Dissection and Live-Imaging of the Late Embryonic &lt;em&gt;Drosophila&lt;/em&gt; Gonad

Nelson, Kara; Warder, Bailey N.; DiNardo, Stephen; Anllo, Lauren

doi:10.3791/61872

Cited by 6 publications

(9 citation statements)

References 27 publications

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“…We first wanted to define the features of niche compaction using a combination of live– and fixed-imaging approaches. To observe the dynamics of compaction, we carried out ex vivo live-imaging on gonads dissected from embryos at a stage when the niche had assembled at the gonad anterior but had not compacted 13,16 . To highlight changes in the contour of the niche, the gonads carried a marker for filamentous actin (F-actin) expressed solely in somatic cells (Six4-Moe::GFP), as well as a ubiquitously expressed nuclear marker (Histone H2::RFP) to visualize cell movements.…”

Section: Resultsmentioning

confidence: 99%

“…To observe initial formation of this niche during gonadogenesis, which happens late in embryonic development 14 , we developed live-imaging techniques to capture the movement and behavior of niche cells and prospective stem cells 16 . This, together with prior work using fixed tissue, has revealed two dynamic processes during niche formation: 1) assembly and 2) compaction 13,14 .…”

Section: Introductionmentioning

confidence: 99%

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An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

Warder,

Nelson,

Sui

et al. 2023

Preprint

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SummaryStem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed, and how morphology impacts function. To address this, we leverage theDrosophilagonadal niche, combining genetic tractability with live-imaging. We have previously shown that proper positioning of niche cells relies on signaling from visceral mesoderm. Here, we show that once positioned, niche cells robustly polarize filamentous actin and Non-muscle Myosin II (MyoII) towards neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells, and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

Warder,

Nelson,

Sui

et al. 2023

Preprint

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“…This improvisation allows the isolated testis to be imaged for an extended period of time (4–6 h) on the confocal microscope, with minimal peristalsis of the tissue. Previously used imaging techniques require the use of an insect culture medium with nutrient supplements ( Sheng and Matunis, 2011 )( Nelson et al., 2020 ), which leads to a high rate of peristalsis of the testis muscle layer, making it challenging to monitor rapid actin dynamics. The technique described by Noguchi and Miller (2003) requires the isolation of the cyst from the testis.…”

Section: Before You Beginmentioning

confidence: 99%

Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

2022

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Summary Here we describe a simple step-by-step protocol for collecting high-resolution, time-lapse images of intact Drosophila testis ex vivo for a limited period using a confocal microscope, with minimum photo-toxic damage, to monitor spermatid individualization, coiling, and release. The F-actin dynamics during spermatid morphogenesis can be further investigated through laser ablations, Fluorescence-Recovery-After-Photobleaching, and drug treatments, using this protocol. For complete details on the use and execution of this protocol, please refer to Dubey et al. (2016) , Dubey et al. (2019) , and Kapoor et al. (2021) .

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“…We study development of the Drosophila testis niche, as this niche is well defined and has served as a paradigm for understanding niche-stem cell interactions. Our recent work pioneered live imaging this niche during its formation, which has enhanced its strength as a model system (Anllo et al, 2019; Nelson et al, 2020). Appropriate placement of the niche within the testis is important for polarizing the tissue, and for enabling tissue function (Fuller, 1993; Lee et al, 2008; Tanentzapf et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…We refer to the testis niche, or niche cells, as the cells that emit signals to support neighboring stem cells. Our recent work pioneered live-imaging formation of this niche, enhancing its strength as a model (Anllo et al, 2019; Nelson et al, 2020). Appropriate placement of this niche is important for polarizing the testis, and enabling tissue function (Fuller, 1993; Lee et al, 2008; Tanentzapf et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

Anllo

DiNardo

2021

Preprint

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Tissue homeostasis often requires a properly placed niche to support stem cells. The morphogenetic processes that position a niche are just being described. We recently showed that Drosophila testis pro niche cells, specified at disparate positions during early gonadogenesis, must assemble in one collective at the gonad anterior. Here, we identify Slit and FGF signals emanating from adjacent visceral mesoderm (Vm) that regulate assembly. In response to signaling, niche cells express islet, which we find is also required for positioning the niche. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of the dispersed niche is severely disrupted, with pro-niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

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Dissection and Live-Imaging of the Late Embryonic <em>Drosophila</em> Gonad

Cited by 6 publications

References 27 publications

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

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