Figure 3. FoxG1 down-regulation promotes an early to late multipolar phase transition through induction of Unc5D.

Migrating pyramidal neuron precursors that failed to down-regulate FoxG1 were delayed within the lower part of the intermediate zone (Figure 2). These cells shut off Tbr2 (A) but ectopically maintained NeuroD1 expression (B) (in a region above the normal expression domain, asterisk), suggesting that they become arrested at the early multipolar phase. (C, C’) Control cells within the intermediate zone expressed Unc5D (arrowheads in C’). (D, D’) FoxG1 gain-of-function cells, although they possess multipolar morphology, failed to express Unc5D protein (open arrowheads). (E) As we have previously shown, three days after electroporation at E13.5, FoxG1 gain-of-function cells (with pCAG-FoxG1-IRES EGFP) remained within the lower part of the intermediate zone. Note that mCherry was expressed in NeuroD1-expressing cells by co-introducing a pNeuroD1-IRES mCherry control vector. (F) When Unc5D expression was restored in NeuroD1-expressing FoxG1 gain-of-function cells (by using a pNeuroD1-Unc5D-IRES mCherry vector), a subset of these pyramidal neuron precursors migrated normally into the cortical plate after three days (green and red cells) and after 13 days at P7 (G, G’, H, I), we observed cells located in both layers II/III and layer IV (see summary in J). The partial restoration in the laminar location of this population at P7 was consistent with the degree of rescue in migration we observed after three days (F, compare with Figure 2A). In FoxG1 gain-of-function cells, both rescued (in layer IV) and non-rescued (in layers II/III) populations express molecular signatures appropriate to their laminar locations. Specifically, Unc5D-rescued cells in layer IV showed molecular expression profiles consistent with them being layer IV cells, i.e., Cux1-on (G’), Brn2-low (H) and RORβ-on (I). (J) Schematized layer distribution of Control (pCAG-IRES EGFP) (Figures 2E to 2G), FoxG1 gain-of-function (pCAG-FoxG1-IRES EGFP) (Figures 2H to 2J) and Unc5D-rescued FoxG1 gain-of-function (pCAG-FoxG1-IRES EGFP + pNeuroD1-Unc5D-IRES mCherry) experiments. Note that the numbers of EGFP-labeled cells (including the low-expressors) within the cortical plate is normalized (20 cells) and represented in this scheme. (K) Similar to FoxG1 gain-of-function, Dcc over-expression (pCAG-Dcc-IRES EGFP) delays cell migration at the intermediate zone. (L) Unc5D over-expression (pCAG-Unc5D-IRES EGFP) rescues the impaired migration phenotype observed by Dcc over-expression. Thus, a precise balance between Dcc versus Unc5D expression is important for cells to migrate through the intermediate zone (See also Supplemental Figures S4). This balance appears to be critically controlled by transient FoxG1 down-regulation as FoxG1 gain-of-function affects Unc5D (D, D’) but not Dcc expression (data not shown).

Scale bars: 50µm, except for C’ and D’: 20µm