(A) Cortical region shown in (B) and source of cortical neurons shown in (C).

(B) X-gal staining of FLRT1-3 expression on coronal sections of E15.5 cortex from Flrt1-3lacZ/lx reporter lines. Cortical layers were identified by DAPI and immunostained for Pax6 and Tbr2.

(C) Flrt1lacZ/+ cortical cultures at E15.5 (2 days in vitro), immunostained for FLRT3 (surface staining, green in merge), X-gal (for labeling Flrt1+ cells, red in merge), and phalloidin. Yellow arrowheads/outlines indicate FLRT1/3 double-positive neurons. Magenta arrowheads/outlines show FLRT3 single-positive neurons. White outlines show FLRT3-negative neurons.

(D) X-gal staining of coronal sections from E15.5 Flrt3 heterozygous (Flrt3lox/lacZ), Flrt3 CKO and Flrt1/3 DKO embryos. Areas in dashed rectangles are shown with higher magnification on the right. Normalized intensity plots are shown, obtained from the areas delineated with a dashed rectangle. Arrowheads indicate regions containing cell clusters, and green boxes highlight lateral and red boxes mediolateral portions of the cortex at intermediate-caudal levels.

(E) Quantification of the intensity fluctuations in those portions of the neocortex; n = 3–5 mice/group, 2 sections/mouse; ∗p < 0.05, one-way ANOVA test with Tukey’s post hoc analysis.

(F) 3D mouse brain (template from the Allen Mouse Brain Atlas) with cortical areas displaying cell clustering in Flrt3 CKO (green area) and Flrt1/3 DKO (blue area) at E15.5.

(G) X-gal staining of coronal sections from E15.5 Flrt3 heterozygous and Flrt1/3 DKO embryos. Cell clusters in the upper CP were identified based on the position of individual X-gal+ neurons (areas in dashed rectangles are shown with higher magnification on the right) using a distance-based clustering method (a cluster was defined as a minimum of 25 cells spaced less than 20 μm). Cluster identification is as follows. Neuron clusters in Flrt1/3 DKO embryos are marked with different colors (red, green, and blue; “Clus”), and centroids of each cluster (cnt) are shown in the same color. Neurons that are not clustered are colored in gray.

(H) Quantification of the number of clusters and the distance between them from the data shown in (G); n = 3–5 mice/group; ∗∗∗p < 0.001, unpaired Student’s t test.

Whiskers in the box plot represent minimum and maximum. The data are presented as mean ± SEM. Scale bars represent 150 μm (B), 14 μm (C), 300 μm (D), and 50 μm (G).