Nessys: A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures

Blin, Guillaume; Sadurska, Daina; Migueles, Rosa Portero; Chen, Naiming; Watson, Julia A.; Lowell, Sally

doi:10.1371/journal.pbio.3000388

Cited by 29 publications

(35 citation statements)

References 80 publications

(85 reference statements)

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“…Immunofluorescence was quantified by nuclear segmentation based on DAPI signal and manual editing of segmentation results using Nessys software, as detailed by Blin et al (2019).…”

Section: Quantification Of Immunostainingmentioning

confidence: 99%

The transcription factor E2A drives neural differentiation in pluripotent cells

et al. 2020

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The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under nonpermissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

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“…Immunofluorescence was quantified by nuclear segmentation based on DAPI signal and manual editing of segmentation results using Nessys software, as detailed by Blin et al (2019).…”

Section: Quantification Of Immunostainingmentioning

confidence: 99%

The transcription factor E2A drives neural differentiation in pluripotent cells

et al. 2020

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“…Sample shrinkage is a further factor that affects the segmentation accuracy adversely since the cell nuclei are more densely packed with more overlapping objects. This may lead to under-segmentation, and hence to a lower number of detected nuclei compared to the original sample (Blin et al, 2019). It is likely, that the high variance in cell numbers between different clearing protocols as observed in the present study is at least partially due to sample shrinkage in combination with high cell density.…”

Section: Effects Of Clearing On Segmentationmentioning

confidence: 76%

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward

Nürnberg

Vitacolonna

Klicks

et al. 2020

Front. Mol. Biosci.

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Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid-and chip-based threedimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-tonoise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols-in particular, for screening purposes-clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

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“…Immunofluorescence was quantified by nuclear segmentation based on DAPI signal and manual editing of segmentation results using NesSys software, as detailed in (Blin et al, 2019).…”

Section: Quantification Of Immunostainingmentioning

confidence: 99%

The transcription factor E2A drives neural differentiation in pluripotent cells

Rao

Malaguti

Mason

et al. 2019

Preprint

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The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of Inhibitor of Differentiation (Id) factors. We have previously identified that the major binding partner of Id proteins in pluripotent cells is the basic helix-loop-helix (bHLH) transcription factor (TF), E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

show abstract

Nessys: A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures

Cited by 29 publications

References 80 publications

The transcription factor E2A drives neural differentiation in pluripotent cells

The transcription factor E2A drives neural differentiation in pluripotent cells

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward

The transcription factor E2A drives neural differentiation in pluripotent cells

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