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1.
Elife ; 122023 02 16.
Article in English | MEDLINE | ID: mdl-36795469

ABSTRACT

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.


Subject(s)
Adult Stem Cells , Testis , Animals , Male , Testis/metabolism , Drosophila , RNA-Seq , Semen
2.
Elife ; 112022 04 25.
Article in English | MEDLINE | ID: mdl-35468055

ABSTRACT

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. Here we find that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. We propose that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.


Subject(s)
Cysts , Drosophila Proteins , Animals , Cell Transdifferentiation , Cysts/metabolism , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , ErbB Receptors/metabolism , Male , Receptors, Invertebrate Peptide/genetics , Receptors, Invertebrate Peptide/metabolism , Stem Cells/physiology , Testis/metabolism , Tumor Microenvironment
3.
Sci Rep ; 9(1): 19368, 2019 12 18.
Article in English | MEDLINE | ID: mdl-31852969

ABSTRACT

Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.


Subject(s)
Chromatin Assembly and Disassembly/genetics , DNA Helicases/genetics , Drosophila Proteins/genetics , Endocytosis/genetics , Homeodomain Proteins/genetics , Synaptic Vesicles/genetics , Acyltransferases/genetics , Animals , DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Histone Deacetylase 1/genetics , Humans , Neurons/metabolism , Synaptic Vesicles/metabolism , Transcription Factors/genetics , Transcription Initiation Site/drug effects , Vesicular Glutamate Transport Proteins/genetics , Vesicular Transport Proteins/genetics , rab GTP-Binding Proteins/genetics
4.
Dev Neurobiol ; 79(8): 805-818, 2019 08.
Article in English | MEDLINE | ID: mdl-31581354

ABSTRACT

The Class I basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors that are ubiquitously expressed. A wealth of literature on Class I bHLH proteins has shown that these proteins must homodimerize or heterodimerize with tissue-specific HLH proteins in order to bind DNA at E-box consensus sequences to control tissue-specific transcription. Due to its ubiquitous expression, Class I bHLH proteins are also extensively regulated posttranslationally, mostly through dimerization. Previously, we reported that in addition to its role in promoting neurogenesis, the Class I bHLH protein daughterless also functions in mature neurons to restrict axon branching and synapse number. Here, we show that part of the molecular logic that specifies how daughterless functions in neurogenesis is also conserved in neurons. We show that the Type V HLH protein extramacrochaetae (Emc) binds to and represses daughterless function by sequestering daughterless to the cytoplasm. This work provides initial insights into the mechanisms underlying the function of daughterless and Emc in neurons while providing a novel understanding of how Emc functions to restrict daughterless activity within the cell.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cytoplasm/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental/genetics , Neurons/metabolism , Repressor Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Proliferation/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Neurogenesis/physiology , Presynaptic Terminals/metabolism , Repressor Proteins/genetics
5.
iScience ; 16: 79-93, 2019 Jun 28.
Article in English | MEDLINE | ID: mdl-31153043

ABSTRACT

Axon pruning is critical for sculpting precise neural circuits. Although axon pruning has been described in the literature for decades, relatively little is known about the molecular and cellular mechanisms that govern axon pruning in vivo. Here, we show that the epigenetic reader Kismet (Kis) is required for developmental axon pruning in Drosophila mushroom bodies. Kis binds to cis-regulatory elements of the steroid hormone receptor ecdysone receptor (ecr) gene and is necessary for activating expression of EcR-B1. Kis promotes the active H3K36 di- and tri-methylation and H4K16 acetylation histone marks at the ecr locus. We show that transgenic EcR-B1 can rescue axon pruning and memory defects associated with loss of Kis and that the histone deacetylase inhibitor SAHA also rescues these phenotypes. EcR protein abundance is the cell-autonomous, rate-limiting step required to initiate axon pruning in Drosophila, and our data suggest this step is under the epigenetic control of Kis.

6.
Mol Cell Neurosci ; 87: 77-85, 2018 03.
Article in English | MEDLINE | ID: mdl-29249293

ABSTRACT

We are beginning to appreciate the complex mechanisms by which epigenetic proteins control chromatin dynamics to tightly regulate normal development. However, the interaction between these proteins, particularly in the context of neuronal function, remains poorly understood. Here, we demonstrate that the activity of histone deacetylases (HDACs) opposes that of a chromatin remodeling enzyme at the Drosophila neuromuscular junction (NMJ). Pharmacological inhibition of HDAC function reverses loss of function phenotypes associated with Kismet, a chromodomain helicase DNA-binding (CHD) protein. Inhibition of HDACs suppresses motor deficits, overgrowth of the NMJ, and defective neurotransmission associated with loss of Kismet. We hypothesize that Kismet and HDACs may converge on a similar set of target genes in the nervous system. Our results provide further understanding into the complex interactions between epigenetic protein function in vivo.


Subject(s)
DNA Helicases/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/drug effects , Neuromuscular Junction/drug effects , Synaptic Transmission/drug effects , Animals , Chromatin , DNA Helicases/genetics , Histone Deacetylases/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Neuromuscular Junction/genetics , Synapses/drug effects , Synapses/metabolism , Synaptic Transmission/genetics
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