The temporal and spatial origins of cortical interneurons predict their physiological subtype

Interneurons of the cerebral cortex represent a heterogeneous population of cells with important roles in network function. At present, little is known about how these neurons are specified in the developing telencephalon. To explore whether this diversity is established in the early progenitor populations, we conducted in utero fate-mapping of the mouse medial and caudal ganglionic eminences (MGE and CGE, respectively), from which most cortical interneurons arise. Mature interneuron subtypes were assessed by electrophysiological and immunological analysis, as well as by morphological reconstruction. At E13.5, the MGE gives rise to fast-spiking (FS) interneurons, whereas the CGE generates predominantly regular-spiking interneurons (RSNP). Later at E15.5, the CGE produces RSNP classes distinct from those generated from the E13.5 CGE. Thus, we provide evidence that the spatial and temporal origin of interneuron precursors in the developing telencephalic eminences predicts the intrinsic physiological properties of mature interneurons.     

Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon

We have examined the role of Tlx, an orphan nuclear receptor, in dorsal-ventral patterning of the mouse telencephalon. Tlx is expressed broadly in the ventricular zone, with the exception of the dorsomedial and ventromedial regions. The expression spans the pallio-subpallial boundary, which separates the dorsal (i.e. pallium) and ventral (i.e. subpallium) telencephalon. Despite being expressed on both sides of the pallio-subpallial boundary, Tlx homozygous mutants display alterations in the development of this boundary. These alterations include a dorsal shift in the expression limits of certain genes that abut at the pallio-subpallial boundary as well as the abnormal formation of the radial glial palisade that normally marks this boundary. The Tlx mutant phenotype is similar to, but less severe than, that seen in Small eye (i.e. Pax6) mutants. Interestingly, removal of one allele of Pax6 on the homozygous Tlx mutant background significantly worsens the phenotype. Thus Tlx and Pax6 cooperate genetically to regulate the establishment of the pallio-subpallial boundary. The patterning defects in the Tlx mutant telencephalon result in a loss of region-specific gene expression in the ventral-most pallial region. This correlates well with the malformation of the lateral and basolateral amygdala in Tlx mutants, both of which have been suggested to derive from ventral portions of the pallium.     

Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain

Dorsoventral (DV) specification is a crucial step for the development of the vertebrate telencephalon. Clarifying the origin of this mechanism will lead to a better understanding of vertebrate central nervous system (CNS) evolution. Based on the lamprey, a sister group of the gnathostomes (jawed vertebrates), we identified three lamprey Hedgehog (Hh) homologues, which are thought to play central signalling roles in telencephalon patterning. However, unlike in gnathostomes, none of these genes, nor Lhx6/7/8, a marker for the migrating interneuron subtype, was expressed in the ventral telencephalon, consistent with the reported absence of the medial ganglionic eminence (MGE) in this animal. Homologues of Gsh2, Isl1/2 and Sp8, which are involved in the patterning of the lateral ganglionic eminence (LGE) of gnathostomes, were expressed in the lamprey subpallium, as in gnathostomes. Hh signalling is necessary for induction of the subpallium identity in the gnathostome telencephalon. When Hh signalling was inhibited, the ventral identity was disrupted in the lamprey, suggesting that prechordal mesoderm-derived Hh signalling might be involved in the DV patterning of the telencephalon. By blocking fibroblast growth factor (FGF) signalling, the ventral telencephalon was suppressed in the lamprey, as in gnathostomes. We conclude that Hh- and FGF-dependent DV patterning, together with the resultant LGE identity, are likely to have been established in a common ancestor before the divergence of cyclostomes and gnathostomes. Later, gnathostomes would have acquired a novel Hh expression domain corresponding to the MGE, leading to the obtainment of cortical interneurons.     

The homeobox gene Gsh2 is required for retinoid production in the embryonic mouse telencephalon

We have examined the role of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer DARPP-32 neurons than wild types. We show that the expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, we found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Previously, we had shown that removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. As RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, we examined whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, our results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon.     

Characterization of a new Gsx2‐cre line in the developing mouse telencephalon

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis‐regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior–posterior neuraxis. By crossing this transgenic line with the Rosa^tdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE.     

The Gsh2 homeodomain gene controls multiple aspects of telencephalic development

Homeobox genes have recently been demonstrated to be important for the proper patterning of the mammalian telencephalon. One of these genes is Gsh2, whose expression in the forebrain is restricted to the ventral domain. In this study, we demonstrate that Gsh2 is a downstream target of sonic hedgehog and that lack of Gsh2 results in profound defects in telencephalic development. Gsh2 mutants have a significant decrease in the expression of numerous genes that mark early development of the lateral ganglionic eminence, the striatal anlage. Accompanying this early loss of patterning genes is an initial expansion of dorsal telencephalic markers across the cortical-striatal boundary into the lateral ganglionic eminence. Interestingly, as development proceeds, there is compensation for this early loss of markers that is coincident with a molecular re-establishment of the cortical-striatal boundary. Despite this compensation, there is a defect in the development of distinct subpopulations of striatal neurons. Moreover, while our analysis suggests that the migration of the ventrally derived interneurons to the developing cerebral cortex is not significantly affected in Gsh2 mutants, there is a distinct delay in the appearance of GABAergic interneurons in the olfactory bulb. Taken together, our data support a model in which Gsh2, in response to sonic hedgehog signaling, plays a crucial role in multiple aspects of telencephalic development.     

Hindbrain rhombic lip is comprised of discrete progenitor cell populations allocated by Pax6

The lower rhombic lip (LRL) is a germinal zone in the dorsal hindbrain productive of tangentially migrating neurons, streaming extramurally (mossy fiber neurons) or intramurally (climbing fiber neurons). Here we show that LRL territory, operationally defined by Wnt1 expression, is parceled into molecular subdomains predictive of cell fate. Progressing dorsoventrally, Lmx1a and Gdf7 expression identifies the primordium for hindbrain choroid plexus epithelial cells; Math1, for mossy fiber neurons; and immediately ventral to Math1 yet within Wnt1(+) territory, a climbing fiber primordium dominated by Ngn1-expressing cells. Elimination of Pax6 results in expansion of this Ngn1(+) progenitor pool and reduction in the Math1(+) pool, with accompanying later enlargement of the climbing fiber nucleus and reductions in mossy fiber nuclei. Pax6 loss also disrupts Msx expression cell-nonautonomously, suggesting Pax6 may influence LRL progenitor identity indirectly through potentiating BMP signaling. These studies suggest that underlying the diversity and proportions of fates produced by the LRL is a precise suborganization regulated by Pax6.     

Comparison of the mammalian and avian telencephalon from the perspective of gene expression data.

Pallial and subpallial morphological subdivisions of the mouse and chicken telencephalon were examined from the new perspective given by gene markers expressed in these territories during development. The rationale of this approach is that common gene expression patterns may underlie similar histogenetic specification and, consequently, comparable morphological nature. The nested expression domains of the genes Dlx-2 and Nkx-2.1 are characteristic for the subpallium (lateral and medial ganglionic eminences). Similar expression of these markers in parts of the mouse septum and amygdala suggests that such parts may be considered subpallial. The genes Pax-6, Tbr-1 and Emx-1 are expressed in the pallium. Complementary areas of the septum and amygdala shared expression of these genes, suggesting these are the pallial parts of these units. Differences in the relative topography of pallial marker genes also define different regions of the pallium, which can be partially traced into the amygdala. Importantly, there is evidence of a novel "ventral pallium" subdivision, which is a molecularly distinct pallial territory intercalated between the striatum and the lateral pallium. Its derivatives in the mouse apparently belong to the claustroamygdaloid complex. Chicken genes homologous sequence-wise to these mouse developmental genes are expressed in topologically comparable patterns during development. The avian subpallium -the paleostriatum- expresses Dlx-2 and Nkx-2.1; expression extends as well into the septum and anterior and medial parts of the archistriatum. The avian pallium expresses Pax-6, Tbr-1 and Emx-1 and also contains a distinct ventral pallium, formed by the neostriatum and ventral intermediate parts of the archistriatum. The lateral pallium comprises the hyperstriatum ventrale, overlying temporo-parieto-occipital corticoid layer and piriform cortex, plus dorsal intermediate and posterior archistriatum. The dorsal pallium includes the dorsal, intercalated and accessory hyperstriatum, plus the dorsolateral corticoid area. The medial pallium contains the hippocampus and parahippocampal area. A dorsal part of the septum shares pallial molecular markers. Gene markers thus suggest common sets of molecular developmental determinants in either pallial or subpallial domains of the mouse and chicken telencephalon, extending all the way from the posterior pole (amygdala) to the septum. Ventral pallial derivatives identified as claustroamygdaloid in the mouse correlate with avian neostriatum and parts of the archistriatum.     

Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain.

Regional patterning in the developing mammalian brain is partially regulated by restricted gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progeny. Whether or not neural stem cells, which are considered at the top of the neural lineage hierarchy, are regionally specified remains unknown. Here we show that the cardinal properties of neural stem cells (self-renewal and multipotentiality) are conserved among embryonic cortex, ganglionic eminence and midbrain/hindbrain, but that these different stem cells express separate molecular markers of regional identity in vitro, even after passaging. Neural stem cell progeny derived from ganglionic eminence but not from other regions are specified to respond to local environmental cues to migrate ventrolaterally, when initially deposited on the germinal layer of ganglionic eminence in organotypic slice cultures. Cues exclusively from the ventral forebrain in a 5 day co-culture paradigm could induce both early onset and late onset marker gene expression of regional identity in neural stem cell colonies derived from both the dorsal and ventral forebrain as well as from the midbrain/hindbrain. Thus, neural stem cells and their progeny are regionally specified in the developing brain, but this regional identity can be altered by local inductive cues.     

Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene

The processes that specify early regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The mouse open brain (opb) gene plays an essential role in dorsal neural patterning: in the caudal spinal cord of opb mutants, dorsal cell types are absent and markers of ventral fates, including Shh, expand into dorsal regions. Analysis of the opb mutant phenotype and of opb/opb <--> wild-type chimeric embryos reveals that early in neural development, the wild-type opb gene (opb(+)) is required cell autonomously for the expression of Pax7 in dorsal cells and Pax6 in lateral cells. Thus the opb(+) gene product acts intracellularly in the reception or interpretation of signals that determine cell types in the dorsal 80% of the neural tube. At later stages, the lack of opb(+) causes a non-cell-autonomous expansion of ventral cell types into dorsal regions of the neural tube, revealing that opb(+) controls the production of a diffusible molecule that defines the domain of Shh expression. The data indicate that opb(+) could act as either a novel component of a dorsalizing pathway or a novel intracellular negative regulator of the Shh signal transduction pathway.     

PROX1: A Lineage Tracer for Cortical Interneurons Originating in the Lateral/Caudal Ganglionic Eminence and Preoptic Area

The homeobox-encoding gene Prox1 and its Drosophila homologue prospero are key regulators of cell fate-specification. In the developing rodent cortex a sparse population of cells thought to correspond to late-generated cortical pyramidal neuron precursors expresses PROX1. Using a series of transgenic mice that mark cell lineages in the subcortical telencephalon and, more specifically, different populations of cortical interneurons, we demonstrate that neurons expressing PROX1 do not represent pyramidal neurons or their precursors but are instead subsets of cortical interneurons. These correspond to interneurons originating in the lateral/caudal ganglionic eminence (LGE/CGE) and a small number of preoptic area (POA)-derived neurons. Expression within the cortex can be detected from late embryonic stages onwards when cortical interneurons are still migrating. There is persistent expression in postmitotic cells in the mature brain mainly in the outer cortical layers. PROX1^+ve interneurons express neurochemical markers such as calretinin, neuropeptide Y, reelin and vasoactive intestinal peptide, all of which are enriched in LGE/CGE- and some POA-derived cells. Unlike in the cortex, in the striatum PROX1 marks nearly all interneurons regardless of their origin. Weak expression of PROX1 can also be detected in oligodendrocyte lineage cells throughout the forebrain. Our data show that PROX1 can be used as a genetic lineage tracer of nearly all LGE/CGE- and subsets POA-derived cortical interneurons at all developmental and postnatal stages in vivo.     

Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos.

The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organised into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signalling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signalling is still unclear, owing to overlapping functions of Fgf family genes. To address this, we have examined, in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signalling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signalling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. We then examine the function of Fgf signalling in telencephalic development by use of several inhibitors to Fgf signalling cascade, including dominant-negative forms of Ras (Ras(N17)) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeded but the development of the subpallial telencephalon was suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development.     

Cell cycle regulation and interneuron production

The regulation of progenitor proliferation in developing brain in has been extensively studied in the cerebral cortex, but relatively little is known about progenitor divisions in ventral germinal zones. Recent observations pertinent to interneuron genesis in the ventral forebrain, especially in the medial ganglionic eminence, indicate similarities to cerebral cortical neurogenesis and hint at some interesting differences between ventral and dorsal telencephalon progenitors. Proliferation within the ganglionic eminences is discussed from the vantage point of neural precursor cell cycles, especially G1-phase, and current models of neurogenic divisions in cortex that may apply to ventral forebrain as well.