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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

Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.A. Basal and apical RS are associated with rosette size. E-RG rosettes (Left, Pearson: apical Rho = -0.81, p = 5.77E-07; basal Rho = -0.77, p = 5.61E-06). M-RG rosettes (Right, Pearson: apical Rho = -0.67, p = 0.0088; basal Rho = -0.70, p = 0.0048). Linear fit: dashed line for apical, solid for basal motion. B. RS of basal and apical motion are associated (E-RG: Pearson Rho = 0.947, p = 7.883E-13; M-RG: Pearson Rho = 0.899, p = 1.238E-05). Black line y = x, values above this line reflect reduced radial organization (increased RS) of apical motions. C. Left, RS of basal motions in M-RG rosettes were significantly increased (reduced radial organization) compared to E-RG rosettes. Boxplots showing signed distances between RS of basal motion in E-RG and M-RG rosettes to the linear fit between RS of basal motions for E-RG rosettes (Wilcoxon rank sum test, p = 0.039). Right, apical RS values in M-RG rosettes were not found to be significantly farther from ERG’s apical score linear model (Wilcoxon rank sum test, p = 0.1173).
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pcbi.1004453.g004: Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.A. Basal and apical RS are associated with rosette size. E-RG rosettes (Left, Pearson: apical Rho = -0.81, p = 5.77E-07; basal Rho = -0.77, p = 5.61E-06). M-RG rosettes (Right, Pearson: apical Rho = -0.67, p = 0.0088; basal Rho = -0.70, p = 0.0048). Linear fit: dashed line for apical, solid for basal motion. B. RS of basal and apical motion are associated (E-RG: Pearson Rho = 0.947, p = 7.883E-13; M-RG: Pearson Rho = 0.899, p = 1.238E-05). Black line y = x, values above this line reflect reduced radial organization (increased RS) of apical motions. C. Left, RS of basal motions in M-RG rosettes were significantly increased (reduced radial organization) compared to E-RG rosettes. Boxplots showing signed distances between RS of basal motion in E-RG and M-RG rosettes to the linear fit between RS of basal motions for E-RG rosettes (Wilcoxon rank sum test, p = 0.039). Right, apical RS values in M-RG rosettes were not found to be significantly farther from ERG’s apical score linear model (Wilcoxon rank sum test, p = 0.1173).

Mentions: Previous in vivo studies have shown differences in speed between nuclei migrating apically and basally [16,33]. We hypothesized that radial organization may also differ between apical and basal motion. To test this hypothesis we classified each motion vector as moving apically (inward) or basally (outward) with respect to rosette center, reflective of apical and basal nuclei migration during INM, and examined apical and basal motion independently. We partitioned the motion vectors of each rosette into basal and apical groups and calculated each group’s RS. We found that similarly to general RS, basal or apical RS were associated with rosette size (Fig 4A). However, basal motion tends to be more radially organized (i.e., lower RS) than apical motion (Fig 4B, most points above the y = x line), regardless of rosette stage (Fig 4B) or size (Fig 4A, basal RS tends to be lower than apical RS for all rosette sizes). This was also judged by the basal RS values for M-RG rosettes, which were higher (less organized) than the basal RS values predicted by the linear fit of E-RG rosettes (Fig 4C). These results led to the hypothesis that enhanced radial organization of basal motions contributes to the overall elevated radial organization observed for E-RG rosettes.


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)

Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.A. Basal and apical RS are associated with rosette size. E-RG rosettes (Left, Pearson: apical Rho = -0.81, p = 5.77E-07; basal Rho = -0.77, p = 5.61E-06). M-RG rosettes (Right, Pearson: apical Rho = -0.67, p = 0.0088; basal Rho = -0.70, p = 0.0048). Linear fit: dashed line for apical, solid for basal motion. B. RS of basal and apical motion are associated (E-RG: Pearson Rho = 0.947, p = 7.883E-13; M-RG: Pearson Rho = 0.899, p = 1.238E-05). Black line y = x, values above this line reflect reduced radial organization (increased RS) of apical motions. C. Left, RS of basal motions in M-RG rosettes were significantly increased (reduced radial organization) compared to E-RG rosettes. Boxplots showing signed distances between RS of basal motion in E-RG and M-RG rosettes to the linear fit between RS of basal motions for E-RG rosettes (Wilcoxon rank sum test, p = 0.039). Right, apical RS values in M-RG rosettes were not found to be significantly farther from ERG’s apical score linear model (Wilcoxon rank sum test, p = 0.1173).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608579&req=5

pcbi.1004453.g004: Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.A. Basal and apical RS are associated with rosette size. E-RG rosettes (Left, Pearson: apical Rho = -0.81, p = 5.77E-07; basal Rho = -0.77, p = 5.61E-06). M-RG rosettes (Right, Pearson: apical Rho = -0.67, p = 0.0088; basal Rho = -0.70, p = 0.0048). Linear fit: dashed line for apical, solid for basal motion. B. RS of basal and apical motion are associated (E-RG: Pearson Rho = 0.947, p = 7.883E-13; M-RG: Pearson Rho = 0.899, p = 1.238E-05). Black line y = x, values above this line reflect reduced radial organization (increased RS) of apical motions. C. Left, RS of basal motions in M-RG rosettes were significantly increased (reduced radial organization) compared to E-RG rosettes. Boxplots showing signed distances between RS of basal motion in E-RG and M-RG rosettes to the linear fit between RS of basal motions for E-RG rosettes (Wilcoxon rank sum test, p = 0.039). Right, apical RS values in M-RG rosettes were not found to be significantly farther from ERG’s apical score linear model (Wilcoxon rank sum test, p = 0.1173).
Mentions: Previous in vivo studies have shown differences in speed between nuclei migrating apically and basally [16,33]. We hypothesized that radial organization may also differ between apical and basal motion. To test this hypothesis we classified each motion vector as moving apically (inward) or basally (outward) with respect to rosette center, reflective of apical and basal nuclei migration during INM, and examined apical and basal motion independently. We partitioned the motion vectors of each rosette into basal and apical groups and calculated each group’s RS. We found that similarly to general RS, basal or apical RS were associated with rosette size (Fig 4A). However, basal motion tends to be more radially organized (i.e., lower RS) than apical motion (Fig 4B, most points above the y = x line), regardless of rosette stage (Fig 4B) or size (Fig 4A, basal RS tends to be lower than apical RS for all rosette sizes). This was also judged by the basal RS values for M-RG rosettes, which were higher (less organized) than the basal RS values predicted by the linear fit of E-RG rosettes (Fig 4C). These results led to the hypothesis that enhanced radial organization of basal motions contributes to the overall elevated radial organization observed for E-RG rosettes.

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