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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

Inhibition of ACTIN or NMII reduces INM.Treatment with Blebbistatin or Cytochalasin-B reduces INM measures. E-RG rosettes were treated with or without indicated inhibitors immediately prior to live imaging and throughout the experiment. A. INM measures: Left: RS—signed distances of RS of treated rosettes from control E-G linear model (Blebbistatin: Wilcoxon rank sum test, p = 8.6841e-04; Cytochalasin-B: p = 1.5890e-04). Middle: B/A ratio (Blebbistatin: p = 3.1994e-04; Cytochalasin-B: p = 6.0475e-05). Right: Speed (Blebbistatin: p = 0.2370; Cytochalasin-B: p = 0.0434). Note that all measures were significantly reduced for drug-treated cells excluding speed for Blebbistatin-treatment. 10 control E-RG rosettes, 8 treated with Blebbistatin and 14 with Cytochalasin-B were analyzed in panels A-C. B. Treatment with Blebbistatin or Cytochalasin-B disrupts the ordered spatial distribution of cell cycle markers within rosettes. E-RG rosettes were labeled with BrdU immediately following the experiment and then fixed and immunostained for BrdU and PHH3 marking DNA replication (green) and mitosis (red) phases, respectively. DAPI marks nuclei. Sites of mitosis (PHH3+, arrows) are less confined to rosette centers (arrowheads) under inhibitor treatments.
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pcbi.1004453.g007: Inhibition of ACTIN or NMII reduces INM.Treatment with Blebbistatin or Cytochalasin-B reduces INM measures. E-RG rosettes were treated with or without indicated inhibitors immediately prior to live imaging and throughout the experiment. A. INM measures: Left: RS—signed distances of RS of treated rosettes from control E-G linear model (Blebbistatin: Wilcoxon rank sum test, p = 8.6841e-04; Cytochalasin-B: p = 1.5890e-04). Middle: B/A ratio (Blebbistatin: p = 3.1994e-04; Cytochalasin-B: p = 6.0475e-05). Right: Speed (Blebbistatin: p = 0.2370; Cytochalasin-B: p = 0.0434). Note that all measures were significantly reduced for drug-treated cells excluding speed for Blebbistatin-treatment. 10 control E-RG rosettes, 8 treated with Blebbistatin and 14 with Cytochalasin-B were analyzed in panels A-C. B. Treatment with Blebbistatin or Cytochalasin-B disrupts the ordered spatial distribution of cell cycle markers within rosettes. E-RG rosettes were labeled with BrdU immediately following the experiment and then fixed and immunostained for BrdU and PHH3 marking DNA replication (green) and mitosis (red) phases, respectively. DAPI marks nuclei. Sites of mitosis (PHH3+, arrows) are less confined to rosette centers (arrowheads) under inhibitor treatments.

Mentions: Finally, to further strengthen the validity of our method and to shed some light on mechanisms of INM in vitro, we quantified the effects of pharmacological perturbation on INM. Different molecular motors are thought to mediate nuclei migration [12,34]. Such motors are believed to be a part of the cytoskeletal structural machinery such as actin, or motor proteins such as NMII, both shown to be involved in INM movements [35]. We treated rosettes with Blebbistatin or Cytochalasin-B, two agents known to alter INM by inhibiting NMII ATPase activity or depolymerizing actin, respectively. Quantifying INM dynamics in these rosettes following live imaging (S4–S6 Movies) showed decrease in INM measures (Fig 7A). This was further supported by demonstrating a loss of the ordered spatial composition of cell cycle components within rosettes. This was judged by immunostaining for mitosis (PHH3) and DNA synthesis (BrdU)–two distinct phases in the cell cycle that are spatially distributed to lumen and periphery, respectively (Fig 7B) (See also Ref. [20]). These findings further validate the ability of our quantitative approach to distinguish between rosettes under different molecular perturbations and further provide new evidence for possible roles of these molecular motors in driving INM in human radial glial cells.


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)

Inhibition of ACTIN or NMII reduces INM.Treatment with Blebbistatin or Cytochalasin-B reduces INM measures. E-RG rosettes were treated with or without indicated inhibitors immediately prior to live imaging and throughout the experiment. A. INM measures: Left: RS—signed distances of RS of treated rosettes from control E-G linear model (Blebbistatin: Wilcoxon rank sum test, p = 8.6841e-04; Cytochalasin-B: p = 1.5890e-04). Middle: B/A ratio (Blebbistatin: p = 3.1994e-04; Cytochalasin-B: p = 6.0475e-05). Right: Speed (Blebbistatin: p = 0.2370; Cytochalasin-B: p = 0.0434). Note that all measures were significantly reduced for drug-treated cells excluding speed for Blebbistatin-treatment. 10 control E-RG rosettes, 8 treated with Blebbistatin and 14 with Cytochalasin-B were analyzed in panels A-C. B. Treatment with Blebbistatin or Cytochalasin-B disrupts the ordered spatial distribution of cell cycle markers within rosettes. E-RG rosettes were labeled with BrdU immediately following the experiment and then fixed and immunostained for BrdU and PHH3 marking DNA replication (green) and mitosis (red) phases, respectively. DAPI marks nuclei. Sites of mitosis (PHH3+, arrows) are less confined to rosette centers (arrowheads) under inhibitor treatments.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004453.g007: Inhibition of ACTIN or NMII reduces INM.Treatment with Blebbistatin or Cytochalasin-B reduces INM measures. E-RG rosettes were treated with or without indicated inhibitors immediately prior to live imaging and throughout the experiment. A. INM measures: Left: RS—signed distances of RS of treated rosettes from control E-G linear model (Blebbistatin: Wilcoxon rank sum test, p = 8.6841e-04; Cytochalasin-B: p = 1.5890e-04). Middle: B/A ratio (Blebbistatin: p = 3.1994e-04; Cytochalasin-B: p = 6.0475e-05). Right: Speed (Blebbistatin: p = 0.2370; Cytochalasin-B: p = 0.0434). Note that all measures were significantly reduced for drug-treated cells excluding speed for Blebbistatin-treatment. 10 control E-RG rosettes, 8 treated with Blebbistatin and 14 with Cytochalasin-B were analyzed in panels A-C. B. Treatment with Blebbistatin or Cytochalasin-B disrupts the ordered spatial distribution of cell cycle markers within rosettes. E-RG rosettes were labeled with BrdU immediately following the experiment and then fixed and immunostained for BrdU and PHH3 marking DNA replication (green) and mitosis (red) phases, respectively. DAPI marks nuclei. Sites of mitosis (PHH3+, arrows) are less confined to rosette centers (arrowheads) under inhibitor treatments.
Mentions: Finally, to further strengthen the validity of our method and to shed some light on mechanisms of INM in vitro, we quantified the effects of pharmacological perturbation on INM. Different molecular motors are thought to mediate nuclei migration [12,34]. Such motors are believed to be a part of the cytoskeletal structural machinery such as actin, or motor proteins such as NMII, both shown to be involved in INM movements [35]. We treated rosettes with Blebbistatin or Cytochalasin-B, two agents known to alter INM by inhibiting NMII ATPase activity or depolymerizing actin, respectively. Quantifying INM dynamics in these rosettes following live imaging (S4–S6 Movies) showed decrease in INM measures (Fig 7A). This was further supported by demonstrating a loss of the ordered spatial composition of cell cycle components within rosettes. This was judged by immunostaining for mitosis (PHH3) and DNA synthesis (BrdU)–two distinct phases in the cell cycle that are spatially distributed to lumen and periphery, respectively (Fig 7B) (See also Ref. [20]). These findings further validate the ability of our quantitative approach to distinguish between rosettes under different molecular perturbations and further provide new evidence for possible roles of these molecular motors in driving INM in human radial glial cells.

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