The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

The prosomeric model proposes that the hypothalamus is a rostral forebrain entity, placed ventral to the telencephalon and rostral to the diencephalon. Gene expression markers differentially label molecularly distinct dorsoventral progenitor domains, which represent continuous longitudinal bands across the hypothalamic alar and basal regions. There is also circumstantial support for a rostrocaudal subdivision of the hypothalamus into transverse peduncular (caudal) and terminal (rostral) territories (PHy, THy). In addition, there is evidence for a specialized acroterminal domain at the rostral midline of the terminal hypothalamus (ATD). The PHy and THy transverse structural units are presently held to form part of two hypothalamo-telencephalic prosomeres (hp1 and hp2, respectively), which end dorsally at the telencephalic septocommissural roof. PHy and THy have distinct adult nuclei, at all dorsoventral levels. Here we report the results of data mining from the Allen Developing Mouse Brain Atlas database, looking for genes expressed differentially in the PHy, Thy, and ATD regions of the hypothalamus at several developmental stages. This search allowed us to identify additional molecular evidence supporting the postulated fundamental rostrocaudal bipartition of the mouse hypothalamus into the PHy and THy, and also corroborated molecularly the singularity of the ATD. A number of markers were expressed in Thy (Fgf15, Gsc, Nkx6.2, Otx1, Zic1/5), but were absent in PHy, while other genes showed the converse pattern (Erbb4, Irx1/3/5, Lmo4, Mfap4, Plagl1, Pmch). We also identified markers that selectively label the ATD (Fgf8/10/18, Otx2, Pomc, Rax, Six6). On the whole, these data help to explain why, irrespective of the observed continuity of all dorsoventral molecular hypothalamic subdivisions across PHy and THy, different nuclear structures originate within each of these two domains, and also why singular structures arise at the ATD, e.g., the suprachiasmatic nuclei, the arcuate nucleus, the median eminence and the neurohypophysis.

Little is known about genetic mechanisms that regulate the ratio of cortical excitatory and inhibitory neurons. We show that NPAS1 and NPAS3 transcription factors (TFs) are expressed in progenitor domains of the mouse basal ganglia (subpallium, MGE, and CGE). NPAS1(-/-) mutants had increased proliferation, ERK signaling, and expression of Arx in the MGE and CGE. NPAS1(-/-) mutants also had increased neocortical inhibition (sIPSC and mIPSC) and generated an excess of somatostatin(+) (SST) (MGE-derived) and vasoactive intestinal polypeptide(+) (VIP) (CGE-derived) neocortical interneurons, but had a normal density of parvalbumin(+) (PV) (MGE-derived) interneurons. In contrast, NPAS3(-/-) mutants showed decreased proliferation and ERK signaling in progenitors of the ganglionic eminences and had fewer SST(+) and VIP(+) interneurons. NPAS1 repressed activity of an Arx enhancer, and Arx overexpression resulted in increased proliferation of CGE progenitors. These results provide insights into genetic regulation of cortical interneuron numbers and cortical inhibitory tone.

The development of the cortex is an elaborate process that integrates a plethora of finely tuned molecular processes ranging from carefully regulated gradients of transcription factors, dynamic changes in the chromatin landscape, or formation of protein complexes to elicit and regulate transcription. Combined with cellular processes such as cell type specification, proliferation, differentiation, and migration, all of these developmental processes result in the establishment of an adult mammalian cortex with its typical lamination and regional patterning. By examining in-depth the role of one transcription factor, Pax6, on the regulation of cortical development, its integration in the regulation of chromatin state, and its regulation by cis-regulatory elements, we aim to demonstrate the importance of integrating each level of regulation in our understanding of cortical development.

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Here we define the expression of approximately 100 transcription factors in progenitors and neurons of the developing basal ganglia. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum.

Here we analyze the role of the Lhx6 lim-homeobox transcription factor in regulating the development of subsets of neocortical, hippocampal, and striatal interneurons. An Lhx6 loss-of-function allele, which expresses placental alkaline phosphatase (PLAP), allowed analysis of the development and fate of Lhx6-expressing interneurons in mice lacking this homeobox transcription factor. There are Lhx6+;Dlx+ and Lhx6-;Dlx+ subtypes of tangentially migrating interneurons. Most interneurons in Lhx6(PLAP/PLAP) mutants migrate to the cortex, although less efficiently, and exhibit defects in populating the marginal zone and superficial parts of the neocortical plate. By contrast, migration to superficial parts of the hippocampus is not seriously affected. Furthermore, whereas parvalbumin+ and somatostatin+ interneurons do not differentiate, NPY+ interneurons are present; we suggest that these NPY+ interneurons are derived from the Lhx6-;Dlx+ subtype. Striatal interneurons show deficits distinct from pallial interneurons, including a reduction in the NPY+ subtype. We provide evidence that Lhx6 mediates these effects through promoting expression of receptors that regulate interneuron migration (ErbB4, CXCR4, and CXCR7), and through promoting the expression of transcription factors either known (Arx) or implicated (bMaf, Cux2, and NPAS1) in controlling interneuron development.

Embryonic medial ganglionic eminence (MGE) cells transplanted into the adult brain can disperse, migrate, and differentiate to neurons expressing GABA, the primary inhibitory neurotransmitter. It has been hypothesized that grafted MGE precursors could have important therapeutic applications increasing local inhibition, but there is no evidence that MGE cells can modify neural circuits when grafted into the postnatal brain. Here we demonstrate that MGE cells grafted into one location of the neonatal rodent brain migrate widely into cortex. Grafted MGE-derived cells differentiate into mature cortical interneurons; the majority of these new interneurons express GABA. Based on their morphology and expression of somatostatin, neuropeptide Y, parvalbumin, or calretinin, we infer that graft-derived cells integrate into local circuits and function as GABA-producing inhibitory cells. Whole-cell current-clamp recordings obtained from MGE-derived cells indicate firing properties typical of mature interneurons. Moreover, patch-clamp recordings of IPSCs on pyramidal neurons in the host brain, 30 and 60 d after transplantation, indicated a significant increase in GABA-mediated synaptic inhibition in regions containing transplanted MGE cells. In contrast, synaptic excitation is not altered in the host brain. Grafted MGE cells, therefore, can be used to modify neural circuits and selectively increase local inhibition. These findings could have important implications for reparative cell therapies for brain disorders.

The frontal cortex (FC) is the seat of higher cognition. The genetic mechanisms that control formation of the functionally distinct subdivisions of the FC are unknown. Using a set of gene expression markers that distinguish subdivisions of the newborn mouse FC, we show that loss of Fgf17 selectively reduces the size of the dorsal FC whereas ventral/orbital FC appears normal. These changes are complemented by a rostral shift of sensory cortical areas. Thus, Fgf17 functions similar to Fgf8 in patterning the overall neocortical map but has a more selective role in regulating the properties of the dorsal but not ventral FC.

Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1+ RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.

One long-standing model of striatal function divides the striatum into compartments called striosome and matrix. While some anatomical evidence suggests that these populations represent distinct striatal pathways with differing inputs and outputs, functional investigation has been limited by the methods for identifying and manipulating these populations. Here, we utilize hs599CreER mice as a new tool for targeting striosome projection neurons and testing their functional connectivity. Extending anatomical work, we demonstrate that striosome neurons receive greater synaptic input from prelimbic cortex, whereas matrix neurons receive greater input from primary motor cortex. We also identify functional differences in how striosome and matrix neurons process excitatory input, providing the first electrophysiological method for delineating striatal output neuron subtypes. Lastly, we provide the first functional demonstration that striosome neurons are the predominant striatal output to substantia nigra pars compacta dopamine neurons. These results identify striosome and matrix as functionally distinct striatal pathways.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

The globus pallidus (GP) of primates is divided conventionally into distinct internal and external parts. The literature repeats since 1930 the opinion that the homolog of the primate internal pallidum in rodents is the hypothalamic entopeduncular nucleus (embedded within fiber tracts of the cerebral peduncle). To test this idea, we explored its historic fundaments, checked the development and genoarchitecture of mouse entopeduncular and pallidal neurons, and examined relevant comparative connectivity data. We found that the extratelencephalic mouse entopeduncular structure consists of four different components arrayed along a dorsoventral sequence in the alar hypothalamus. The ventral entopeduncular nucleus (EPV), with GABAergic neurons expressing Dlx5&6 and Nkx2-1, lies within the hypothalamic peduncular subparaventricular area. Three other formations-the dorsal entopeduncular nucleus (EPD), the prereticular entopeduncular nucleus (EPPRt ), and the preeminential entopeduncular nucleus (EPPEm )-lie within the overlying paraventricular area, under the subpallium. EPD contains glutamatergic neurons expressing Tbr1, Otp, and Pax6. The EPPRt has GABAergic cells expressing Isl1 and Meis2, whereas the EPPEm population expresses Foxg1 and may be glutamatergic. Genoarchitectonic observations on relevant areas of the mouse pallidal/diagonal subpallium suggest that the GP of rodents is constituted as in primates by two adjacent but molecularly and hodologically differentiable telencephalic portions (both expressing Foxg1). These and other reported data oppose the notion that the rodent extratelencephalic entopeduncular nucleus is homologous to the primate internal pallidum. We suggest instead that all mammals, including rodents, have dual subpallial GP components, whereas primates probably also have a comparable set of hypothalamic entopeduncular nuclei. Remarkably, there is close similarity in some gene expression properties of the telencephalic internal GP and the hypothalamic EPV. This apparently underlies their notable functional analogy, sharing GABAergic neurons and thalamopetal connectivity.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.

Cortical GABAergic interneurons in rodents originate from subpallial progenitors and tangentially migrate to the cortex. While the majority of mouse neocortical interneurons are derived from the medial and caudal ganglionic eminence (MGE and CGE, respectively), it remains unknown whether the lateral ganglionic eminence (LGE) also contributes to a subpopulation of cortical interneurons. Here, we show that the transcription factor Sp8 is expressed in one-fifth of adult cortical interneurons, which appear to be derived from both the dorsal LGE and the dorsal CGE (dLGE and dCGE, respectively). Compared with the MGE-derived cortical interneurons, dLGE/dCGE-derived Sp8-expressing (Sp8+) ones are born at later embryonic stages with peak production occurring at embryonic day 15.5. They tangentially migrate mainly along the subventricular/intermediate zone (SVZ/IZ) route; some continue to express mitotic markers (Ki67 and PH3) in the neonatal cortical SVZ/IZ. Sp8+ interneurons continue to radially migrate from the SVZ/IZ into the cortical layers at early postnatal stages. In contrast to MGE-derived interneurons, dLGE/dCGE-derived Sp8+ interneurons follow an outside-in layering pattern, preferentially occupying superficial cortical layers.

Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ∼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage that underlie processes central to development and disease.

The genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV+) and somatostatin (SST+) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST+ cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development.

The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.