ARHGEF6-dependent cytoskeletal regulation underlies a conserved program of forebrain interneuron development

Department of Molecular Biotechnologies and Health Sciences, University of Turin, Turin, Italy
Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, United States
Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, United States
Rita Levi-Montalcini Department of Neuroscience, University of Turin, Turin, Italy
Neuroscience Institute Cavalieri Ottolenghi University of Turin, Orbassano, Italy
Human Technopole, Milan, Italy
Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Sciences (DINOGMI), University of Genoa, Genoa, Italy
Department of Clinical and Biological Sciences, University of Turin, Turin, Italy
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy
Department of Drug Science, NIS Center, University of Turin, Turin, Italy
Faculty of Medicine, Department of Biophysics, Izmir Bakircay University, Izmir, Turkiye
Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy

https://doi.org/10.7554/eLife.111288.1

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Figures and data

ARHGEF6 expression in the ventral telencephalic lineage and reduced GABAergic IN number in the Arhgef6-KO murine telencephalon.
A. ARHGEF6 expression across brain regions between 8-9 post-conception weeks from bulk RNA-seq data. 30 total samples from 2 unique donors. The dashed line indicates the minimum level of expression detected in the ganglionic eminences (GEs). PFC, prefrontal cortex (areas: dorsolateral prefrontal cortex, anterior [rostral] cingulate [medial prefrontal] cortex, orbital frontal cortex, ventrolateral prefrontal cortex); SMC, sensorimotor cortex (areas: primary motor cortex [area M1, area 4], primary somatosensory cortex [area S1, areas 3, 1, 2], primary motor-sensory cortex); TCX, temporal cortex (areas: temporal neocortex, posterior [caudal] superior temporal cortex, inferolateral temporal cortex [area TEv, area 20], primary auditory cortex [core]); OCX, occipital cortex (areas: occipital neocortex, primary visual cortex [striate cortex, area V1/17]); PCX, parietal cortex (areas: parietal neocortex, posteroventral [inferior] parietal cortex); HIP, hippocampus (areas: hippocampus [hippocampal formation]); AMY, amygdala (areas: amygdaloid complex); STR, striatum (areas: striatum); THAL, thalamus (areas: dorsal thalamus, mediodorsal nucleus of thalamus); CBL/URL, cerebellum, upper rhombic lip (areas: cerebellum, cerebellar cortex, upper [rostral] rhombic lip). B. UMAP of single-nucleus RNA-sequencing (snRNA-seq) data from human postnatal donors (3 months - 13 years) colored by cell type. UMAP of snRNA-seq data with each nuclei’s ARHGEF6 expression. Expression values were clipped at the 99th percentile to improve color scale contrast and reduce the influence of outlier cells. 114,216 cells included from 18 samples across 9 unique subjects. EN-IT-Immature, intratelencephalic excitatory immature neurons; EN-L2_3-IT, layer 2/3 intratelencephalic excitatory neurons; EN-L4-IT, layer 4 intratelencephalic excitatory neurons; EN-L5-ET, layer 5 extratelencephalic excitatory neurons; EN-L5-IT, layer 5 intratelencephalic excitatory neurons; EN-L5_6-NP, layer 5/6 near-projecting excitatory neurons; EN-L6-CT, layer 6 corticothalamic excitatory neurons; EN-L6-IT, layer 6 intratelencephalic excitatory neurons; EN-L6b, layer 6b excitatory neurons; EN-Newborn, Newborn excitatory neurons; EN-Non-IT-Immature, immature non-intratelencephalic excitatory neuron;. IN-CGE-Immature, immature caudal ganglionic eminence-derived inhibitory neurons; IN-CGE-SNCG, immature caudal ganglionic eminence-derived gamma-synuclein inhibitory neurons; IN-CGE-VIP, caudal ganglionic eminence-derived vasoactive intestinal polypeptide inhibitory neurons; IN-MGE-Immature, immature medial ganglionic eminence-derived inhibitory neurons; IN-MGE-PV, medial ganglionic eminence-derived parvalbumin inhibitory neurons; IN-MGE-SST, medial ganglionic eminence-derived somatostatin inhibitory neurons; IN-Mix-LAMP5, mixed lysosomal-associated membrane protein family member 5 inhibitory neurons; IN-NCx_dGE-Immature, immature neocortex and dorsal ganglionic eminence-derived inhibitory neurons; IPC-EN, intermediate progenitor cell for excitatory neurons; OPC, oligodendrocyte precursor cells; RG-oRG, outer radial glial cells; RG-tRG, truncated radial glial cells; RG-vRG, ventricular radial glial cells; Tri-IPC, tripotential intermediate progenitor cells. C. UMAP of single-cell RNA-sequencing (scRNA-seq) data from ∼8 week old (∼P56) mice colored by cell type. UMAP of scRNA-seq data with Arhgef6 expression for each cell cluster. 73347 cells included. PV, parvalbumin; SST, somatostatin; SST CHOLD, somatostatin and chondrolectin; LAMP5, lysosomal-associated membrane protein family member 5; SNCG, gamma-synuclein; VIP, vasoactive intestinal polypeptide; MEIS2, meis homeobox 2; CR, cajal-retzius cell; L2 IT ENTl, layer 2 intratelencephalic lateral entorhinal area; L2 IT ENTm, layer 2 intratelencephalic medial entorhinal area; L2/3 IT CTX, layer 2/3 intratelencephalic isocortex; L2/3 IT ENTl, layer 2/3 intratelencephalic lateral entorhinal area; L2/3 IT PPP, layer 2/3 intratelencephalic postsubiculum-presubiculum-parasubiculum; L2/3 IT RHP, layer 2/3 intratelencephalic retrohippocampal region; L3 IT ENT, layer 3 intratelencephalic entorhinal area; L4 RSP-ACA, layer 4 retrosplenial area-anterior cingulate area; L4/5 IT CTX, layer 4/5 intratelencephalic isocortex; L5 IT CTX, layer 5 intratelencephalic isocortex; L5 PPP, layer 5 postsubiculum-presubiculum-parasubiculum; L5 PT CTX, layer 5 pyramidal tract isocortex; L5/6 IT TPE-ENT, layer 5/6 intratelencephalic temporal association areas-perirhinal area-ectorhinal area-entorhinal area; L5/6 NP CTX, layer 5/6 near-projecting isocortex; L6 CT CTX, layer 6 corticothalamic isocortex; L6 IT CTX, layer 6 intratelencephalic isocortex; L6 IT ENTl, layer 6 intratelencephalic lateral entorhinal area; L6b CTX, layer 6b isocortex; L6b/CT ENT, layer 6b corticothalamic entorhinal area; NP PPP, near-projecting postsubiculum-presubiculum-parasubiculum; NP SUB, near-projecting subiculum; CT SUB, corticothalamic subiculum; Car3, carbonic anhydrase 3; SUB-Pros, subiculum-prosubiculum; CA1-ProS, field cornu ammonis 1-prosubiculum; CA2-IG-FC, field cornu ammonis 2-fasciola cinerea-indusium griseum; CA3, field cornu ammonis 3; DG, dentate gyrus; Astro, astrocyte; Oligo, oligodendrocyte; Micro-PVM, microglia/perivascular macrophage; Endo, endothelial cell; SMC-Peri, smooth muscle cell perivascular area; VLMC, vascular leptomeningeal cell. D. Representative in situ hybridization (ISH) images using an Arhgef6 antisense RNA probe on coronal sections of embryonic (E) 14.5 wild-type (WT) mouse brains. LV, lateral ventricle; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; CGE, caudal ganglionic eminence. Scale bar: 400 µm. E. Representative maximum intensity projection of z-stack images (30 serial image planes, z-step size = 1 µm) of the somatosensory cortex of postnatal (P) 45 GAD67-eGFP (left) and GAD67-eGFP;Arhgef6-KO mice (right). Scale bars, 200 µm. F. Average density of eGFP⁺ GABAergic INs in the adult cortex in each of the 10 bins (shown in E). At least 3 distinct sections distributed along the anteroposterior axis from 3 different mice per genotype were analyzed. p-values (from bin 1 to 10) = (1) 0.004; (2) 0.058; (3) 0.032; (4) 0.012; (5) 0.033; (6) 0.004; (7) 0.005; (8) 0.005; (9) 0.043; (10) 0.044; (TOT) 0.005. G. Average percentage of eGFP⁺ GABAergic INs in the adult cortex over the total number of cells. At least 3 distinct sections distributed along the anteroposterior axis from 3 different mice per genotype were analyzed. p-values = 0.008. H. Representative maximum intensity projection of z-stack images (30 serial image planes, z-step size = 1 µm) of the hippocampus of P45 GAD67-eGFP (left) and GAD67-eGFP;Arhgef6-KO (right) mice. Scale bar, 100 µm. I. Average density of eGFP⁺ GABAergic INs in the cornu ammonis region 1, 2, and 3 (CA1, CA2, CA3), and dentate gyrus (DG) regions and whole hippocampus. At least 3 distinct sections distributed along the anteroposterior axis from 3 different mice per genotype were analyzed. p-values = 0.097 (CA1); 0.00004 (CA2); 0.005 (CA3); 0.12 (DG); 0.044 (TOT). p-values were calculated using unpaired multiple t-tests corrected for False Discovery Rate (<1%) (F) and Holm-Šídák method (H). * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot represents one animal.

Murine embryonic INs show altered migratory patterns and reduced survival.
A. Representative maximum intensity projection of z-stack images (30 serial image planes, z-step size = 1 µm) of telencephalic coronal sections from E14.5 WT (left) and Arhgef6-KO (right) mouse stained for apoptotic nuclei using the TUNEL assay; insets show the corresponding DAPI staining highlighting overall cytoarchitecture. NCX, neocortex; LV, lateral ventricle; hem, cortical hem; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence. Scale bars, 500 µm. B, C. Average percentage of TUNEL punctae over the total number of nuclei. At least 3 distinct sections distributed along the anteroposterior axis from 3 different mice per genotype were analyzed. p-values = 0.1871 (E14.5 NCX); 0.0129 (E14.5 GE). D. Representative maximum intensity projection of z-stack images (30 serial image planes, z-step size = 1 µm) of hippocampal coronal sections from E18.5 WT (left) and Arhgef6-KO (right) mouse stained for apoptotic nuclei using the TUNEL assay; insets show the corresponding DAPI staining highlighting overall cytoarchitecture. NCX, neocortex; DG, dentate gyrus. Scale bars, 500 µm. E. Average percentage of TUNEL punctae over the total number of nuclei. At least 3 distinct sections distributed along the anteroposterior axis from 4 different mice per genotype were analyzed. p-value = 0.029 (E18.5 HIPP). F. Representative maximum intensity projection of z-stack images of neocortical coronal sections from E14.5 GAD67-eGFP (left) and GAD67-eGFP;Arhgef6-KO (right) stained for DAPI. The GAD67-eGFP shows the division of the neocortex into four areas of equal size (1, 2, 3, 4) used for the analysis (G). Scale bars, 200 µm. G. Average percentage of eGFP⁺ GABAergic INs over the total number of cells in each of the four areas. At least 3 distinct sections distributed along the anteroposterior axis from 4 different mice per genotype were analyzed. p-value (from area 1 to 4) = 0.008 (1); 0.006 (2); 0.008 (3); 0.0119 (4). H. Representative maximum intensity projection of z-stack images of neocortices from E14.5 GAD67-eGFP (left) and GAD67-eGFP;Arhgef6-KO (right) embryos; the inset in the GAD67-eGFP shows a schematic representation of the four directions analysed: tangential-dorsal (1), pial (2), ventral (3), ventricular (4). Scale bars, 150 µm. Below, higher magnification of the regions outlined by dashed white boxes, showing different orientations of the leading processes of INs. Scale bar, 50 µm. I. Percentage of eGFP⁺ GABAergic INs with the leading processes oriented in each of the 4 directions. p-values (from direction 1 to 4) = 0.0001 (1); 0.002 (2); 0.003 (3); 0.00004 (4). J. Average length (µm) of leading processes of migrating eGFP⁺ GABAergic INs. p-value < 0.001. p-values were calculated using unpaired t-test (B, C, E, J), unpaired multiple t-tests corrected for False Discovery Rate (<1%) (G, I). * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot represents one animal. Violin plots show the distribution of values with median and quartiles indicated.

Arhgef6-KO INs exhibit simpler branching and reduced excitability.
A. Representative micrographs of eGFP⁺ primary murine hippocampal INs from GAD67-eGFP (left) and GAD67-eGFP;Arhgef6-KO (right) after 10 days in vitro (DIV). Scale bars, 20 µm. B. Sholl analysis showing the overall complexity of arborization in GAD67-eGFP and GAD67-eGFP;Arhgef6-KO primary INs after 10 DIV. p-values (from 20 to 200 µm) = 0.023 (20 µm), 0.054 (40 µm), 0.019 (60 µm), 0.0009 (80 µm), 0.0003 (100 µm), 0.0003 (120 µm), 0.0003 (140 µm), 0.0009 (160 µm), 0.0007 (180 µm), 0.0003 (200 µm). Around 30 neurons from 2 independent primary cultures were analyzed for each genotype. C. Representative whole-cell current clamp recordings of action potentials evoked by 100 pA step current for GAD67-eGFP (top) and GAD67-eGFP;Arhgef6-KO (bottom) INs. D. Average firing frequency vs. current relationships recorded in GAD67-eGFP and GAD67-eGFP;Arhgef6-KO INs in response to a set of injected current steps (from 0 to 300 pA; 10 pA steps). p-values (from 0 to 300 pA) = 0.999 (0 pA), >0.999 (10 pA), 0.999 (20 pA), 0.708 (30 pA), 0.368 (40 pA), 0.102 (50 pA), 0.008 (60 pA), 0.002 (70 pA), 0.0004 (80 pA), 0.0004 (90 pA), 0.0006 (100 pA), 0.0004 (110 pA), 0.0004 (120 pA), 0.0002 (130 pA), 0.0003 (140 pA), 0.0003 (150 pA), 0.0003 (160 pA), 0.0002 (170 pA), 0.0002 (180 pA), 0.00002 (190 pA), 0.0002 (200 pA), 0.00001 (210 pA), 0.00002 (220 pA), 0.00001 (230 pA), 0.000002 (240 pA), 0.00002 (250 pA), 0.00001 (260 pA), 0.000002 (270 pA), 0.000002 (280 pA), 0.0004 (290 pA). Patch-clamp recordings were obtained from 11 cells from 4 GAD67-eGFP mice and 12 cells from 4 GAD67-eGFP;Arhgef6-KO mice. E. Mean firing frequency at steady state (f_ss) in response to current injections (120, 180, and 280 pA) in GAD67-eGFP and GAD67-eGFP;Arhgef6-KO INs. p-values: 0.039 (120 pA), 0.039 (180 pA), 0.024 (280 pA). F. Mean initial firing frequency (f₀) measured at the onset of current injection in GAD67-eGFP and GAD67-eGFP;Arhgef6-KO INs at 120, 180, and 280 pA. p-values: 0.034 (120 pA), 0.0098 (180 pA), 0.127 (280 pA). p-values were calculated using unpaired multiple t-tests corrected for False Discovery Rate (<1%) (B), and Holm-Šídák method (D, E, F). * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Violin plots show the distribution of values with median and quartiles indicated.

Characterization of ventral forebrain organoids from ARHGEF6-KO hiPSCs.
A. Representative brightfield images of RNP negative (control) and ARHGEF6-KO human induced pluripotent stem cell (hiPSC)–derived organoids at 2 months (2M) of differentiation. Scale bars, 4 mm. B–E. Quantification of organoid size, density, and shape descriptors. 3 different batches of differentiation were analyzed. p-values = <0.0001 (area), <0.0001 (integrated density), <0.0001 (aspect ratio), <0.0001 (roundness). F. Immunofluorescence staining of 1-month ventral forebrain organoids showing expression of the neural progenitor marker SOX2 and the ventral telencephalic marker NKX2.1, confirming ventral forebrain identity. Nuclei are labeled with DAPI. Scale bars, 500 µm. G. Detection of apoptotic nuclei by TUNEL assay in RNP negative and ARHGEF6-KO 1-month organoids. Right panels show higher magnification of the boxed regions. Nuclei are labeled with DAPI. Scale bars, 500 µm. H. Average percentage of TUNEL punctae over the total number of nuclei. p-value = 0.015. At least 5 distinct sections from 5 different organoids per genotype from 3 different batches of differentiation were analyzed. I. Immunofluorescence staining for the neural progenitor marker SOX2 and the neuronal marker NEUN, indicating neuronal differentiation in RNP negative and ARHGEF6-KO 1-month organoids. Nuclei are labeled with DAPI. Scale bars, 500 µm. J, K. Quantification of SOX2⁺ progenitors and NEUN⁺ neurons normalized to the total number of nuclei in RNP negative and ARHGEF6-KO organoids. p-values = 0.028 (J), 0.0008 (K). At least 5 distinct sections from 6 different organoids per genotype from 3 different batches of differentiation were analyzed. p-values were calculated using unpaired t-test. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot represents one organoid.

Impaired migration dynamics of DLX1/2b-GFP–labeled INs in ARHGEF6-deficient dorsal–ventral forebrain assembloids.
A. Characterization of a fused dorsal–ventral assembloids by immunofluorescence. The dorsal compartment is labeled by SATB2 (red), whereas the ventral compartment expresses lenti-DLX1/2b-eGFP (green), marking ventral telencephalic IN progenitors. Nuclei are counterstained with DAPI (blue). Insets (1–2) show higher magnification of regions at the dorsal–ventral interface where GFP⁺ INs migrate from the ventral to the dorsal compartment. Scale bars, 500 µm, 150 (insets) µm. B. Representative frames from time-lapse imaging of migrating DLX1/2b-eGFP+ INs in fused assembloids, showing the saltatory migration behavior of individual INs in RNP negative (control) and ARHGEF6-KO assembloids. The tracked neuron is highlighted by dashed boxes in each frame. Scale bars, 100 µm. C. Quantification of the percentage of DLX1/2b-eGFP⁺ INs in the ventral compartment migrating toward the dorsal compartment. p-value = 0.624. D–H. Quantitative analysis of migration parameters of individual DLX1/2b-eGFP⁺ INs in RNP negative and ARHGEF6-KO assembloids. p-values = 0.016 (directness), 0.021 (average velocity), 0.038 (saltation frequency), 0.039 (jump (saltation event) duration), 0.043 (saltation length). At least 32 neurons from 3 independent assembloids per genotype from 2 different batches of differentiation were analyzed. p-values were calculated using unpaired t-test. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot represents one tracked neuron. Violin plots show the distribution of values with median and quartiles indicated.

Impaired neuritogenesis and disrupted growth cone morphology in ARHGEF6-KO human neurons.
A. Representative fluorescence images of reconstructed individual DLX1/2b-eGFP⁺ INs (purple skeletonization) from 4 months (4M) RNP negative (left) and ARHGEF6-KO (right) forebrain ventral organoids. Scale bar, 70 µm. B. Sholl analysis quantifying neurite length/complexity as the number of intersections across concentric radii (10–220 µm). p-values (from 10 to 220 µm) = 0.000003 (10 µm), 0.00003 (20 µm), 0.0003 (30 µm), 0.002 (40 µm), 0.008 (50 µm), 0.027 (60 µm), 0.070 (70 µm), 0.148 (80 µm), 0.263 (90 µm), 0.403 (100 µm), 0.548 (110 µm), 0.615 (120 µm), 0.569 (130 µm), 0.615 (140 µm), 0.679 (150 µm), 0.783 (160 µm), 0.814 (170 µm), 0.835 (180 µm), 0.848 (190 µm), 0.848 (200 µm), 0.848 (210 µm), 0.848 (220 µm). C. Representative image of neurites extending from a 1-month forebrain organoids transduced with LifeAct-GFP lentivirus. The inset contains a transmitted-light (TL) image of the organoid (org) plated showing migrating neurons and neuronal extensions. LifeAct-GFP⁺ growth cones are indicated by purple arrowheads. Scale bars, 50 µm, 200 µm (inset). D. Representative frames from time-lapse recordings of growth cones from RNP negative (top) and ARHGEF6-KO (bottom) LifeAct-GFP⁺ growth cones at 0’’, 50’’, 100’’, 150’’, 200’’, and 250’’. Scale bar, 5 µm. E–G. Quantification of growth cone shape descriptors and dynamics. p-values = < 0.001 (average circularity index), 0.003 (average solidity index), 0.299 (average rate of change in size). A total of 43 growth cones from 6 RNP negative organoids and 60 growth cones from 6 ARHGEF6-KO organoids, derived from 2 independent differentiation batches, were analyzed. Each dot represents one tracked growth cone. Data are shown as mean ± SEM. Statistical comparisons were performed using unpaired t-tests (E-G) and unpaired multiple t-tests corrected for False Discovery Rate (<1%) (B). *p < 0.05, **p < 0.01, ***p < 0.001.

Morphological and intrinsic electrophysiological properties of GAD67-eGFP and GAD67-eGFP;Arhgef6-KO INs.
A–C. Quantification of morphological parameters of reconstructed hippocampal primary INs after 10 days in vitro (DIV). p-values = 0.055 (soma diameter), 0.76 (number of primary neurites), 0.385 (longest neurite length). D–H. Passive membrane properties measured by whole-cell patch-clamp recordings in eGFP⁺ INs from control and Arhgef6-KO cultures. p-values = 0.105 (resting membrane potential, V_rest), 0.464 (input resistance, R_in), 0.287 (membrane capacitance), 0.275 (rheobase). I–L. Active membrane properties of recorded INs. p-values = 0.829 (action potential overshoot), 0.928 (after-hyperpolarization amplitude, AHP), 0.553 (maximum rate of rise of the action potential, Max. rate of rise), 0.182 (action potential half-width). p-values were calculated using unpaired t-test (F-I, K,L) and Mann-Whitney test (A-C, D, J). * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot represents an individual recorded neuron; box plots show median and interquartile range with whiskers indicating the data range.

Mutagenesis of ARHGEF6 in hiPSC and characterization of the line.
A. Chromatogram of Sanger sequencing of human induced pluripotent stem cells (hiPSCs) ATCC-DYS0100, edited clone ARHGEF6-KO (top) and RNP negative isogenic control (bottom). The dashed line indicates the site of the CRISPR/Cas9 double-strand break. The mutation resulted in the deletion of a single cytosine, causing a frameshift mutation. B. Immunofluorescence staining for the expression of hiPSCs markers OCT4 (red) and SOX2 (green) in ARHGEF6-KO iPSCs. Nuclei were stained with DAPI (blue). Scale bars, 50 µm. C. Chromatograms from Sanger sequencing of the edited hiPSC clone showing no sequence changes at the top 3 predicted crRNA off-target sites. D. Immunofluorescence staining of marker genes for all three germ layers in embryoid bodies (EBs) obtained from ARHGEF6-KO hiPSCs. ACTA2 (mesoderm derivative, top, red), TUJ1/MAP2 (neuroectoderm, green), GATA4 (endoderm, bottom, red) and nuclei were stained with DAPI (blue). Scale bars, 200 µm. E. Immunofluorescence staining of neuroepithelial markers (top) Nestin (red), SOX2 (green), and radial glia markers (bottom) PAX6 (red) and FABP7 (green) in neural progenitor cells (NPCs) obtained from ARHGEF6-KO hiPSCs. Scale bars, 80 µm, 200 µm. F. Phalloidin-FITC staining of NPCs obtained from RNP negative (control) and ARHGEF6-KO hiPSC. Scale bars, 50 µm. G. Quantification of the anisotropy of F-Actin fibers in NPCs stained with Phalloidin-FITC. p-value = 0.0464. H. Quantification of polymerized F-actin by measuring the corrected total cell fluorescence (CTCF) of Phalloidin–FITC staining. p-value = 0.0056. p-values were calculated using unpaired t-test. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Data are presented as mean ± SEM. Each dot corresponds to a cell derived from 3 distinct batches of differentiation.

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