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• W2000966516 abstract "Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). Herein we demonstrate that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome). Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). Herein we demonstrate that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome). Zfhx1b expression is directly regulated by Dlx2 via two distinct Zfhx1b enhancers Zfhx1b mutants overproduce a subtype of striatal interneuron Zfhx1b is upstream of Cxcr7, MafB, and cMaf in cortical (not striatal) interneurons This study provides a mechanism for epilepsy in humans with Zfhx1b mutations Cell type specification within the embryonic basal ganglia is regulated at multiple levels. Distinct subdivisions within this region generate distinct neurons. For instance, the lateral ganglionic eminence (LGE) generates striatal projection neurons whereas the medial ganglionic eminence (MGE) generates pallidal projection neurons. Domains within the MGE are biased toward generating different cell types, whereas the rostrodorsal MGE largely produces cortical and striatal interneurons, the caudoventral MGE largely produces pallidal projection neurons (Flandin et al., 2010Flandin P. Kimura S. Rubenstein J.L.R. The progenitor zone of the ventral medial ganglionic eminence requires Nkx2-1 to generate most of the globus pallidus but few neocortical interneurons.J. Neurosci. 2010; 30: 2812-2823Crossref PubMed Scopus (136) Google Scholar; Nobrega-Pereira et al., 2010Nobrega-Pereira S. Gelman D. Bartolini G. Pla R. Pierani A. Marin O. Origin and molecular specification of globus pallidus neurons.J. Neurosci. 2010; 30: 2824-2834Crossref PubMed Scopus (103) Google Scholar). Distinct MGE-derived cortical interneuron subtypes appear to be generated from the same progenitors, perhaps in a temporal sequence (Brown et al., 2011Brown K.N. Chen S. Han Z. Lu C.-H. Tan X. Zhang X.-J. Ding L. Lopez-Cruz A. Saur D. Anderson S.A. et al.Clonal production and organization of inhibitory interneurons in the neocortex.Science. 2011; 334: 480-486Crossref PubMed Scopus (120) Google Scholar). Cortical and striatal interneurons are both generated from the MGE (Marin et al., 2000Marin O. Anderson S.A. Rubenstein J.L. Origin and molecular specification of striatal interneurons.J. Neurosci. 2000; 20: 6063-6076Crossref PubMed Google Scholar). The Nkx2-1 homeobox transcription factor has a central role in specifying their identity. While Nkx2-1 is initially required for both of these cell types, Nkx2-1 expression is repressed soon after immature cortical interneurons tangentially migrate from the MGE, while it is maintained in striatal interneurons (Butt et al., 2008Butt S.J.B. Sousa V.H. Fuccillo M.V. Hjerling-Leffler J. Miyoshi G. Kimura S. Fishell G. The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes.Neuron. 2008; 59: 722-732Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar; Marin et al., 2000Marin O. Anderson S.A. Rubenstein J.L. Origin and molecular specification of striatal interneurons.J. Neurosci. 2000; 20: 6063-6076Crossref PubMed Google Scholar; Nóbrega-Pereira et al., 2008Nóbrega-Pereira S. Kessaris N. Du T. Kimura S. Anderson S.A. Marín O. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors.Neuron. 2008; 59: 733-745Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar; Sussel et al., 1999Sussel L. Marin O. Kimura S. Rubenstein J.L. 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: 3359-3370PubMed Google Scholar). Forced expression of Nkx2-1 in cortical interneurons changes their migration so that they settle in the striatum (Nóbrega-Pereira et al., 2008Nóbrega-Pereira S. Kessaris N. Du T. Kimura S. Anderson S.A. Marín O. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors.Neuron. 2008; 59: 733-745Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), providing additional evidence that repression of Nkx2-1 is a key step in generating cortical interneurons. How Nkx2-1 expression is repressed in these cells is unknown. Herein, we provide evidence that the Zfhx1b (Sip1, Zeb2) zinc-finger homeobox transcription factor is required to repress Nkx2-1 in the generation of cortical interneurons. In its absence, we find a decrease in cortical interneurons concomitant with increased striatal nNOS/NPY/Sst GABAergic interneurons. We provide evidence that expression of the cMaf transcription factor is a highly specific marker of the cortical interneuron lineage, and discovered that its expression is lost in Zfhx1b mutants. Previous analysis of Zfhx1b mouse mutants has shed light on its functions in the development of cortical projection neurons (Miquelajauregui et al., 2007Miquelajauregui A. Van De Putte T. Polyakov A. Nityanandam A. Boppana S. Seuntjens E. Karabinos A. Higashi Y. Huylebroeck D. Tarabykin V. Smad-interacting protein-1 (Zfhx1b) acts upstream of Wnt signaling in the mouse hippocampus and controls its formation.Proc. Natl. Acad. Sci. USA. 2007; 104: 12919-12924Crossref PubMed Scopus (73) Google Scholar; Seuntjens et al., 2009Seuntjens E. Nityanandam A. Miquelajauregui A. Debruyn J. Stryjewska A. Goebbels S. Nave K.-A. Huylebroeck D. Tarabykin V. Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors.Nat. Neurosci. 2009; 12: 1373-11380Crossref PubMed Scopus (171) Google Scholar). In humans, mutations of Zfhx1b result in Mowat-Wilson syndrome, a developmental disorder characterized by mental retardation, epilepsy, and defects of neural crest-derived tissues, including craniofacial and enteric nervous system (Mowat et al., 2003Mowat D.R. Wilson M.J. Goossens M. Mowat-Wilson syndrome.J. Med. Genet. 2003; 40: 305-310Crossref PubMed Scopus (170) Google Scholar). Our results that demonstrate Zfhx1b is required to generate cortical interneurons suggest a mechanism for the epilepsy observed in Mowat-Wilson syndrome. Zfhx1b prenatal expression has been noted in migrating cortical interneurons and the subpallial telencephalon (Batista-Brito et al., 2008Batista-Brito R. Machold R. Klein C. Fishell G. Gene expression in cortical interneuron precursors is prescient of their mature function.Cereb. Cortex. 2008; 18: 2306-2317Crossref PubMed Scopus (98) Google Scholar; Seuntjens et al., 2009Seuntjens E. Nityanandam A. Miquelajauregui A. Debruyn J. Stryjewska A. Goebbels S. Nave K.-A. Huylebroeck D. Tarabykin V. Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors.Nat. Neurosci. 2009; 12: 1373-11380Crossref PubMed Scopus (171) Google Scholar). We found that Zfhx1b RNA is expressed in E12.5 MGE-derived cells that are tangentially migrating through the LGE and into the cortex by performing fluorescent in situ hybridization (FISH) on a brain in which MGE-derived cells expressed EGFP (expressed due to Nkx2.1-Cre induced recombination of the CAG:CAT-EGFP Cre reporter allele) (see Figures S1A–S1A″ available online). To determine the role of Zfhx1b in the development of the basal ganglia, we used a conditional mutagenesis approach. Using an allele of Zfhx1b, in which exon 7 is floxed (Higashi et al., 2002Higashi Y. Maruhashi M. Nelles L. Van de Putte T. Verschueren K. Miyoshi T. Yoshimoto A. Kondoh H. Huylebroeck D. Generation of the floxed allele of the SIP1 (Smad-interacting protein 1) gene for Cre-mediated conditional knockout in the mouse.Genesis. 2002; 32: 82-84Crossref PubMed Scopus (85) Google Scholar), we removed Zfhx1b expression using two different Cre alleles. Deletion of exon 7 creates a frameshift mutation and premature truncation of the protein. Previous analysis failed to detect the truncated mutant protein in Zfhx1b mutant tissues, providing evidence that this is a null allele (Higashi et al., 2002Higashi Y. Maruhashi M. Nelles L. Van de Putte T. Verschueren K. Miyoshi T. Yoshimoto A. Kondoh H. Huylebroeck D. Generation of the floxed allele of the SIP1 (Smad-interacting protein 1) gene for Cre-mediated conditional knockout in the mouse.Genesis. 2002; 32: 82-84Crossref PubMed Scopus (85) Google Scholar). To remove Zfhx1b in the early progenitors of the MGE, we used the Nkx2.1-Cre allele (Xu et al., 2008Xu Q. Tam M. Anderson S.A. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.J. Comp. Neurol. 2008; 506: 16-29Crossref PubMed Scopus (390) Google Scholar), which drives Cre expression in the ventricular zone (VZ) of the MGE beginning around E9.5 (later it also drives expression in the subventricular and mantle zones [SVZ and MZ]). To differentiate between the role of Zfhx1b in the VZ and the SVZ/MZ, we used the DlxI1/2b-Cre allele (Potter et al., 2008Potter G.B. Petryniak M.A. Shevchenko E. Mckinsey G.L. Ekker M. Rubenstein J.L.R. Generation of Cre-transgenic mice using Dlx1/Dlx2 enhancers and their characterization in GABAergic interneurons.Mol. Cell. Neurosci. 2008; 40: 167-186Crossref PubMed Scopus (89) Google Scholar), which drives Cre expression in the SVZ and MZ of the entire subpallium beginning around E10.5. To examine the pattern of recombination, we used an antisense riboprobe designed against Zfhx1b exon 7. By E12.5, Cre activity from both the Nkx2.1-Cre and DlxI1/2b-Cre alleles removed Zfhx1b RNA expression in the expected patterns (Figures 1A–1C). As previously reported, the Nkx2-1 allele did not express Cre in the dorsal-most portion of the MGE, thus explaining the persistence of Zfhx1b in that location (Figure 1B). Of note, in the Nkx2.1-Cre; Zfhx1b conditional mutant brains, Zfhx1b RNA expression was not observed in the cells that appear to be migrating from the dorsal MGE into the mantle of the LGE, suggesting that the Zfhx1b+ cells in the mantle of the E12.5 LGE are likely to be MGE-derived cells (e.g., cortical and/or striatal interneurons) (X in Figure 1B). Also, note that DlxI1/2b-Cre leads to recombination in the SVZ and MZ of the LGE, MGE and CGE (white arrowhead, Figure 1C, and data not shown). Next, we examined the expression of Zfhx1b’s closely related homolog, Zfhx1a, in the E12.5 control and mutant telencephalon. Both Zfhx1a and Zfhx1b are expressed in the subpallial VZ, whereas only Zfhx1b is clearly expressed in the SVZ (Figures 1A and S1B). Zfhx1a’s expression did not clearly change in the Nkx2.1-Cre mediated Zfhx1b mutant (Figures S1B–S1S1D′). Thus, in the Nkx2.1-Cre conditional Zfhx1b mutant, only the VZ of the MGE continued to strongly express a Zfhx homolog. We analyzed the effect of deleting Zfhx1b, using Nkx2.1-Cre at multiple developmental stages, including E12.5, E15.5, and P0. To track the fate of Zfhx1b mutant cells, we used the CAG:CAT-EGFP Cre reporter allele (Kawamoto et al., 2000Kawamoto S. Niwa H. Tashiro F. Sano S. Kondoh G. Takeda J. Tabayashi K. Miyazaki J. A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination.FEBS Lett. 2000; 470: 263-268Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Mutant brains had the following genotype: Nkx2.1-Cre;Zfhx1bF/−;CAG:CAT-EGFP; whereas controls had the following genotype: Nkx2.1-Cre;Zfhx1bF/+;CAG:CAT-EGFP (on occasion, some were: Nkx2.1-Cre; Zfhx1bF/+). At E12.5, while the control brain showed a robust stream of EGFP+ cells migrating into the cortex, the mutant’s EGFP+ MGE derivatives failed to migrate to the cortex, and many were detected in the LGE mantle (Figures 1D–1G′). Next, we analyzed the phenotype using molecular markers of MGE-derived cells including Nkx2-1 and Lhx6. While Nkx2-1 RNA and protein is expressed throughout the VZ and SVZ of the MGE, its expression thereafter is restricted to specific neuronal lineages. MGE-derived cortical interneurons repress Nkx2.1 expression as they migrate out of the MGE while most, but not all, classes of striatal interneurons maintain Nkx2.1 expression. (Flandin et al., 2010Flandin P. Kimura S. Rubenstein J.L.R. The progenitor zone of the ventral medial ganglionic eminence requires Nkx2-1 to generate most of the globus pallidus but few neocortical interneurons.J. Neurosci. 2010; 30: 2812-2823Crossref PubMed Scopus (136) Google Scholar; Marin et al., 2000Marin O. Anderson S.A. Rubenstein J.L. Origin and molecular specification of striatal interneurons.J. Neurosci. 2000; 20: 6063-6076Crossref PubMed Google Scholar; Nóbrega-Pereira et al., 2008Nóbrega-Pereira S. Kessaris N. Du T. Kimura S. Anderson S.A. Marín O. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors.Neuron. 2008; 59: 733-745Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar; Sussel et al., 1999Sussel L. Marin O. Kimura S. Rubenstein J.L. 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: 3359-3370PubMed Google Scholar). In the mutants, there was a subtle increase in Nkx2-1 RNA expression in the LGE and CGE (Figures 1H–1J′). This increase was more apparent at higher magnification when analyzing NKX2-1 protein expression (Figures 1G and 1G′) and at later stages (E13.5 and E15.5) (Figures 1N–1P′ and 2A–2F′). EGFP and NKX2-1 protein expression in control brains colocalized in a subset of cells derived from the MGE. EGFP/NKX2-1+ cells were observed in the MGE VZ and SVZ progenitors and a subset of their derived neurons, including the globus pallidus, and striatal interneurons (Xu et al., 2008Xu Q. Tam M. Anderson S.A. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.J. Comp. Neurol. 2008; 506: 16-29Crossref PubMed Scopus (390) Google Scholar; Figure 1G, solid arrowheads), while interneurons migrating to the cerebral cortex showed little to no NKX2-1 protein expression (Figure 1G, open arrowheads). In mutant brains, however, most if not all EGFP labeled cells had detectable levels of NKX2-1 protein, with many cells strongly coexpressing NKX2-1 and EGFP in the LGE MZ, and in a region lateral to the globus pallidus (Figure 1G′, solid arrowheads). Thus, Zfhx1b mutants had a defect in their ability to repress Nkx2-1 RNA and protein expression, concomitant with failure of MGE-derived migration to the cerebral cortex. While Zfhx1b was required to repress Nkx2-1 expression, we did not find evidence that Nkx2-1 regulated Zfhx1b expression; this conclusion was based on in situ hybridization analysis of Zfhx1b expression in mice lacking Nkx2-1 in newly born MGE neurons at E15.5 (Nkx2-1 conditional mutant with Dlx5/6-Cre) (Figure S6). Lhx6 RNA is expressed in tangentially migrating cells that are immature cortical and striatal interneurons, as well as cell types that remain in the subpallium (Flandin et al., 2011Flandin P. Zhao Y. Vogt D. Jeong J. Long J. Potter G. Westphal H. Rubenstein J.L. Lhx6 and Lhx8 coordinately induce neuronal expression of Shh that controls the generation of interneuron progenitors.Neuron. 2011; 70: 939-950Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar; Lavdas et al., 1999Lavdas A.A. Grigoriou M. Pachnis V. Parnavelas J.G. The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex.J. Neurosci. 1999; 19: 7881-7888PubMed Google Scholar; Liodis et al., 2007Liodis P. Denaxa M. Grigoriou M. Akufo-Addo C. Yanagawa Y. Pachnis V. Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes.J. Neurosci. 2007; 27: 3078-3089Crossref PubMed Scopus (281) Google Scholar; Sussel et al., 1999Sussel L. Marin O. Kimura S. Rubenstein J.L. 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: 3359-3370PubMed Google Scholar; Zhao et al., 2008Zhao Y. Flandin P. Long J.E. Cuesta M.D. Westphal H. Rubenstein J.L.R. Distinct molecular pathways for development of telencephalic interneuron subtypes revealed through analysis of Lhx6 mutants.J. Comp. Neurol. 2008; 510: 79-99Crossref PubMed Scopus (167) Google Scholar). In the Zfhx1b mutant, Lhx6+ cells failed to be detected in the pallium, whereas they continued to be densely located throughout the MGE, and as a scattered population in the LGE and CGE (Figures 1K–1M′). On the other hand, Lhx8 and Gbx2 RNA expression was not appreciably changed in the mutants (Figures S1H–S1J′). Thus, Zfhx1b mutants may have a selective defect in cells fated to become pallial interneurons, but not cholinergic striatal interneurons. To explore this hypothesis we studied the phenotype at later developmental stages. By E15.5, the tangential migration of immature cortical interneurons can be readily visualized by expression of Lhx6, Somatostatin (Sst), and EGFP (in Nkx2.1-Cre;CAG-EGFP brains) (Figures 2 and S2D–S2F). By contrast, in Zfhx1b mutants (Nkx2.1-Cre), pallial expression of Lhx6, Sst, and EGFP was strongly attenuated (Figures 2 and S2D′–S2F′). On the other hand, subpallial expression of these markers was increased in two locations: the striatum (asterisks, Figures 2A–2C′, 2G–2I′, and S2D–S2F′) and a region contiguous with the caudoventral striatum, which we believe corresponds to the anlage of the central nucleus of the amygdala (labeled e, for ectopia, Figures 2E′’, 2H′, and S2D′; note that Figure S2T shows Dlx5 expression labeling the central nucleus of the amygdala, CeA). The ectopia in these regions also contained increased expression of Nkx2-1 and Sox6 (Figures 2D–2F′ and 2J–2L′). These genes are normally expressed in the subpallial projection neurons such as the globus pallidus, striatal interneurons, and cortical interneurons (Sox6 only) (Azim et al., 2009Azim E. Jabaudon D. Fame R.M. Macklis J.D. SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development.Nat. Neurosci. 2009; 12: 1238-1247Crossref PubMed Scopus (151) Google Scholar; Batista-Brito et al., 2008Batista-Brito R. Machold R. Klein C. Fishell G. Gene expression in cortical interneuron precursors is prescient of their mature function.Cereb. Cortex. 2008; 18: 2306-2317Crossref PubMed Scopus (98) Google Scholar). Next, we tested whether the mutant cells that failed to migrate to the pallium had features of the globus pallidus or striatal interneurons. We examined expression of several globus pallidus markers including Kcnmb4, Kctd12, Gbx2, and Lhx8. Unlike the abnormal expression of Lhx6, Sst, Nkx2-1, and Sox6, expression of Kcnmb4, Kctd12, Gbx2, and Lhx8 appeared normal in the Zfhx1b mutants (Figures 2M–2O′, 2S–2U′, and S2J–S2O′), providing evidence that the abnormal collections of cells correspond either to abnormally migrated cortical interneurons or to striatal interneurons, and not globus pallidus neurons. Furthermore, as Gbx2 and Lhx8 expression and function are linked to the development of striatal cholinergic interneurons (Chen et al., 2010Chen L. Chatterjee M. Li J.Y.H. The mouse homeobox gene Gbx2 is required for the development of cholinergic interneurons in the striatum.J. Neurosci. 2010; 30: 14824-14834Crossref PubMed Scopus (33) Google Scholar; Zhao et al., 2003Zhao Y. Marín O. Hermesz E. Powell A. Flames N. Palkovits M. Rubenstein J.L.R. Westphal H. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain.Proc. Natl. Acad. Sci. USA. 2003; 100: 9005-9010Crossref PubMed Scopus (187) Google Scholar), these results provided evidence that increased striatal Nkx2-1 expression did not correspond to cells destined to become striatal cholinergic interneurons. To distinguish whether the abnormal collections of cells in the mutant striatum were cortical or striatal interneurons, we examined expression of Cxcr7 and NPY. At E15.5, Cxcr7 marked migrating cortical interneurons and few cells in the striatum (Figures 7J, 7K, and 7L), suggesting that it is a relatively specific cortical interneuron marker (Wang et al., 2011Wang Y. Li G. Stanco A. Long J.E. Crawford D. Potter G.B. Pleasure S.J. Behrens T. Rubenstein J.L.R. CXCR4 and CXCR7 have distinct functions in regulating interneuron migration.Neuron. 2011; 69: 61-76Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In the mutant, there was a robust reduction of Cxcr7 expression in the pattern of migrating cortical interneurons, without a substantive increase in striatal expression (Figures 7J′, 7K′,and 7L′); a similar result was seen for Cux2 (not shown). On the other hand, at E15.5, NPY expression strongly marks scattered striatal cells (probably interneurons), and relatively few migrating cortical interneurons (note, most of the cortical expression at this age resembles that of immature projection neurons in the cortical plate). In the mutant, there was a robust increase in NPY expression in the striatum (Figures 2P–2R′), in a pattern closely resembling the pattern of ectopic Nkx2-1, Lhx6 and Sox6 (Figures 2D–2L′). Thus, we propose that the mutant cortical interneurons are transformed toward GABAergic striatal interneurons. Toward defining the stage of differentiation when Zfhx1b is required for programming interneurons to migrate to the cortex, and not the striatum, we used the DlxI12b-Cre allele (Potter et al., 2008Potter G.B. Petryniak M.A. Shevchenko E. Mckinsey G.L. Ekker M. Rubenstein J.L.R. Generation of Cre-transgenic mice using Dlx1/Dlx2 enhancers and their characterization in GABAergic interneurons.Mol. Cell. Neurosci. 2008; 40: 167-186Crossref PubMed Scopus (89) Google Scholar). DlxI1/2b-Cre expression begins in subpallial SVZ cells that express the mitotic marker Ki67 (Figures S1T–S1T″), suggesting that Cre recombination occurs in secondary progenitor cells that are mitotically active. Thus, DlxI12b-Cre induces recombination beginning in the SVZ of the entire subpallium, whereas Nkx2.1-Cre induces recombination in the VZ of the MGE and preoptic area (Figures 1D–1F′). We analyzed the effect of deleting Zfhx1b using DlxI12b-Cre at E12.5 and E15.5. In general, all of the phenotypes of MGE-derived cells observed with the Nkx2.1-Cre were recapitulated with the DlxI12b-Cre (Figures 3, S1, S3), including the strong reduction of tangential migration to the cortex, indicated by analysis of Cre-dependent reporter EGFP expression, and Lhx6, Sst, and CXCR7 expression. Like Nkx2.1-Cre mutants, DlxI1/2b-Cre mutants showed increased numbers of striatal cells that expressed Lhx6, Nkx2-1, NPY, Sst, and Sox6 (Figures 3A–3I′, 3M–3O′, and S3J–S3L′). Furthermore, these mutants did not show an increase in the number of cells that expressed markers of the globus pallius (Lhx8, Gbx2, Kcnmb4) or striatal cholinergic interneurons (Lhx8, Gbx2) (Figures 3J–3L′, 3P–3R′, and S1Q′–S1S′, and data not shown). Nkx2.1-Cre conditional mutants died between P17 and P21; at P15, mutants weighed ∼30% less than their control littermates, a phenotype that was exacerbated by litter size. We did not observe seizures or other neurological/behavioral phenotypes. We analyzed postnatal day 0 (P0) and P15 Nkx2.1-Cre;Zfhx1bF/− conditional mutants to better understand the nature and extent of their cortical and striatal interneuron defects. In the P0 neocortex there was an ∼90% reduction in the number of EGFP+ Cre-reporter marked cells, as well as a decrease in Calbindin (CB), Sst, and Lhx6 expressing interneurons (Figures 4A–4C′ and 4S ). Likewise, at P15 there was a >90% reduction in number of neocortical EGFP+ cells (Figures 4D–4F′ and 4S). Next, we counted the number cortical interneurons in the Nkx2-1-Cre lineage that expressed Parvalbumin (PV) or Sst, which are the two main MGE-derived subtypes (Rudy et al., 2011Rudy B. Fishell G. Lee S. Hjerling-Leffler J. Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons.Dev. Neurobiol. 2011; 71: 45-61Crossref PubMed Scopus (837) Google Scholar). We saw a strong reduction in double labeled neurons, with the numbers of EGFP+ interneurons expressing Sst or PV reduced by >90% or more (Figure 4S). The expression of cortical Calretinin (CR), which predominantly marks CGE-derived cortical interneurons, showed little to no change in the Zfhx1b; Nkx2.1-Cre conditional mutant (Figures 4F and 4F′). In the striatum at P0, as we saw at E15.5, there was an increase in the number of cells expressing EGFP (Cre reporter), Sst, and Lhx6 (Figures 4G–4I′); consistent with the hypothesis that Zfhx1b mutant cells that were destined to go to the neocortex, instead migrated to the striatum. Additionally, there was a clear increase in the number of striatal cells expressing nNos, NPY, and Nkx2-1 (Figures 4J–4K′, S4B, and S4B′), while we observed no change in Lhx8, a marker for striatal cholinergic interneurons (Figures S4A and S4A′). As NPY, Sst, and nNos are also expressed in subsets of cortical interneurons, their increased striatal expression does not provide unequivocal information about whether supernumerary cells correspond to cortical interneurons that failed to correctly migrate, or to interneurons that changed fate due to the mutation. To this end, we searched for a marker that is expressed in striatal, but not cortical interneurons. Substance P receptor (TacR1) is robustly expressed in striatal interneurons (Ardelt et al., 1996Ardelt A.A. Karpitskiy V.V. Krause J.E. Roth K.A. The neostriatal mosaic: basis for the changing distribution of neurokinin-1 receptor immunoreactivity during development.J. Comp. Neurol. 1996; 376: 463-475Crossref PubMed Scopus (18) Google Scholar). We found that TacR1 is almost exclusively expressed in striatal and not cortical interneurons at E15.5, P0, and P15 (Figures 4L and 4L′, and 4Q and 4Q′, and S4D–S4F′, and data not shown). In Zfhx1b-Nkx2.1-Cre mutants at P0, there was increased striatal TacR1 expression (Figures 4Q and 4Q′), supporting the idea that at least some of the mutant cells are adopting a striatal interneuron identity. At P15 the number of mutant cells (EGFP+) was roughly the same as in controls, and they were evenly dispersed within the striatum, lacking the cell clusters and ectopia (striatal and caudal amygdala) that were apparent at younger ages (Figures 4M, 4M′, and 4T). The elimination of the excess mutant striatal cells appears to occur through apoptosis, which is robust at P0 (expression of activated cleaved-caspase 6), particularly in the ectopia (Figures S4C–S4C′). Despite the cell death, Zfhx1b conditional mutants at P15 continued to have significantly increased numbers of striatal nNOS, NPY, Sst, and TacR1 expressing cells (183%, 230%, 225%, and 164%; Figures 4N–4Q′ and 4T). Importantly, total striatal PV+ cells were decreased by 58% (Figure 4S). Furthermore, there was no detectable change in TrkA expression (Figures 4R, 4R′, and 4T), which marks striatal cholinergic interneurons. We saw very few CR+ cells in the control striatum (1–3 cells per section), which did not noticeably change in the Zfhx1b conditional mutant (data not shown). Thus, Nkx2.1-Cre;Zfhx1b mutants have a selective increase in striatal interneurons expressing nNos, NPY, Sst, and TacR1 but have reduced PV interneurons, and no change in cholinergic or CR interneurons. We also analyzed the gross morphological properties of nNos/NPY/Sst striatal interneurons in the Zfhx1b mutant and found that, like control brains, Zfhx1b conditional mutants had Sst processes restricted to the matrisomes (Chesselet and Graybiel, 1986Chesselet M.F. Graybiel A.M. Striatal neurons expressing somatostatin-like immunoreactivity: evidence for a peptidergic interneuronal system in the cat.Neuroscience. 1986; 17: 547-571Crossref PubMed Scopus (111) Google Scholar), in a lateral to medial gradient (Figures S4G–S4I′, arrowheads mark CB-poor striosomes), suggesting that the overproduced nNos/NPY/Sst interneurons in the Zfhx1b conditional mutant share grossly similar morphological properties with wild-type striatal interneurons. Dlx1 and Dlx2 are necessary for subpallial development, including interneuron migration to the cortex (Anderson et al., 1997aAnderson S.A. Eisenstat D.D. Shi L. Rubenstein J.L. Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes.Science. 1997; 278: 474-476Crossref PubMed Scopus (1240) Google Scholar; Long et al., 2009aLong J.E. Cobos I. Potter G.B. Rubenstein J.L.R. Dlx1&2 and Mash1 transcription factors control MGE and CGE patterning and differentiation through parallel and overlapping pathways.Cereb. Cortex. 2009; 1" @default.
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