Fig. 7. Slit2^-/-;Ntn1^-/- MECs are unable to form bi-layered aggregates in vitro. (A-J) Aggregates are stained with SMA (red) and DAPI (blue). Bi-layered is defined as having one or more MECs surrounding the LEC aggregate. (A) Wild-type aggregates are bi-layered, with MECs surrounding an LEC aggregate. (B) Thirty percent of Slit2^-/-;Ntn1^-/- aggregates are bi-layered compared with wild type. Addition of (C) 3 µg/ml or (D) 6 µg/ml of SLIT2 and NTN1 restores bi-layered structure of the aggregates. Addition of (E) 3 µg/ml, (F) 6 µg/ml or (G) 12 µg/ml SLIT2 alone partially restores bi-layered aggregate structure. Bi-layered aggregation is not restored in the presence of (H) 3 µg/ml, (I) 6 µg/ml or (J) 12 µg/ml NTN1 alone. Quantification of percentage bi-layered aggregation is below each representative aggregate picture. (K) Quantification of aggregate size in the absence or presence of SLIT2 and NTN1. Slit2^-/-;Ntn1^-/- cells (gray) form greater numbers of smaller aggregates compared with wild-type cells (black). In the presence of 3 µg/ml or 6 µg/ml of SLIT2 and NTN1, Slit2^-/-;Ntn1^-/- cells form larger aggregates (purple). They also form larger aggregates in the presence of SLIT2 alone (blue), but with NTN1 alone (orange), the aggregates remain small. Scale bar: 10 µm.