Limits...
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

Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.E-RG rosettes (top panel) correspond to early cortical radial glial cells (NSCs; green colored) that hold strong epithelial characteristics and hence organize in a radial manner with apical sites adjoining at rosette lumens—similarly to the ventricular zone of the developing cortex. M-RG rosettes (bottom) are characterized by decreased numbers of epithelial radial glial cells and elevated number of neurons (blue colored) and intermediate progenitors (red colored), which are both non-epithelial—hence decreasing rosette epithelial integrity and eventually lead to rosette disassembly at later stages. Both E-RG and M-RG rosettes perform INM—the hallmark of cortical radial glial development. INM of E-RG rosettes is characterized by basal (blue phase, right) and apical (red phase, right) motions that are faster (higher frequency of blue and red phases) and more radially organized (less twisted pattern of blue and red phases), compared to M-RG rosettes. However, for all rosettes regardless of developmental stage (top or bottom panels), basal motions (blue) are always slower yet more organized than apical motions (red). The enhanced radial organization of E-RG rosettes can be explained by enhanced radial organization of basal motions as well as inherent mechanism that increases both basal and apical motions, possibly due to the strong confining structure and high NSC abundance within E-RG rosettes. B→A, basal to apical; A→B, apical to basal.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608579&req=5

pcbi.1004453.g008: Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.E-RG rosettes (top panel) correspond to early cortical radial glial cells (NSCs; green colored) that hold strong epithelial characteristics and hence organize in a radial manner with apical sites adjoining at rosette lumens—similarly to the ventricular zone of the developing cortex. M-RG rosettes (bottom) are characterized by decreased numbers of epithelial radial glial cells and elevated number of neurons (blue colored) and intermediate progenitors (red colored), which are both non-epithelial—hence decreasing rosette epithelial integrity and eventually lead to rosette disassembly at later stages. Both E-RG and M-RG rosettes perform INM—the hallmark of cortical radial glial development. INM of E-RG rosettes is characterized by basal (blue phase, right) and apical (red phase, right) motions that are faster (higher frequency of blue and red phases) and more radially organized (less twisted pattern of blue and red phases), compared to M-RG rosettes. However, for all rosettes regardless of developmental stage (top or bottom panels), basal motions (blue) are always slower yet more organized than apical motions (red). The enhanced radial organization of E-RG rosettes can be explained by enhanced radial organization of basal motions as well as inherent mechanism that increases both basal and apical motions, possibly due to the strong confining structure and high NSC abundance within E-RG rosettes. B→A, basal to apical; A→B, apical to basal.

Mentions: We propose three quantitative measures as means to quantify rosette dynamics: RS, B/A ratio and speed. These measures were used to assess differences in dynamics between early (E-RG) and late (M-RG) rosettes, revealing that INM of early rosettes is more efficient. This may well reflect the situation in the developing cortex: E-RG rosettes correspond to symmetrically dividing NSCs during early cortical development [20], which exhibit high self-replication rates and low levels of differentiation, resulting in increased number of cells undergoing INM within the ventricular zone. This implies that radial glial cells during early cortical development hold inherently elevated radial organization that may be required for accommodating the high traffic and orchestrating cell motion and cell division. In this regard, the radial expansion of the ventricular zone, which can be mirrored in vitro by the emergence of larger rosettes, may add greater mechanical constrains that ultimately contribute as well to enhance radial organization. At more advanced stages of cortical development, which are reflected by the M-RG stage in vitro [20], the production of neurons and intermediate progenitors—both non-polarized cells—is prevalent due to increase in asymmetric cell division of the corresponding radial glial cells. This occurs on the expanse of polarized NSCs adjacent to apical sites, which still perform INM. Thus, the accumulation of differentiated progeny increases non-NSC ratios, which in turn disrupt radial organization performance (Fig 8). To conclude, our analyses provide a first link between function and 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)

Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.E-RG rosettes (top panel) correspond to early cortical radial glial cells (NSCs; green colored) that hold strong epithelial characteristics and hence organize in a radial manner with apical sites adjoining at rosette lumens—similarly to the ventricular zone of the developing cortex. M-RG rosettes (bottom) are characterized by decreased numbers of epithelial radial glial cells and elevated number of neurons (blue colored) and intermediate progenitors (red colored), which are both non-epithelial—hence decreasing rosette epithelial integrity and eventually lead to rosette disassembly at later stages. Both E-RG and M-RG rosettes perform INM—the hallmark of cortical radial glial development. INM of E-RG rosettes is characterized by basal (blue phase, right) and apical (red phase, right) motions that are faster (higher frequency of blue and red phases) and more radially organized (less twisted pattern of blue and red phases), compared to M-RG rosettes. However, for all rosettes regardless of developmental stage (top or bottom panels), basal motions (blue) are always slower yet more organized than apical motions (red). The enhanced radial organization of E-RG rosettes can be explained by enhanced radial organization of basal motions as well as inherent mechanism that increases both basal and apical motions, possibly due to the strong confining structure and high NSC abundance within E-RG rosettes. B→A, basal to apical; A→B, apical to basal.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004453.g008: Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.E-RG rosettes (top panel) correspond to early cortical radial glial cells (NSCs; green colored) that hold strong epithelial characteristics and hence organize in a radial manner with apical sites adjoining at rosette lumens—similarly to the ventricular zone of the developing cortex. M-RG rosettes (bottom) are characterized by decreased numbers of epithelial radial glial cells and elevated number of neurons (blue colored) and intermediate progenitors (red colored), which are both non-epithelial—hence decreasing rosette epithelial integrity and eventually lead to rosette disassembly at later stages. Both E-RG and M-RG rosettes perform INM—the hallmark of cortical radial glial development. INM of E-RG rosettes is characterized by basal (blue phase, right) and apical (red phase, right) motions that are faster (higher frequency of blue and red phases) and more radially organized (less twisted pattern of blue and red phases), compared to M-RG rosettes. However, for all rosettes regardless of developmental stage (top or bottom panels), basal motions (blue) are always slower yet more organized than apical motions (red). The enhanced radial organization of E-RG rosettes can be explained by enhanced radial organization of basal motions as well as inherent mechanism that increases both basal and apical motions, possibly due to the strong confining structure and high NSC abundance within E-RG rosettes. B→A, basal to apical; A→B, apical to basal.
Mentions: We propose three quantitative measures as means to quantify rosette dynamics: RS, B/A ratio and speed. These measures were used to assess differences in dynamics between early (E-RG) and late (M-RG) rosettes, revealing that INM of early rosettes is more efficient. This may well reflect the situation in the developing cortex: E-RG rosettes correspond to symmetrically dividing NSCs during early cortical development [20], which exhibit high self-replication rates and low levels of differentiation, resulting in increased number of cells undergoing INM within the ventricular zone. This implies that radial glial cells during early cortical development hold inherently elevated radial organization that may be required for accommodating the high traffic and orchestrating cell motion and cell division. In this regard, the radial expansion of the ventricular zone, which can be mirrored in vitro by the emergence of larger rosettes, may add greater mechanical constrains that ultimately contribute as well to enhance radial organization. At more advanced stages of cortical development, which are reflected by the M-RG stage in vitro [20], the production of neurons and intermediate progenitors—both non-polarized cells—is prevalent due to increase in asymmetric cell division of the corresponding radial glial cells. This occurs on the expanse of polarized NSCs adjacent to apical sites, which still perform INM. Thus, the accumulation of differentiated progeny increases non-NSC ratios, which in turn disrupt radial organization performance (Fig 8). To conclude, our analyses provide a first link between function and 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