchers in the Rosbash and Griffith labs are pursuing the question in the fruit fly Drosophila melanogaster, to take advantage of the genetic tools in the model system and the excellent understanding of circadian rhythms in the fly.

Like other animals, the fruit fly displays a robust activity/sleep pattern, which consists of a morning (M) activity peak, a middle-day siesta, an evening (E) activity peak and nighttime sleep. M and E peaks are controlled by different subgroups of circadian neurons such as wake-promoting M and E clock cells.

In a paper just published in Nature, Brandeis postdoctoral fellow Fang Guo and coworkers identify a small group of circadian neurons, a subset of the glutamatergic DN1 (gDN1s) cells, which have a critical role in both types of regulation. The authors manipulated the gDN1s activity by using recently developed optogenetics tools, and found activity of those neurons is both necessary and sufficient to promote sleep.

circadian-feedback

The cartoon model illustrates how the circadian neuron negative feedback set the timing of activity and siesta of Drosophila. The arousal-promoting M cells (sLNv) release pigment-dispersing factor (PDF) peptide to promote M activity at dawn. PDF peptide can activate gDN1s, which release glutamate to inhibit arousal-promoting M and E (LNds) cells and cause a middle-day siesta. At evening, the gDN1s activity is reduced to trough levels and release E cell activity from inhibition.

DN1s enhance baseline sleep by acting as feedback inhibitors of previously identified wake-promoting M and E clock cells, making them the first known sleep-promoting neurons in this circadian circuit. It is already known that M cell can activate gDN1s at dawn. Thus the daily activity-sleep pattern of Drosophila is timed by the circadian neuron negative feedback circuitry (see Figure).  More interestingly, by using in vivo calcium reporters, the authors reveal that the activity of the gDN1s is also shown to be sexually dimorphic, explaining the well-known difference in daytime sleep between males and females. DN1s also have a key role in mediating the effects of temperature on daytime sleep. The circadian and environmental responsiveness of gDN1s positions them to be key players in shaping sleep to the needs of the individual animal.

Authors on the paper include postdocs Guo, Junwei Yu and Weifei Luo, staff member Kate Abruzzi, and Brandeis graduate Hyung Jae Jung ’15 (Biology/HSSP).

Guo F, Yu J, Jung HJ, Abruzzi KC, Luo W, Griffith LC, Rosbash M. Circadian neuron feedback controls the Drosophila sleep-activity profile. Nature. 2016.

Fruit flies alter their sleep to beat the heat

Do you have trouble sleeping at night in the summer when it is really hot?

Does a warm sunny day make you want to take a nap?

You are not alone — fruit flies also experience changes in their sleep patterns when ambient temperature is high. In a new paper in Current Biology, research scientist Katherine Parisky and her co-workers from the Griffith lab show that hot temperatures cause animals to sleep more during the day and less at night, and then investigate the mechanisms governing the behavior.

The increase in daytime sleep is caused by a complex interplay between light and the circadian clock. The balance between daytime gains and nighttime losses at high temperatures is also influenced by homeostatic processes that work to keep total daily sleep amounts constant. This study shows how the nervous system deals with changes caused by environmental conditions to maintain normal operations.

Parisky KM, Agosto Rivera JL, Donelson NC, Kotecha S, Griffith LC. Reorganization of Sleep by Temperature in Drosophila Requires Light, the Homeostat, and the Circadian Clock. Curr Biol. 2016.

Data Diving for Genomics Treasure

Laboratories around the world and here at Brandeis are generating a tsunami of deep-sequencing data from organisms large and small, past and present. These sequencing data range from genomes to segments of chromatin to RNA transcripts. To explore this “big data” ocean, one can navigate the portals of the National Computational Biotechnology Institute’s (NCBI’s) two signature repositories, the Sequencing Read Archive (SRA) and the Gene Expression Omnibus (GEO).  With the right bioinformatics tools, scientists can explore and discover freely-available data that can lead to new biological insights.

Nelson Lau’s lab in the Department of Biology at Brandeis has recently completed two such successful voyages of genomics data mining, with studies published in the Open Access journals of Nucleic Acids Research (NAR) and the Public Library of Science Genetics (PLoSGen).   Publication of both these two studies was supported by the Brandeis University LTS Open Access Fund for Scholarly Communications.

In this scientific journey, the Lau lab made use of important collaborations from across the globe. The NAR study employed openly shared genomics data from the United Kingdom (Casey Bergman lab) and Germany (Björn Brembs lab).  The PlosGen study employed contributions from Austria (Daniel Gerlach), Australia (Benjamin Kile’s lab), Nebraska (Mayumi Naramura’s lab), and next door neighors (Bonnie Berger’s lab at MIT).  This collaborative effort has been noted at Björn Bremb’s blog, who has been a vocal advocate for Open Access and Open Data Sharing to improve the speed and accessibility of communicating scientific research.

tidal fly banner

In the NAR study, postdoctoral fellow Reazur Rahman and the Lau team devised a program called TIDAL (Transposon Insertion and Depletion AnaLyzer) that scoured over 360 fly genome sequences publicly accessible in the SRA portal.  Their study discovered that transposons (jumping genetic parasites) formed different genome patterns in every fly strain.  Common fly strains with the same name but living in different laboratories turn out to have very different patterns of transposons. Simply noting “Canton-S” or “Oregon-R” strains are used may not be enough to fully characterize a strain.  The Lau lab hopes to utilize the TIDAL tool to study how expanding transposon patterns might alter genomes in aging fly brains.

animals

The piRNAs from these animals were compared in the PLoS Genetics story

In the PLoSGen study, visiting scientist Gung-wei Chirn and the Lau team developed a novel small RNA tracking program that discovered Piwi-interacting RNA loci expression patterns from many mammalian datasets extracted from the GEO portal.  Coupling these datasets with other small RNA datasets created in the Lau lab at Brandeis, the Lau group discovered a remarkable diversity of these RNA loci for each species. For example, the piRNA genomic loci made in humans were quite distinct from other primates like the macaque monkey and the marmoset.  However, a special set of these genomic loci have been conserved in their piRNA expression patterns, extending across humans, through primates, to rodents, and even to dogs, horses and pigs.

These conserved piRNA expression patterns span nearly 100 million years of evolution, which is quite a long time for these types of loci to be maintained for some likely important function in mammals.  To test this hypothesis that evolution preserved these piRNAs for their utility, the Lau lab analyzed two existing mouse mutations in these loci.  They showed that the mutations indeed affected the generation of the piRNAs, and these mice were less fertile because sperm count was reduced.  The future studies from the Lau lab will explore how infertility diseases may be linked to these specific piRNA loci.

FOXO links stress to the innate immune response in flies

Life is tough. Every living thing is constantly dealing with insults that damage or disrupt homeostasis. At the cellular level these insults, or stresses, come in multiple forms: starvation, oxidative stress, heat shock, radiation damage, and infection. In response to these stresses, organisms have evolved numerous mechanisms to promote survival. Broadly speaking, an insult stimulates various signaling cascades that alter gene expression in the cell.

One way this is achieved is through the “turning on” of transcription factors. One such transcription factor is FOXO, which is activated under many types of stress, both metabolic and environmental. Another way gene expression can be accomplished is the post-transcriptional control of gene expression. An important player of post-transcriptional control is the small RNA pathways composed of the RNA interference (RNAi), micro RNA (miRNA), and PIWI RNA (piRNA) branches. In a recent article from the Marr lab titled “FOXO regulates RNA interference in Drosophila and protects from RNA virus infection”, published in PNAS this November, the authors identify a new connection between both the transcriptional and small RNA mediated post-transcriptional mechanisms that respond to stress.

Screen Shot 2015-11-16 at 9.22.43 AM

RNAi efficiency is enhanced in a dFOXO-dependent manner. For full explanations, see Fig. 2 in Spellberg & Marr (2015)

Using Drosophila as a model system, the authors identify FOXO as a transcription factor that regulates important genes in the small RNA pathways in response to stress. This is the first transcription factor identified to control these genes. Despite being a hot and competitive field for over 15 years, work in small RNA pathways had yet to reveal the transcriptional regulation of the core protein machinery that are involved in small RNA biogenesis and utilization. Under stress conditions, FOXO directly binds the promoters of core small RNA pathway genes, such as Ago1, Ago2, and Dicer 2, leading to increases in their expression. As one might expect, this is followed by an increase in RNAi efficiency and post-transcriptional control of gene expression.

A known physiological role for RNAi is to fight off viral infections as part of an innate immune response. The authors find that FOXO is activated by viral infection to promote this anti-viral response. In addition, animals deleted for the FOXO gene are more susceptible to a viral infection. Theses results are consistent with the notion that virally-activated FOXO stimulates RNAi gene transcription as a mechanism to enhance viral immunity.

Finally, the work in this paper identifies integration between metabolic and stress signaling and the innate immune response, with FOXO serving the bridge. There is evidence that acute stress can confer a protective effect against infection in humans. If the identified role of FOXO is conserved, perhaps it can be utilized therapeutically.

Spellberg MJ, Marr MT, 2nd. FOXO regulates RNA interference in Drosophila and protects from RNA virus infection. Proc Natl Acad Sci U S A. 2015

TIDAL-Fly: a new database resource of Transposon Landscapes for understanding animal genome dynamics.

We tend to think of our genomes as nicely-ordered encyclopedias,  curated with only useful information that makes up our genes.  In actuality, nature and evolution is extremely sloppy.  All animal genomes, from us humans to the simple fruit fly, are littered with genetic baggage.  This baggage is sizeable, making up at least 11% of the fly genome and more than 45% of our genome.  The scientific term for this baggage is transposable elements (TEs) or transposons, which are mobile entities that must copy themselves to other places of the genome to ensure their survival during animal evolution.

Because there are so many copies of transposons, they can be difficult to analyze by most standard genetic methods. Brandeis postdoctoral fellow Reazur Rahman and a team in Nelson Lau’s lab have formulated a new tool called the Transposon Insertion and Depletion AnaLyzer (TIDAL). TIDAL aims to provide an accurate and user-friendly program to reveal how frequently transposons can move around in animal genomes.  Currently, the TIDAL tool has been applied to over 360 fruit fly genomes that have been sequenced and deposited in the NIH NCBI Sequencing Read Archive.  The outputs from this program are available to the whole genetics community through the TIDAL-FLY database.

tidal fly banner

The TIDAL-Fly database will allow geneticists to pick their favorite fly strain and see if a transposon has landed near to their gene and perhaps affect gene expression. Fruit flies are key model organisms utilized by many researchers, including here at Brandeis, to study human diseases, from infertility to insulin signaling to aging to sleep disorders.  Since these new transposon insertions are not available in the standard genome databases, this tool and website may provide answers to previously puzzling genetic effects not revealed by typical DNA sequencing studies.  It is Reazur’s and the Lau lab’s goal to continue updating the TIDAL-Fly database with more genomes as fly genome re-sequencing becomes easier and easier to perform.

see also: Rahman R, Chirn GW, Kanodia A, Sytnikova YA, Brembs B, Bergman CM, Lau NC. Unique transposon landscapes are pervasive across Drosophila melanogaster genomes. Nucleic Acids Res. 2015.

Sleep and memory are connected by a pair of neurons in Drosophila

In a recent post on the Fly on the Wall blog, Neuroscience grad student Bethany Christmann talks about recently published research from Leslie Griffith’s lab:

 … [How are sleep and behavior] connected in the brain? Does sleep simply permit memory storage to take place, such that the part of the brain involved in memory just takes advantage of sleep whenever it can? Or are sleep and memory physically connected, and the same mechanism in the brain is involved in both? In a recent study published in eLife, researchers in the Griffith lab may have [uncovered the answer]. They found that a single pair of neurons, known as the DPM neurons, are actively involved in both sleep and memory storage in fruit flies.

Haynes PR, Christmann BL, Griffith LC. A single pair of neurons links sleep to memory consolidation in Drosophila melanogaster. eLife. 2015;4.

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