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

Radial patterns of cell dynamics in neural rosettes.A. Combined HES5::eGFP (green) reporter expression and immunostaining of the cortical neural progenitor marker PAX6 (red) throughout NSC progression from unstructured neuroepithelial cells (top), to early radial glial (E-RG) rosettes (middle) to mid radial glial (M-RG) rosettes (bottom). Nuclei are stained with DAPI (blue). Rosette contours are marked in white. Scale bars: 25 μm. HES5::eGFP co-localizes with PAX6+ nuclei, attesting a NSC stage. E-RG rosettes contain multiple radially organized GFP+/PAX6+ nuclei, whereas M-RG rosettes harbor GFP+/PAX6+ cells only close to rosette lumens, reflective of enhanced or reduced NSC numbers, respectively. Many cells in M-RG rosettes are not associated to apical sites (e.g., rosette lumens), reflecting the beginning of rosette disassembly. B. Representative HES5::eGFP and its matched phase contrast image from time-lapse imaging of an E-RG stage neural rosette (left panels) or a non-rosette area adjacent to a rosette (right panels). Rosette contours and center were manually annotated (white dashed marking). Scale bars: 25 μm. An image was acquired every 5 minutes for a total of 250 minutes. Rosette annotation for M-RG rosettes is shown in S1A Fig. C. Motion patterns follow the expected radial angle. Average patch velocity orientation over time for an E-RG rosette (left, corresponding to panel B) and a non-rosette. Color code is illustrated in panel E (bottom). Radial organization is subjectively observed in E-RG rosettes, for both GFP and phase contrast, but not in non-rosettes. D. Distributions of angular alignment of all patches over the entire time course. INM patterns (tendency to follow the expected angle) are found for E-RG rosettes (left, mean angle of 29° for GFP, 36.4° for phase contrast) but not for non-rosettes (right, mean angle of 44.7° for GFP, 45.7° for phase contrast). E. Schematic sketch of angular alignment γ, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004453.g001: Radial patterns of cell dynamics in neural rosettes.A. Combined HES5::eGFP (green) reporter expression and immunostaining of the cortical neural progenitor marker PAX6 (red) throughout NSC progression from unstructured neuroepithelial cells (top), to early radial glial (E-RG) rosettes (middle) to mid radial glial (M-RG) rosettes (bottom). Nuclei are stained with DAPI (blue). Rosette contours are marked in white. Scale bars: 25 μm. HES5::eGFP co-localizes with PAX6+ nuclei, attesting a NSC stage. E-RG rosettes contain multiple radially organized GFP+/PAX6+ nuclei, whereas M-RG rosettes harbor GFP+/PAX6+ cells only close to rosette lumens, reflective of enhanced or reduced NSC numbers, respectively. Many cells in M-RG rosettes are not associated to apical sites (e.g., rosette lumens), reflecting the beginning of rosette disassembly. B. Representative HES5::eGFP and its matched phase contrast image from time-lapse imaging of an E-RG stage neural rosette (left panels) or a non-rosette area adjacent to a rosette (right panels). Rosette contours and center were manually annotated (white dashed marking). Scale bars: 25 μm. An image was acquired every 5 minutes for a total of 250 minutes. Rosette annotation for M-RG rosettes is shown in S1A Fig. C. Motion patterns follow the expected radial angle. Average patch velocity orientation over time for an E-RG rosette (left, corresponding to panel B) and a non-rosette. Color code is illustrated in panel E (bottom). Radial organization is subjectively observed in E-RG rosettes, for both GFP and phase contrast, but not in non-rosettes. D. Distributions of angular alignment of all patches over the entire time course. INM patterns (tendency to follow the expected angle) are found for E-RG rosettes (left, mean angle of 29° for GFP, 36.4° for phase contrast) but not for non-rosettes (right, mean angle of 44.7° for GFP, 45.7° for phase contrast). E. Schematic sketch of angular alignment γ, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).

Mentions: We used the HES5::eGFP Notch activation reporter human embryonic stem cell (hESC) line, expressing cytoplasmic GFP in Notch active cells [22]. HES5 is a major and direct downstream target of Notch activation pathway (for review, see Ref. [23]) and specifically marks NSCs in vivo. We recently showed that neural rosettes correspond to NSCs of the developing cortex based on their strong apico-basal epithelial polarity and the expression of cortex associated genes such as PAX6 together with the NSC marker HES5::eGFP [20] (Fig 1A). When newly formed E-RG rosettes appear in culture on day 14, most rosette cells express the cortical marker PAX6 and the NSC marker HES5::eGFP (Fig 1A, middle panel) (>80%; see Ref. [20]) in accordance with their high proliferative capacity and lack of differentiation in culture. In contrast, continued culture of E-RG rosettes results in their progression towards M-RG rosettes around day 35, and this is marked by significant loss in the NSC marker HES5::eGFP and the cortical marker PAX6 in rosette cells (Fig 1A, bottom panel) (<30%; see Ref. [20]). Importantly, PAX6 expression is now limited only to the regions adjacent to rosette lumens, reflecting the limited area where stem cells reside at that stage. Since the ability of neural progenitors to radially organize in rosettes is correlated with increased ratios of polarized epithelial NSCs [17,18,20], we hypothesized that this difference in NSC numbers among early and advanced rosettes would be phenotypically reflected in rosette dynamics, specifically that of INM.


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)

Radial patterns of cell dynamics in neural rosettes.A. Combined HES5::eGFP (green) reporter expression and immunostaining of the cortical neural progenitor marker PAX6 (red) throughout NSC progression from unstructured neuroepithelial cells (top), to early radial glial (E-RG) rosettes (middle) to mid radial glial (M-RG) rosettes (bottom). Nuclei are stained with DAPI (blue). Rosette contours are marked in white. Scale bars: 25 μm. HES5::eGFP co-localizes with PAX6+ nuclei, attesting a NSC stage. E-RG rosettes contain multiple radially organized GFP+/PAX6+ nuclei, whereas M-RG rosettes harbor GFP+/PAX6+ cells only close to rosette lumens, reflective of enhanced or reduced NSC numbers, respectively. Many cells in M-RG rosettes are not associated to apical sites (e.g., rosette lumens), reflecting the beginning of rosette disassembly. B. Representative HES5::eGFP and its matched phase contrast image from time-lapse imaging of an E-RG stage neural rosette (left panels) or a non-rosette area adjacent to a rosette (right panels). Rosette contours and center were manually annotated (white dashed marking). Scale bars: 25 μm. An image was acquired every 5 minutes for a total of 250 minutes. Rosette annotation for M-RG rosettes is shown in S1A Fig. C. Motion patterns follow the expected radial angle. Average patch velocity orientation over time for an E-RG rosette (left, corresponding to panel B) and a non-rosette. Color code is illustrated in panel E (bottom). Radial organization is subjectively observed in E-RG rosettes, for both GFP and phase contrast, but not in non-rosettes. D. Distributions of angular alignment of all patches over the entire time course. INM patterns (tendency to follow the expected angle) are found for E-RG rosettes (left, mean angle of 29° for GFP, 36.4° for phase contrast) but not for non-rosettes (right, mean angle of 44.7° for GFP, 45.7° for phase contrast). E. Schematic sketch of angular alignment γ, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004453.g001: Radial patterns of cell dynamics in neural rosettes.A. Combined HES5::eGFP (green) reporter expression and immunostaining of the cortical neural progenitor marker PAX6 (red) throughout NSC progression from unstructured neuroepithelial cells (top), to early radial glial (E-RG) rosettes (middle) to mid radial glial (M-RG) rosettes (bottom). Nuclei are stained with DAPI (blue). Rosette contours are marked in white. Scale bars: 25 μm. HES5::eGFP co-localizes with PAX6+ nuclei, attesting a NSC stage. E-RG rosettes contain multiple radially organized GFP+/PAX6+ nuclei, whereas M-RG rosettes harbor GFP+/PAX6+ cells only close to rosette lumens, reflective of enhanced or reduced NSC numbers, respectively. Many cells in M-RG rosettes are not associated to apical sites (e.g., rosette lumens), reflecting the beginning of rosette disassembly. B. Representative HES5::eGFP and its matched phase contrast image from time-lapse imaging of an E-RG stage neural rosette (left panels) or a non-rosette area adjacent to a rosette (right panels). Rosette contours and center were manually annotated (white dashed marking). Scale bars: 25 μm. An image was acquired every 5 minutes for a total of 250 minutes. Rosette annotation for M-RG rosettes is shown in S1A Fig. C. Motion patterns follow the expected radial angle. Average patch velocity orientation over time for an E-RG rosette (left, corresponding to panel B) and a non-rosette. Color code is illustrated in panel E (bottom). Radial organization is subjectively observed in E-RG rosettes, for both GFP and phase contrast, but not in non-rosettes. D. Distributions of angular alignment of all patches over the entire time course. INM patterns (tendency to follow the expected angle) are found for E-RG rosettes (left, mean angle of 29° for GFP, 36.4° for phase contrast) but not for non-rosettes (right, mean angle of 44.7° for GFP, 45.7° for phase contrast). E. Schematic sketch of angular alignment γ, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).
Mentions: We used the HES5::eGFP Notch activation reporter human embryonic stem cell (hESC) line, expressing cytoplasmic GFP in Notch active cells [22]. HES5 is a major and direct downstream target of Notch activation pathway (for review, see Ref. [23]) and specifically marks NSCs in vivo. We recently showed that neural rosettes correspond to NSCs of the developing cortex based on their strong apico-basal epithelial polarity and the expression of cortex associated genes such as PAX6 together with the NSC marker HES5::eGFP [20] (Fig 1A). When newly formed E-RG rosettes appear in culture on day 14, most rosette cells express the cortical marker PAX6 and the NSC marker HES5::eGFP (Fig 1A, middle panel) (>80%; see Ref. [20]) in accordance with their high proliferative capacity and lack of differentiation in culture. In contrast, continued culture of E-RG rosettes results in their progression towards M-RG rosettes around day 35, and this is marked by significant loss in the NSC marker HES5::eGFP and the cortical marker PAX6 in rosette cells (Fig 1A, bottom panel) (<30%; see Ref. [20]). Importantly, PAX6 expression is now limited only to the regions adjacent to rosette lumens, reflecting the limited area where stem cells reside at that stage. Since the ability of neural progenitors to radially organize in rosettes is correlated with increased ratios of polarized epithelial NSCs [17,18,20], we hypothesized that this difference in NSC numbers among early and advanced rosettes would be phenotypically reflected in rosette dynamics, specifically that of INM.

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