Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development

<div><p>Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (<i>cytoarchitecture</i>) 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 <i>Neural Rosettes—</i>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 <i>radial organization</i>, 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.</p></div

Radial patterns of cell dynamics in neural rosettes.

<p><b>A.</b> Combined <i>HES5</i>::<i>eGFP</i> (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>B.</b> Representative <i>HES5</i>::<i>eGFP</i> 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 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004453#pcbi.1004453.s008" target="_blank">S1A Fig</a>. <b>C.</b> 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. <b>D.</b> 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). <b>E.</b> Schematic sketch of angular alignment <b>γ</b>, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).</p

Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.

<p><b>A.</b> 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>B.</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. <b>C.</b> 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).</p

Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.

<p>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.</p

Functional instability of M-RG rosettes.

<p>Temporal variance of RS <b>(A)</b>, B/A <b>(B)</b> and Speed <b>(C)</b> was calculated for each rosette over time. <b>A.</b> E-RG rosettes are more stable in RS compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0009). <b>B.</b> E-RG rosettes are more stable in B/A compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.0006). <b>C.</b> E-RG rosettes are more stable in Speed compared to M-RG rosettes (Wilcoxon rank sum test, p = 0.03). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.</p

Inhibition of ACTIN or NMII reduces INM.

<p>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. <b>A.</b> 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>B.</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.</p

Radial score is associated with rosette size, and enhanced for E-RG rosettes.

<p><b>A.</b> Radial score is associated with rosette size. RS of E-RG (Pearson Rho = -0.8, p = 1.55E-06) and M-RG (Pearson Rho = -0.68, p = 0.0112) are associated with rosette size. RS of most M-RG rosettes are above the linear fit of E-RG RS and rosette size (black line), implying reduced radial organization. <b>B.</b> Radial score is elevated for E-RG rosettes. Boxplots showing signed distances between RS of E-RG and M-RG rosettes to the linear fit of E-RG RS and rosette size. Value of 0 implies perfect fit, positive values indicate reduced radial organization. E-RG rosettes are characterized by enhanced radial organization (reduced RS) than M-RG rosettes (Wilcoxon rank-sum test, p = 0.048). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.</p

Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.

<p><b>A.</b> Two fold increase in percent of highly motile cells (above speed of 15 μm hr^-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). <b>B.</b> Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. <b>C.</b> Apical speed of E-RG rosettes (mean = 38.81μm hour^-1) > basal speed of E-RG rosettes (mean = 35.27μm hour^-1) > apical speed of M-RG rosettes (mean = 30.25μm hour^-1) > basal speed of M-RG rosettes (mean = 27.12μm hour^-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.</p

Quantitative measures for rosette dynamics.

<p>Schematic sketch (top, circular structure represent a neural rosette) and patches visualization (bottom) for radial score <b>(A)</b>, basal-to-apical ratio <b>(B)</b> and speed <b>(C)</b>, depicted for a representative E-RG rosette. Each patch is assigned its average value over time.</p