Figure 5.
Inhibition of Myosin and ROCK Activity Disrupts the Self-Organization of Apically Convex VSX2+ Epithelium but Does Not Affect the Apoptosis
(A–R) Myosin inhibitor blebbistatin (50 μM) and ROCK inhibitor Y27632 (10 μM) were supplemented to the medium 1 hr before, during, and following the cell detachment for the time as indicated. After blebbistatin or Y27632 treatment, pMYL2 was reduced or undetectable (arrows in A–C) at D13 + 2 hr; at D13 + 2D, TJP1 was reduced (arrowheads in D–F), VSX2+ epithelium was less evident or absent (arrowheads in G–I), but activated CASP3 (act CASP3) was unaffected (arrowheads in J–L) (n = 4/4, independent aggregates). At D16 + 10D, VSX2+ cells failed to sort out and self-organize to form apically convex epithelium after blebbistatin or Y27632 treatment (arrowheads in M–R; n = 4/4 for Y27632, n = 3/4 for blebbistatin, independent aggregates). In the control, VSX2+ RPCs formed apically convex epithelium (arrowheads in M and P), whereas VSX2– cells were not (arrows in M and P). An internal VSX2+ epithelium with TJP1 at the lumen (∗ in P) deposited LAMB1 at the basal surface (ˆ in P). The fusing region lacked TJP1 at the outer surface (diamond arrow in P). In the blebbistatin-treated aggregates, TJP1 was at luminal surfaces of internal vesicles (∗ in Q). In the Y27632-treated aggregates, TJP1 expression was reduced and irregular (diamond arrowhead in R); LAMB1 pattern was irregular (ˆ in R).