Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Sheng, Xiaowei; Matunis, Erika

doi:10.1242/dev.065797

Cited by 142 publications

(163 citation statements)

References 60 publications

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“…To date, several protocols for ex vivo cultivation of Drosophila organs and tissues including testes and germ-line cysts have been developed (e.g., see 4,14,17,20,39,40 ). The cultivation medium and the general conditions used in the protocol presented here were established in 1979 17 .…”

Section: Discussionmentioning

confidence: 99%

“…This includes especially research focusing on germ-line stem cells. It has been shown that stem cell development can be followed by live imaging of cultured adult testes 20,43 . However, the peristaltic movement of the mature testis sheath interferes with image acquisition.…”

Section: Discussionmentioning

confidence: 99%

“…However, the peristaltic movement of the mature testis sheath interferes with image acquisition. Therefore, either imaging is done using fragmented testes 43 or the number of imaging experiments performed has to be increased to account for lost datasets 20 . Early pupal testes (24 hr APF) are not yet enclosed with muscle cells.…”

Section: Discussionmentioning

confidence: 99%

“…One advantage of Drosophila that can be exploited in such cases is the ability of dissected intact testes and even cysts of germ cells to develop ex vivo in culture medium 4,12,[17][18][19][20] . The dissection and cultivation of testes or germ-line cysts allows not only microscopic observation of germ-cell development in living cells, but also the use of pharmacological assays with, e.g., inhibitors of enzyme complexes such as histone acetyltransferases (HATs), histone deacetylases (HDACs), topoisomerases, or the proteasome.…”

Section: Introductionmentioning

confidence: 99%

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Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Gärtner

Rathke

Renkawitz‐Pohl

et al. 2014

JoVE

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During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin-the histone-toprotamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre-and post-meiotic spermatogenesis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Gärtner

Rathke

Renkawitz‐Pohl

et al. 2014

JoVE

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“…In addition, recent success of long-term live imaging of testis organ culture visualized interesting behaviors of stem cell daughters (Sheng & Matunis 2011): although a stem cell always divided in a geographically asymmetric manner, namely perpendicular to the niche (hub), as had been observed (Yamashita & Fuller 2005, Losick et al 2011, the subsequent fates of daughter cells were not always asymmetric. According to Sheng and Matunis (2011), w7 and w13% of stem cell divisions produced daughter pairs that both self-renew (symmetric renewal) and daughter pairs that both differentiate (symmetric differentiation) respectively. The probabilities of symmetric renewal and symmetric differentiation were increased and decreased, respectively, and the reversion from syncytia also takes place when regeneration was induced.…”

Section: Flexibility and Stochasticity May Be Common Features Of Stemmentioning

confidence: 96%

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis

Yoshida¹

2012

Reproduction

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Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A single (A s ) model, which was originally established in 1971, proposes that singly isolated A s spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A s and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.Reproduction (2012) 144 293-302

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Cycling progenitors maintain epithelia while diverse cell types contribute to repair

Doupé

Jones

2013

BioEssays

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It has recently been shown that stem and progenitor cells undergo population self-renewal to maintain epithelial homeostasis. The fate of individual cells is stochastic but the production of proliferating and differentiating cells is balanced across the population. This new paradigm, originating in mouse epidermis and since extended to mouse oesophagus and mouse and Drosophila intestine, is in contrast to the long held model of epithelial maintenance by exclusively asymmetric division of stem cells. Recent lineage tracing studies have now shown that wound responses vary between tissues, and that a stem cell reserve is not essential as cycling progenitors and even differentiating cells contribute to regeneration.

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Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Cited by 142 publications

References 60 publications

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis

Cycling progenitors maintain epithelia while diverse cell types contribute to repair

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