one-step-crispr-based-strategy-for-endogenous-gene-tagging-drosophila

One-step CRISPR-based Strategy for Endogenous Gene Tagging in <em>Drosophila melanogaster</em>

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

University of Michigan

Adult stem cells continuously supply differentiated cells throughout the life of organisms. This increases the risk of replicative senescence or neoplastic transformation due to mutations that accumulate over many rounds of DNA replication. The immortal strand hypothesis proposes that stem cells reduce the accumulation of replication-induced mutations by retaining the older template DNA strands. Other models have also been proposed in which stem cells asymmetrically segregate chromosome strands for other reasons, such as retention of epigenetic memories. Recently, the idea has emerged that the mother centrosome, which is stereotypically retained within some asymmetrically dividing stem cells, might be utilized as a means of asymmetrically segregating chromosome strands. We have tested this hypothesis in germline stem cells (GSCs) from Drosophila melanogaster testis, which undergo asymmetric divisions marked by the asymmetric segregation of centrosomes and the acquisition of distinct daughter cell fates (stem cell self-renewal versus differentiation). Using 5-bromo-2-deoxyuridine labeling combined with direct visualization of GSC-gonialblast (differentiating daughter) pairs, we directly scored the outcome of chromosome strand segregation. Our data show that, in male GSCs in the Drosophila testis, chromosome strands are not asymmetrically segregated, despite asymmetrically segregating centrosomes. Our data demonstrate that asymmetric centrosome segregation in stem cells does not necessarily lead to asymmetric chromosome strand segregation.

Drosophila male germline stem cells do not asymmetrically segregate chromosome strands.

Adult stem cells undergo asymmetric cell division to self-renew and give rise to differentiated cells that comprise mature tissue. Sister chromatids may be distinguished and segregated nonrandomly in asymmetrically dividing stem cells, although the underlying mechanism and the purpose it may serve remain elusive. Here we develop the CO-FISH (chromosome orientation fluorescence in situ hybridization) technique with single-chromosome resolution and show that sister chromatids of X and Y chromosomes, but not autosomes, are segregated nonrandomly during asymmetric divisions of Drosophila male germline stem cells. This provides the first direct evidence, to our knowledge, that two sister chromatids containing identical genetic information can be distinguished and segregated nonrandomly during asymmetric stem-cell divisions. We further show that the centrosome, SUN-KASH nuclear envelope proteins and Dnmt2 (also known as Mt2) are required for nonrandom sister chromatid segregation. Our data indicate that the information on X and Y chromosomes that enables nonrandom segregation is primed during gametogenesis in the parents. Moreover, we show that sister chromatid segregation is randomized in germline stem cell overproliferation and dedifferentiated germline stem cells. We propose that nonrandom sister chromatid segregation may serve to transmit distinct information carried on two sister chromatids to the daughters of asymmetrically dividing stem cells.

Chromosome-specific nonrandom sister chromatid segregation during stem-cell division.

Stem cells are the source of differentiated cells that constitute tissues and organs. Two fundamental characteristics of stem cells are their abilities to self-renew stem cell identity and to produce differentiated cells, the balance of which can be achieved by asymmetric stem cell division. Many stem cells have been shown to reside in a stem cell niche, the home of stem cells that regulates the stem cell behavior. Recent studies have revealed the critical contribution of cytoskeletons in achieving asymmetric stem cell division: mitotic spindles in dividing stem cells are often oriented with respect to the stem cell niche, which is supported by concerted actions of microtubule networks and components at the cell membrane such as adherens junctions, the actin cytoskeleton, and the extracellular matrix. In this article, we review the mechanism of stem cell spindle orientation, with emphasis on its relationship with the stem cell niche, and discuss how it contributes to tissue development and homeostasis.

Spindle positioning in the stem cell niche.

The immortal strand hypothesis proposes that stem cells retain a template copy of genomic DNA (i.e. an 'immortal strand') to avoid replication-induced mutations. An alternative hypothesis suggests that certain cells segregate sister chromatids non-randomly to transmit distinct epigenetic information. However, this area of research has been highly controversial, with conflicting data even from the same cell types. Moreover, historically, the same term of 'non-random sister chromatid segregation' or 'biased sister chromatid segregation' has been used to indicate distinct biological processes, generating a confusion in the biological significance and potential mechanism of each phenomenon. Here, we discuss the models of non-random sister chromatid segregation, and we explore the strengths and limitations of the various techniques and experimental model systems used to study this question. We also describe our recent study on Drosophila male germline stem cells, where sister chromatids of X and Y chromosomes are segregated non-randomly during cell division. We aim to integrate the existing evidence to speculate on the underlying mechanisms and biological relevance of this long-standing observation on non-random sister chromatid segregation.

DNA asymmetry in stem cells - immortal or mortal?

How past experiences reshape behavior is not well understood. In this issue, two studies (Schild et al., 2014; Yu et al., 2014) dissected the molecular mechanisms underlying experience-dependent plasticity in thermosensory behavior. They show that Ca(2+)/calmodulin-dependent kinase I (CaMKI) regulates thermal preferences according to past experience.

Past experience resets behavior: CaMK takes the heat.

Temperature-sensing neurons in the  brain cooperate with the central circadian clock to help regulate body temperature.

How a brain keeps its cool.

Circadian clocks coordinate behaviour, physiology and metabolism with Earth's diurnal cycle. These clocks entrain to both light and temperature cycles, and daily environmental temperature oscillations probably contribute to human sleep patterns. However, the neural mechanisms through which circadian clocks monitor environmental temperature and modulate behaviour remain poorly understood. Here we elucidate how the circadian clock neuron network of Drosophila melanogaster processes changes in environmental temperature. In vivo calcium-imaging techniques demonstrate that the posterior dorsal neurons 1 (DN1s), which are a discrete subset of sleep-promoting clock neurons, constantly monitor modest changes in environmental temperature. We find that these neurons are acutely inhibited by heating and excited by cooling; this is an unexpected result when considering the strong correlation between temperature and light, and the fact that light excites clock neurons. We demonstrate that the DN1s rely on peripheral thermoreceptors located in the chordotonal organs and the aristae. We also show that the DN1s and their thermosensory inputs are required for the normal timing of sleep in the presence of naturalistic temperature cycles. These results identify the DN1s as a major gateway for temperature sensation into the circadian neural network, which continuously integrates temperature changes to coordinate the timing of sleep and activity.

Circadian clock neurons constantly monitor environmental temperature to set sleep timing.

Significant recent evidence suggests that metabolism is intricately linked to the regulation and dysfunction of complex cellular and physiological responses ranging from altered metabolic programs in cancers and aging to circadian rhythms and molecular clocks. While the metabolic pathways and their fundamental control mechanisms are well established, the precise cellular mechanisms underpinning, for example, enzymatic pathway control, substrate preferences or metabolic rates, remain far less certain. Comprehensive, continuous metabolic studies on model organisms, such as the fruit fly Drosophila melanogaster, may provide a critical tool for deciphering these complex physiological responses. Here, we describe the development of a high-resolution calorimeter, which combines sensitive thermometry with optical imaging to concurrently perform measurements of the metabolic rate of ten individual flies, in real-time, with ~100 nW resolution. Using this calorimeter we have measured the mass-specific metabolic rates of flies of different genotypes, ages, and flies fed with different diets. This powerful new approach enables systematic studies of the metabolic regulation related to cellular and physiological function and disease mechanisms.

Parallelized, real-time, metabolic-rate measurements from individual Drosophila.

Calorimetry has been widely used in metabolic studies, but direct measurements from individual small biological model organisms such as C. elegans or isolated single cells have been limited by poor sensitivity of existing techniques and difficulties in resolving very small heat outputs. Here, by careful thermal engineering, we developed a robust, highly sensitive and bio-compatible calorimetric platform that features a resolution of ~270 pW-more than a 500-fold improvement over the most sensitive calorimeter previously used for measuring the metabolic heat output of C. elegans. Using this calorimeter, we demonstrate time-resolved metabolic measurements of single C. elegans worms from larval to adult stages. Further, we show that the metabolic output is significantly lower in long-lived C. elegans daf-2 mutants. These demonstrations clearly highlight the broad potential of this tool for studying the role of metabolism in disease, development and aging of small model organisms and single cells.

Sub-nanowatt resolution direct calorimetry for probing real-time metabolic activity of individual C. elegans worms.

Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic and molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood are the intracellular dynamics of circadian clock components and how they affect circadian rhythms. Here, we elucidate how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in  clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that  clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, we show that core clock genes,  and , are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, we show that loss of Lamin B receptor, a nuclear envelope protein, leads to disruption of PER foci and  gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. Our results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.

Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms.

Circadian clocks are cell autonomous timekeepers that regulate ∼24-h oscillations in the expression of many genes and control rhythms in nearly all our behavior and physiology. Almost every cell in the human body has a molecular clock and networks of cells containing clock proteins orchestrate daily rhythms in many physiological processes, from sleep-wake cycles to metabolism to immunity. All eukaryotic circadian clocks are based on transcription-translation delayed negative feedback loops in which activation of core clock genes is negatively regulated by their cognate protein products. Our current understanding of circadian clocks has been accumulated from decades of genetic and biochemical experiments, however, what remains poorly understood is how clock proteins, genes, and mRNAs are spatiotemporally organized within live clock cells and how such subcellular organization affects circadian rhythms at the single cell level. Here, we review recent progress in understanding how clock proteins and genes are spatially organized within clock cells over the circadian cycle and the role of such organization in generating circadian rhythms and highlight open questions for future studies.

The role of spatiotemporal organization and dynamics of clock complexes in circadian regulation.

Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating ∼24-h circadian rhythms. Here we describe a streamlined single molecule Fluorescence  Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of , a core circadian transcription factor, in individual clock neurons in whole mount  adult brains. Specifically, we used ∼48 fluorescent oligonucleotide probes that can bind to an individual  mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect  mRNA spots in individual clock neurons. Our results demonstrate that the number of  mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4 h after lights are turned on) with ∼80 molecules/neuron and trough levels at ZT16 with ∼30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in  brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms.

Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount  brains.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. We observed significant circadian oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in  null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Article	Year
Drosophila male germline stem cells do not asymmetrically segregate chromosome strands. Journal of cell science\| PubMed ID: 21325028	2011
Chromosome-specific nonrandom sister chromatid segregation during stem-cell division. Nature\| PubMed ID: 23644460	2013
Spindle positioning in the stem cell niche. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23801437	2012
DNA asymmetry in stem cells - immortal or mortal? Journal of cell science\| PubMed ID: 23970416	2013
Past experience resets behavior: CaMK takes the heat. Neuron\| PubMed ID: 25475181	2014
How a brain keeps its cool. eLife\| PubMed ID: 28556778	2017
Circadian clock neurons constantly monitor environmental temperature to set sleep timing. Nature\| PubMed ID: 29466329	2018
Parallelized, real-time, metabolic-rate measurements from individual Drosophila. Scientific reports\| PubMed ID: 30262912	2018
Sub-nanowatt resolution direct calorimetry for probing real-time metabolic activity of individual C. elegans worms. Nature communications\| PubMed ID: 32532993	2020
Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34234015	2021
The role of spatiotemporal organization and dynamics of clock complexes in circadian regulation. Current opinion in cell biology\| PubMed ID: 36126370	2022
Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount brains. Frontiers in physiology\| PubMed ID: 36439243	2022
Clock-dependent chromatin accessibility rhythms regulate circadian transcription. bioRxiv : the preprint server for biology\| PubMed ID: 37645872	2023
Regulation of circadian rhythms by clock protein nuclear bodies. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 38232300	2024

Swathi Yadlapalli

Biography

JoVE Journal Publications

Other Publications