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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

Quantitative measures for rosette dynamics.Schematic sketch (top, circular structure represent a neural rosette) and patches visualization (bottom) for radial score (A), basal-to-apical ratio (B) and speed (C), depicted for a representative E-RG rosette. Each patch is assigned its average value over time.
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pcbi.1004453.g002: Quantitative measures for rosette dynamics.Schematic sketch (top, circular structure represent a neural rosette) and patches visualization (bottom) for radial score (A), basal-to-apical ratio (B) and speed (C), depicted for a representative E-RG rosette. Each patch is assigned its average value over time.

Mentions: Based on our initial observations we devised three objective measures to study cell dynamics in rosettes to enable functional quantification. Each measure was defined as a scalar readout per rosette that quantifies different aspects in its dynamics throughout time. The first measure, Radial Score (RS), was defined as the average angular alignment (γ) of all motions in each rosette over the entire time course (Fig 2A). RS quantifies the mean alignment between observed and expected radial angles. Thus, lower scores correspond to better alignment, reflecting a more organized radial migration (denoted radial organization henceforth). The second measure, Basal to Apical ratio (B/A ratio), was defined as the ratio between the number of basal (distal) motions to apical (luminal) motions within rosettes along the entire time course (Fig 2B). RS and B/A ratio were designed to quantify INM in vitro, which corresponds to the basal to apical migration observed for the developing neuroepithelium in vivo [16,19]. The third measure, Speed was defined as the average magnitude of velocity for all patches across time (Fig 2C), a measure that was quantified in vivo [16,19]. When calculated for each time frame over time, these three measures fluctuated around a mean value, validating that the progressive rosette-disassembly in culture is much slower than the four hour imaging course (S2E Fig), thus allowing us to focus on the mean measures as our readouts.


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)

Quantitative measures for rosette dynamics.Schematic sketch (top, circular structure represent a neural rosette) and patches visualization (bottom) for radial score (A), basal-to-apical ratio (B) and speed (C), depicted for a representative E-RG rosette. Each patch is assigned its average value over time.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004453.g002: Quantitative measures for rosette dynamics.Schematic sketch (top, circular structure represent a neural rosette) and patches visualization (bottom) for radial score (A), basal-to-apical ratio (B) and speed (C), depicted for a representative E-RG rosette. Each patch is assigned its average value over time.
Mentions: Based on our initial observations we devised three objective measures to study cell dynamics in rosettes to enable functional quantification. Each measure was defined as a scalar readout per rosette that quantifies different aspects in its dynamics throughout time. The first measure, Radial Score (RS), was defined as the average angular alignment (γ) of all motions in each rosette over the entire time course (Fig 2A). RS quantifies the mean alignment between observed and expected radial angles. Thus, lower scores correspond to better alignment, reflecting a more organized radial migration (denoted radial organization henceforth). The second measure, Basal to Apical ratio (B/A ratio), was defined as the ratio between the number of basal (distal) motions to apical (luminal) motions within rosettes along the entire time course (Fig 2B). RS and B/A ratio were designed to quantify INM in vitro, which corresponds to the basal to apical migration observed for the developing neuroepithelium in vivo [16,19]. The third measure, Speed was defined as the average magnitude of velocity for all patches across time (Fig 2C), a measure that was quantified in vivo [16,19]. When calculated for each time frame over time, these three measures fluctuated around a mean value, validating that the progressive rosette-disassembly in culture is much slower than the four hour imaging course (S2E Fig), thus allowing us to focus on the mean measures as our readouts.

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