• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC May 3, 2011.
Published in final edited form as:
PMCID: PMC3071646
NIHMSID: NIHMS250395

The mouse homeobox gene Gbx2 is required for the development of cholinergic interneurons in the striatum

Abstract

Mammalian forebrain cholinergic neurons are composed of local circuit neurons in the striatum and projection neurons in the basal forebrain. These neurons are known to arise from a common pool of progenitors that primarily resides in the medial ganglionic eminence (MGE). However, little is known about the genetic programs that differentiate these two types of cholinergic neurons. Using inducible genetic fate mapping, here we have examined the developmental fate of cells that express the homeodomain transcription factor Gbx2 in the MGE. We show that the Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to almost all cholinergic interneurons in the striatum, while those undergoing radial migration mainly produce non-cholinergic neurons in the basal forebrain. Deletion of Gbx2 throughout the mouse embryo or specifically in the MGE results in abnormal distribution and significant reduction of cholinergic neurons in the striatum. We show that early-born cholinergic interneurons preferentially populate the lateral aspect of the striatum and mature earlier than late-born neurons, which normally reside in the medial part of the striatum. In the absence of Gbx2, early-born striatal cholinergic precursors display abnormal neurite outgrowth and increased complexity, and abnormally contribute to the medial of the caudate-putamen, while late-born (after embryonic day 12.5) striatal cholinergic interneurons are largely missing. Together, our data demonstrate that Gbx2 is required for development of striatal cholinergic interneurons, perhaps by regulating tangential migration of the striatal cholinergic precursors.

Keywords: medial ganglionic eminence, striatum, cholinergic neuron, interneurons, cell fate, mice, transcription factor, Gbx2

Introduction

The mammalian forebrain contains two main groups of cholinergic neurons, which differ in their location and connectivity. One group consists of local circuit neurons (interneurons), which are located in the striatum and receive synaptic input from midbrain dopaminergic neurons and GABAergic projection neurons in the striatum (Gerfen, 1992; Kaneko et al., 2000). The other group of cholinergic neurons is distributed in different nuclei in the so-called Ch1-4 areas in the basal forebrain (Mesulam et al., 1983). These cholinergic neurons are projection neurons and innervate cortical and subcortical structures (Mesulam et al., 1983). Although they share similar biochemical properties, these two types of forebrain cholinergic neurons have very different functions (Berger-Sweeney, 2003; Smythies, 2005; Pisani et al., 2007).

All forebrain cholinergic neurons are generated in the ventral telencephalon, including the medial ganglionic eminence (MGE) and preoptic area (POA) (Olsson et al., 1998; Marin et al., 2000). Cholinergic precursors from the MGE/POA undergo radial migration to form projection neurons in the basal forebrain, whereas those that undergo tangential migration form interneurons in the striatum (Marin et al., 2000). Although much progress has been made in understanding the development of the ventral telencephalon (Marin and Rubenstein, 2001; Wonders and Anderson, 2006), the molecular mechanism that differentiates striatal cholinergic interneurons from the cholinergic projection neurons in Ch1-4 remains to be elucidated.

In the striatum, cholinergic interneurons are differentially distributed in distinct functional and chemical compartments (Gerfen, 1992; Bernacer et al., 2007). Although striatal cholinergic interneurons, which are born between E12 and E17 in rat, are the earliest born neurons in the striatum (Semba and Fibiger, 1988; Phelps et al., 1989), maturation of these neurons takes place after birth (Mobley et al., 1989; Gould et al., 1991). It has been shown that choline acetyltransferase (ChAT), the key enzyme for acetylcholine synthesis, is expressed in caudal-to-rostral and lateral-to-medial gradients in the dorsal striatum in rat during postnatal development (Semba and Fibiger, 1988; Phelps et al., 1989). However, little is known about the relationship among neurogenesis, distribution, and maturation of striatal cholinergic interneurons.

In the ventral telencephalon, the mouse homeobox gene Gbx2 is expressed in the mantle zone (MZ) of the MGE (Bulfone et al., 1993). Interestingly, the expression of Gbx2 is reduced in the MGE of Lhx8-deficient mice, in which the formation of forebrain cholinergic neurons is severely disrupted, suggesting that Gbx2 may act downstream of Lhx8 to regulate the development of cholinergic neurons (Zhao et al., 2003). In this study, using inducible genetic fate mapping, we show that Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to striatal cholinergic interneurons, whereas Gbx2-derived cells that undergo radial migration mainly give rise to GABAergic and other non-cholinergic neurons in the basal forebrain. Inactivation of Gbx2 disrupts the migration of striatal cholinergic precursors, resulting in significant reduction and abnormal distribution of cholinergic interneurons in the striatum.

Materials and Methods

Animals and tissue preparation

The generation and genotyping of Gbx2CreER knock-in and Gbx2 conditional mutation, Gbx2F, have been described previously (Li et al., 2002; Chen et al., 2009). Nkx2.1-Cre BAC transgenic mice were reported previously (Qing Xu, 2008). Mice were maintained on an outbred CD1 genetic background (Charles River Lab, Wilmington, MA). All animal procedures described herein were approved by the Animal Care Committee at the University of Connecticut Health Center.

Noon of the day on which the vaginal plug was found was designated as embryonic day 0.5 (E0.5). For inducible genetic fate mapping, Gbx2CreER/+; R26R−/− males, homozygous for the Cre reporter R26R (Soriano, 1999), were bred with wild-type (WT) or Gbx2+/− females (Wassarman et al., 1997). 4–6 mg of tamoxifen (Sigma) in corn oil (20 mg/ml) was administered by oral gavage to pregnant females as described previously (Li and Joyner, 2001). For tissue preparation, embryonic brains were fixed by immersion in 4% paraformaldehyde (PFA) at 4°C for 3–16 hr. Postnatal mice were deeply anesthetized and transcardially perfused with 4% PFA. Brains were postfixed in the same fixative overnight at 4°C. For frozen tissue sections, brains were cryoprotected in 30% sucrose in PBS, rapidly frozen in OCT compound (Sakura Finetek USA, Torrance, CA), and sectioned with freezing microtome.

In situ hybridization, β-galactosidase, NADPH-diaphorase histochemistry and immunohistochemistry

Embryos or brains were processed for in situ hybridization as described previously (Guo and Li, 2007). Standard X-gal staining was used to examine β-galactosidase (β-gal) activity (Nagy, 2003). To perform NADPH-diaphorase histochemistry, free-floating tissue sections were incubated with freshly-made staining solution (0.5 mg/ml β-NADPH, 0.2 mg/ml nitroblue tetrazolium in 0.3% PBST) for 30 minutes to 2 hours at 37 °C. Primary antibodies used in the study: mouse anti-BrdU (BD Biosciences, San Jose, CA), goat anti-ChAT and rabbit anti-DARPP-32 (Chemicon, Billerica, MA), rabbit anti-GABA (Sigma, St. Louis, MO), rabbit anti-EGFP (Invitrogen, Carlsbad, CA), rat anti-EGFP (Nacalai Tesque, San Diego, CA), mouse anti-Islet1 (DSHB, Iowa City, IA), rat anti-Ki67 (DAKO, CA), rabbit anti-Olig2 (IBL, Minneapolis, MN), rabbit anti-pH3 (Upstate, Billerica, MA), rabbit anti-Casp3 (cleaved) (Cell Signaling Technology, Danvers, MA), rabbit anti-TrkA (courtesy of Dr. Louis Reichardt, UCSF, CA). Secondary antibodies: Alexa fluorescent secondary antibodies (Invitrogen, Carlsbad, CA), and biotinylated rabbit anti-goat and biotinylated horse anti-rabbit (Vector Laboratories, Burlingame, CA). Detailed protocols are available on the Li lab website (http://lilab.uchc.edu/Pages/Protocols.html).

Cell counts and statistical analysis

The striatum in its entirety, including the caudate-putamen (CPu), the nucleus accumbens (Acb), and olfactory tubercle (Tu), was determined using external anatomical landmarks based on a mouse atlas (Paxinos and Franklin, 2004). For cell profile counting in postnatal day 10 (P10) brains, sections from rostral, intermediate and caudal levels of the corresponding structures were collected from three different animals. ChAT or GABA positive cells together with EGFP positive cells were counted, and the ratio of double positive cells was calculated. To estimate the number of striatal cholinergic neurons in P42 brains, ChAT positive cells were counted on every fifth coronal section (30 µm thick) throughout the whole CPu, and every third coronal section throughout Acb and Tu. To quantify the ratio of BrdU and ChAT double positive cells to total ChAT positive cells in the striatum, comparable coronal sections from both control and conditional knockout mice were collected at P10. At least three control and three mutant brains were analyzed for each experiment, and five sections from the rostral to caudal striatum were collected from each brain. Statistical significance was determined by Student’s t tests between mutant and control littermates using Microsoft Excel.

Morphometric analysis

Confocal images of GFP-immunofluorescence of cholinergic precursor cells were captured by Z sectioning on a Zeiss LSM510 Meta. 3D images were produced using LSM Software Zen (Zeiss) and the morphology of individual GFP+ neurons was manually traced in Adobe Photoshop (Adobe Systems, San Jose, CA). Sholl analysis was carried out by counting the number of neurite that cross a series of concentric circles at 5 µm intervals from the center of soma. Statistical analysis for the Sholl dendritic analysis was carried out with the Mann-Whitney U Test using Prism 4.0 (GraphPad, La Jolla, CA).

In vitro migration assays

In vitro migration assay using matrix gel was performed as described previously {Liodis, 2007 #986}. Gbx2+/− females were mated with Gbx2CreER/+; R26RRFP/− males, which carried a tdTomato Cre reporter (Madisen et al., 2009), and time-pregnant females were given tamoxifen at E10.5. Brains were dissected from E12.5 or E13.5 embryos in Leibovitz’s L-15 medium, and brain slices (250 µm thickness) were prepared using a vibrotome (VT1000/Leica, Nussloch, Germany). Small tissue fragments containing RFP+ striatal cholinergic precursors at the LGE/MGE junction were dissected using tungsten needles and incubated for 1 h in L15/10 FCS at 37°C. Explants were placed in a three-dimensional Matrigel (BD Biosciences, San Jose, CA) and cultured for 48 h in Neurobasl (supplemented with B27 and N2) medium in 3-cm plates. Explants were analyzed using an epifluorescence microscope (Axiovert 40 CFL/Zeiss, Oberkochen, Germany). To compare the migration of control and Gbx2-null cholinergic precursors in vitro, 24 RFP+ cells positioned furthest away from the center of each explant were selected and their distance from its edge was measured (three embryos for each genotype and 3–4 explants for each embryos). The average value of these measurements represented the maximum distance of cell migration from each explants. Statistical significance was determined by Student’s t tests.

Results

Gbx2 is expressed in a subset of Lhx8-positive cells after they exit the cell cycle in the ventral telencephalon

Gbx2 expression is initiated in the ventral telencephalon of mouse embryos at E10.5 (Waters et al., 2003) (Fig. 1A). To examine the expression in greater detail, we used EGFP to follow Gbx2 expression in a Gbx2CreER knock-in mouse line. This mouse line contains an insertion of CreER-ires-Egfp cassette in the 5’ untranslated region of Gbx2, so that the expression of both CreER and Egfp recapitulates the endogenous Gbx2 expression (Chen et al., 2009) (Fig. S1). Double immunofluorescence for EGFP and BrdU or Ki67, which marks cells in active cell cycle, showed that EGFP+ cells in the MGE were completely segregated from Ki67+ or BrdU+ progenitor cells in the ventral telencephalon at E11.5 and E12.5 (Fig. 1B, Fig. S2 and data not shown). Lhx8 is expressed in the subventricular zone (SVZ) and MZ of the MGE at E11.5 and E12.5, and is necessary for the expression of Gbx2 (Zhao et al., 2003). Analysis of Gbx2 and Lhx8 transcripts on adjacent sections showed that the expression domain of Lhx8 encompasses Gbx2+ cells, which are restricted to the ventral and medial part of the Lhx8-positive region in the MGE at E12.5 (Fig. 1C and 1D). Therefore, Gbx2 is probably induced in a subset of Lhx8-expressing cells after they exit the cell cycle in the MGE.

Figure 1
Gbx2-expressing cells contribute to the striatum and basal forebrain

Gbx2-expressing cells in the ventral telencephalon contribute to the striatum and the basal forebrain

It is known that MGE cells undergo radial and tangential migration and give rise to neurons in the basal forebrain, striatum, and cortex (Marin and Rubenstein, 2001). To determine the structures to which Gbx2-expressing cells contribute, we performed inducible genetic fate mapping. Tamoxifen was administered to pregnant females carrying Gbx2CreER/+; R26R+/− embryos at E10.5 to induce Cre-mediated recombination and to permanently mark Gbx2-expressing cells with β-gal, which is expressed from the R26R allele (Soriano, 1999). As it has been shown that tamoxifen-induced labeling of CreER-expressing cells occurs in a window of 6–36 hr (Joyner and Zervas, 2006), we deduced that administration of tamoxifen at E10.5 would likely label the Gbx2-expressing cells between E11.0 and E12.0. At E12.5, a single cohort of X-gal+ cells was detected in the MGE, recapitulating the endogenous Gbx2 expression (Fig. 1E and H). A few X-gal+ cells, which are probably the frontrunners of tangentially migrating MGE cells, were found in the MZ of the lateral ganglionic eminence (LGE) (Fig. 1E and G). From E13.5 onward, the labeled cells were segregated into two groups: the first one was tightly packed in the MZ of the MGE/POA, while the second group progressively moved toward the LGE with streams of X-gal+ cells extending into the MZ of the LGE (Fig. 1F and H). By postnatal day 20 (P20), Gbx2-derived X-gal+ cells were found in the striatum, the basal magnocellular complex (Ch4), medial preoptic area (MPA), and to a lesser degree, in the horizontal limb of the diagonal band of Broca (HDB, Ch3), the vertical limb of the diagonal band of Broca (VDB, Ch2), the medial septum (MS, Ch1), and the anterior amyloid area (AAD) (Fig. 1I and J). X-gal+ cells were rarely detected in the cortex of Gbx2CreER/+; R26R+/− mice that received tamoxifen at E10.5 (Fig. 1I). Fate-mapped Gbx2-expressing cells labeled by administering tamoxifen at E12.5 or E14.5 were found in the striatum and basal forebrain as those fate-mapped at E10.5 (data not shown). In summary, we show that the Gbx2-expressing cells in the ventral telencephalon contribute to the striatum and the basal forebrain, but not to the cortex.

Gbx2-expressing cells give rise to cholinergic interneurons in the striatum, but not to cholinergic projection neurons in the basal forebrain

To determine the identity of neurons derived from the Gbx2-lineage, we first examined cholinergic neurons by immunohistochemistry for ChAT. In P42 Gbx2CreER/+; R26R+/− mice that received tamoxifen at E10.5, all X-gal+ cells were positive for ChAT in the CPu and the Acb (Fig. 2A–C). In the Tu, many X-gal+ cells were positive for ChAT [ChAT+/X-gal+ ± standard deviation (number of neurons analyzed): 36.7 ± 4.9% (171)](Fig. 2B and D). In contrast to the striatum, all X-gal+ cells in Ch1-Ch4 areas were negative for ChAT, except for the ventral pallidium (VP), where some X-gal+ cells were positive for ChAT [25.8 ± 4.0% (146)] (Fig. 3A–G). These data demonstrate that Gbx2-expressing cells at E10.5 give rise to cholinergic neurons in the striatum, but non-cholinergic neurons in the basal forebrain.

Figure 2
Striatal cholinergic interneurons are derived from the Gbx2 lineage
Figure 3
Forebrain cholinergic projection neurons are not derived from the Gbx2 lineage

Because tamoxifen does not induce recombination in all CreER-expressing cells (Joyner and Zervas, 2006), inducible genetic fate mapping does not allow us to ascertain whether all striatal cholinergic neurons are derived from the Gbx2 lineage. To address this question, we explored EGFP as a possible lineage tracer to mark Gbx2-expressing cells and their descendents in Gbx2CreER/+ mice. We detected EGFP immunofluorescence in the striatum and basal forebrain in Gbx2CreER/+ mice as late as P10, and CreER transcripts in the same regions at P4 (data not shown). Importantly, all X-gal+ cells were immunoreactive for EGFP in the striatum and basal forebrain in P10 Gbx2CreER/+; R26R+/− mice that received tamoxifen at E10.5, demonstrating that the expression of EGFP persists and thus marks cells derived from the Gbx2-lineage in the ventral telencephalon of Gbx2CreER/+ mice (data not shown). Double immunofluorescence for ChAT and EGFP revealed that virtually all ChAT positive cells were positive for EGFP in the CPu and Acb [95.5 ± 0.4% (524) and 91.3 ± 9.1% (328), respectively] of Gbx2CreER/+ mice (n=3, Fig. 2E–J). Significantly, all EGFP+ cells expressed ChAT (534 GFP+ cells analyzed from 3 mice), indicating that the Gbx2-lineage exclusively gives rise to all cholinergic neurons in the CPu and Acb. In the Tu, although the majority of ChAT+ cells were positive for EGFP [77.4 ± 7.8% (217)], about half of the EGFP+ cells were negative for ChAT [50.2 ± 11.2% (160)](Fig. 2K–M). By contrast, all ChAT positive neurons were negative for EGFP in Ch4 area, except for the VP, where many ChAT+ neurons were positive for EGFP [65.4 ± 4.1% (124)] (Fig. 3H–N).

Next, we examined if Gbx2-expressing cells contribute to GABAergic neurons by double immunofluorescence for GABA and EGFP. Many EGFP+ cells in Ch4 were immunoreactive for GABA (Fig. 3O–R) [68.9 ± 5.5% (63) in MCPO, 76.6 ± 7.3% (54) in NB, 72.2 ± 13.7% (68) in SI, 33.2 ± 4.5% (63) in VP, 3 mice]. Taken together, out data show that the Gbx2-lineage produces almost all cholinergic interneurons in the striatum. In the basal forebrain, however, the Gbx2-lineage gives rise to GABAergic and other non-cholinergic neurons.

Contribution of the Gbx2 lineage to the striatum is dependent on Gbx2 function

The specific expression of Gbx2 in striatal cholinergic interneurons suggests that Gbx2 may play a role in the development of this population of cells. To investigate the function of Gbx2, we first examined if Gbx2 inactivation alters expression of Nkx2.1, Dlx5, Lhx6, Lhx8, Isl1, and Olig2, which are required for the specification, migration, and differentiation of MGE-derived cells, particularly development of cholinergic neurons (Sussel et al., 1999; Zhao et al., 2003; Alifragis et al., 2004; Furusho et al., 2006; Cobos et al., 2007; Elshatory and Gan, 2008). No discernable difference in the expression of these molecules was detected between the control littermates (Gbx2CreER/+) and Gbx2CreER/− mutants, which carry two different null alleles, Gbx2 and Gbx2CreER (Wassarman et al., 1997; Chen et al., 2009), at E11.5 and E12.5 (Fig. S3). Expression of Gbx1, which is closely related with Gbx2 (Waters et al., 2003), in the MGE is unaffected in Gbx2CreER/− embryos at E11.5 and E12.5 (Fig. S4 and data not shown). These findings suggest that the initial formation of cholinergic precursors is unaffected in Gbx2-deficient embryos.

We next examined whether the differentiation of the striatal cholinergic precursors was affected in Gbx2CreER/− mice by taking advantage of the persistent expression of EGFP in these cells. The expression of EGFP was indistinguishable between Gbx2CreER/+ and Gbx2CreER/− embryos at E11.5 and E12.5, demonstrating that the expression of EGFP in the MGE from the Gbx2CreER allele is independent on Gbx2 function (Fig. S2, and S3I–N). By E13.5, EGFP+ cells were found throughout the LGE in Gbx2CreER/+ embryos (Fig. 4A). In contrast, the majority of EGFP+ cells were found near the junction between the LGE and MGE and there was a noticeable reduction of EGFP+ cells in the dorsal and lateral-most areas of the LGE in Gbx2CreER/− embryos (Fig. 4B). To rule out that preferential absence of cholinergic precursors in the dorsal and lateral-most of LGE is caused by loss of EGFP expression in these cells deficient for Gbx2, we fate-mapped Gbx2-transcribing cells lacking Gbx2 protein in Gbx2CreER/−; R26R+/− embryos. In Gbx2CreER/+; R26R+/− embryos, the fate-mapped Gbx2-transcribing cells labeled at E10.5 entered the prospective striatum along its lateral part at E14.5, and mainly contributed to the lateral-most area of the CPu at E18.5 (Fig. 4E and G). In the absence of Gbx2, the fate-mapped Gbx2-transcribing cells mainly populated the center of the CPu, avoiding the lateral area of the striatum in Gbx2CreER/−; R26R+/− embryos at E14.5 and E18.5 (Fig. 4F and H). Therefore, inactivation of Gbx2 results in abnormal distribution of cholinergic precursors in the striatum.

Figure 4
Loss of Gbx2 results in abnormal migration and neurite outgrowth of cholinergic interneuron precursors

Between E17.5 and P0, the overall number of EGFP+ cells was significantly reduced and the reduction of EGFP+ cells was more pronounced in the peripheral region, including the subcallosal streak, of the CPu in Gbx2CreER/− embryos (Fig. 4C, D and S5). Compared to those of control littermates, the numbers of EGFP+ neurons on coronal sections of the CPu in Gbx2CreER/− embryos were reduced between 30.7% (E17.5) and 44.8% (P0). To determine if the reduction of EGFP+ is caused by cell death, we examined apoptosis by double immunofluorescence for EGFP and active form of Caspase 3 (Casp3), a marker for apoptotic cells. No obvious increase of Casp3+ cells was detected in the LGE, MGE and developing basal forebrain in Gbx2CreER/− embryos at E12.5, E13.5, E14.5, and E17.5 (data not shown). Furthermore, Casp3 was rarely detected in EGFP+ cells in these brain regions in control or Gbx2CreER/− embryos. Therefore, caspase-mediated apoptosis is not responsible for the reduction of cholinergic precursors in the striatum of Gbx2CreER/− embryos.

To examine the cellular morphology of striatal cholinergic precursors without Gbx2, we marked these neurons using a R26RYFP reporter (Srinivas et al., 2001), which uses YFP instead of β-gal to label Cre-mediated recombinant cells. Although anti-GFP antibodies detect both YFP and EGFP, the expression of YFP is expressed at a higher level than EGFP from the Gbx2CreER locus so that the robust YFP expression clearly marks a subset of Gbx2-expressing cells, facilitating morphological analysis of migrating striatal cholinergic precursors. There was no discernable difference in the morphology of the YFP-labeled neurons between Gbx2-deficient and their control littermates at E12.5 (data not shown). In E13.5 control embryos, the labeled Gbx2-derived cells in transit to the LGE displayed one or two leading processes and a trailing tail, the typical morphology of neurons that undergo tangential migration (Anderson et al., 1999) (Fig. 4I and supplementary Figure S5C). By contrast, the fate-mapped Gbx2-deficient neurons had increased number and branching of neurites in Gbx2CreER/−; R26RYFP/+ embryos at E13.5 (Fig. 4J–L and supplementary Figure S5C).

In summary, our data show that although Gbx2 is not essential for the patterning and specification of MGE cells, Gbx2 is required for the differentiation of striatal cholinergic precursors. Loss of Gbx2 results in reduction in number, and abnormal distribution and morphology of cholinergic precursors in the striatum.

Loss of Gbx2 leads to a significant reduction of striatal cholinergic interneurons

Gbx2-null mice die immediately after birth, precluding our study of striatal cholinergic interneuron differentiation and maturation, which occur between P1 and P35 in mice (Gould et al., 1991; Wassarman et al., 1997). To this end, we specifically removed Gbx2 in the ventral telencephalon by combining a Gbx2 conditional mutation allele, Gbx2F (Li et al., 2002), with a transgenic mouse line that expresses Cre under the control of the Nkx2.1 promoter (Xu et al., 2008). Nkx2.1 is expressed in the ventricular zone of the MGE by E11.5, and the expression of Cre mimics the endogenous Nkx2.1 expression in these transgenic mice (Xu et al., 2008). In mice carrying both Nkx2.1-Cre and R26R transgenes, Cre activity resulted in recombination throughout the ventral telencephalon encompassing the Gbx2-expressing cells at E11.5 (Fig. S7L and M). Moreover, all striatal cholinergic neurons were derived from Nkx2.1-Cre expressing cells (Fig. S7A–K). To examine the specific inactivation of Gbx2, we performed in situ hybridization with a Gbx2 riboprobe corresponding to Gbx2 exon II, which is removed by Cre-mediated recombination. Gbx2 transcripts were largely absent in the MGE of Gbx2CreER/F; Nkx2.1-Cre embryos (designated as Gbx2-CKO) by E11.5, demonstrating that Gbx2 is specifically inactivated in the ventral telencephalon in Gbx2-CKO mice (Fig. S7N and O).

Gbx2-CKO mice are viable and do not display any gross abnormality. We examined striatal cholinergic neurons in Gbx2-CKO mice between P2 and P42 by ChAT immunohistochemistry. Consistent with the reduction of striatal cholinergic precursors (EGFP+) found in Gbx2CreER/− embryos by E17.5 and P0 (Fig. 4D and supplementary figure S5), the number of ChAT+ cells was considerably reduced in Gbx2-CKO mice (n=3) by P2 (Fig. 5D). At P42, there was a significant reduction of cholinergic neurons in the CPu (35 ± 1.2%), Acb (29 ± 4.3%), and the Tu (50 ± 11.0%) in Gbx2-CKO mice (n=3). In addition to the reduction in the overall number, the cholinergic neurons displayed abnormal expression pattern of ChAT and altered distribution within the striatum of Gbx2-CKO mice. Only weak expression of ChAT was detected in cholinergic neurons throughout the striatum in control (Gbx2CreER/+; Nkx2.1-Cre) and Gbx2-CKO mice at P2 (Fig. 5A and B). At P4, cholinergic neurons in the lateral and caudal parts of the CPu displayed significantly higher levels of ChAT than those in the rostral and medial part of the CPu (n=4, Fig. 5F–H). By contrast, robust ChAT expression was detected in cholinergic neurons throughout the CPu of P4 Gbx2-CKO mice (Fig. 5I–K). In the control mice by P6, strong ChAT expression was detected in cholinergic neurons throughout the striatum, suggesting that the maturation of cholinergic neurons is largely complete by P6 (Fig. 5C and E). Compared with control animals, ChAT+ cells were fewer in number, particularly in the lateral and dorsal-most of the CPu in P6 Gbx2-CKO mice (n= 3, Fig. 5E). In the CPu of Gbx2-CKO mice at P42, the loss of cholinergic neurons was more prominent in the mid-segment than the anterior or posterior segment, and the remaining cholinergic neurons were mainly found in the center of the nucleus (Fig. 6B and F). Therefore, inactivation of Gbx2 leads to reduction in number and abnormal distribution of cholinergic neurons in the striatum.

Figure 5
Specific deletion of Gbx2 in the MGE results in significant reduction and aberrant distribution of cholinergic interneurons in the striatum
Figure 6
Significant reduction of striatal cholinergic interneurons in adult Gbx2-CKO mice

To determine whether the defect in the striatum of Gbx2-CKO mutants is specific to the cholinergic system, we examined GABAergic interneurons that express nitric oxide synthase (NOS), which also originate from the MGE, and striatal projection neurons, which are generated in the LGE and express dopamine-and cAMP-regulated phosphoprotein (Darpp-32) (Marin et al., 2000). Examination of these neurons by histochemical staining for NADPH-diaphorase and immunofluorescence for Darpp-32 revealed that there was no difference in the number and the distribution of NOS+ and Darpp-32+ cells in the striatum between the control littermates and Gbx2-CKO mice at P42 (Fig. S5 and S8).

In summary, deletion of Gbx2 in the ventral telencephalon leads to significant reduction in the number and abnormal distribution of striatal cholinergic neurons. Furthermore, defects in the striatum are largely restricted to the cholinergic system in Gbx2-CKO mice.

Development of striatal cholinergic interneurons is independent of Gbx2 after birth

As Gbx2 expression persists in striatal cholinergic interneurons after birth (Fig. 2E–M and data not shown), we next investigated if Gbx2 plays a role during postnatal development of these neurons. No obvious difference was found in the morphology of striatal cholinergic neurons between control and Gbx2-CKO mice at P0, P4, P6, and P42 (insets in Fig. 6A, 6B, and Fig. 7). We examined expression of Isl1 and the tyrosine kinase receptor TrkA, which is known to play an important role in the maturation and survival of striatal cholinergic neurons (Fagan et al., 1997; Elshatory and Gan, 2008). Double immunofluorescence for EGFP and Isl1 or TrkA showed that all EGFP+ cells express Isl1 or TrkA in the CPu of control and Gbx2-CKO mice at P0 and P4 (Fig. 7A–F”). No discernable change in the expression of Isl1 or TrkA was detected in the striatum of Gbx2-CKO mice. Similarly, all EGFP+ cells were positive for ChAT in the striatum of the control littermates and Gbx2-CKO mice at P6 (Fig. 7G–H”). In addition, examination of cell death by immunofluorescence for Casp3 and TUNEL assay did not detect any increase in cell death in the striatum of Gbx2-CKO mice at P2, P4 and P10 (data not shown). Our molecular marker analyses suggest that Gbx2 is not essential for the development of striatal cholinergic interneurons after birth.

Figure 7
Differentiation of striatal cholinergic interneurons during early postnatal stages is unaffected by the loss of Gbx2

Preferential reduction of late-born striatal cholinergic interneurons due to loss of Gbx2

Based on birth-dating analysis in rat (Phelps et al., 1989), we deduced that striatal cholinergic neurons are likely born between E10.5 and E14.5 in mice. We observed that the fate-mapped Gbx2-expressing cells at E10.5 mainly contributed to the lateral part of the striatum, where cholinergic neurons first expressed high level of ChAT at P4 (Fig. 4G and and5G),5G), suggesting that early-born striatal cholinergic neurons may mature first. Interestingly, in Gbx2-CKO mice, all cholinergic neurons uniformly expressed high level of ChAT at P4 (Fig. 5I–K). These findings raise the possibility that, in the absence of Gbx2, the remaining striatal cholinergic interneurons may be mainly born at the early stage, while late-born striatal cholinergic interneurons fail to develop. To test this hypothesis, we carried out birthdating analysis by administering BrdU to pregnant mice carrying Gbx2-CKO embryos between E10.5 and E15.5. Cells that undergo their last S-phase at the time of BrdU injection will retain BrdU, whereas cells that continue to cycle dilute the label over time (Howell et al., 1997). Double immunofluorescence for ChAT and BrdU at P10 revealed that the majority of striatal cholinergic neurons were born between E11.5 and E14.5, and few striatal cholinergic neurons were born before E11.5 and after 14.5 (Fig. 8A–C and data not shown). There was no significant difference in the percentage of cholinergic neurons born at E11.5 between control and Gbx2-CKO (Fig. 8A). However, the striatal cholinergic neurons that are born at E12.5 were significantly reduced in Gbx2-CKO mutants, and virtually no E14.5-born cholinergic neurons were detected in the mutants at P10 (n=3) (Fig. 8A–C). These data demonstrate that the cholinergic neurons that are born after E12.5 are preferentially affected due to the loss of Gbx2.

Figure 8
Late-born striatal cholinergic interneurons are preferentially lost in the absence of Gbx2

Discussion

Gbx2-expressing cells in the MGE differentially contribute to striatal cholinergic interneurons and non-cholinergic neurons in the basal forebrain

Transplantation studies first suggested that striatal cholinergic interneurons are derived from the MGE (Olsson et al., 1998), and this notion was subsequently supported by genetics experiments (Sussel et al., 1999; Marin et al., 2000). Using inducible genetic fate mapping and EGFP as lineage tracer, here we provide definite evidence showing that Gbx2-expressing cells in the MGE that undergo tangential migration exclusively give rise to nearly all cholinergic interneurons in the CPu and Acb. Interestingly, in the Tu, around 20% of cholinergic neurons are not derived from the Gbx2 lineage, whereas some Gbx2-derived cells become non-cholinergic neurons (Fig. 2E–F and O–Q). These results indicate that the Tu has different and heterogeneous developmental ancestry compared with the CPu and Acb.

In contrast to those undergoing tangential migration, the fate-mapped Gbx2-derived cells that remain in the MGE give rise to GABAergic and other non-cholinergic neurons in the basal forebrain, indicating that the Gbx2-expressing cells in the MGE are heterogeneous. Our preliminary study shows that mis-expression of Gbx2 in all Nkx2.1-expressing cells does not affect the specification or differentiation of cholinergic and GABAergic neurons derived from the MGE, indicating that Gbx2 does not act as a determinant for the differentiation of striatal cholinergic interneurons (Sunmonu et al., 2009). Therefore, factors acting upstream of Gbx2 may be required to specify and differentiate cholinergic interneurons and projection neurons. Previous studies identified Lhx8 as the key regulator for the development of both cholinergic interneurons and projection neurons in the ventral forebrain (Zhao et al., 2003; Mori et al., 2004; Fragkouli et al., 2005). Results from our current study suggest that Lhx8+/Gbx2+ cells contribute to the striatum and form cholinergic interneurons, and that Lhx8+/Gbx2 cells contribute to the cholinergic projection neurons in the basal forebrain (see Fig. 8I). Since Lhx8+ cells in the MGE are known to give rise to GABAergic neurons (Fragkouli et al., 2005), Lhx8+/Gbx2+ cells that undergo radial migration may also give rise to GABAergic and other non-cholinergic neurons in the basal forebrain. Interestingly, Gbx1, a paralogue of Gbx2, is apparently expressed in the basal forebrain cholinergic projection neurons (Asbreuk et al., 2002). Therefore, the two major groups of cholinergic neurons in mammalian forebrain appear to be differentially demarcated by Gbx1 and Gbx2, which were duplicated during evolution (Rhinn et al., 2003; Waters et al., 2003; Rhinn et al., 2004).

The temporal order of neurogenesis correlates with the location and progressive maturation of cholinergic interneurons in the striatum

We found that most striatal cholinergic interneurons are generated before E14.5, with more than 50% of the neurons being generated before E12.5 in mice (Fig. 8 and data not shown). As Gbx2 expression is initiated in striatal cholinergic precursors after they exit the cell cycle (Fig. 1B), tamoxifen-induced labeling of Gbx2-expressing cells at E10.5 likely fate-maps the early-born striatal cholinergic interneurons. We found that fate-mapped Gbx2-expressing cells at E10.5 preferentially contribute to the lateral part of CPu (Fig. 4G), suggesting that striatal cholinergic neurons are probably arranged in a lateral-to-medial order in the CPu according to the temporal order of genesis of these neurons. Intriguingly, this orderly deposition of striatal cholinergic neurons in the CPu appears less prominent at 3 weeks after birth (Fig. 1J), suggesting a continued cellular rearrangement during postnatal development.

In agreement with previous findings (Phelps et al., 1989; Gould et al., 1991), we show that striatal cholinergic neurons in the lateral side of the CPu display higher levels of ChAT immunoreactivity than those in the medial CPu at P4, but by P6, all striatal cholinergic interneurons express uniform and robust ChAT expression (Fig. 5). These observations suggest that the maturation of cholinergic interneurons progresses from the lateral-to-medial direction in the CPu, and this maturation order thus correlates with the temporal order of neurogenesis. Members of the neurotrophin family, particular nerve growth factor (NGF), are known to play an important role in promoting maturation and survival of cholinergic neurons in the striatum and basal forebrain (Hefti, 1986; Lucidi-Phillipi et al., 1996). NGF administration increased Chat mRNA levels, and conversely, anti-NGF serum infusion suppressed expression of Chat in the forebrain of rat (Li et al., 1995). Furthermore, in the absence of trkA, a receptor for NGF, striatal cholinergic interneurons have reduced ChAT expression at P7/8 (Fagan et al., 1997). Therefore, the progressive maturation of striatal cholinergic interneurons may be regulated by the spatial or temporal gradient of NGF signaling in the striatum. However, in Gbx2-CKO mice, the remaining cholinergic neurons, which are mostly early-born and abnormally reside in the medial part of the CPu, express robust ChAT similar to the early-born cholinergic neurons, arguing against the notion that a spatial gradient of NGF determines maturation progression of striatal cholinergic interneurons (Fig. 5). Instead, our results suggest that an intrinsic mechanism associated with the temporal order of neurogenesis, probably by controlling the responsiveness to NGF signals, determines the maturation order of striatal cholinergic interneruons.

Different effects on the development of early-born and late-born striatal cholinergic interneurons due to loss of Gbx2

By fate mapping Gbx2-transcribing cells in Gbx2-deficient embryos at E10.5, we show that early-born cholinergic precursors shift their migration route medially contributing to the medial, rather than the lateral, part of the CPu (Fig. 4F, D). As Gbx2-expressing cells that undergo tangential migration are embedded among the developing thalamocortical and corticothalamic axons, which are largely abolished in Gbx2-deficient embryos (Miyashita-Lin et al., 1999; Hevner et al., 2002), it is possible that the abnormal distribution of striatal cholinergic neurons may be related to the loss of these axons. However, similar abnormal distribution of cholinergic interneurons was found in the CPu of the Gbx2-CKO mutants, where thalamocortical and corticothalamic projections develop normally (data not shown), demonstrating that the development of the thalamocortical axons and tangential migration of the Gbx2-expressing cells in the MGE are mutually independent. In addition, we found that tangentially migrating Gbx2-derived cells that were labeled at E10.5 display abnormal neurite outgrowth and increased neurite complexity (Fig. 4I–L). These data suggest that loss of Gbx2 may affect the migration of early-born striatal cholinergic interneurons. The potential defect in migration may account for the abnormal distribution of cholinergic neurons found in the striatum of Gbx2-null and Gbx2-CKO mutants. To investigate the potential defect in migration of striatal cholinergic neurons, we performed migration assay in matrix gel. However, no noticeable difference in the number and distance of migration of control and Gbx2-null cholinergic precursors was detected, suggesting that Gbx2-deficient cells are not impaired in general cell migration (Fig. S9). Future study is necessary to determine the molecular mechanism underlying the function of Gbx2 in regulating migration of striatal cholinergic neurons.

In addition to the abnormal distribution, there is a significant reduction of cholinergic interneurons in the striatum due to the loss of Gbx2. By examining EGFP+ cells in the striatum, we detected a significant reduction in the number of striatal cholinergic precursors in Gbx2CreER/− mutants, 30.7% at E17.5 and 44.8% at P0. The reduction of EGFP+ cells in Gbx2CreER/− embryos at these stages is comparable with the 35% reduction of striatal cholinergic neurons that were examined by ChAT in the CPu of Gbx2-CKO mice, demonstrating that the reduction of striatal cholinergic neurons is caused by a loss cholinergic precursors by E17.5 (Fig. 6). Interestingly, labeling progenitors that undergo their last mitosis by BrdU incorporation showed that there was no significant difference in the number of striatal cholinergic neurons born at E11.5 between control and Gbx2-CKO mice, and that there was a dramatic and disproportionate reduction of the striatal cholinergic neurons that were born after E12.5 in Gbx2-CKO mice (Fig. 8). These observations collectively suggest that development of late-born striatal cholinergic interneurons is preferentially affected in the absence of Gbx2. Because Gbx2 expression is maintained in the migrating cholinergic precursors, we cannot specifically fate map Gbx2-expressing cells after E12.5 without marking the neurons that are born earlier. Therefore, we cannot definitively determine the fate of late-born cholinergic neurons in the Gbx2 mutant mice. Future experiments are required to determine the distinct requirement for Gbx2 in development of the early-born and late-born striatal cholinergic interneruons.

Supplementary Material

Supp1

Acknowledgements

We are grateful to Drs. Sandra Blaess, and lab members for discussion and critical reading of the manuscript. We thank Dr. Kairong Li and Qiuxia Guo for their technical help. We thank Dr. Louise Reichardt and Rashmi Bansal for providing the antibodies against TrkA and Olig2. We thank Drs. Alexandra Joyner, John Rubenstein, and Mark Lewandoski for providing probes for RNA in situ hybridization analysis. J. Li is supported by grants from the NIH and March of Dimes Foundation.

References

  • Alifragis P, Liapi A, Parnavelas JG. Lhx6 regulates the migration of cortical interneurons from the ventral telencephalon but does not specify their GABA phenotype. J Neurosci. 2004;24:5643–5648. [PubMed]
  • Anderson S, Mione M, Yun K, Rubenstein JL. Differential origins of neocortical projection and local circuit neurons: role of Dlx genes in neocortical interneuronogenesis. Cereb Cortex. 1999;9:646–654. [PubMed]
  • Asbreuk CH, van Schaick HS, Cox JJ, Kromkamp M, Smidt MP, Burbach JP. The homeobox genes Lhx7 and Gbx1 are expressed in the basal forebrain cholinergic system. Neuroscience. 2002;109:287–298. [PubMed]
  • Berger-Sweeney J. The cholinergic basal forebrain system during development and its influence on cognitive processes: important questions and potential answers. Neurosci Biobehav Rev. 2003;27:401–411. [PubMed]
  • Bernacer J, Prensa L, Gimenez-Amaya JM. Cholinergic interneurons are differentially distributed in the human striatum. PLoS One. 2007;2:e1174. [PMC free article] [PubMed]
  • Bulfone A, Puelles L, Porteus MH, Frohman MA, Martin GR, Rubenstein JL. Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt- 3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J Neurosci. 1993;13:3155–3172. [PubMed]
  • Chen L, Guo Q, Li JY. Transcription factor Gbx2 acts cell-nonautonomously to regulate the formation of lineage-restriction boundaries of the thalamus. Development. 2009;136:1317–1326. [PMC free article] [PubMed]
  • Cobos I, Borello U, Rubenstein JL. Dlx transcription factors promote migration through repression of axon and dendrite growth. Neuron. 2007;54:873–888. [PubMed]
  • Elshatory Y, Gan L. The LIM-homeobox gene Islet-1 is required for the development of restricted forebrain cholinergic neurons. J Neurosci. 2008;28:3291–3297. [PMC free article] [PubMed]
  • Fagan AM, Garber M, Barbacid M, Silos-Santiago I, Holtzman DM. A role for TrkA during maturation of striatal and basal forebrain cholinergic neurons in vivo. J Neurosci. 1997;17:7644–7654. [PubMed]
  • Fragkouli A, Hearn C, Errington M, Cooke S, Grigoriou M, Bliss T, Stylianopoulou F, Pachnis V. Loss of forebrain cholinergic neurons and impairment in spatial learning and memory in LHX7-deficient mice. Eur J Neurosci. 2005;21:2923–2938. [PubMed]
  • Furusho M, Ono K, Takebayashi H, Masahira N, Kagawa T, Ikeda K, Ikenaka K. Involvement of the Olig2 transcription factor in cholinergic neuron development of the basal forebrain. Dev Biol. 2006;293:348–357. [PubMed]
  • Gerfen CR. The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci. 1992;15:133–139. [PubMed]
  • Gould E, Woolf NJ, Butcher LL. Postnatal development of cholinergic neurons in the rat: I. Forebrain. Brain Res Bull. 1991;27:767–789. [PubMed]
  • Guo Q, Li JY. Distinct functions of the major Fgf8 spliceform, Fgf8b, before and during mouse gastrulation. Development. 2007;134:2251–2260. [PMC free article] [PubMed]
  • Hefti F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections. J Neurosci. 1986;6:2155–2162. [PubMed]
  • Hevner RF, Miyashita-Lin E, Rubenstein JL. Cortical and thalamic axon pathfinding defects in Tbr1, Gbx2, and Pax6 mutant mice: evidence that cortical and thalamic axons interact and guide each other. J Comp Neurol. 2002;447:8–17. [PubMed]
  • Howell BW, Hawkes R, Soriano P, Cooper JA. Neuronal position in the developing brain is regulated by mouse disabled-1. Nature. 1997;389:733–737. [PubMed]
  • Joyner AL, Zervas M. Genetic inducible fate mapping in mouse: establishing genetic lineages and defining genetic neuroanatomy in the nervous system. Dev Dyn. 2006;235:2376–2385. [PubMed]
  • Kaneko S, Hikida T, Watanabe D, Ichinose H, Nagatsu T, Kreitman RJ, Pastan I, Nakanishi S. Synaptic integration mediated by striatal cholinergic interneurons in basal ganglia function. Science. 2000;289:633–637. [PubMed]
  • Li JY, Joyner AL. Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression. Development. 2001;128:4979–4991. [PubMed]
  • Li JY, Lao Z, Joyner AL. Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron. 2002;36:31–43. [PubMed]
  • Li Y, Holtzman DM, Kromer LF, Kaplan DR, Chua-Couzens J, Clary DO, Knusel B, Mobley WC. Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons. J Neurosci. 1995;15:2888–2905. [PubMed]
  • Lucidi-Phillipi CA, Clary DO, Reichardt LF, Gage FH. TrkA activation is sufficient to rescue axotomized cholinergic neurons. Neuron. 1996;16:653–663. [PMC free article] [PubMed]
  • Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2009;13:133–140. [PMC free article] [PubMed]
  • Marin O, Rubenstein JL. A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci. 2001;2:780–790. [PubMed]
  • Marin O, Anderson SA, Rubenstein JL. Origin and molecular specification of striatal interneurons. J Neurosci. 2000;20:6063–6076. [PubMed]
  • Mesulam MM, Mufson EJ, Wainer BH, Levey AI. Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6) Neuroscience. 1983;10:1185–1201. [PubMed]
  • Miyashita-Lin EM, Hevner R, Wassarman KM, Martinez S, Rubenstein JL. Early neocortical regionalization in the absence of thalamic innervation. Science. 1999;285:906–909. [PubMed]
  • Mobley WC, Woo JE, Edwards RH, Riopelle RJ, Longo FM, Weskamp G, Otten U, Valletta JS, Johnston MV. Developmental regulation of nerve growth factor and its receptor in the rat caudate-putamen. Neuron. 1989;3:655–664. [PubMed]
  • Mori T, Yuxing Z, Takaki H, Takeuchi M, Iseki K, Hagino S, Kitanaka J, Takemura M, Misawa H, Ikawa M, Okabe M, Wanaka A. The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons. Eur J Neurosci. 2004;19:3129–3141. [PubMed]
  • Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the Mouse Embryo. Third Edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2003.
  • Olsson M, Bjorklund A, Campbell K. Early specification of striatal projection neurons and interneuronal subtypes in the lateral and medial ganglionic eminence. Neuroscience. 1998;84:867–876. [PubMed]
  • Paxinos G, Franklin KBJ. Amsterdam ; Boston: Elsevier Academic Press; 2004. The mouse brain in stereotaxic coordinates, Compact 2nd Edition.
  • Phelps PE, Brady DR, Vaughn JE. The generation and differentiation of cholinergic neurons in rat caudate-putamen. Brain Res Dev Brain Res. 1989;46:47–60. [PubMed]
  • Pisani A, Bernardi G, Ding J, Surmeier DJ. Re-emergence of striatal cholinergic interneurons in movement disorders. Trends Neurosci. 2007;30:545–553. [PubMed]
  • Qing Xu. MTSAA. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. The Journal of Comparative Neurology. 2008;506:16–29. [PubMed]
  • Rhinn M, Lun K, Werner M, Simeone A, Brand M. Isolation and expression of the homeobox gene Gbx1 during mouse development. Dev Dyn. 2004;229:334–339. [PubMed]
  • Rhinn M, Lun K, Amores A, Yan YL, Postlethwait JH, Brand M. Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling. Mech Dev. 2003;120:919–936. [PubMed]
  • Semba K, Fibiger HC. Time of origin of cholinergic neurons in the rat basal forebrain. J Comp Neurol. 1988;269:87–95. [PubMed]
  • Smythies J. Section I. The cholinergic system. Int Rev Neurobiol. 2005;64:1–122. [PubMed]
  • Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain [letter] Nat Genet. 1999;21:70–71. [PubMed]
  • Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol. 2001;1:4. [PMC free article] [PubMed]
  • Sunmonu NA, Chen L, Li JY. Misexpression of Gbx2 throughout the mesencephalon by a conditional gain-of-function transgene leads to deletion of the midbrain and cerebellum in mice. Genesis. 2009 [PMC free article] [PubMed]
  • Sussel L, Marin O, Kimura S, Rubenstein JL. Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development. 1999;126:3359–3370. [PubMed]
  • Wassarman KM, Lewandoski M, Campbell K, Joyner AL, Rubenstein JL, Martinez S, Martin GR. Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development. 1997;124:2923–2934. [PubMed]
  • Waters ST, Wilson CP, Lewandoski M. Cloning and embryonic expression analysis of the mouse Gbx1 gene. Gene Expr Patterns. 2003;3:313–317. [PubMed]
  • Wonders CP, Anderson SA. The origin and specification of cortical interneurons. Nat Rev Neurosci. 2006;7:687–696. [PubMed]
  • Xu Q, Tam M, Anderson SA. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. J Comp Neurol. 2008;506:16–29. [PubMed]
  • Zhao Y, Marin O, Hermesz E, Powell A, Flames N, Palkovits M, Rubenstein JL, Westphal H. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain. Proc Natl Acad Sci U S A. 2003;100:9005–9010. [PMC free article] [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

  • Pax6 regulates the formation of the habenular nuclei by controlling the temporospatial expression of Shh in the diencephalon in vertebrates[BMC Biology. ]
    Chatterjee M, Guo Q, Weber S, Scholpp S, Li JY. BMC Biology. 1213
  • Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon[Genome Research. 2014]
    Spieler D, Kaffe M, Knauf F, Bessa J, Tena JJ, Giesert F, Schormair B, Tilch E, Lee H, Horsch M, Czamara D, Karbalai N, von Toerne C, Waldenberger M, Gieger C, Lichtner P, Claussnitzer M, Naumann R, Müller-Myhsok B, Torres M, Garrett L, Rozman J, Klingenspor M, Gailus-Durner V, Fuchs H, Hrabě de Angelis M, Beckers J, Hölter SM, Meitinger T, Hauck SM, Laumen H, Wurst W, Casares F, Gómez-Skarmeta JL, Winkelmann J. Genome Research. 2014 Apr; 24(4)592-603
  • The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse[Proceedings of the National Academy of Scie...]
    Ehrman LA, Mu X, Waclaw RR, Yoshida Y, Vorhees CV, Klein WH, Campbell K. Proceedings of the National Academy of Sciences of the United States of America. 2013 Oct 15; 110(42)E4026-E4035
  • GWAS of 126,559 Individuals Identifies Genetic Variants Associated with Educational Attainment[Science (New York, N.Y.). 2013]
    Rietveld CA, Medland SE, Derringer J, Yang J, Esko T, Martin NW, Westra HJ, Shakhbazov K, Abdellaoui A, Agrawal A, Albrecht E, Alizadeh BZ, Amin N, Barnard J, Baumeister SE, Benke KS, Bielak LF, Boatman JA, Boyle PA, Davies G, de Leeuw C, Eklund N, Evans DS, Ferhmann R, Fischer K, Gieger C, Gjessing HK, Hägg S, Harris JR, Hayward C, Holzapfel C, Ibrahim-Verbaas CA, Ingelsson E, Jacobsson B, Joshi PK, Jugessur A, Kaakinen M, Kanoni S, Karjalainen J, Kolcic I, Kristiansson K, Kutalik Z, Lahti J, Lee SH, Lin P, Lind PA, Liu Y, Lohman K, Loitfelder M, McMahon G, Vidal PM, Meirelles O, Milani L, Myhre R, Nuotio ML, Oldmeadow CJ, Petrovic KE, Peyrot WJ, Polašek O, Quaye L, Reinmaa E, Rice JP, Rizzi TS, Schmidt H, Schmidt R, Smith AV, Smith JA, Tanaka T, Terracciano A, van der Loos MJ, Vitart V, Völzke H, Wellmann J, Yu L, Zhao W, Allik J, Attia JR, Bandinelli S, Bastardot F, Beauchamp J, Bennett DA, Berger K, Bierut LJ, Boomsma DI, Bültmann U, Campbell H, Chabris CF, Cherkas L, Chung MK, Cucca F, de Andrade M, De Jager PL, De Neve JE, Deary IJ, Dedoussis GV, Deloukas P, Dimitriou M, Eiriksdottir G, Elderson MF, Eriksson JG, Evans DM, Faul JD, Ferrucci L, Garcia ME, Grönberg H, Gudnason V, Hall P, Harris JM, Harris TB, Hastie ND, Heath AC, Hernandez DG, Hoffmann W, Hofman A, Holle R, Holliday EG, Hottenga JJ, Iacono WG, Illig T, Järvelin MR, Kähönen M, Kaprio J, Kirkpatrick RM, Kowgier M, Latvala A, Launer LJ, Lawlor DA, Lehtimäki T, Li J, Lichtenstein P, Lichtner P, Liewald DC, Madden PA, Magnusson PK, Mäkinen TE, Masala M, McGue M, Metspalu A, Mielck A, Miller MB, Montgomery GW, Mukherjee S, Nyholt DR, Oostra BA, Palmer LJ, Palotie A, Penninx B, Perola M, Peyser PA, Preisig M, Räikkönen K, Raitakari OT, Realo A, Ring SM, Ripatti S, Rivadeneira F, Rudan I, Rustichini A, Salomaa V, Sarin AP, Schlessinger D, Scott RJ, Snieder H, Pourcain BS, Starr JM, Sul JH, Surakka I, Svento R, Teumer A, The LifeLines Cohort Study, Tiemeier H, Rooij FJ, Van Wagoner DR, Vartiainen E, Viikari J, Vollenweider P, Vonk JM, Waeber G, Weir DR, Wichmann HE, Widen E, Willemsen G, Wilson JF, Wright AF, Conley D, Davey-Smith G, Franke L, Groenen PJ, Hofman A, Johannesson M, Kardia SL, Krueger RF, Laibson D, Martin NG, Meyer MN, Posthuma D, Thurik AR, Timpson NJ, Uitterlinden AG, van Duijn CM, Visscher PM, Benjamin DJ, Cesarini D, Koellinger PD. Science (New York, N.Y.). 2013 Jun 21; 340(6139)1467-1471
  • Use of "MGE Enhancers" for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons[PLoS ONE. ]
    Chen YJ, Vogt D, Wang Y, Visel A, Silberberg SN, Nicholas CR, Danjo T, Pollack JL, Pennacchio LA, Anderson S, Sasai Y, Baraban SC, Kriegstein AR, Alvarez-Buylla A, Rubenstein JL. PLoS ONE. 8(5)e61956
See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...