(A–K) Immunofluorescence for PAX6 (A) TBR2 (F) and PROX1 (I) were performed at indicated developmental (E14.5: Ai, ii; E16.5: Aiii, iv; E18.5: Fi-iii and Ii-iii) and postnatal stages (P2: Fiv-vi and Iiv-vi) of DG development. Arrows in Fii-iii,v-vi and Iv-vi point to respectively TBR2+ and PROX1+ cells accumulating close to the ventricle (yellow dashed line). 1ry, 2ry, and 3ry matrices are delineated with white dashed lines. Localization within the developing DG of each cell type analyzed is schematized for PAX6 at E14.5 (B) and E16.5 (C), for TBR2 at E18.5 and P2 (H) and PROX1 for E18.5 and P2 (K), where color intensity in the illustration reflects level of markers expression. (D) shows experimental analysis, total cell number indicate sum of cells in 1ry, 2ry, and 3ry matrix. Quantification of total PAX6+ cells (E), TBR2+ cells (G) and PROX1+ cells (J) is shown at the indicated developmental and postnatal stages. Total cell number analysis shows a reduced number of TBR2+ cells at P2 (G) in Sox9fl/fl;Sox1Cre/+ mutants (2020.31 ± 267.74; Tukey’s multiple comparison test p=0.01190, ANOVA p=0.014) compared to controls (2792 ± 331.72). The same tendency was observed for TBR2+ cells in P2 Sox9fl/fl;Nestin-Cre mutants (G) (2249.75 ± 195.18), and for PROX1+ cells (J) in both Sox9 mutants at P2 (Sox9fl/fl;Sox1Cre: 2225.50 ± 299.24 and Sox9fl/fl;Nestin-Cre: 2538.75 ± 340.30) compared to controls (2895.33 ± 367.51). (L, M) Analysis of PAX6+, TBR2+, and PROX1+ cells distribution along the three matrices, according to the corresponding above schematics where dashed lines indicate areas considered for 1ry, 2ry, and 3ry matrix quantification (also shown in A, F, Iiv-vi). At E14.5 and E16.5 (L), the same amount of PAX6+ cells are found in the 1ry and 2ry matrix in Sox9fl/fl;Sox1Cre/+ mutants compared to controls. At E18.5 (Mi), more TBR2+ cells were found in the 2ry matrix of Sox9fl/fl;Nestin-Cre mutants (276.53 ± 18.96) compared to both Sox9fl/fl;Sox1Cre/+ mutants (207.33 ± 39.85, p=0.03660) and controls (180.07 ± 1.79, Tukey’s multiple comparison test p=0.00850, ANOVA p=0.0090), while less TBR2+ cells were found in the 3ry matrix of both Sox9fl/fl;Sox1Cre/+ mutants (66.93 ± 7.90, p=0.0016) and Sox9fl/fl;Nestin-Cre mutants (84.00 ± 8.50, p=0.0075) compared to controls (132.53 ± 18.29, Tukey’s multiple comparison test, ANOVA p=0.0017). At P2 (Mii) more TBR2+ cells are found in 1ry matrix of Sox9fl/fl;Sox1Cre/+ mutants (79.47+14.59), compared to controls (36.47 ± 9.87, p=0.0101) and Sox9fl/fl;Nestin-Cre mutants (48.13 ± 10.35, Tukey’s multiple comparison test p=0.0399, ANOVA p=0.0106). In Sox9fl/fl;Nestin-Cre mutants, more TBR2+ cells are accumulating in the 2ry matrix (184.07 ± 8.47) compared to Sox9fl/fl;Sox1Cre/+ mutants (127.87 ± 22.72, Tukey’s multiple comparison test p=0.0175, ANOVA p=0.0183). In both Sox9 mutants, less TBR2+ cells are found in the 3ry matrix (Sox9fl/fl;Sox1Cre/+: 201.00 ± 59.44, p=0.0119; Sox9fl/fl;Nestin-Cre: 233.73 ± 27.81, p=0.029) compared to controls (378.93 ± 57.88, Tukey’s multiple comparison test, ANOVA p=0.0109). At P2, PROX1+ cells (Miii) accumulate in the 1ry matrix of Sox9fl/fl;Sox1Cre/+ mutants, (111.00 ± 39.89) compared to controls (17.67 ± 14.15, Tukey’s multiple comparison test p=0.0088, ANOVA p=0.0100), and a significant decrease in the 3ry matrix of both Sox9fl/fl;Sox1Cre/+ mutants (1786.67 ± 266.25, p=0.0117) and Sox9fl/fl;Nestin-Cre mutants (1991.33±260.48, p=0.0329) is observed compared to controls (2758.33 ± 297.16, Tukey’s multiple comparison test, ANOVA p=0.0112). (N) Analysis of the distribution of PROX1+ granule neurons distribution within the upper and lower blade of the forming DG at E18.5: the 3ry matrix was divided in 10 horizonal ventral to dorsal bins spanning the lower to upper blade domain. Cells were then counted within each bin. The percentage of PROX1+ cells present in each bin is represented. In Sox9fl/fl;Sox1Cre/+ mutants, PROX1+ cells are accumulating in the lower blade (18.40 ± 2.29%) compared to controls (13.57 ± 1.29%, p=0.0187), and are reduced in the upper blade (8.57 ± 0.58%) compared to controls (13.13 ± 0.55%, Tukey’s multiple comparison test p=0.0071, Two-way ANOVA interaction p=0.0387, row factor p<0.0001, column factor p=0.9991). A similar tendency was observed in Sox9fl/fl;Nestin-Cre mutants; however, it did not reached statistical significance. DG: dentate gyrus; DNE: dentate neuroepithelium; CH: cortical hem. Scale bar represent 50 µm in (Ai-ii) 100 µm in (Aiii-iv), (F) and (Ii-iii); 200 µm in (Iiv-vi).