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We show that most globus pallidus neurons, but very few neocortical interneurons, are generated from the ventral MGE and dorsal POA based on fate mapping using a Shh-Cre allele. The Shh-expressing subpallial lineage produces parvalbumin+ GABAergic neurons, ChAT+ cholinergic neurons, and oligodendrocytes. Loss of Nkx2-1 function from the Shh-expressing domain eliminated most globus pallidus neurons, whereas most cortical and striatal interneurons continued to be generated, except for striatal cholinergic neurons. Finally, our analysis provided evidence for a novel cellular component (Nkx2-1−;Npas1+) of the globus pallidus.
The embryonic mouse subpallium generates the projection neurons of the basal ganglia, as well as telencephalic GABAergic, cholinergic and dopaminergic interneurons. The subpallium has four principle progenitor domains: lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), preoptic area (POA), and septum (the caudal ganglionic eminence contains caudal parts of the LGE and MGE) (Flames et al., 2007); each of these is further subdivided. For instance, Flames et al. (2007) proposed five MGE progenitor domain subdivisions: pMGE1-5.
Distinct progenitor domains generate distinct neurons and glia based on loss of function mutants, and fate mapping studies. Nkx2-1−/− mutants lack an identifiable globus pallidus and ~50% of cortical interneurons (Lhx6+, parvalbumin+ and somatostatin+) (Sussel et al., 1999; Pleasure et al., 2000; Xu et al., 2004; Butt et al., 2008). The Nkx2-1−/−MGE lacks Lhx6 and Lhx7(8) expression and is transformed towards an LGE identity (Sussel et al., 1999; Corbin et al., 2003; Butt et al., 2008). Lhx6 mutants fail to generate parvalbumin and somatostatin cortical interneurons (Liodis et al., 2007; Zhao et al., 2008), while Lhx8 mutants have fewer subpallial cholinergic neurons (Zhao et al., 2003; Mori et al., 2004; Fragkouli et al., 2005).
Transplantation of dissected MGE subdivisions provided evidence that somatostatin+ interneurons are preferentially derived from dorsal MGE while parvalbumin+ interneurons are preferentially derived from ventral MGE (Flames et al., 2007; Wonders et al., 2008). Cre mediated recombination fate mapping, using Cre driven by Lhx6, Nkx2-1 and Nkx6.2 BAC transgenes, is consistent with the loss of function and transplantation analysis (Fogarty et al., 2007; Xu et al., 2008). Furthermore, the Shh+ domain in the ventral MGE/dorsal POA appears to be a source for oligodendrocytes (Fogarty et al., 2007; Petryniak et al., 2007).
Derivatives of the POA progenitor zones are less well characterized. Fate mapping with Dbx1-Cre provided shows that the POA contributes cells to the preoptic area and to the amygdala (Hirata et al., 2009), whereas Nkx5.1-Cre shows that the POA also generates a specific subtype of NPY+ cortical interneuron (Gelman et al., 2009).
Here, we report on the fate of the dorsal POA and the ventral-most MGE, by performing fate mapping with a Shh-Cre allele (Harfe et al., 2004). These results show that most of the globus pallidus is generated from this region whereas it generates surprisingly few cortical interneurons.
Next, we investigated the function of Nkx2-1 in Shh-expressing telencephalic cells using Shh-Cre and a floxed-Nkx2-1 allele (Kusakabe et al., 2006). Loss of Nkx2-1 function from the Shh-expressing subcortical domain eliminated most globus pallidus neurons, whereas most cortical and striatal interneurons continue to be generated, except for striatal cholinergic neurons. Thus, this analysis demonstrated that the dorsal MGE and caudal parts of the LGE (caudal ganglionic eminence; CGE) generated most of the cortical GABA interneurons whereas the ventral-most MGE and dorsal POA primarily generated most of the globus pallidus and striatal cholinergic neurons. Finally, our analysis provided evidence for an Nkx2-1- and Shh-independent lineage that is Npas1+, which constitutes ~15% of globus pallidus cells.
Mouse colonies were maintained in accordance with the protocols approved by the Committee on Animal Research at University of California, San Francisco. Nkx2-1 floxed (Nkx2-1f) alleles and constitutive null alleles were generated by S. Kimura and genotyped as described (Kimura et al., 1996; Kusakabe et al., 2006; Mastronardi et al., 2006). Shh-Cre mice were provided by C. Tabin and genotyped as described (Harfe et al., 2004). The ROSA26 LacZ reporter mouse (ROSA) was used for Cre fate mapping as described by Soriano, 1999. The Lhx6 BAC-GFP and Dlx1 BACGFP mouse transgenic lines were generated by GENSAT (http://www.gensat.org/index.html) and described in Cobos et al., 2006. For staging of embryos, midday of the vaginal plug was defined as embryonic day 0.5 (E0.5). Mouse colonies were maintained in accordance with the protocols approved by the Committee on Animal Research at University of California, San Francisco. Breeding was performed as follows: Nkx2-1+/−;ShhCre/+ males were crossed with Nkx2-1f/f;ROSAR26R/+ females. Nkx2-1f/−;Shh+/+ or Nkx2-1f/+;ShhCre/+ mice were use as controls (HET) and Nkx2-1f/−;ShhCre/+ mice were used as mutants. Adults were deeply anesthetized in a CO2 chamber and sacrificed by cervical dislocation. Embryos were removed by cesarean section. Embryos and P0 were anesthetized by cooling; the brain was removed and immersion fixed in 4% paraformaldehyde (PFA) in phosphate-buffered solution (PB 0.1 M, pH 7.4) for 4-12 hours. 2 month old mice were anesthetized with Avertin (0.2 cc/10 g body weight) and perfused intracardially with 4% PFA in PB prior to removing their brains. Samples were cryoprotected in a gradient of sucrose 30%, frozen in embedding medium (OCT; Tissue–Tek, Torrance, CA) and sectioned using a cryostat.
Nonradioactive in situ RNA hybridization was performed using digoxigenin-labeled riboprobes on 20 μm frozen sections as described on the Rubenstein laboratory website (http://physio.ucsf.edu/rubenstein/protocols/index.asp; Cobos DIG ISH protocol). Probes sequences have been previously described (Long et al., 2009a; Zhao et al., 2008). We thank the following people for the cDNAs: CoupTF1 (Ming Tsai), Dbx1 (Sanwei Lu), ER81 (Tom Jessell), Gad1 (Brian Condie), Gbx1 (Mike Frohman), Gsx2 (Gsh2) (Steve Potter), Golf (Richard Axel), Ikaros (Katia Georgopoulos), Islet1 (Tom Jessell), Lhx6 and Lhx7(8) (Vassilis Pachnis), Lmo3 (TH Rabbitts), Lmo4 (Gordon Gill), Npas1 (Steve McKnight), Olig2 (David Anderson), Pou3f1 (Oct6) (MG Rosenfeld), Shh (Andy McMahon), Somatostatin (Thomas Lufkin) and Zic1 (Jun Aruga). Nkx2-1 exon 2 (Nkx2-1-E), Nkx2-1 full length (Nkx2-1-FL), Nkx6.2, Dlx1 and Dlx5 were generated in the Rubenstein laboratory. For more complete information about these cDNAs, see Long et al., 2009a.
Brains sections were cut using a cryostat at 20 μm thickness for embryonic ages and P0, or at 40 μm for 2 month old mouse brains. 20 μm sections were mounted on glass slides (Superfrost Plus, VWR, West Chester, PA) and 40 μm sections were kept free floating in PB buffer. Immunostaining were performed according to Zhao et al., 2008. The following primary antibodies were used: rabbit anti-MAFb (1:1000) (Bethyl laboratories, Montgomery, TX); rabbit anti-ER81 (1:20,000) (Covance,CA), mouse anti-Nkx2-1 (1:50) (Novocastra Laboratories, UK); rabbit anti-Nkx2-1 (1:1000) (BIOPAT immunotechnologies s.r.l, Italy); rabbit anti-Parvalbumin (PV) (1:4000) (Swant, Bellinzona, Switzerland); chicken anti-Green fluorescent protein (GFP) (1:1000) (Aves labs inc., OR); rabbit anti-Olig2 (1:20,000) (kindly provided by. John Alberta, Dana-Farber Cancer Institute, Boston, MA); goat anti-Choline acetyltransferase (ChAT) (1:250) (Chemicon, Temecula, CA); rabbit anti-Npas1 (1:1000) (kindly provided by Steve Mc Knight, University of Texas Southwestern, Dallas, TX); rabbit anti-Phosphohistone 3 (PH-3) (1:200) (Upstate/Millipore, Billerica, MA); rabbit anti-Neuropeptide Y (NPY) (1:2000) (Immunostar, Hudson, WI); rat anti-Somatostatin (SS) (1:250) (Chemicon, Temecula, CA ); rabbit anti-Calretinin (CR) (1:2000) (Chemicon, Temecula, CA); guinea pig anti-beta-galactosidase (bGAL) (1:1000) (kindly provided by Thomas Finger); mouse anti-β-III-tubulin (TUBIII) (1:1000) (clone TUJ1; Covance, CA); mouse anti-Bromodeoxyuridine (BrdU) (1:500) (clone B44; Becton Dickinson, Franklin Lakes, NJ); rabbit anti-Caspase-3 (1:500) (BD Pharmigen, Franklin Lakes, NJ); rabbit anti-Calbindin (CB) (1:4000) (Swant, Bellinzona, Switzerland); mouse anti-Nestin (1:200) (Chemicon, Temecula, CA); rabbit anti-Brain lipid binding protein (BLBP) (1:1000)(Chemicon, Temecula, CA); rabbit anti-DARPP32 (1:2000) (Cell signaling); rabbit anti-TrkA (1:1000) (kindly provided by Louis Reichardt, University of California, San Francisco, CA). Alexa 488 and Alexa 594 secondary antibodies (Invitrogen) were used accordingly to the primary antibody species. Sections were counterstained with DAPI and mounted with Vectashield mounting media (Vector laboratories, Burlingame, CA). Immunoperoxidase method was used for ChAT staining with a biotinylated anti-goat IgG secondary antibody, and the ABC elite kit (Vector Laboratories, Burlingame, CA).
Brightfield images were taken for in situ RNA hybridization data using an SZX7 microscope (Olympus, Japan) and DP70 camera (Olympus, Japan). Immunolabeling images were taken using Nikon Eclipse 80i microscope (Nikon), CoolSnap camera (Photometrics) and NIS-Elements BR 3.00 software (Nikon). Confocal images were taken using a confocal microscope (LSM510, Zeiss) (pinhole: 2 μm). Contrast and brightness of these images were adjusted for better visualization using Photoshop CS2 software (Adobe Systems, San Jose, CA).
Confocal images were used for the quantification of data in Figure 1 and figure 3 (P0 and P60 analysis). The whole image area was used for counting. For Figure 1A’’ (E13.5), Figure 4 (E18.5) and Supplemental Fig. 2 (E18.5), counting was performed on 10x magnification image. Boxes were drawn within the boundaries of the NKX2-1+ globus pallidus; the area of the boxes represented ~1/4 of the globus pallidus area, and had ~100 DAPI+ cells for the E13.5 data, and ~150-200 DAPI+ cells for the E18.5 data. For both confocal and 10x data, an image with red, green and blue channels were created using Photoshop CS2 software. Only cells with clear DAPI staining were counted. Total numbers of red, green and yellow cells were counted.
The quantification of cortical interneurons on E18.5 heterozygote and the mutant were performed on 10x images as in Figure 10. The cortex and striatum were used as anatomical landmarks to match the rostrocaudal levels. We drew boxes (500 μm wide) from the ventricular zone to the pial surface. For the ventral pallium region, we used Gad1 staining on the heterozygote to define the size of the boxes (3.6 mm2 surface area). The surface area of the boxes counting was similar between heterozygote and mutant.
Nkx2-1 is expressed in the progenitor cells of the entire MGE and POA (Lazzaro et al., 1991; Shimamura et al., 1995; Sussel et al., 1999; Puelles et al., 2000; Flames et al., 2007; García-López et al., 2008). Its expression persists in subsets of postmitotic neurons, including those of the globus pallidus (GP; pallidum) and striatal interneurons (Figs. 1A,B and and2;2; Sussel et al., 1999; Marin et al., 2000). Furthermore, fate-mapping using Nkx2-1-Cre, confirms that the Nkx2-1-expressing cells give rise to these neurons (Xu et al., 2008). However, it is unclear what parts of the subpallium contribute to the GP and striatal interneurons.
Expression of ER81 at E10.5 and E11.5 suggests that ventral regions of the basal telencephalon (ventral MGE and dorsal POA) contribute to the GP (Flames et al., 2007). Here we used Shh-GFP-Cre (Harfe et al., 2004) to determine the contribution of the POA and ventral MGE to the globus pallidus, and interneuron populations. Cre expression from the Shh locus provides an approach to follow the fate of these cells following recombination of a reporter gene, such as LacZ, which encodes a β-galactosidase bGAL) (Soriano, 1999).
Analysis of the Shh-Cre-mediated recombination pattern at E11.5 and E13.5 showed a high density of bGAL+ ventricular zone (VZ) cells in the dorsal preoptic area and the ventral-most MGE (pPOA1 and pMGE5 of Flames et al., 2007) and was very similar to the expression of Shh RNA; there was scattered recombination in the dorsal MGE regions, and occasional bGAL+ cells in the LGE, CGE and cortex (Figs. 1A’; Sup. Fig. 1 and not shown). In addition, there was migration of bGAL+ cells that appeared to be emanating from the ventral MGE and populating the deep and superficial MGE mantle zone, as well as cells migrating through the LGE and into the cortex (Figs. 1A’; Sup. Fig. 1A”-D” and not shown). At E11.5, GFP expression from the Shh-GFP-Cre allele closely matched the pattern of recombination except in two regions: 1) the region just dorsal to the zone of Shh expression in the VZ expressed more bGAL+ than GFP, suggesting that at an earlier developmental stage Shh was expressed in these progenitors, and subsequently was down-regulated (Sup. Fig. 1B’’’); 2) in a superficial part of the MGE mantle zone the cells were GFP+ but bGAL− (i.e. had not undergone recombination) (Sup. Fig. 1A’’’-C’’’).
Analysis of the Shh-Cre recombination pattern at E11.5, E13.5 and P0 showed that extensive regions of the subcortical telencephalon had large numbers of cells derived from Shh-expressing cells, many of which continued to co-express NKX2-1 protein (Figs. 1A’’,B’’, ,2;2; Sup. Figs. 1A”-D” and not shown). This included substantial parts of the septum, diagonal band, ventral pallidum, preoptic area, and globus pallidus, and small domains of the bed nucleus stria terminalis and amygdala. Recombination within the hypothalamus will be discussed elsewhere. In most regions, there was a high concordance between NKX2-1 protein expression and Shh-Cre recombination (particularly in the globus pallidus, see below), whereas in other regions, such as the medial amygdala, they appeared discordant; the ventral medial nucleus was derived from the Shh expressing cells, whereas the dorsal medial nucleus was NKX2-1+ and not derived from the Shh lineage (Fig. 2H). In addition, there were scattered cells in the striatum and pallium (discussed below).
Most globus pallidus cells expressed NKX2-1 prenatally (E11.5, E13.5 and E15.5) and postnatally (P0), and were in the Shh lineage (Figs. 1A’’,B’’, 2D,E and not shown). At E13.5, ~80% of the NKX2-1+ cells in the region of the developing globus pallidus were derived from the Shh-Cre lineage (Fig. 1A-A’’). Analysis at P0 confirmed that the majority (~70%) of NKX2-1+ globus pallidus cells were derived from Shh-Cre+ descendents (Fig. 2D,E). In the adult (P60), we found that ~70% of parvalbumin+ (PV+) globus pallidus neurons were derived from Shh-Cre descendents (Fig. 3D,E). PV+ projection neurons constitute a major fraction of globus pallidus neurons.
Other derivatives of the Shh-Cre lineage included cortical and striatal GABAergic interneurons (largely PV+) (Fig. 3), striatal TrkA+ cholinergic neurons (not shown), and Olig2+ cells, presumably oligodendrocytes, populating several regions, but especially parts of the septum and telencephalic axon tracts (corpus callosum, hippocampal commissure and anterior commissure; Fig. 2J,K).
The Shh-expressing progenitor domain of the basal telencephalon generated most of the PV+ neurons of the globus pallidus (~70%) (Fig. 3D) and a substantial fraction of PV+ striatal interneurons (~30%; Fig. 3C’”), but only a small fraction of PV+ cortical and hippocampal (~5-10%) interneurons (Fig. 3A”’,B”’). Thus, this Shh-domain is a rich source for subpallial, but not pallial, PV+ neurons. However, some neurons in the globus pallidus were not descended from Shh-expressing cells, suggesting heterogeneity of its developmental origins and perhaps neuronal subtypes. This hypothesis was confirmed by analysis of transcription factor expression (Fig. 4) and of the Nkx2-1 mutants described below.
Our fate mapping analysis suggested that not all globus pallidus neurons were derived from Shh-expressing cells, implying that there may be molecularly/developmentally distinct neuronal subtypes. To investigate this further, we compared the expression of the Dlx1, ER81, Lhx6, Nkx2-1 and Npas1 transcription factors in the globus pallidus at E15.5, E18.5, P0 and the adult ages. We did not have Dlx1 or Lhx6 antibodies, and thus used mice expressing GFP from BAC transgenes to label these cells (Cobos et al., 2006).
At E18.5 ~80% of globus pallidus cells (DAPI+) expressed NKX2-1 protein (Fig. 4A-A’’). Double labeling of NKX2-1 with NPAS1 and OLIG2 showed that the globus pallidus had the following cell subtypes: 81% expressed NKX2-1 (54% were NKX2-1+;NPAS1− ; 27% NKX2-1+;NPAS1+); 42% expressed NPAS1 (27% NPAS1+;NKX2-1+; 15% NPAS1+ ;NKX2-1−)(Fig. 4B-B’’); and 13% expressed OLIG2 (probably oligodendrocytes; Petryniak et al., 2007)(Fig. 4C-C”). While none of the NKX2-1+ cells co-expressed OLIG2, we do not know whether there were any NPAS1+;OLIG2+ cells because both antisera were rabbit, precluding double-labeling.
Dlx1 (BAC-GFP), ER81 and Lhx6 (BAC-GFP) were co-expressed with ~60-90% of NKX2-1+ cells (Sup. Fig. 2A’’,C’’,E’’). Likewise most of the cells expressing these genes also expressed NKX2-1 (Sup. Fig. 2A’’,C’’,E’’). While ~90% NKX2-1+ globus pallidus cells co-express Dlx1-GFP, only 65% of the NPAS1+ globus pallidus cells expressed Dlx1-GFP (Sup. Fig. 2D’’,E’’). Prenatally (E15.5), we observed similar results (data not shown).
Fate mapping analysis (Shh-Cre) at P0 showed that the majority of NKX2-1+ (~70%) and ER81+ (~80%) GP cells were derived from Shh-Cre+ descendents (Fig. 1B’’,C’’). On the other hand, only ~35% of globus pallidus cells expressing NPAS1 were derived from the Shh-Cre lineage (Fig. 1D’’). Thus, while most globus pallidus cells expressed NKX2-1, Dlx1, ER81 and Lhx6, and were derived from Shh-Cre lineage, a large fraction of NPAS1+ globus pallidus cells represented a distinct population.
Mice constitutively lacking Nkx2-1 failed to generate a globus pallidus expressing ER81 and Lhx6 (Sussel et al., 1999). On the other hand, these mutants continued to express NPAS1 in the region of the globus pallidus (Fig. 9L,L’), providing further evidence that a subset of these NPAS1+ neurons were independent of Nkx2-1 expression and function.
To establish which basal telencephalic progenitor zones require Nkx2-1 function to generate the globus pallidus, we selectively removed Nkx2-1 function in cells expressing Shh-Cre using a floxed allele of Nkx2-1 (Kusakabe et al., 2006). We used the following cross: Nkx2-1f/f × Nkx2-1+/−; ShhCre/+ (with or without the ROSA LacZ Cre reporter), and compared the telencephalic phenotype of mutant (Nkx2-1f/−; ShhCre/+) and control (Nkx2-1+/−) offspring at E11.5, E13.5, E15.5 and E18.5 (they do not survive postnatally).
To establish the efficiency of Cre-mediated deletion, we studied the expression of Nkx2-1 RNA by in situ hybridization at E11.5, E13.5, E15.5 and E18.5. We used two different probes; one probe was complementary to the region that should be deleted by Cre-excision (exon 2 probe); the other probe was complementary to the 5’ region of the transcript (full-length probe). Analysis with the exon 2 probe showed that Cre efficiently eliminated exon 2 expression in the progenitor domains of the ventral MGE (pMGE5) and the POA (pPOA1-2) throughout the rostrocaudal extent of the basal telencephalon (arrows, Figs. 5B,B’, 6B,B’; Sup. Figs. 3-6). On the other hand, analysis with the full-length Nkx2-1 probe provided evidence that these progenitor domains continued to express Nkx2-1, albeit at lower levels, suggesting that they maintain some degree of their initial specification as MGE and POA progenitors (Figs. 5C,C’, 6C,C’; Sup. Figs. 3-6).
Next we followed the fate of the recombined cells by analyzing bGAL expression (the ROSA LacZ Cre reporter was included in the cross). At E11.5, bGAL expression in the VZ was greatly reduced, providing evidence that these cells failed to be generated, proliferate at the normal rate and/or survive; likewise there was a large reduction of bGAL+ cells in the MGE mantle zone suggesting that very few neurons in this lineage were produced by this age (Fig. 6A,A’).
At E13.5 and E18.5 bGAL fate mapping analysis of the Nkx2-1f/−; ShhCre/+ mutant showed that Shh-expressing descendents failed to generate a globus pallidus and the tangentially migrating stream of SVZ cells of the MGE; rather these cells accumulated in a deep mantle zone adjacent to the Shh+ progenitor zone (Figs. 5A,A’, 7A-C’; Sup. Fig. 7A’-C’).
In wild type mice there was a robust cell migration parallel to radial glial processes expressing brain lipid binding protein (BLBP) and Nestin; these processes emanated from the Shh+ progenitor domain and extended through the SVZ of the MGE (Fig. 5E,F and not shown), and were continuous with cells tangentially migrating through the SVZ of the MGE, LGE and cortex (data not shown). Note that the radial processes of the Shh-domain extended perpendicularly to the radial processes emanating from the adjacent MGE. On the other hand, in the Nkx2-1f/−;ShhCre/+ mutant, these radial glial processes appeared to have an altered trajectory from the Shh+ progenitor domain (Fig. 5E’,F’).
The reduced numbers of mantle zone cells in the Shh-Cre lineage was not accounted for by increased cell death, as we did not detect an increase in the number of apoptotic cells at E11.5 and E13.5 (based on caspase-3+ cells)(data not shown). There were defects in the progenitor zones of the caudoventral MGE and POA of the Nkx2-1f/−;ShhCre/+ mutant; the subventricular zone (SVZ) showed a ~2-fold reduced proliferative index at E13.5 based on phosphohistone 3+ M-phase cells (Fig. 5H,H’). While we did not detect a phenotype in ventricular zone (VZ) proliferation (Fig. 5H,H’), many bGAL+ cells accumulated as radial clone-like clusters in the progenitor zone (Figs. 5A’, 7B’,C’). These cells were not ectopic immature neurons, as they did not express β-III-tubulin (Fig. 5D,D’, and not shown). The morphology of the VZ in this region was altered; the bGAL+ progenitor domain was wider and extended further along the dorsoventral axis (Figs. 5A’, 7B’,C’). To explore whether these VZ and SVZ defects reflected an alteration in the regional identity of this progenitor zone, we performed in situ RNA hybridization.
To assess whether selective deletion of Nkx2-1 from the Shh-domain altered the molecular properties of the VZ, we used a panel of molecular markers that have expression boundaries within this region at E11.5 and E13.5 (Flames et al., 2007; Zhao et al. 2008). The exon 2 Nkx2-1 probe identified where recombination deleted Nkx2-1; this domain closely matched the VZ domain of Shh expression (Figs. 5A’,B’, 6A’,B’; Sup. Fig. 1). Hybridization with the full-length Nkx2-1 probe showed that this region maintained Nkx2-1 transcripts (albeit at lower levels), suggesting that transcription and some aspects of fate were maintained (Figs. 5C’, 6C’, 8E’). Note that normal Nkx2-1 expression was maintained in the dorsal MGE along its rostrocaudal extent (Sup. Figs. 3, 4).
Next we studied the effect of removing Nkx2-1 from the Shh-expression zone (ventral MGE and POA) at E11.5. Shh expression was reduced in most of the VZ (arrows; Fig. 6D,D’; Sup. Fig. 3), whereas Shh expression was maintained in the mantle zone (MZ) of the rostrodorsal MGE (Fig. 6D,D’). The region with preserved Shh MZ expression probably corresponds to the GFP+ Shh-expression zone where Cre recombination did not occur (Sup. Fig. 1A”’-D”’). Likewise, the Nkx2-1f/−;ShhCre/+ mutants showed reduced expression of ER81, Lhx6 and Lhx7(8) in the region where Nkx2-1 was deleted (Sup. Fig. 3). MGE expression of Lhx6 and Lhx7(8) was maintained both rostrodorsally and caudodorsally (Sup. Fig. 3).
Deletion of Nkx2-1 by E11.5 did not show a major effect on the ventral POA, based on preserved expression of Dbx1, Nkx5.1 and Nkx6.2 at E11.5 and E13.5 (Fig. 6; Sup. Figs. 3,4). On the other hand, the dorsal POA and ventral MGE showed ectopic expression of two of these ventral POA markers (Dbx1 and Nkx6.2) (arrowheads, Fig. 6E’,F’; Sup. Figs. 3, 4); in the ventral MGE, there were radial clusters of cells showing this ectopic expression. Despite, these changes, the expression of COUP-TF1, Nkx5.1, Olig2 and Zic1 in this region did not show major changes, although there were subtle changes in COUP-TF1 and Zic1 in the VZ of the POA (Sup. Fig.3).
Deletion of Nkx2-1 in the Shh domain led to a loss of most of the globus pallidus based on several criteria. In control brains at E13.5 and E15.5, we detected the globus pallidus based on its location and its expression of Dlx1, Gbx1, ER81, Lhx6, Lhx7(8), Lmo3, Nkx2-1 and Zic1 (Fig. 8; Sup. Figs. 4, 5). At E13.5 the mutant lacked expression of most of these markers in the region where the globus pallidus was normally present (Fig. 8; Sup. Fig. 4). Expression of NPAS1, a marker of a subtype of globus pallidus neurons (Fig. 4B’’; Sup. Fig. 2D’’), and cortical interneurons (Zhao et al., 2008) was not affected in the mutant at E13.5 (Fig. 8I,I’; Sup. Fig. 4). At this age however, NPAS1 was not detected in the position of the globus pallidus, but rather showed expression in a pattern that could reflect a tangential migration from a progenitor zone near pMGE5 or pPOA1.
At E18.5 molecular features of the globus pallidus continued to be deficient in the mutant, showing only scattered expression of ER81, Lmo3 and Nkx2-1 (full-length, exon 2) (Fig. 9; Sup. Fig. 6). On the other hand, the mutant had clusters of cells expressing Lhx6, Lhx7(8), Dlx1 and Npas1 in the general region where the globus pallidus should form. The cells expressing Npas1 and Dlx1 (Sup. Fig. 6) could correspond to the Npas1+;Dlx1+;Nkx2-1− globus pallidus neurons observed in normal mice (Fig. 4; Sup. Fig. 2). While the Lhx6 and Lhx7(8) expressing cells could represent some globus pallidus cells, perhaps produced by the dorsal MGE, they are found in the same region where we detect cholinergic (ChAT+) neurons (Fig. 9; Sup. Figs. 6, 8).
Expression of Nkx2-1-regulated genes was not affected (or only modestly altered) in regions where Nkx2-1 was not deleted. This included the entire rostrocaudal extent of the dorsal MGE (Sup. Figs. 3--6).6). For instance, in rostral sections containing the immature anterior BNST and ventral pallidum (VP), expression of Gbx1, ER81, Lhx6, Lhx7(8), Nkx2-1 and Zic1 appeared normal. In addition, in most regions, there was expression of these genes in MZ superficial to the GP. Many of the neurons that populate these layers are produced at early developmental stages. In the mutant, we found no major change in the numbers of early born neurons in these regions generated by BrdU incorporation at E10.5 (data not shown), and as assessed by βIII-tubulin expression (Fig. 5D,D’). Thus, we conclude that most early neurogenesis (through ~E10.5) in the basal telencephalon was not disrupted by deleting Nkx2-1 in the Shh-Cre lineage.
Surprisingly, Lhx6 expression in the mutant's BNST appeared partially preserved (Figs. 8B’, 9E’; Sup. Figs. 3-6). We postulate that the remaining Lhx6+ BNST expression domain was generated by a ventral migration from the dorsal MGE.
The MGE in constitutive Nkx2-1 null mice showed evidence of a fate transformation towards the LGE (Sussel et al., 1999), although further analysis showed that constitutive Nkx2-1 nulls maintain expression of Npas1 in the globus pallidus (Fig. 9L,L’). Here we assessed whether deletion of Nkx2-1 in the ventral MGE/dorsal POA in the Nkx2-1f/−; ShhCre/+ mutant, roughly 1 day after Nkx2-1 expression had been established, resulted in this fate switch. Above, we have shown that the ventral MGE and the dorsal POA lacked Nkx2-1 expression and failed to generate a GP. In addition to the dorsal expansion of POA markers (Dbx1 and Nkx6.2), the ventral MGE and the dorsal POA now expressed LGE/striatal markers in the SVZ [Lmo4 and Oct6 (Pou3f1)] and MZ (Golf, Ikaros and Lmo4) at E13.5, E15.5 and E18.5 (Figs. 8, ,9;9; Sup. Figs. 3-6). Cre-fate mapping at E18.5 showed that the same MGE region with ectopic Golf, Ikaros and Lmo4 expression also contained derivatives of the Shh-Cre lineage (Fig. 9). Some of the cells derived from the Shh-Cre lineage expressed the striatal marker DARPP32 (Sup. Fig. 7D,D’).
BrdU birthdating analysis provided evidence that this region of ectopic striatal tissue was produced around E13.5. BrdU administered at E13.5, and analyzed at E15.5, showed many BrdU-labeled cells ectopically collected in the deep MZ region of the Nkx2-1f/−; ShhCre/+ mutant caudoventral MGE (data not shown).
Constitutive Nkx2-1 mutants failed to generate ~50% of neocortical and hippocampal interneurons; paleocortical interneurons may have been even more reduced (Sussel et al., 1999; Pleasure et al., 2000; Xu et al., 2004). As discussed above, fate mapping with Shh-Cre showed that only a very small fraction of neocortical and hippocampal interneurons were derived from ventral MGE/dorsal POA (Fig. 3), providing evidence that most MGE-derived interneurons are produced by the dorsal MGE. To further assess this, we compared the numbers of neocortical interneurons at E13.5, E14.5, E15.5 and E18.5 in control and Nkx2-1f/−;ShhCre/+ mutants.
At E13.5 and E14.5 the mutants showed a reduction in neocortical interneurons expressing Dlx1+ (~35% and ~20% reduction) and Lhx6+ (~45% and ~48% reduction). More severe reductions were found in the paleocortex (data not shown).
By E18.5, however, the interneuron reductions found in the mutants were smaller compared to the earlier stages. Dlx1+ and Lhx6+ neocortical interneurons were reduced ~10% (Fig. 10A,A’,B,B’). These decreases were consistent with the Shh-Cre fate mapping data suggesting that less than 10% of adult cortical interneurons were derived from the Shh-domain (Fig. 3). On the other hand, MAFb, Npas1 and somatostatin expressing interneurons showed greater reductions (MAFb: ~35%; calbindin: ~22%; Npas1: ~25%; Somatostatin: ~40%) (Fig. 10C,C’,D,D’ and not shown), suggesting either that these molecular subtypes were preferentially affected, or that alterations in Lhx6 expression levels affected gene expression in these cell types. The mutant ventral pallium (paleo/piriform cortex, claustrum and endopiriform nucleus) also showed interneuron defects, with reductions of 25% of Lhx6+ and 40% of GAD1+ interneurons (Fig. 10E,E’,F,F’). Likewise, there was a large reduction of bGAL+ cells (Shh-Cre lineage) in the ventral pallium (Sup. Fig. 6A,A’).
Many striatal interneurons, (including ~30% of parvalbumin+) are derived from Shh-expressing cells based on Shh-Cre fate mapping (Fig. 3). In E18.5 Nkx2-1f/−;ShhCre/+ mutants showed striatal interneuron reductions. There was roughly a ten-fold reduction in the number of Lhx7(8) expressing cells, and consistent with its known function, ChAT+ cholinergic striatal interneurons were reduced ~4-fold (Sup. Fig. 8). Furthermore, Lhx6+ striatal interneurons were reduced, particularly in the ventral striatum/accumbens core (dorsal: ~15% reduction; ventral: ~50%). On the other hand, there was not an obvious reduction in the number of NPY+ and somatostatin+ striatal interneurons (data not shown); PV expression does not begin until postnatally, and therefore could not be assessed. Medial septal expression of Lhx6 and Lhx7(8) were greatly reduced, consistent with Shh-Cre fate mapping (Fig. 2), whereas their expression in the accumbens shell appeared preserved (Sup. Fig. 8).
Herein, we used the Shh-GFP-Cre allele, which makes a GFP-Cre fusion protein (Harfe et al., 2004), to perform fate mapping and to eliminate Nkx2-1 function in the ventral MGE and POA. Cre-mediated recombination in the ventricular zone was more extensive in the MGE and POA than either GFP or Shh RNA expression (Sup. Fig. 1) suggesting that Shh expression was down-regulated as development proceeds. Shh RNA expression wanes as development proceeds (Zhao et al., 2008), which explains why Flames et al, 2007, included Shh expression at E13.5 only in pPOA1.
Shh-Cre mediated recombination was robust in most Shh-GFP+ cells. The only exception was in a superficial part of the MGE mantle zone, where the cells were GFP+ but were bGAL− (i.e. no recombination) (Sup. Fig. 1). We do not understand why these cells fail to show Cre-mediated recombination. Consistent with this result, the Nkx2-1f/−;ShhCre/+ mutant continues to express Shh RNA in the MGE mantle zone (Fig. 6D,D’; Sup. Fig. 3).
Previous studies suggest that the dorsal MGE is the predominant source for somatostatin+ cortical interneurons, whereas the ventral MGE is the predominant source for parvalbumin+ interneurons (Flames et al., 2007; Wonders et al., 2008). Fate mapping from the ventral MGE and the POA using Shh-Cre surprisingly showed that these regions produce very few neocortical and hippocampal interneurons, although there was a strong bias to generate the parvalbumin+ subtype (Fig. 3E). The morphological and gene expression evidence suggests that Shh-Cre expressing cells induce robust recombination in pMGE5 and perhaps pMGE4, as well as pPOA2 and pPOA1 (domains defined by Flames et al., 2007). The results imply pMGE1-3 (perhaps pMGE4) generate most parvalbumin+ and somatostatin+ neocortical and hippocampal interneurons. The Shh-Cre domain may also produce interneurons that migrate to the ventral pallium (Fig. 2).
Furthermore, the ventral MGE and the dorsal POA produced a substantial fraction of striatal interneurons (GABAergic and cholinergic) (Fig. 3; Sup. Fig. 8). However, among the GABAergic subtypes, parvalbumin+ cells predominated. Thus, the Shh-Cre+ progenitors were biased towards generating parvalbumin+ neurons. This concept is further supported in the globus pallidus.
The Shh-Cre domain generates cells that contribute to the medial septum, diagonal band, pallidal complex (globus pallidus, and ventral pallidum), preoptic complex (medial and lateral), the ventral medial amygdala and ventral pallial structures (Fig. 2). We focused on the cellular and molecular complexity of the globus pallidus and found that NPAS1+;NKX2-1− cells are a novel cell type (Fig. 4).
Evidence supporting the existence of NPAS1+;NKX2-1− cells comes from analysis of Nkx2-1 constitutive and conditional mutants; both have NPAS1+ globus pallidus cells (Fig. 9H,H’,L,L’). Fate mapping analysis at P0 showed that ~70% of NKX2-1+ are derived from Shh-Cre+ descendents (Figs. 1, ,2).2). On the other hand, only ~35% of NPAS1+ globus pallidus cells were derived from the Shh-Cre lineage (Fig. 1). Thus, NKX2-1+ and NPAS1+;NKX2-1− cells represent distinct subtypes of globus pallidus neurons.
While the NKX2-1+ cells were largely derived from the Shh-Cre domain, the origin of the NPAS1+;NKX2-1− neurons is unclear. The ventral POA is probably not a source, based on fate mapping with Dbx1-Cre (Hirata et al., 2009). At E13.5 there were Npas1+ SVZ cells between the MGE and dorsal POA; this expression is maintained in the Nkx2-1f/−; ShhCre/+ mutant (Fig. 8I,I’). Perhaps these cells later migrate to the globus pallidus. We are uncertain about the location of the progenitors of these cells.
Finally, the fate mapping shows that ~70% of globus pallidus neurons are parvalbumin+ (Fig. 3D,E). Thus, the majority of GABAergic neurons generated from the Shh-expression domain, destined for the globus pallidus, striatum and cortex, express parvalbumin.
Previous studies of the constitutive and conditional Nkx2-1 null mouse demonstrated that Nkx2-1 is essential for specifying MGE, and repressing LGE (and dorsal CGE) identity (Sussel et al., 1999; Corbin et al., 2003; Butt et al., 2008). Herein we deleted Nkx2-1 from the Shh-expressing progenitors in the ventral MGE and POA, beginning around E9.5 (Shh expression in the MGE begins shortly after Nkx2-1; Crossley et al., 2001; Shimamura et al., 1995). Perdurance of Nkx2-1 transcripts in the ventral MGE and POA, showed that these regions were initially specified to express Nkx2-1 (Fig. 6C,C’, 8E,E’; Sup. Figs. 3-6). Continued Nkx2-1 function in the ventral MGE was required to: 1) repress dorsal spread of POA properties (Dbx1, Nkx6.2, Islet1) (Fig. 6E,E’,F,F’; Sup. Figs. 3-6); 2) maintain normal levels of Shh expression in the VZ (Fig. 6D,D’; Sup. Fig. 3); 3) repress induction of LGE/striatal properties (DARRP32, Dlx5, Golf, Ikaros, Islet1, Lmo4, Pou3f1) (Fig. 8D,D’,H,H’, 9I,I’,J,J’,K,K’; Sup. Figs. 5-7); 4) repress induction of dCGE properties (COUP-TFI) (Sup. Fig. 3). Thus, persistent Nkx2-1 function represses expression of both dorsal (LGE/dCGE) and ventral (POA) properties in the ventral MGE.
The reduced expression of Shh (Fig. 6D,D’; Sup. Fig. 3) could cause cell-non-autonomous effects (Xu et al., 2005; Gulacsi and Anderson, 2006), which may extend the phenotype to adjacent regions of the MGE and POA. This could contribute to the decrease in proliferative index (Fig. 5H,H’).
Many facets of MGE and POA development were not derailed in the Nkx2-1f/−;ShhCre/+ mutant. For instance, the ventral POA continues to express Nkx5.1 (Sup. Figs. 3, 4). Furthermore, Nkx2-1 expression in the rostral and caudal MGE appeared normal, consistent with the localized domain of Shh-Cre expression in the middle of the basal telencephalon. The progenitor model of Flames et al. (2007) posited that dorsal MGE domains are present in the rostral (pMGE1-3) and caudal (pMGE3) MGE. We suggest that Shh-Cre expression is centered in MGE5 and POA1; MGE4 and POA2 may also be affected by the action of Shh-Cre. Expression of Nkx2-1-dependent genes continued in the dorsal MGE domains [Gbx1, Lhx6 and Lhx7(8)], showing that these regions maintained their fate (Fig. 8B,B’,F,F’,G,G’; Sup. Figs. 3-6). We propose that pMGE1-3 generate the lion-share of MGE-derived neocortical and hippocampal interneurons.
Loss of Nkx2-1 from the Shh-domain reduced the number of striatal interneurons. Striatal Lhx7(8), ChAT, P75 and TrkA expression was almost eliminated (Sup. Fig. 8, and not shown), consistent with: Zhao et al. 2003; Mori et al., 2004; Fragkouli et al., 2005. Lhx6 expression in striatal interneurons was also reduced (Sup. Fig. 8), particularly in the ventral striatum and the core of the nucleus accumbens, suggesting the ventral MGE/POA preferentially produces interneurons for ventral striatal structures. Likewise, we saw a trend for a greater reduction of ventral pallial interneurons (~25%) than dorsal pallial (~10%) interneurons (Fig. 10).
While the number of Lhx6+ and Dlx1+ neocortical interneurons was only reduced ~10% at E18.5, their numbers were more severely reduced (~50%) at E13.5 and E14.5 (data not shown). This suggests that the ventral MGE/POA has a prominent role in producing early-born interneurons, whereas the dorsal MGE is more important for late-born interneurons.
Most globus pallidus neurons were derived from Shh+ and Nkx2-1+ progenitors (Figs. 1B’’, 3D,E; Xu et al., 2008). Nkx2-1 mutants have reduced expression of transcription factors in the globus pallidus: Dlx1, ER81, Gbx1, Npas1, Lhx6, Lhx7(8), Lmo3 and Zic1 (Figs. 8, ,9;9; Sup. Figs. 3-6). Thus, we propose Nkx2-1-positive regulation of ER81, Lhx6, Lhx7(8), Lmo3 and Gbx1. We also propose at least three Nkx2-1 independent pathways: 1) general pathways for subcortical GABAergic neuronal development requiring Dlx1/2/5/6, Gsx2 and Ascl1/Mash1 in Nkx2-1+ cells (Long et al., 2009a,b; Wang, Campbell and Rubenstein, unpublished); 2) Npas1+ globus pallidus neurons that are not in the Nkx2-1-dependent and Shh-Cre lineage; 3) Zic1, whose expression is not lost in the Nkx2-1f/−;ShhCre/+ mutant; Zic1+ migrating cells fail to populate the globus pallidus.
The Shh+ progenitor domain generates radial processes that are oriented tangentially to the radial glial in more dorsal parts of the MGE (Figs. 5E,F). This perpendicular orientation is probably related to the orthogonal orientation of the ventricular zones in these adjacent regions. We speculate that Shh+ progenitor domain's “tangential” radial glial processes contribute to the migratory pathway of globus pallidus neurons. Radial glial processes that extend from the Nkx2-1f/−; ShhCre/+ mutant's Shh+ progenitor domain appeared to have an altered trajectory (Fig. 5E’,F’), and most of the bGAL+ cells generated from the Shh-domain failed to migrate to their correct subcortical and cortical destinations (Fig. 5A,A’, 7A-C’, 9A,A’). Furthermore, Shh+ progenitor domain marked the location whether the anterior commissure crosses the midline (Garcia-Lopez et al., 2008). The anterior commissure fails to form in the Nkx2-1f/−;ShhCre/+ mutants (Fig. 9 I-J’; Sup. Figs. 5,6); perhaps the “tangential” radial glia processes participate in guiding the commissural axons.
This work was supported by the research grants to JLRR from: Nina Ireland, Weston Havens Foundation, NIMH R37 Grant MH049428 and R01 MH081880; to PF from the Swiss National Science Foundation Fellowship PBGE33-112882 and PA0033-117463.