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

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

Requirement for Slit-1 and Robo-2 in zonal segregation of olfactory sensory neuron axons in the main olfactory bulb

The formation of precise stereotypic connections in sensory systems is critical for the ability to detect and process signals from the environment. In the olfactory system, olfactory sensory neurons (OSNs) project axons to spatially defined glomeruli within the olfactory bulb (OB). A spatial relationship exists between the location of OSNs within the olfactory epithelium (OE) and their glomerular targets along the dorsoventral axis in the OB. The molecular mechanisms underlying the zonal segregation of OSN axons along the dorsoventral axis of the OB are poorly understood. Using robo-2/ (roundabout) and slit-1/ mice, we examined the role of the Slit family of axon guidance cues in the targeting of OSN axons during development. We show that a subset of OSN axons that normally project to the dorsal region of the OB mistarget and form glomeruli in the ventral region in robo-2/ and slit-1/ mice. In addition, we show that the Slit receptor, Robo-2, is expressed in OSNs in a high dorsomedial to low ventrolateral gradient across the OE and that Slit-1 and Slit-3 are expressed in the ventral region of the OB. These results indicate that the dorsal-to-ventral segregation of OSN axons are not solely defined by the location of OSNs within the OE but also relies on axon guidance cues

Both doublecortin and doublecortin-like kinase play a role in cortical interneuron migration

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubule-associated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration

Identification of Arx targets unveils new candidates for controlling cortical interneuron migration and differentiation

Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc 12a5, Ets2, Phlda1, Egr1, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, and Napb) and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b, and Slit2) in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy

Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves.

The mesenchymal cushions lining the early embryonic heart undergo complex remodelling to form the membranous ventricular septum as well as the atrioventricular and semilunar valves in later life. Disruption of this process underlies the most common congenital heart defects. Here, we identified a novel role for Slit-Robo signalling in the development of the murine membranous ventricular septum and cardiac valves

Semaphorin3A-neuropilin1 signalling is involved in the generation of cortical interneurons

Cortical interneurons are generated predominantly in the medial ganglionic eminence of the ventral telencephalon and migrate to the cortex during embryonic development. These cells express neuropilin (Nrp1 and Nrp2) receptors which mediate their response to the chemorepulsive class 3 semaphorin (Sema) ligands. We show here that semaphorins Sema3A and Sema3F are expressed in layers adjacent to cortical interneuron migratory streams as well as in the striatum, suggesting they may have a role in guiding these cells throughout their journey. Analysis of Sema3A (-/-) and Sema3F (-/-) mice during corticogenesis showed that absence of Sema3A, but not Sema3F, leads to aberrant migration of cortical interneurons through the striatum. Reduced number of cortical interneurons was found in the cortex of Sema3A (-/-), Nrp1 (-/-) and Nrp2 (-/-) mice, as well as altered distribution in Sema3F (-/-), Nrp1 (-/-), Nrp2 (-/-) animals and especially in neuropilin double mutants. The observed decrease in interneurons in Sema3A (-/-) and Nrp1 (-/-) mice was due to altered proliferative activity of their progenitors highlighted by changes in their mitotic spindle positioning and angle of cleavage plane during cell division. These findings point to a novel role for Sema3A-Nrp1 signalling in progenitor cell dynamics and in the generation of interneurons in the ventral telencephalon

Mutations in ARX Result in Several Defects Involving GABAergic Neurons

Genetic investigations of X-linked mental retardation have demonstrated the implication of ARX in a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities, but with associated features of dystonia and epilepsy. These investigations have in recent years directed attention to the role of this gene in brain development. Analysis of its spatio-temporal localization profile revealed expression in telencephalic structures at all stages of development, mainly restricted to populations of GABA-containing neurons. Furthermore, studies of the effects of ARX loss of function either in humans or in lines of mutant mice revealed varying defects, suggesting multiple roles of this gene during development. In particular, Arx has been shown to contribute to almost all fundamental processes of brain development: patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. In this review, we will present and discuss recent findings concerning the role of ARX in brain development and how this information will be useful to better understand the pathophysiological mechanisms of mental retardation and epilepsy associated with ARX mutations

Molecules and mechanisms involved in the generation and migration of cortical interneurons

The GABA (γ-aminobutyric acid)-containing interneurons of the neocortex are largely derived from the ganglionic eminences in the subpallium. Numerous studies have previously defined the migratory paths travelled by these neurons from their origins to their destinations in the cortex. We review here results of studies that have identified many of the genes expressed in the subpallium that are involved in the specification of the subtypes of cortical interneurons, and the numerous transcription factors, motogenic factors and guidance molecules that are involved in their migration

Altered proliferative ability of neuronal progenitors in PlexinA1 mutant mice

Cortical interneurons are generated predominantly in the medial ganglionic eminence (MGE) and migrate through the ventral and dorsal telencephalon before taking their final positions within the developing cortical plate. Previously we demonstrated that interneurons from Robo1 knockout (Robo1(-/-) ) mice contain reduced levels of neuropilin 1 (Nrp1) and PlexinA1 receptors, rendering them less responsive to the chemorepulsive actions of semaphorin ligands expressed in the striatum and affecting their course of migration (Hernandez-Miranda et al. [2011] J. Neurosci. 31:6174-6187). Earlier studies have highlighted the importance of Nrp1 and Nrp2 in interneuron migration, and here we assess the role of PlexinA1 in this process. We observed significantly fewer cells expressing the interneuron markers Gad67 and Lhx6 in the cortex of PlexinA1(-/-) mice compared with wild-type littermates at E14.5 and E18.5. Although the level of apoptosis was similar in the mutant and control forebrain, proliferation was significantly reduced in the former. Furthermore, progenitor cells in the MGE of PlexinA1(-/-) mice appeared to be poorly anchored to the ventricular surface and showed reduced adhesive properties, which may account for the observed reduction in proliferation. Together our data uncover a novel role for PlexinA1 in forebrain development. J. Comp. Neurol., 2015. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc

Protective role of Cadherin 13 in interneuron development

Cortical interneurons are generated in the ganglionic eminences and migrate through the ventral and dorsal telencephalon before finding their final positions within the cortical plate. During early stages of migration, these cells are present in two well-defined streams within the developing cortex. In an attempt to identify candidate genes which may play a role in interneuron stream specification, we previously carried out a microarray analysis which identified a number of cadherin receptors that were differentially expressed in these streams, including Cadherin-13 (Cdh13). Expression analysis confirmed Cdh13 to be present in the preplate layer at E13.5 and, later in development, in some cortical interneurons and pyramidal cells. Analysis of Cdh13 knockout mice at E18.5, but not at E15.5, showed a reduction in the number of interneurons and late born pyramidal neurons and a concomitant increase in apoptotic cells in the cortex. These observations were confirmed in dissociated cell cultures using overexpression and short interfering RNAs (siRNAs) constructs and dominant negative inhibitory proteins. Our findings identified a novel protective role for Cdh13 in cortical neuron development