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Developmental Cell Oct 2023The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses....
The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of ex vivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. We suggest that some of these "high-threshold" genes dominantly suppress the activity of the pro-proliferation genes. We then show mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone.
Topics: Animals; Drosophila; Drosophila Proteins; Ligands; Receptors, Steroid; Hormones; Cell Proliferation; Ecdysone
PubMed: 37769663
DOI: 10.1016/j.devcel.2023.08.032 -
Neuron Jan 2024Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways...
Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here, we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior.
Topics: Animals; Astrocytes; Drosophila; Drosophila Proteins; Phospholipids; Receptors, G-Protein-Coupled; Sphingosine-1-Phosphate Receptors; Zebrafish
PubMed: 38096817
DOI: 10.1016/j.neuron.2023.11.008 -
The Journal of Cell Biology Oct 2023Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC)...
Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.
Topics: Animals; Centrosome; Cytosol; Drosophila; Microtubule-Organizing Center; Microtubules; Tubulin; Cytoskeletal Proteins; Drosophila Proteins; Homeodomain Proteins
PubMed: 37698931
DOI: 10.1083/jcb.202212043 -
Proceedings of the National Academy of... Aug 2023Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large...
Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the community.
Topics: Animals; Transcription Factors; Drosophila; Drosophila Proteins; Genetic Techniques; Sequence Analysis, RNA; Drosophila melanogaster
PubMed: 37523539
DOI: 10.1073/pnas.2307451120 -
Current Biology : CB Dec 2023The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe...
The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe the morphogenetic events generating an extracellular lipid lining of the Drosophila airways required for their gas filing and animal survival. We show that sequential Rab39/Syx1A/Syt1-mediated secretion of lysosomal acid sphingomyelinase (Drosophila ASM [dASM]) and Rab11/35/Syx1A/Rop-dependent exosomal secretion provides distinct components for lipid film assembly. Tracheal inactivation of Rab11 or Rab35 or loss of Rop results in intracellular accumulation of exosomal, multi-vesicular body (MVB)-derived vesicles. On the other hand, loss of dASM or Rab39 causes luminal bubble-like accumulations of exosomal membranes and liquid retention in the airways. Inactivation of the exosomal secretion in dASM mutants counteracts this phenotype, arguing that the exosomal secretion provides the lipid vesicles and that secreted lysosomal dASM organizes them into a continuous film. Our results reveal the coordinated functions of extracellular vesicle and lysosomal secretions in generating a lipid layer crucial for airway gas filling and survival.
Topics: Animals; Drosophila; Surface-Active Agents; Endosomes; Trachea; Lipids; Nerve Tissue Proteins; Drosophila Proteins
PubMed: 37992718
DOI: 10.1016/j.cub.2023.10.058 -
Biological Research Sep 2023The endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that...
BACKGROUND
The endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes' lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. We hypothesize that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila.
RESULTS
We show that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and glutamate vesicular transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1-43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, our hypothesis that BMP signaling is not involved is supported. We also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements.
CONCLUSIONS
Our findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. Our data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.
Topics: Animals; Female; Male; Biological Transport; Drosophila; Drosophila Proteins; Receptors, Cell Surface; Synaptic Transmission; Synaptic Vesicles
PubMed: 37710314
DOI: 10.1186/s40659-023-00462-1 -
BMC Genomics Sep 2023Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by...
BACKGROUND
Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown.
RESULTS
Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes.
CONCLUSIONS
The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.
Topics: Adult; Humans; Animals; Amyotrophic Lateral Sclerosis; Drosophila; Muscular Atrophy, Spinal; Motor Neurons; RNA; DNA-Binding Proteins; Drosophila Proteins
PubMed: 37759179
DOI: 10.1186/s12864-023-09562-4 -
The Journal of Biological Chemistry Aug 2023Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral...
Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in in vitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in in vitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.
Topics: Animals; Actins; Kinetics; Myosin Type I; Drosophila Proteins; Protein Domains; Drosophila melanogaster; Functional Laterality
PubMed: 37380077
DOI: 10.1016/j.jbc.2023.104961 -
International Journal of Molecular... Jul 2023Ecdysone signaling in remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind...
Ecdysone signaling in remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.
Topics: Animals; Drosophila; Ecdysone; Drosophila Proteins; Ecdysterone; Receptors, Steroid; Transcriptional Activation; Drosophila melanogaster; Microtubule-Associated Proteins; Nuclear Proteins
PubMed: 37511602
DOI: 10.3390/ijms241411844 -
PloS One 2023In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila...
In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. Here, we investigate the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. We document a heretofore underappreciated rapid evolutionary rate for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation.
Topics: Male; Female; Animals; Drosophila; Drosophila melanogaster; Seeds; Reproduction; Drosophila Proteins; Cell Count; Sexual Behavior, Animal
PubMed: 37878630
DOI: 10.1371/journal.pone.0278811