Dlx Homeobox Genes Promote Cortical Interneuron Migration from the Basal Forebrain by Direct Repression of the Semaphorin Receptor Neuropilin-2

Abstract

Dlx homeobox genes play an important role in vertebrate forebrain development. Dlx1/Dlx2 null mice die at birth with an abnormal cortical phenotype, including impaired differentiation and migration of GABAergic interneurons to the neocortex. However, the molecular basis for these defects downstream of loss of Dlx1/Dlx2 function is unknown. Neuropilin-2 (NRP-2) is a receptor for Class III semaphorins, which inhibit neuronal migration. Herein, we show that Neuropilin-2 is a specific DLX1 and DLX2 transcriptional target by applying chromatin immunoprecipitation to embryonic forebrain tissues. Both homeobox proteins repress Nrp-2 expression in vitro, confirming the functional significance of DLX binding. Furthermore, the homeodomain of DLX1 and DLX2 is necessary for DNA binding and this binding is essential for Dlx repression of Nrp-2 expression. Of importance, there is up-regulated and aberrant expression of NRP-2 in the forebrains of Dlx1/Dlx2 null mice. This is the first report that DLX1 or DLX2 can function as transcriptional repressors. Our data show that DLX proteins specifically mediate the repression of Neuropilin-2 in the developing forebrain. As well, our results support the hypothesis that down-regulation of Neuropilin-2 expression may facilitate tangential interneuron migration from the basal forebrain.Members of the Dlx homeobox gene family, orthologs of Distal-less in Drosophila melanogaster, are expressed in the developing brain (1, 2), retina (3), craniofacial structures, and limbs (4). Four Dlx genes, Dlx1, Dlx2, Dlx5, and Dlx6, are expressed in overlapping domains in the subcortical telencephalon and diencephalon, including the ventral thalamus and the ganglionic eminences. There are distinct boundaries of Dlx1 and Dlx2 expression at the pallial-subpallial boundary (1). Insights into the functional role of Dlx genes in development have been primarily gained from analysis of the phenotypes of mice with targeted deletions of Dlx1/Dlx2 (5-8), Dlx5 (9), and Dlx5/Dlx6 (10). The single Dlx1 and Dlx2 knockouts have relatively normal forebrain development at birth, which is consistent with functional redundancy between Dlx1 and Dlx2 in this anatomic region (1, 4). However, postnatal Dlx1 mutants show specific differentiation defects in interneuron subclasses (11,12). In the absence of both Dlx1 and Dlx2 function, there is abnormal development of the subventricular zone (SVZ) 2 of the ganglionic eminences. There is an almost complete loss of tangential migration of GABAergic interneurons from the medial ganglionic eminence (MGE) to the neocortex and from the lateral ganglionic eminence (LGE) to the olfactory bulb (5,13) 3 . These migrations comprise the major source of cortical inhibitory interneurons in the murine telencephalon (5, 14, 15). Furthermore, it is evident that there are both Dlx-dependent and Dlxindependent pathways affecting the differentiation and subsequent migration of interneurons to the striatum (6), olfactory bulb (13), and hippocampus (16).Our understanding of Dlx gene function is limit...

Bidirectional EphrinB3/EphA4 Signaling Mediates the Segregation of Medial Ganglionic Eminence- and Preoptic Area-Derived Interneurons in the Deep and Superficial Migratory Stream

“…1 F). The hypothesis that Nrp-1-and Nrp-2-positive interneurons represent distinct interneuron subsets is supported by a study of Le et al (2007) that provided evidence that Dlx1/Dlx2 specifically repress the transcription of Nrp-2 but not Nrp-1. In agreement with this finding, Dlx1 and Dlx2 expression was reported in the SVZ of the eminences coincident with Nrp-1, but complementary to what we found for Nrp-2 (Long et al, 2009b;Wang et al, 2011).…”

Arx Is a Direct Target of Dlx2 and Thereby Contributes to the Tangential Migration of GABAergic Interneurons

“…In addition, Dlx1/2 can promote GABAergic neuronal cell fate (Stühmer, 2002) while repressing oligodendrocyte precursor cell formation by negatively regulating Olig2 expression (Petryniak et al, 2007). During interneuron migration, Dlx1/2 proteins sustain cell motility by repressing neurite development through the inhibition of the Pak3 kinase, and lead the cellular path by controlling the expression of guidance molecules such as Neuropilin-2 (Le et al, 2007). How Dlx proteins tightly control these different processes at the molecular level is unknown.…”

“…PCR primers used are the following: mArx-F2, 5Ј-GTCTATAAGTACA-ATGGTGACAC-3Ј; mArx-R2, 5Ј-CTCCATC-AAGATCCTTCTC-3Ј (amplification product, 240 bp); interleukin-1␤ (IL-1␤)-F, 5Ј-AC-CTATCTTCTTCGACACATGGG-3Ј; IL-1␤-R, 5Ј-GGGCTTATCATCTTTCAACACGC-3Ј (amplification product 200 bp). PCR primers for Neuropilin-2 (Npn2) regulatory sequences were used as described by Le et al (2007). PCR products were analyzed on 2% agarose gels in 0.5ϫ TBE buffer.…”

“…Subsequently, to determine whether this interaction also occurred in vivo, we performed chromatin immunoprecipitation assays. As a positive control, we used a previously reported Dlx2-binding sequence on the Npn2 promoter, whereas an IL-1␤ promoter element was chosen as unspecific sequence (Le et al, 2007). The chromatin was isolated from E14.5 mouse forebrains and precipitated with a highly specific Dlx2 antibody (Kuwajima et al, 2006).…”

Transcriptional Regulation of Cortical Interneuron Development: Figure 1.

“…Dlx1 and Dlx2 may promote interneuron migration by directly repressing the expression of neuropilin 2 (Le et al, 2007), a known regulator of interneuron migration (Marin et al, 2001). Dlx1 and Dlx2 are also required for Arx1 to be expressed at normal levels (Cobos et al, 2005a).…”