Lhx6 regulates the migration of cortical interneurons from the ventral telencephalon but does not specify their GABA phenotype

Abstract

The LIM homeodomain family of transcription factors is involved in many processes in the developing CNS, ranging from cell fate specification to connectivity. A member of this family of transcription factors, lhx6, is expressed in the medial ganglionic eminence (MGE) of the ventral telencephalon, where the vast majority of cortical interneurons are generated. Its expression in the GABA-containing MGE cells that migrate to the cortex suggests that this gene uniquely or in combination with other transcription factors may play a role in the neurochemical identity and migration of these neurons. We performed loss of function studies for lhx6 in mouse embryonic day 13.5 brain slices and dissociated MGE neuronal cultures using Lhx6-targeted small interfering RNA produced by a U6 promoter-driven vector. We found that silencing lhx6 impeded the tangential migration of interneurons into the cortex, although it did not obstruct their dispersion within the ganglionic eminence. Blocking lhx6 expression in dissociated MGE cultured neurons did not interfere with the production of GABA or its synthesizing enzyme. These results indicate that lhx6, unlike the closely related member lhx7, does not regulate neurotransmitter choice in interneurons but plays an important role in their migration from the ventral telencephalon to the neocortex.

Figure 1.

RNAi effectively blocks the expression of lhx6 in GE neurons. A-D, Dissociated MGE cells taken from E13 mice were transfected with pEGFP and control vector (A, B) or pEGFP and the siRNA vector (C, D) and immunostained for Lhx6 (red). A high proportion of MGE cells transfected with pEGFP/control vector were also positive for Lhx6 (A, B, arrows). Dissociated MGE cells transfected with pEGFP/siRNA vector did not exhibit Lhx6 immunoreactivity, indicating that the production of RNAi in these cells inhibited lhx6 expression (n = 5 experiments). E, F, siRNA transfection did not affect cell viability as shown with DAPI nuclear staining (arrows). Scale bars, 50 μm.

Figure 2.

Electroporation of lhx6 silencing constructs in E13.5 mouse brain slices. A-C, The GE was electroporated with control vector/pEGFP, and tangential migration was visualized after 24 hr (A), 48 hr (B), and 72 hr (C). More GFP cells were found in the cortex with time. Electroporation of siRNA vector/pEGFP resulted in labeled neurons dispersing within the GE but not entering the cortex (D). Sometimes, neurons (arrow) would accumulate at the corticostriatal boundary. Scale bars, 200 μm. E, Graph depicts quantification of cell migration from the GE electroporated with control vector (31 ± 4.7 cells within the cortex) and siRNA vector (4 ± 1.5 cells at corticostriatal boundary; Student's t test; p < 0.0001).

Figure 3.

Expression of Lhx6 in dissociated cultures from mouse E13.5 brain. A, The graph depicts percentage colocalization of Lhx6 with various markers in dissociated cultures (n = 3 litters). B, C, Lhx6 (B, red) colocalizes with GABA (C, green) in some cells in dissociated cortical cultures (arrows). GABA cells that do not colocalize with Lhx6 and many Lhx6 cells that do not express GABA are also observed (arrowheads). D, E, Lhx6 (D, red) in cortical dissociated cultures colocalizes (arrows) with MAP2 (E, green). F, G, Lhx6 (red) colocalizes with GABA in MGE cell cultures (arrows), but there are also GABA cells that do not express Lhx6 and vice versa (arrowheads). Scale bar, 50 μm.

Figure 4.

lhx6 silencing in dissociated cultures from mouse E13.5 brain. Dissociated MGE cultures were transfected with lhx6-directed siRNA-producing vector and pEGFP. A-D, Silencing of lhx6 did not affect expression of GABA (A, B, red) or GAD65/67 (C, D, red), because several siRNA-transfected cells (B, D, green) were positive for these markers (arrows). Scale bars, 50 μm. n = 3 litters.