In vivo single-cell labeling by confined primed conversion

Dempsey, William P.; Georgieva, Lada; Helbling, Patrick M.; Sonay, Ali Y.; Truong, Thai V.; Haffner, Michel; Pantazis, Periklis

doi:10.1038/nmeth.3405

Cited by 64 publications

(117 citation statements)

References 34 publications

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“…To limit excitation in the Z-plane, we attempted to use a two-photon system. However, we observed that Dendra2 photoconversion did not occur under a two-photon regime, consistent with other reports (Dempsey et al, 2015). …”

Section: Resultssupporting

confidence: 93%

Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation

Rosu

Cohen-Fix

2017

Developmental Biology

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The C. elegans adult hermaphrodite contains a renewable pool of mitotically dividing germ cells that are contained within the progenitor zone (PZ), at the distal region of the germline. From the PZ, cells enter meiosis and differentiate, ensuring the continued production of oocytes. In this study, we investigated the proliferation strategy used to maintain the PZ pool by using a photoconvertible marker to follow the fate of selected germ cells and their descendants in live worms. We found that the most distal pool of 6–8 rows of cells in the PZ (the distal third) behave similarly, with a fold expansion corresponding to one cell division every 6 hours on average. Proximal to this region, proliferation decreases, and by the proximal third of the PZ, most cells have stopped dividing. In addition, we show that all the descendants of cells in rows 3 and above move proximally and leave the PZ over time. Combining our data with previous studies, we propose a stochastic model for the C. elegans PZ proliferation, where a pool of proliferating stem cells divide symmetrically within the distal most 6–8 rows of the germline and exit from this stem cell niche occurs by displacement due to competition for limited space.

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Section: Resultssupporting

confidence: 93%

Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation

Rosu

Cohen-Fix

2017

Developmental Biology

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“…Spatiotemporal quantitative imaging in vivo is not merely limited to early embryonic development but can also be exploited as a tool to study development and regeneration in whole organisms with potential benefits for future biomedical applications (Dempsey et al, 2012a(Dempsey et al, , 2015. The additional information acquired using these quantitative imaging techniques is critical for our understanding of the regulation of developmental processes in healthy and diseased states such as cancer.…”

Section: Discussionmentioning

confidence: 99%

“…Yet, until recently, spatially confined photoconversion using high-power, pulsed laser illumination was extremely inefficient. Now, Dempsey and colleagues have reported a unique optical mechanism, termed primed conversion, where dual-wavelength continuous-wave illumination results in pronounced photoconversion of fluorescent proteins (Dempsey et al, 2015). Confined primed conversion can be implemented on a conventional confocal microscope and succeeds in precise targeting of single cells in complex 3D structures.…”

Section: Quantitative Imaging Of Transcription Factor Dynamicsmentioning

confidence: 99%

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

2015

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In recent years, advances in imaging probes, cutting-edge microscopy techniques and powerful bioinformatics image analysis have markedly expanded the imaging toolbox available to developmental biologists. Apart from traditional qualitative studies, embryonic development can now be investigated in vivo with improved spatiotemporal resolution, with more detailed quantitative analyses down to the single-cell level of the developing embryo. Such imaging tools can provide many benefits to investigate the emergence of the asymmetry in the early mammalian embryo. Quantitative single-cell imaging has provided a deeper knowledge of the dynamic processes of how and why apparently indistinguishable cells adopt separate fates that ensure proper lineage allocation and segregation. To advance our understanding of the mechanisms governing such cell fate decisions, we will need to address current limitations of fluorescent probes, while at the same time take on challenges in image processing and analysis. New discoveries and developments in quantitative, single-cell imaging analysis will ultimately enable a truly comprehensive, multidimensional and multi-scale investigation of the dynamic morphogenetic processes that work in concert to shape the embryo.

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“…After tail flick imaging, zebrafish were kept overnight at 28°C within the 96-well plate. At 2 dpf, zebrafish injected with the spinal cord neuron expressing GFE construct were anesthetized with 0.015–0.03% MS-222 (Argent Laboratories), embedded in 1–1.5% SeaPlaque agarose within a 30X Danieau’s solution (17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO 4 , 0.18 mM Ca(NO 3 ) 2 , 1.5 mM HEPES), and imaged using a Plan Apochromat ×20 objective on a commercial LSM 700 confocal microscope (Carl Zeiss AG), as similarly described 45 . Mosaic-labeled GFP positive cells were visualized throughout the zebrafish spinal cord using the tiled confocal stack feature of the microscope.…”

Section: Methodsmentioning

confidence: 99%

An E3-ligase-based method for ablating inhibitory synapses

et al. 2016

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Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to Gephyrin. Expression of GFE3 leads to a strong and specific reduction of Gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.

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In vivo single-cell labeling by confined primed conversion

Cited by 64 publications

References 34 publications

Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation

Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

An E3-ligase-based method for ablating inhibitory synapses

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