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1.
Elife ; 122024 Jun 06.
Article in English | MEDLINE | ID: mdl-38842917

ABSTRACT

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila. Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth.


Subject(s)
Cadherins , Cell Cycle , Drosophila Proteins , Drosophila melanogaster , Signal Transduction , Wings, Animal , Animals , Wings, Animal/growth & development , Wings, Animal/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Cadherins/metabolism , Larva/growth & development , Larva/metabolism , Cell Proliferation , Cell Adhesion Molecules
2.
Genetics ; 2024 May 03.
Article in English | MEDLINE | ID: mdl-38701221

ABSTRACT

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111 bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into four independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.

3.
bioRxiv ; 2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38645118

ABSTRACT

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila. Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth.

4.
Development ; 151(1)2024 Jan 01.
Article in English | MEDLINE | ID: mdl-38063847

ABSTRACT

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that, in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions.


Subject(s)
Drosophila Proteins , Repressor Proteins , Animals , Repressor Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/genetics , Drosophila/metabolism , Energy Metabolism/genetics , Gene Expression , ErbB Receptors/genetics , ErbB Receptors/metabolism
5.
bioRxiv ; 2023 Oct 26.
Article in English | MEDLINE | ID: mdl-37961620

ABSTRACT

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions.

6.
Development ; 150(8)2023 04 15.
Article in English | MEDLINE | ID: mdl-36942737

ABSTRACT

Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. Here, we investigate how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein, despite expressing both proteins with pulsatile dynamics. A rapid and sustained twofold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a twofold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how twofold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program.


Subject(s)
Drosophila Proteins , Transcription Factors , Animals , Transcription Factors/metabolism , Drosophila/metabolism , DNA-Binding Proteins/genetics , Repressor Proteins/metabolism , Drosophila Proteins/metabolism , Eye Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Nerve Tissue Proteins/metabolism , DNA
7.
iScience ; 25(10): 105097, 2022 Oct 21.
Article in English | MEDLINE | ID: mdl-36157584

ABSTRACT

Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make during sensory organ differentiation has been described as such. We extended these studies by focusing on the Senseless protein which orchestrates sensory cell fate transitions. Wing cells contain intermediate Senseless numbers before their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are inconsistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the states are stable over time, the number of molecules in each state vary with time. The fold change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.

8.
Methods Mol Biol ; 2540: 135-156, 2022.
Article in English | MEDLINE | ID: mdl-35980576

ABSTRACT

The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Drosophila/genetics , Gene Editing/methods , Recombinational DNA Repair
9.
Cell Rep ; 39(12): 110976, 2022 06 21.
Article in English | MEDLINE | ID: mdl-35732126

ABSTRACT

dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. Here, we show that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, our results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.


Subject(s)
DNA, Viral , Drosophila , Animals , Antiviral Agents , DNA Viruses/genetics , Drosophila/metabolism , Iridovirus , RNA Interference , RNA Polymerase II/metabolism , RNA, Double-Stranded/genetics , RNA, Small Interfering/metabolism , RNA, Viral/metabolism
10.
Elife ; 112022 01 17.
Article in English | MEDLINE | ID: mdl-35037852

ABSTRACT

Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. In this study, we investigate the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. Here, we find that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting, and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven.


Subject(s)
Drosophila melanogaster/anatomy & histology , Retina/cytology , Animals , Cell Division , Cell Movement , Drosophila melanogaster/physiology , Retina/growth & development
11.
Elife ; 102021 06 28.
Article in English | MEDLINE | ID: mdl-34180394

ABSTRACT

Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robustness implies the existence of hidden simplicities in developmental programs. Here, using the Drosophila wing as a model system, we develop a new quantitative strategy that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population and variation generated by weak perturbations in genetic and environmental conditions, we observe a highly constrained set of wing phenotypes. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. Our results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, we introduce a general strategy for finding such invariances in other developmental contexts.


Subject(s)
Biological Variation, Population , Drosophila melanogaster/growth & development , Phenotype , Wings, Animal/growth & development , Animals , Drosophila melanogaster/genetics
12.
Nat Rev Mol Cell Biol ; 22(6): 425-438, 2021 06.
Article in English | MEDLINE | ID: mdl-33772227

ABSTRACT

In animals, systemic control of metabolism is conducted by metabolic tissues and relies on the regulated circulation of a plethora of molecules, such as hormones and lipoprotein complexes. MicroRNAs (miRNAs) are a family of post-transcriptional gene repressors that are present throughout the animal kingdom and have been widely associated with the regulation of gene expression in various contexts, including virtually all aspects of systemic control of metabolism. Here we focus on glucose and lipid metabolism and review current knowledge of the role of miRNAs in their systemic regulation. We survey miRNA-mediated regulation of healthy metabolism as well as the contribution of miRNAs to metabolic dysfunction in disease, particularly diabetes, obesity and liver disease. Although most miRNAs act on the tissue they are produced in, it is now well established that miRNAs can also circulate in bodily fluids, including their intercellular transport by extracellular vesicles, and we discuss the role of such extracellular miRNAs in systemic metabolic control and as potential biomarkers of metabolic status and metabolic disease.


Subject(s)
Glucose/metabolism , MicroRNAs/metabolism , Animals , Humans , Lipid Metabolism/genetics , Lipid Metabolism/physiology , Metabolic Diseases/metabolism
13.
Trends Genet ; 37(4): 389-400, 2021 04.
Article in English | MEDLINE | ID: mdl-33092903

ABSTRACT

It is recognized that cell metabolism is tightly connected to other cellular processes such as regulation of gene expression. Metabolic pathways not only provide the precursor molecules necessary for gene expression, but they also provide ATP, the primary fuel driving gene expression. However, metabolic conditions are highly variable since nutrient uptake is not a uniform process. Thus, cells must continually calibrate gene expression to their changing metabolite and energy budgets. This review discusses recent advances in understanding the molecular and biophysical mechanisms that connect metabolism and gene regulation as cells navigate their growth, proliferation, and differentiation. Particular focus is given to these mechanisms in the context of organismal development.


Subject(s)
Energy Metabolism/genetics , Gene Expression Regulation/genetics , Metabolic Networks and Pathways/genetics , Adenosine Triphosphate/genetics , Animals , Biophysical Phenomena/genetics , Humans
14.
Fly (Austin) ; 14(1-4): 34-48, 2020.
Article in English | MEDLINE | ID: mdl-33016195

ABSTRACT

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Drosophila melanogaster/genetics , Gene Editing/methods , RNA, Guide, Kinetoplastida/metabolism , Ribonucleoproteins/metabolism , Animals , Chromosomes , Drosophila melanogaster/embryology , RNA, Guide, Kinetoplastida/genetics , Ribonucleoproteins/genetics
16.
Elife ; 92020 06 22.
Article in English | MEDLINE | ID: mdl-32568073

ABSTRACT

Morphogen signaling contributes to the patterned spatiotemporal expression of genes during development. One mode of regulation of signaling-responsive genes is at the level of transcription. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. Although the effects of many gene regulatory mechanisms on transcriptional bursting have been studied, it remains unclear how morphogen gradients affect this dynamic property of downstream genes. Here we have adapted single molecule fluorescence in situ hybridization (smFISH) for use in the Drosophila wing imaginal disc in order to measure nascent and mature mRNA of genes downstream of the Wg and Dpp morphogen gradients. We compared our experimental results with predictions from stochastic models of transcription, which indicated that the transcription levels of these genes appear to share a common method of control via burst frequency modulation. Our data help further elucidate the link between developmental gene regulatory mechanisms and transcriptional bursting.


Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Imaginal Discs/growth & development , Wings, Animal/growth & development , Wnt1 Protein/genetics , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Imaginal Discs/metabolism , In Situ Hybridization, Fluorescence , Larva/genetics , Larva/growth & development , RNA, Messenger/analysis , Signal Transduction , Single Molecule Imaging , Transcriptional Activation , Wnt1 Protein/metabolism
17.
PLoS Comput Biol ; 16(3): e1007406, 2020 03.
Article in English | MEDLINE | ID: mdl-32126077

ABSTRACT

Mosaic analysis provides a means to probe developmental processes in situ by generating loss-of-function mutants within otherwise wildtype tissues. Combining these techniques with quantitative microscopy enables researchers to rigorously compare RNA or protein expression across the resultant clones. However, visual inspection of mosaic tissues remains common in the literature because quantification demands considerable labor and computational expertise. Practitioners must segment cell membranes or cell nuclei from a tissue and annotate the clones before their data are suitable for analysis. Here, we introduce Fly-QMA, a computational framework that automates each of these tasks for confocal microscopy images of Drosophila imaginal discs. The framework includes an unsupervised annotation algorithm that incorporates spatial context to inform the genetic identity of each cell. We use a combination of real and synthetic validation data to survey the performance of the annotation algorithm across a broad range of conditions. By contributing our framework to the open-source software ecosystem, we aim to contribute to the current move toward automated quantitative analysis among developmental biologists.


Subject(s)
Computational Biology/methods , Data Curation/methods , Mosaicism/embryology , Animals , Developmental Biology/methods , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental/genetics , Imaginal Discs/metabolism , Larva/metabolism , Loss of Function Mutation/genetics , Microscopy, Confocal , Software , Wings, Animal/embryology
18.
Elife ; 92020 02 26.
Article in English | MEDLINE | ID: mdl-32101167

ABSTRACT

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. We investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.


Subject(s)
Gene Expression Regulation/physiology , Sensory Receptor Cells/metabolism , Alleles , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Drosophila melanogaster/physiology , Female , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Sensory Receptor Cells/physiology , Stochastic Processes , Transcription Factors/metabolism , Transcription Factors/physiology , Transcription, Genetic
19.
Proc Natl Acad Sci U S A ; 117(3): 1312-1320, 2020 01 21.
Article in English | MEDLINE | ID: mdl-31900365

ABSTRACT

Spherical nucleic acids (SNAs) are nanostructures formed by chemically conjugating short linear strands of oligonucleotides to a nanoparticle template. When made with modified small interfering RNA (siRNA) duplexes, SNAs act as single-entity transfection and gene silencing agents and have been used as lead therapeutic constructs in several disease models. However, the manner in which modified siRNA duplex strands that comprise the SNA lead to gene silencing is not understood. Herein, a systematic analysis of siRNA biochemistry involving SNAs shows that Dicer cleaves the modified siRNA duplex from the surface of the nanoparticle, and the liberated siRNA subsequently functions in a way that is dependent on the canonical RNA interference mechanism. By leveraging this understanding, a class of SNAs was chemically designed which increases the siRNA content by an order of magnitude through covalent attachment of each strand of the duplex. As a consequence of increased nucleic acid content, this nanostructure architecture exhibits less cell cytotoxicity than conventional SNAs without a decrease in siRNA activity.


Subject(s)
Nanoparticles/chemistry , RNA Interference , RNA, Small Interfering/chemistry , Animals , Cell Line, Tumor , Drosophila melanogaster , Humans , Nanoparticles/metabolism , Nanoparticles/toxicity , RNA, Small Interfering/metabolism , Ribonuclease III/metabolism
20.
Endocrinology ; 161(2)2020 02 01.
Article in English | MEDLINE | ID: mdl-31875904

ABSTRACT

The insulin/insulin-like growth factor (IGF) pathway is essential for linking nutritional status to growth and metabolism. MicroRNAs (miRNAs) are short RNAs that are players in the regulation of this process. The miRNA miR-7 shows highly conserved expression in insulin-producing cells across the animal kingdom. However, its conserved functions in regulation of insulin-like peptides (ILPs) remain unknown. Using Drosophila as a model, we demonstrate that miR-7 limits ILP availability by inhibiting its production and secretion. Increasing miR-7 alters body growth and metabolism in an ILP-dependent manner, elevating circulating sugars and total body triglycerides, while decreasing animal growth. These effects are not due to direct targeting of ILP mRNA, but instead arise through alternate targets that affect the function of ILP-producing cells. The Drosophila F-actin capping protein alpha (CPA) is a direct target of miR-7, and knockdown of CPA in insulin-producing cells phenocopies the effects of miR-7 on ILP secretion. This regulation of CPA is conserved in mammals, with the mouse ortholog Capza1 also targeted by miR-7 in ß-islet cells. Taken together, these results support a role for miR-7 regulation of an actin capping protein in insulin regulation, and highlight a conserved mechanism of action for an evolutionarily ancient microRNA.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , MicroRNAs/metabolism , Neuropeptides/metabolism , Animals , Drosophila melanogaster/growth & development , Female , Male
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