Inma Cobos

Origins of cortical interneuron subtypes.

Cerebral cortical functions are conducted by two general classes of neurons: glutamatergic projection neurons and GABAergic interneurons. Distinct interneuron subtypes serve distinct roles in modulating cortical activity and can be differentially affected in cortical diseases, but little is known about the mechanisms for generating their diversity. Recent evidence suggests that many cortical interneurons originate within the subcortical telencephalon and then migrate tangentially into the overlying cortex. To test the hypothesis that distinct interneuron subtypes are derived from distinct telencephalic subdivisions, we have used an in vitro assay to assess the developmental potential of subregions of the telencephalic proliferative zone (PZ) to give rise to neurochemically defined interneuron subgroups. PZ cells from GFP+ donor mouse embryos were transplanted onto neonatal cortical feeder cells and assessed for their ability to generate specific interneuron subtypes. Our results suggest that the parvalbumin- and the somatostatin-expressing interneuron subgroups originate primarily within the medial ganglionic eminence (MGE) of the subcortical telencephalon, whereas the calretinin-expressing interneurons appear to derive mainly from the caudal ganglionic eminence (CGE). These results are supported by findings from primary cultures of cortex from Nkx2.1 mutants, in which normal MGE fails to form but in which the CGE is less affected. In these cultures, parvalbumin- and somatostatin-expressing cells are absent, although calretinin-expressing interneurons are present. Interestingly, calretinin-expressing bipolar interneurons were nearly absent from cortical cultures of Dlx1/2 mutants. By establishing spatial differences in the origins of interneuron subtypes, these studies lay the groundwork for elucidating the molecular bases for their distinct differentiation pathways.

Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy

Dlx homeodomain transcription factors are essential during embryonic development for the production of forebrain GABAergic interneurons. Here we show that Dlx1 is also required for regulating the functional longevity of cortical and hippocampal interneurons in the adult brain. We demonstrate preferential Dlx1 expression in a subset of cortical and hippocampal interneurons which, in postnatal Dlx1 mutants, show a time-dependent reduction in number. This reduction preferentially affects calretinin+ (bipolar cells) and somatostatin+ subtypes (for example, bitufted cells), whereas parvalbumin+ subpopulations (basket cells and chandelier cells) seem to be unaffected. Cell transplantation analysis demonstrates that interneuron loss reflects cell-autonomous functions of Dlx1. The decrease in the number of interneurons was associated with a reduction of GABA-mediated inhibitory postsynaptic current in neocortex and hippocampus in vitro and cortical dysrhythmia in vivo. Dlx1 mutant mice show generalized electrographic seizures and histological evidence of seizure-induced reorganization, linking the Dlx1 mutation to delayed-onset epilepsy associated with interneuron loss.

Dlx Transcription Factors Promote Migration through Repression of Axon and Dendrite Growth

In the mouse telencephalon, Dlx homeobox transcription factors are essential for the tangential migration of subpallial-derived GABAergic interneurons to neocortex. However, the mechanisms underlying this process are poorly understood. Here, we demonstrate that Dlx1/2 has a central role in restraining neurite growth of subpallial-derived immature interneurons at a stage when they migrate tangentially to cortex. In Dlx1-/-;Dlx2-/- mutants, neurite length is increased and cells fail to migrate. In Dlx1-/-;Dlx2+/- mutants, while the tangential migration of immature interneurons appears normal, they develop dendritic and axonal processes with increased length and decreased branching, and have deficits in their neocortical laminar positions. Thus, Dlx1/2 is required for coordinating programs of neurite maturation and migration. In this regard, we provide genetic evidence that in immature interneurons Dlx1/2 repression of the p21-activated serine/threonine kinase PAK3, a downstream effector of the Rho family of GTPases, is critical in restraining neurite growth and promoting tangential migration.

FGF15 promotes neurogenesis and opposes FGF8 function during neocortical development

BackgroundGrowth, differentiation and regional specification of telencephalic domains, such as the cerebral cortex, are regulated by the interplay of secreted proteins produced by patterning centers and signal transduction systems deployed in the surrounding neuroepithelium. Among other signaling molecules, members of the fibroblast growth factor (FGF) family have a prominent role in regulating growth, differentiation and regional specification. In the mouse telencephalon the rostral patterning center expresses members of the Fgf family (Fgf8, Fgf15, Fgf17, Fgf18). FGF8 and FGF17 signaling have major roles in specification and morphogenesis of the rostroventral telencephalon, whereas the functions of FGF15 and FGF18 in the rostral patterning center have not been established.ResultsUsing Fgf15-/- mutant mice, we provide evidence that FGF15 suppresses proliferation, and that it promotes differentiation, expression of CoupTF1 and caudoventral fate; thus, reducing Fgf15 and Fgf8 dosage have opposite effects. Furthermore, we show that FGF15 and FGF8 differentially phosphorylate ERK (p42/44), AKT and S6 in cultures of embryonic cortex. Finally, we show that FGF15 inhibits proliferation in cortical cultures.ConclusionFGF15 and FGF8 have distinct signaling properties, and opposite effects on neocortical patterning and differentiation; FGF15 promotes CoupTF1 expression, represses proliferation and promotes neural differentiation.

Dlx1&2 and Mash1 Transcription Factors Control MGE and CGE Patterning and Differentiation through Parallel and Overlapping Pathways

Here we define the expression of approximately 100 transcription factors (TFs) in progenitors and neurons of the developing mouse medial and caudal ganglionic eminences, anlage of the basal ganglia and pallial interneurons. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most gamma-aminobutyric acidergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 basic helix-loop-helix (b-HLH) TF. These analyses define core transcriptional components that differentially specify the identity and differentiation of the globus pallidus, basal telencephalon, and pallial interneurons.

Dlx1&2 and Mash1 Transcription Factors Control Striatal Patterning and Differentiation Through Parallel and Overlapping Pathways

Here we define the expression of ≈100 transcription factors in progenitors and neurons of the developing basal ganglia. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Dlx‐dependent and Dlx‐independent pathways. The Dlx‐independent pathway depends in part on the function of the Mash1 b‐HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum. J. Comp. Neurol. 512:556–572, 2009.

The vertebrate ortholog of Aristaless is regulated by Dlx genes in the developing forebrain.

The Dlx transcription factors have a central role in controlling the development of γ‐aminobutyric acid (GABA)‐ergic neurons in the forebrain. However, little is known about how they control the properties of GABAergic neurons. One candidate is the Aristaless (Arx) homeobox gene, which lies genetically downstream of the fly Dlx gene (Distal‐less, Dll). The expression of Arx in the mouse forebrain includes Dlx‐expressing territories, such us the ventral thalamus, parts of the hypothalamus, and the ganglionic eminences and their derivatives in the subpallial telencephalon, and is expressed, as with the Dlx genes, in cortical GABAergic neurons. By using gain‐of‐function and loss‐of‐function assays in mouse and chicken embryos, we show that the Dlx genes have a conserved role in regulating the expression of Arx in the forebrain of vertebrates. Ectopic expression of Dlx genes with electroporation in brain slices from mouse embryos and in the neural tube of chick embryos shows that Dlx genes are sufficient to induce Arx ectopically. Moreover, we provide evidence that the Dlx genes exert a functionally relevant role in regulating Arx in vivo, as shown by the severe reduction in the expression of Arx in Dlx1/2 double‐knockout mice. Therefore, our results suggest evolutionarily conserved functions of Dlx genes in regulating Arx expression between Drosophila and vertebrates. J. Comp. Neurol. 483:292–303, 2005.

Graded phenotypic response to partial and complete deficiency of a brain-specific transcript variant of the winged helix transcription factor RFX4.

One line of mice harboring a cardiac-specific epoxygenase transgene developed head swelling and rapid neurological decline in young adulthood, and had marked hydrocephalus of the lateral and third ventricles. The transgene was found to be inserted into an intron in the mouse Rfx4 locus. This insertion apparently prevented expression of a novel variant transcript of RFX4 (RFX4_v3), a member of the regulatory factor X family of winged helix transcription factors. Interruption of two alleles resulted in profound failure of dorsal midline brain structure formation and perinatal death, presumably by interfering with expression of downstream genes. Interruption of a single allele prevented formation of the subcommissural organ, a structure important for cerebrospinal fluid flow through the aqueduct of Sylvius, and resulted in congenital hydrocephalus. These data implicate the RFX4_v3 variant transcript as being crucial for early brain development, as well as for the genesis of the subcommissural organ. These findings may be relevant to human congenital hydrocephalus, a birth defect that affects ∼0.6 per 1000 newborns.

Transcriptional regulation of cortical interneuron development.

GABAergic interneurons modulate both the development and function of the cerebral cortex through the actions of a variety of subtypes. Despite the relevance to cortical function and dysfunction, including seizure disorders and neuropsychiatric illnesses, relatively little is known about the

Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border.

The avian lateral septal organ (LSO) is a telencephalic circumventricular specialization with liquor‐contacting neurons (Kuenzel and van Tienhoven [1982] J. Comp. Neurol. 206:293–313). We studied the topological position of the chicken LSO relative to molecular borders defined previously within the telencephalic subpallium (Puelles et al. [2000] J. Comp. Neurol. 424:409–438). Differential expression of Dlx5 and Nkx2.1 homeobox genes, or the Shh gene encoding a secreted morphogen, allows distinction of striatal, pallidal, and preoptic subpallial sectors. The chicken LSO complex was characterized chemoarchitectonically from embryonic to posthatching stages, by using immunohistochemistry for calbindin, tyrosine hydroxylase, NKX2.1, and BEN proteins and in situ hybridization for Nkx2.1, Nkx2.2, Nkx6.1, Shh, and Dlx5 mRNA. Medial and lateral parts of LSO appear, respectively, at the striatal part of the septum and adjacent bottom of the lateral ventricle (accumbens), in lateral continuity with another circumventricular organ that forms along a thin subregion of the entire striatum, abutting the molecular striatopallidal boundary; we called this the “striatopallidal organ” (SPO). The SPO displays associated distal periventricular cells, which are lacking in the LSO. Moreover, the SPO is continuous caudomedially with a thin, linear ependymal specialization found around the extended amygdala and preoptic areas. This differs from SPO and LSO in some molecular aspects. We tentatively identified this structure as being composed of an “extended amygdala organ” (EAO) and a “preoptohypothalamic organ” (PHO). The position of LSO, SPO, EAO, and PHO within a linear Dlx5‐expressing ventricular domain that surrounds the Nkx2.1‐expressing pallidopreoptic domain provides an unexpected insight into possible common and differential causal mechanisms underlying their formation. J. Comp. Neurol. 499:745–767, 2006.