Figure 4. Mef2c and Snap25 are required downstream of Mafb and Maf to promote neurite outgrowth.

A) Schema showing the in vitro culture assay testing Maf and Mafb’s prenatal function on neurite outgrowth. P0 WT and cDKO neocortical tissues were dissociated (INs were labeled with Nkx2.1-cre-driven tdTomato) and diluted 5-fold with reporter negative dissociated P0 WT neocortical tissues before plating on culture slides. (B–C) Representative traces of cultured WT and cDKO INs. (D–F) Representative traces of cultured cDKO INs transfected with Maf, Mef2c or Snap25-expressing plasmids; each showed a rescue phenotype. (G–I) Sholl analysis showing the neurite complexity and the rescue effects of Maf (G), Mef2c (H) and Snap25 (I). (J) Schema showing the in vitro culture assay testing Maf and Mafb’s postnatal function on neurite outgrowth. (K–O) Representative traces of cultured INs using Maf^f/f;Mafb^f/f; RosaT⁺ P0 neocortices to look at the effect of postnatal deletion of Maf and Mafb on neurite outgrowth and if Maf, Mef2c and Snap25 expression showed rescue effects. Thus, postnatal loss of Maf and Mafb (cDKO^POST) leads to defects in neurite outgrowth (K–L). (P–R) Sholl analysis showing the neurite complexity and the rescue effects of Maf (P), Mef2c (Q) and Snap25 (R) on cDKO^POST INs. Scale bar in (F) and (O) = 100 um. N = 3–4 per groups. The quantity of cells used for analysis was included in (G–I) and (P–R).

Figure 4—figure supplement 1. Individual CINs from transplanted WT and Maf cDKO MGE cells grown in vivo for 30 days.

Here we provide a collection of single cell images from the transplant experiment. Top three rows show WT CINs that developed in a WT neocortex in vivo for 30 days, while the bottom four rows show Maf cDKO CINs that developed in a WT neocortex in vivo for 30 days. We quantified the number of dendrites extending from the soma in each genotype, and used unpaired t-test to conduct statistical comparison. The data suggest that Maf cDKO CINs have decreased neurite numbers, which is consistent with our in vitro finding (Figure 4). Each data point represents 1 cells. ****p<0.0001. WT: 15 cells from three animals; Maf cDKO: 20 cells from three animals.

Figure 4—figure supplement 2. Maf cDKO^Sst-IRES-Cre showed defects in neurite outgrowth We used in vitro culture assay to test Mafb and c-Maf’s post-mitotic function on neurite outgrowth.

Briefly, P0 Sst-IRES-Cre generated WT and Maf cDKO neocortical tissues were dissociated (INs were labeled with Cre-dependent tdTomato expression) and diluted 5-fold with reporter negative dissociated P0 WT neocortical tissues before plating on culture slides. Cells were let grow for 14 days in vitro before analysis. (A–B) Representative traces of cultured WT^Sst-IRES-Cre and cDKO^Sst-IRES-Cre INs. (C) Sholl analysis suggested that Maf cDKO^Sst-IRES-Cre INs have less complex neurite growing pattern, suggesting Mafs roles in the post-mitotic stage for IN morphogenesis. N = 3 for both groups. 6–8 neurons per group were used for quantification. Scale bar in (B) = 50 um.