The transcription factor Zfp503 promotes the D1 MSN identity and represses the D2 MSN identity.

The striatum is primarily composed of two types of medium spiny neurons (MSNs) expressing either D1- or D2-type dopamine receptors. However, the fate determination of these two types of neurons is not fully understood. Here, we found that D1 MSNs undergo fate switching to D2 MSNs in the absence of Zfp503. Furthermore, scRNA-seq revealed that the transcription factor Zfp503 affects the differentiation of these progenitor cells in the lateral ganglionic eminence (LGE). More importantly, we found that the transcription factors Sp8/9, which are required for the differentiation of D2 MSNs, are repressed by Zfp503. Finally, sustained Zfp503 expression in LGE progenitor cells promoted the D1 MSN identity and repressed the D2 MSN identity. Overall, our findings indicated that Zfp503 promotes the D1 MSN identity and represses the D2 MSN identity by regulating Sp8/9 expression during striatal MSN development.

Transcription factor Sp9 is a negative regulator of D1-type MSN development.

The striatum is the main input structure of the basal ganglia, receiving information from the cortex and the thalamus and consisting of D1- and D2- medium spiny neurons (MSNs). D1-MSNs and D2-MSNs are essential for motor control and cognitive behaviors and have implications in Parkinson's Disease. In the present study, we demonstrated that Sp9-positive progenitors produced both D1-MSNs and D2-MSNs and that Sp9 expression was rapidly downregulated in postmitotic D1-MSNs. Furthermore, we found that sustained Sp9 expression in lateral ganglionic eminence (LGE) progenitor cells and their descendants led to promoting D2-MSN identity and repressing D1-MSN identity during striatal development. As a result, sustained Sp9 expression resulted in an imbalance between D1-MSNs and D2-MSNs in the mouse striatum. In addition, the fate-changed D2-like MSNs survived normally in adulthood. Taken together, our findings supported that Sp9 was sufficient to promote D2-MSN identity and repress D1-MSN identity, and Sp9 was a negative regulator of D1-MSN fate.

Dlx1/2-dependent expression of Meis2 promotes neuronal fate determination in the mammalian striatum.

The striatum is a central regulator of behavior and motor function through the actions of D1 and D2 medium-sized spiny neurons (MSNs), which arise from a common lateral ganglionic eminence (LGE) progenitor. The molecular mechanisms of cell fate specification of these two neuronal subtypes are incompletely understood. Here, we found that deletion of murine Meis2, which is highly expressed in the LGE and derivatives, led to a large reduction in striatal MSNs due to a block in their differentiation. Meis2 directly binds to the Zfp503 and Six3 promoters and is required for their expression and specification of D1 and D2 MSNs, respectively. Finally, Meis2 expression is regulated by Dlx1/2 at least partially through the enhancer hs599 in the LGE subventricular zone. Overall, our findings define a pathway in the LGE whereby Dlx1/2 drives expression of Meis2, which subsequently promotes the fate determination of striatal D1 and D2 MSNs via Zfp503 and Six3.

Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons.

Medium spiny neurons (MSNs) in the striatum, which can be divided into D1 and D2 MSNs, originate from the lateral ganglionic eminence (LGE). Previously, we reported that Six3 is a downstream target of Sp8/Sp9 in the transcriptional regulatory cascade of D2 MSN development and that conditionally knocking out Six3 leads to a severe loss of D2 MSNs. Here, we showed that Six3 mainly functions in D2 MSN precursor cells and gradually loses its function as D2 MSNs mature. Conditional deletion of Six3 had little effect on cell proliferation but blocked the differentiation of D2 MSN precursor cells. In addition, conditional overexpression of Six3 promoted the differentiation of precursor cells in the LGE. We measured an increase of apoptosis in the postnatal striatum of conditional Six3-knockout mice. This suggests that, in the absence of Six3, abnormally differentiated D2 MSNs are eliminated by programmed cell death. These results further identify Six3 as an important regulatory element during D2 MSN differentiation.

Transcription Factor VAX1 Regulates the Regional Specification of the Subpallium Through Repressing Gsx2.

Specification of the progenitors' regional identity is a pivotal step during development of the cerebral cortex and basal ganglia. The molecular mechanisms underlying progenitor regionalization, however, are poorly understood. Here we showed that the transcription factor Vax1 was highly expressed in the developing subpallium. In its absence, the RNA-Seq analysis, in situ RNA hybridization, and immunofluorescence staining results showed that the cell proliferation was increased in the subpallium, but the neuronal differentiation was blocked. Moreover, the dLGE expands ventrally, and the vLGE, MGE, and septum get smaller. Finally, overexpressed VAX1 in the LGE progenitors strongly inhibits Gsx2 expression. Taken together, our findings show that Vax1 is crucial for subpallium regionalization by repressing Gsx2.

Transcriptional profiling reveals the transcription factor networks regulating the survival of striatal neurons.

The striatum is structurally highly diverse, and its organ functionality critically depends on normal embryonic development. Although several studies have been conducted on the gene functional changes that occur during striatal development, a system-wide analysis of the underlying molecular changes is lacking. Here, we present a comprehensive transcriptome profile that allows us to explore the trajectory of striatal development and identify the correlation between the striatal development and Huntington's disease (HD). Furthermore, we applied an integrative transcriptomic profiling approach based on machine learning to systematically map a global landscape of 277 transcription factor (TF) networks. Most of these TF networks are linked to biological processes, and some unannotated genes provide information about the corresponding mechanisms. For example, we found that the Meis2 and Six3 were crucial for the survival of striatal neurons, which were verified using conditional knockout (CKO) mice. Finally, we used RNA-Seq to speculate their downstream targets.

Decoding Cortical Glial Cell Development.

Mouse cortical radial glial cells (RGCs) are primary neural stem cells that give rise to cortical oligodendrocytes, astrocytes, and olfactory bulb (OB) GABAergic interneurons in late embryogenesis. There are fundamental gaps in understanding how these diverse cell subtypes are generated. Here, by combining single-cell RNA-Seq with intersectional lineage analyses, we show that beginning at around E16.5, neocortical RGCs start to generate ASCL1+EGFR+ apical multipotent intermediate progenitors (MIPCs), which then differentiate into basal MIPCs that express ASCL1, EGFR, OLIG2, and MKI67. These basal MIPCs undergo several rounds of divisions to generate most of the cortical oligodendrocytes and astrocytes and a subpopulation of OB interneurons. Finally, single-cell ATAC-Seq supported our model for the genetic logic underlying the specification and differentiation of cortical glial cells and OB interneurons. Taken together, this work reveals the process of cortical radial glial cell lineage progression and the developmental origins of cortical astrocytes and oligodendrocytes.

Zfhx3 is required for the differentiation of late born D1-type medium spiny neurons.

The striatum, the major component of the basal ganglia, consists of the caudate-putamen, nucleus accumbens and olfactory tubercle. The striatal principal projection neurons are comprised of medium spiny neurons (MSNs) with two dopamine receptors: DRD1 (D1 MSNs) and DRD2 (D2 MSNs). In the present study, we demonstrate that Zfhx3 is strongly expressed in the boundary of the subventricular zone (SVZ)/mantle zone (MZ) of the lateral ganglionic eminence (LGE), and its expression in the striatum is downregulated during the first postnatal week. At the cellular level, Zfhx3 is selectively expressed in immature D1 MSNs. In Zfhx3 conditional knockouts, we observed an accumulation of progenitors in the LGE SVZ at E16.5 and P0, and an increase in apoptosis in the postnatal striatum. BrdU birthdating experiments revealed that late born D1 MSN production was compromised. Accordingly, we observed a significant reduction in the number of D1 MSNs, whereas the number of D2 MSNs remained unaffected in the striatum of Zfhx3 conditional knockouts at P11. We concluded that Zfhx3 plays a critical role in the differentiation and survival of late born D1 MSNs.

Transcription factors Sp8 and Sp9 regulate the development of caudal ganglionic eminence-derived cortical interneurons.

Cortical interneurons are derived from the subcortical medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE) and preoptic area (POA). CGE-derived cortical interneurons, which comprise around 30% of all cortical interneurons, mainly express Htr3a, calretinin (CR), Reelin (RELN) and vasoactive intestinal polypeptide (VIP). In the present study, we show that transcription factors Sp8 and Sp9 are co-expressed in the subventricular zone (SVZ) of the dorsal CGE. Conditional knockout of Sp8/9 using the Gsx2-Cre transgenic line results in severe loss of CGE-derived cortical interneurons. We observed migration defects of Sp8/9 double mutant CGE-derived cortical interneurons as they had longer leading processes than controls and they ectopically accumulated in the CGE. Dlx5/6-CIE conditional deletion of Sp8/9 also leads to a significant reduction in the CGE-derived cortical interneurons. We provide evidence that Sp8/9 coordinately regulate CGE-derived cortical interneuron development in part through repressing the expression of Pak3, Robo1, and Slit1. Finally, we show that Cxcl14, a member of the CXC chemokine family, is mainly expressed in CGE-derived interneurons in cortical layers I and II, and its expression is critically dependent on SP8.

The PROK2/PROKR2 signaling pathway is required for the migration of most olfactory bulb interneurons.

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricle generate new interneurons, which migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB). The PROK2 (prokineticin 2) and PROKR2 (prokineticin receptor 2) signaling pathway has been identified to cause human Kallmann syndrome, a developmental disease that associates hypogonadism with anosmia (OB developmental defects). However, the identities and properties of PROK2+ and PROKR2+ cells in the SVZ-RMS-OB remain largely unknown. Here we examine the expression patterns of Prok2 and Prokr2 in the SVZ-RMS-OB using Prok2EGFP transgenic and Prokr2LacZ/+ knockin mice. Our results show that Prokr2 is expressed in postmitotic immature interneurons in the SVZ-RMS-OB. Prok2 is not expressed in the SVZ, but a few PROK2+ cells are found in the medial part of the RMS; they are not neural progenitors or migrating neuroblasts. In the OB, Prok2 is expressed in a subset of granule cells and tufted cells, but no coexpression of Prok2 and Prokr2 in the OB cells is observed. In Prok2 and Prokr2 mutant mice, severe tangential and radial migration defects of neuroblasts in the SVZ-RMS-OB result in loss of ~75% of GABAergic interneurons in the OB. These analyses demonstrate that PROK2/PROKR2 signaling is crucial for the tangential and radial migration of OB interneurons.

Dlx1/2 are Central and Essential Components in the Transcriptional Code for Generating Olfactory Bulb Interneurons.

Generation of olfactory bulb (OB) interneurons requires neural stem/progenitor cell specification, proliferation, differentiation, and young interneuron migration and maturation. Here, we show that the homeobox transcription factors Dlx1/2 are central and essential components in the transcriptional code for generating OB interneurons. In Dlx1/2 constitutive null mutants, the differentiation of GSX2+ and ASCL1+ neural stem/progenitor cells in the dorsal lateral ganglionic eminence is blocked, resulting in a failure of OB interneuron generation. In Dlx1/2 conditional mutants (hGFAP-Cre; Dlx1/2F/- mice), GSX2+ and ASCL1+ neural stem/progenitor cells in the postnatal subventricular zone also fail to differentiate into OB interneurons. In contrast, overexpression of Dlx1&2 in embryonic mouse cortex led to ectopic production of OB-like interneurons that expressed Gad1, Sp8, Sp9, Arx, Pbx3, Etv1, Tshz1, and Prokr2. Pax6 mutants generate cortical ectopia with OB-like interneurons, but do not do so in compound Pax6; Dlx1/2 mutants. We propose that DLX1/2 promote OB interneuron development mainly through activating the expression of Sp8/9, which further promote Tshz1 and Prokr2 expression. Based on this study, in combination with earlier ones, we propose a transcriptional network for the process of OB interneuron development.