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Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

Ziv O, Zaritsky A, Yaffe Y, Mutukula N, Edri R, Elkabetz Y - PLoS Comput. Biol. (2015)

Bottom Line: In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization.Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures.Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

ABSTRACT
Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

No MeSH data available.


Related in: MedlinePlus

Functional instability of M-RG rosettes.Temporal variance of RS (A), B/A (B) and Speed (C) was calculated for each rosette over time. A. E-RG rosettes are more stable in RS compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0009). B. E-RG rosettes are more stable in B/A compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0006). C. E-RG rosettes are more stable in Speed compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.03). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
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pcbi.1004453.g006: Functional instability of M-RG rosettes.Temporal variance of RS (A), B/A (B) and Speed (C) was calculated for each rosette over time. A. E-RG rosettes are more stable in RS compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0009). B. E-RG rosettes are more stable in B/A compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0006). C. E-RG rosettes are more stable in Speed compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.03). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.

Mentions: The reduced dynamics and the broader cellular heterogeneity of M-RG rosettes suggest that these rosettes are mechanically compromised, reflecting the progressive disassembly of rosettes, which culminates around day 55 [20]. We therefore tested whether rosette disassembly is reflected functionally in INM measures also at the shorter range of time, i.e. throughout the time-lapse experiment. This was quantified by calculating each measure for each rosette at every time point independently and then recording its temporal variance (Methods). Importantly, no temporal trend was observed during the 4-hour imaging course of an experiment (S2E Fig). This implies that the variance encodes the fluctuations in a certain rosette measure over the imaging time course, a measure we term functional instability. Indeed, temporal variance of RS, B/A and speed was significantly higher for M-RG rosettes (Fig 6), suggesting functional instability as an indicator for the stage of progression in rosette disassembly. Importantly, these high temporal variances observed for M-RG rosettes measure their reduced ability in consistently performing INM, arguably due to their compromised structure. They do not measure the actual rosette disassembly process, which occurs in a longer time scale (from day 14 to day 35, and culminating towards day 55). Taken together, our results validate functional instability as a reliable readout for rosette organized dynamics.


Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

Ziv O, Zaritsky A, Yaffe Y, Mutukula N, Edri R, Elkabetz Y - PLoS Comput. Biol. (2015)

Functional instability of M-RG rosettes.Temporal variance of RS (A), B/A (B) and Speed (C) was calculated for each rosette over time. A. E-RG rosettes are more stable in RS compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0009). B. E-RG rosettes are more stable in B/A compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0006). C. E-RG rosettes are more stable in Speed compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.03). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608579&req=5

pcbi.1004453.g006: Functional instability of M-RG rosettes.Temporal variance of RS (A), B/A (B) and Speed (C) was calculated for each rosette over time. A. E-RG rosettes are more stable in RS compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0009). B. E-RG rosettes are more stable in B/A compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0006). C. E-RG rosettes are more stable in Speed compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.03). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
Mentions: The reduced dynamics and the broader cellular heterogeneity of M-RG rosettes suggest that these rosettes are mechanically compromised, reflecting the progressive disassembly of rosettes, which culminates around day 55 [20]. We therefore tested whether rosette disassembly is reflected functionally in INM measures also at the shorter range of time, i.e. throughout the time-lapse experiment. This was quantified by calculating each measure for each rosette at every time point independently and then recording its temporal variance (Methods). Importantly, no temporal trend was observed during the 4-hour imaging course of an experiment (S2E Fig). This implies that the variance encodes the fluctuations in a certain rosette measure over the imaging time course, a measure we term functional instability. Indeed, temporal variance of RS, B/A and speed was significantly higher for M-RG rosettes (Fig 6), suggesting functional instability as an indicator for the stage of progression in rosette disassembly. Importantly, these high temporal variances observed for M-RG rosettes measure their reduced ability in consistently performing INM, arguably due to their compromised structure. They do not measure the actual rosette disassembly process, which occurs in a longer time scale (from day 14 to day 35, and culminating towards day 55). Taken together, our results validate functional instability as a reliable readout for rosette organized dynamics.

Bottom Line: In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization.Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures.Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

ABSTRACT
Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

No MeSH data available.


Related in: MedlinePlus