Fig. 4.

CHRFAM7A-dependent adaptation to mechanical properties of the extracellular environment. a. Schematic depicting Young's modulus of rodent and human brain. Hydrogel concentrations corresponding to the Young's modulus in kPa (gold) is contrasted to the standard procedure of Matrigel coating (blue). b. Changes in growth cone morphology in response to hydrogel stiffness: representative images of MGE progenitors derived from the null and CHRFAM7A KI lines. c. G-LISA demonstrating activation of small GTPases (top panel) on 2 kPa hydrogel; Rac1/CDC42 ratio (bottom panel) in MGE progenitors derived from the null (grey) and CHRFAM7A KI (orange) lines (n = 3; independent two-tailed T-test. Multiple testing correction was performed by Bonferroni). d. Atomic Force microscopy (AFM): intracellular stiffness of MGE progenitors derived from null (grey) and CHRFAM7A KI (orange) iPSC lines and seeded for 2 h on 0.03 and 0.1 stiffness hydrogels in the absence or presence of Rac1 inhibitor, EHT1864. Total of 50 cells were analyzed for each condition from 5 independent experiments. Averages were compared by an independent two-tailed T-test. Multiple testing correction was performed by Bonferroni. e. Representative images of β-III-tubulin immunofluorescent microscopy of null and CHRFAM7A KI isogenic iPSC derived MGE progenitors 48 h after plating on 0.03% and 0.1% hydrogels. f. Arborization of null (grey) and CHRFAM7A KI (orange) isogenic iPSC derived MGE progenitors on 0.03% and 0.1% hydrogels (left panel) (n = 3 independent experiments, 10 images of total 50 MGE progenitors counted; independent two-tailed T-test). g. Percentage of unipolar (Uni), Bipolar (Bi) and Multipolar (Multi) cells in the total population of MGE progenitors plated on the soft and stiff hydrogel (right panel) h. G-LISA of small GTPases CDC42, Rac1 and RhoA in null (grey) and CHRFAM7A KI (orange) isogenic iPSC derived MGE progenitors on 0.03% and 0.1% hydrogels (n = 3 independent experiments; independent two-tailed T-test). i. Representative images showing differences in axon guidance of MGE progenitors plated on 0.1% stiffness hydrogel. j. MMP2 and MMP9 gene expression levels of null (grey) and CHRFAM7A KI (orange) MGE progenitors upon exposure for 1 h to fibronectin coated stiff hydrogel (0.1%) in the absence or presence of Rac1 inhibitor, EHT1864. PCR data from 3 to 5 independent experiments are depicted as bar graphs with SD. Statistical analysis was performed by an independent two-tailed T-test.