Protocol for evaluating autophagy using LysoTracker staining in the epithelial follicle stem cells of the Drosophila ovary

STAR Protocols, Journal Year: 2021, Volume and Issue: 2(2), P. 100592 - 100592

Published: June 1, 2021

We have outlined the approach of visualizing autophagy specifically in epithelial follicle stem cells Drosophila ovary using LysoTracker dye. The advantage this protocol is that it details several techniques, including dissection, immunofluorescence, and western blotting, positively identify changes a very small population cells. One limitations needs to be combined with other genetic manipulations positive markers pathway. For complete on use execution protocol, please refer Singh et al., (2018).

Language: Английский

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Mechanism of piR-1245/PIWI-like protein-2 regulating Janus kinase-2/signal transducer and activator of transcription-3/vascular endothelial growth factor signaling pathway in retinal neovascularization

Neural Regeneration Research, Journal Year: 2022, Volume and Issue: 18(5), P. 1132 - 1132

Published: Oct. 27, 2022

Inhibiting retinal neovascularization is the optimal strategy for treatment of retina-related diseases, but there currently no effective neovascularization. P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) a type small non-coding implicated in variety diseases. In this study, we found that expression piR-1245 and interacting protein PIWIL2 were remarkably increased human endothelial cells cultured hypoxic environment, cell apoptosis, migration, tube formation proliferation enhanced these cells. Knocking down inhibited above phenomena. After intervention by p-JAK2 activator, decreased hypoxia inducible factor-1α vascular growth factor through JAK2/STAT3 pathway. For vivo analysis, 7-day-old newborn mice raised 75 ± 2% hyperoxia 5 days then retina was knocked down. mice, number newly formed vessels decreased, expressions inflammation-related proteins reduced, apoptotic pathway inhibited, decreased. Injection JAK2 inhibitor JAK2/TYK2-IN-1 into vitreous cavity reduced factor. These findings suggest activates pathway, regulates factor, promotes Therefore, may be new therapeutic target

Language: Английский

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Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation

Development, Journal Year: 2021, Volume and Issue: 148(22)

Published: Nov. 15, 2021

ABSTRACT A major goal in the study of adult stem cells is to understand how cell fates are specified at proper time and place facilitate tissue homeostasis. Here, we found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles Drosophila ovarian epithelial follicle (FSC) lineage. First, Ben part JNK signaling pathway, it, as well other pathway genes, essential for differentiation FSC daughter cells. Our data suggest promotes by suppressing activation EGFR effector, ERK. Also, loss ben, but not kinase hemipterous, resulted upregulation hedgehog signaling, increased proliferation niche competition. Lastly, demonstrate hypercompetition phenotype caused ben suppressed decreasing rate or knockdown Smoothened (Smo). Taken together, our findings reveal a new layer regulation which single gene influences stages early

Language: Английский

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Spatial regulation of Drosophila ovarian Follicle Stem Cell division rates and cell cycle transitions

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: June 26, 2022

Summary Drosophila ovarian Follicle Stem Cells (FSCs) present a favorable paradigm for understanding how stem cell division and differentiation are balanced in communities where they can be regulated independently. Many key extracellular signals FSCs have been identified, including inversely graded Wnt JAK-STAT pathway activators. also exhibit interesting functional spatial heterogeneity; posterior become proliferative Cells, while anterior quiescent Escort at much lower rate. Here, by using live imaging FUCCI cell-cycle reporters, we measured absolute rates found that cycle 3-4 times faster than their neighbors, matching increased We evidence FSC G2/M cycling restrictions G1/S restriction increases more anteriorly, especially beyond the domain. signaling promotes both transitions but alone does not explain of FSCs. Genetic interaction tests reporter assays suggest acts partly through Yorkie largely substitute stimulation Hh signaling. PI3 kinase signaling, contrast to independently induction stimulates transition.

Language: Английский

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JAK activity regulates mesoderm cell fate by controlling MESP1 expression

European Journal of Cell Biology, Journal Year: 2024, Volume and Issue: 103(4), P. 151452 - 151452

Published: Aug. 23, 2024

Cardiac development requires precise gene expression programs at each developmental stage guided by multiple signaling pathways and transcription factors (TFs). MESP1 is transiently expressed in mesoderm, essential for subsequent cardiac development, while the mechanism regulating its own mesoderm cell fate not fully understood. Therefore, we developed a high content screen assay to identify regulators of mesodermal cells differentiated from human pluripotent stem (hPSCs). The identified CYT387, JAK1/JAK2 kinase inhibitor, as potent activator expression, which was also found promote cardiomyocyte differentiation vitro. Mechanistic studies that JAK inhibition promotes reducing cytoplasmic calcium concentration subsequently activating canonical WNT signaling. Our study role early cells, sheds light on connection between JAK-STAT pathway transcriptional regulation MESP1, expands our understanding development.

Language: Английский

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Adult Stem Cells and Niche Cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2020, Volume and Issue: unknown

Published: June 26, 2020

Summary Adult stem cell function relies on the prior development of appropriate numbers and spatial organization cells supportive niche cells. Drosophila Follicle Stem Cells (FSCs) present a favorable paradigm for understanding those developmental processes. About sixteen FSCs in an adult germarium produce transit-amplifying (FCs) from their posterior face quiescent Escort (ECs) to anterior. Both ECs FCs also act as Here we show that ECs, derive common precursors intermingled with germline during pupal development, progeny single precursor commonly including FSCs, FCs, or all three types. Precursors become first engulf largely naked cyst projected out form egg chamber set up key signaling center regulating FSC behavior.

Language: Английский

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Extracellular spreading of Wingless is required forDrosophilaoogenesis

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2020, Volume and Issue: unknown

Published: Dec. 8, 2020

Abstract Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or they only in a juxtacrine manner to neighboring cells. The original evidence for long-range signaling arose Wg, Drosophila protein, which patterns wing disc over several cell diameters central Wg ligand. However, requirement patterning was called into question when it reported that replacing secreted protein with membrane-tethered version, NRT-Wg, results flies normally patterned wings. We others previously spreads ovary about 50 μm 5 diameters, cap cells follicle stem (FSCs) stimulates FSC proliferation. used NRT-wg analyze consequence tethering homozygous are sickly, but we found hemizygous NRT-wg/null flies, carrying one copy tethered Wingless, were significantly healthier. Despite overall improved health, these displayed dramatic reductions fertility Further, proliferation nearly undetectable wg locus converted adults, resulting germarium phenotype consistent loss-of-function phenotype. conclude its promote

Language: Английский

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Determination of expression profiles for Drosophila ovarian Follicle Stem Cells (FSCs) using single-cell RNA sequencing

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: June 27, 2022

Summary Drosophila ovarian Follicle Stem Cells (FSC) present an excellent paradigm for understanding how a community of active stem cells maintained by population asymmetry is regulated. Here we describe single-cell RNA sequencing studies pre-sorted that include FSCs and the neighboring cell types, Escort (ECs) (FCs), which they support. Cell-type assignment relies on anterior-posterior (AP) location within germarium. We clarify previously determined use spatially targeted lineage as further confirmation. The scRNA profiles among four clusters are consistent with AP progression from anterior ECs through posterior then FSCs, to early FCs. Several genes graded FCs highlighted candidate effectors inverse gradients two principal signaling pathways, Wnt JAK-STAT, guide FSC differentiation division.

Language: Английский

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Sequential events during the quiescence to proliferation transition establish patterns of follicle cell differentiation in the Drosophila ovary

Biology Open, Journal Year: 2022, Volume and Issue: 12(1)

Published: Dec. 16, 2022

Stem cells cycle between periods of quiescence and proliferation to promote tissue health. In Drosophila ovaries, transitions follicle stem (FSCs) are exquisitely feeding-dependent. Here, we demonstrate feeding-dependent induction cell differentiation markers, eyes absent (Eya) castor (Cas) in FSCs, a patterning process that does not depend on induction. Instead, FSCs extend micron-scale cytoplasmic projections dictate Eya-Cas patterning. We identify still life sickie as necessary sufficient for FSC projection growth Our results suggest sequential, interdependent events establish long-term patterns precursors, independently

Language: Английский

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