Figure 1.

VSX2⁺ RPCs and PAX6^high RPE Progenitors Form Patterned Epithelial Monolayer Sheets upon Spontaneous Attachment and Spreading of the Cysts

(A–D) In a procedure shown in the scheme, the cysts spontaneously attached to a culture surface and spread, forming monolayer colonies. Adherent cultures on days (D) 12–16 were used for immunocytochemistry. In a well of 24-well plates, 4–7 patches of VSX2⁺ RPCs were found (n = 5/5, independent wells). VSX2⁺ RPCs assembled as a disk on D12 (B, bright field in A) and as a ring on D13 (arrowhead in D, bright field in C). Double-headed arrow (D) indicates the spreading of VSX2⁺ RPCs.

(E–J) Immunostaining of boxed areas in (D). VSX2⁺ RPCs co-expressed SIX3 and PAX6 (E–G). PAX6^high progenitors were peripheral to VSX2⁺ RPCs, forming a pattern in the center-periphery axis (arrowheads and diamond arrowhead in F and G). Both VSX2⁺ RPCs and PAX6^high progenitors expressed TJP1 (F–J).

(K–P) In mouse retinal development, polarized expression of PRKCZ, TJP1, and LAMB1 peaked in E10.5 RPCs (arrows and arrowheads in K–P).

c, center; p, periphery. Scale bars, 100 μm (A–J) and 20 μm (K–P). See also Figures S1 and S2.