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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

Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.A. Two fold increase in percent of highly motile cells (above speed of 15 μm hr-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). B. Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. C. Apical speed of E-RG rosettes (mean = 38.81μm hour-1) > basal speed of E-RG rosettes (mean = 35.27μm hour-1) > apical speed of M-RG rosettes (mean = 30.25μm hour-1) > basal speed of M-RG rosettes (mean = 27.12μm hour-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
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pcbi.1004453.g005: Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.A. Two fold increase in percent of highly motile cells (above speed of 15 μm hr-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). B. Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. C. Apical speed of E-RG rosettes (mean = 38.81μm hour-1) > basal speed of E-RG rosettes (mean = 35.27μm hour-1) > apical speed of M-RG rosettes (mean = 30.25μm hour-1) > basal speed of M-RG rosettes (mean = 27.12μm hour-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.

Mentions: The measures RS and B/A ratio were calculated based on the orientation of the velocity vectors. Next we considered the speed—the magnitude of these vectors—as a third measure for rosette dynamics (See Fig 2). We found a two fold increase in the fraction of cells moving at speed of 15 μm hr-1 or faster in E-RG rosettes compared to M-RG rosettes (Fig 5A). This strikingly fits our recent findings according to which there are twice as much NSCs (i.e., GFP+ cells) in E-RG rosettes compared to M-RG rosettes [20], further drawing a correlation between the actual number of NSCs measured within rosette cultures and the computed quantification of their INM motions. In contrast to RS, rosette speed was not correlated to rosette size (S4 Fig), suggesting that molecular motors driving nuclei motions are less affected by the rosette confining structure. When considering apical and basal motions independently, we found that apical motion was consistently faster than basal motion in a striking linear relation, and with higher speed for E-RG rosettes (Fig 5B). Higher apical (vs. basal) nuclear migration speeds were previously reported in time-lapse ex vivo cultured embryonic cortical slices [16] as well as in vivo in zebrafish retina and brain [33], providing further validation to our quantitative approach as an in vitro platform for investigating INM. Direct comparison further revealed an ordered relation for rosette speed as follows: apical speed of E-RG rosettes > basal speed of E-RG rosettes > apical speed of M-RG rosettes > basal speed of M-RG rosettes (Fig 5C). Inclusively, these data suggest that during early human cortical development, high NSC numbers are accompanied by elevated INM speed towards apical sites, similar to as shown for radial glial cells in cultured cortical slices ex vivo [16].


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)

Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.A. Two fold increase in percent of highly motile cells (above speed of 15 μm hr-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). B. Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. C. Apical speed of E-RG rosettes (mean = 38.81μm hour-1) > basal speed of E-RG rosettes (mean = 35.27μm hour-1) > apical speed of M-RG rosettes (mean = 30.25μm hour-1) > basal speed of M-RG rosettes (mean = 27.12μm hour-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608579&req=5

pcbi.1004453.g005: Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.A. Two fold increase in percent of highly motile cells (above speed of 15 μm hr-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). B. Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. C. Apical speed of E-RG rosettes (mean = 38.81μm hour-1) > basal speed of E-RG rosettes (mean = 35.27μm hour-1) > apical speed of M-RG rosettes (mean = 30.25μm hour-1) > basal speed of M-RG rosettes (mean = 27.12μm hour-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.
Mentions: The measures RS and B/A ratio were calculated based on the orientation of the velocity vectors. Next we considered the speed—the magnitude of these vectors—as a third measure for rosette dynamics (See Fig 2). We found a two fold increase in the fraction of cells moving at speed of 15 μm hr-1 or faster in E-RG rosettes compared to M-RG rosettes (Fig 5A). This strikingly fits our recent findings according to which there are twice as much NSCs (i.e., GFP+ cells) in E-RG rosettes compared to M-RG rosettes [20], further drawing a correlation between the actual number of NSCs measured within rosette cultures and the computed quantification of their INM motions. In contrast to RS, rosette speed was not correlated to rosette size (S4 Fig), suggesting that molecular motors driving nuclei motions are less affected by the rosette confining structure. When considering apical and basal motions independently, we found that apical motion was consistently faster than basal motion in a striking linear relation, and with higher speed for E-RG rosettes (Fig 5B). Higher apical (vs. basal) nuclear migration speeds were previously reported in time-lapse ex vivo cultured embryonic cortical slices [16] as well as in vivo in zebrafish retina and brain [33], providing further validation to our quantitative approach as an in vitro platform for investigating INM. Direct comparison further revealed an ordered relation for rosette speed as follows: apical speed of E-RG rosettes > basal speed of E-RG rosettes > apical speed of M-RG rosettes > basal speed of M-RG rosettes (Fig 5C). Inclusively, these data suggest that during early human cortical development, high NSC numbers are accompanied by elevated INM speed towards apical sites, similar to as shown for radial glial cells in cultured cortical slices ex vivo [16].

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