Susan Lindtner

Dlx1&2-Dependent Expression of Zfhx1b (Sip1, Zeb2) Regulates the Fate Switch between Cortical and Striatal Interneurons

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).

Transcriptional Regulation of Enhancers Active in Protodomains of the Developing Cerebral Cortex

Elucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreER(T2) and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define a comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6, and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions.

Pbx Regulates Patterning of the Cerebral Cortex in Progenitors and Postmitotic Neurons

We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2(+/-) sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.

OTX2 Transcription Factor Controls Regional Patterning within the Medial Ganglionic Eminence and Regional Identity of the Septum

The Otx2 homeodomain transcription factor is essential for gastrulation and early neural development. We generated Otx2 conditional knockout (cKO) mice to investigate its roles in telencephalon development after neurulation (approximately embryonic day 9.0). We conducted transcriptional profiling and in situ hybridization to identify genes de-regulated in Otx2 cKO ventral forebrain. In parallel, we used chromatin immunoprecipitation sequencing to identify enhancer elements, the OTX2 binding motif, and de-regulated genes that are likely direct targets of OTX2 transcriptional regulation. We found that Otx2 was essential in septum specification, regulation of Fgf signaling in the rostral telencephalon, and medial ganglionic eminence (MGE) patterning, neurogenesis, and oligodendrogenesis. Within the MGE, Otx2 was required for ventral, but not dorsal, identity, thus controlling the production of specific MGE derivatives.

Coup-TF1 and coup-TF2 control subtype and laminar identity of mge-derived neocortical interneurons

Distinct cortical interneuron (CIN) subtypes have unique circuit functions; dysfunction in specific subtypes is implicated in neuropsychiatric disorders. Somatostatin- and parvalbumin-expressing (SST+ and PV+) interneurons are the two major subtypes generated by medial ganglionic eminence (MGE) progenitors. Spatial and temporal mechanisms governing their cell-fate specification and differential integration into cortical layers are largely unknown. We provide evidence that Coup-TF1 and Coup-TF2 (Nr2f1 and Nr2f2) transcription factor expression in an arc-shaped progenitor domain within the MGE promotes time-dependent survival of this neuroepithelium and the time-dependent specification of layer V SST+ CINs. Coup-TF1 and Coup-TF2 autonomously repress PV+ fate in MGE progenitors, in part through directly driving Sox6 expression. These results have identified, in mouse, a transcriptional pathway that controls SST-PV fate. Summary: The regulation of somatostatin (SST) and parvalbumin (PV) mouse cortical interneurons is mediated by Coup-TF1- and Coup-TF2-driven Sox6 expression, identifying a transcriptional pathway that controls SST-PV fate.

Dlx1 and Dlx2 Promote Interneuron GABA Synthesis, Synaptogenesis, and Dendritogenesis.

The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.

Sp9 Regulates Medial Ganglionic Eminence-Derived Cortical Interneuron Development.

Immature neurons generated by the subpallial MGE tangentially migrate to the cortex where they become parvalbumin-expressing (PV+) and somatostatin (SST+) interneurons. Here, we show that the Sp9 transcription factor controls the development of MGE-derived cortical interneurons. SP9 is expressed in the MGE subventricular zone and in MGE-derived migrating interneurons. Sp9 null and conditional mutant mice have approximately 50% reduction of MGE-derived cortical interneurons, an ectopic aggregation of MGE-derived neurons in the embryonic ventral telencephalon, and an increased ratio of SST+/PV+ cortical interneurons. RNA-Seq and SP9 ChIP-Seq reveal that SP9 regulates MGE-derived cortical interneuron development through controlling the expression of key transcription factors Arx, Lhx6, Lhx8, Nkx2-1, and Zeb2 involved in interneuron development, as well as genes implicated in regulating interneuron migration Ackr3, Epha3, and St18. Thus, Sp9 has a central transcriptional role in MGE-derived cortical interneuron development.

SP8 and SP9 coordinately promote D2-type medium spiny neuron production by activating Six3 expression

ABSTRACT Dopamine receptor DRD1-expressing medium spiny neurons (D1 MSNs) and dopamine receptor DRD2-expressing medium spiny neurons (D2 MSNs) are the principal projection neurons in the striatum, which is divided into dorsal striatum (caudate nucleus and putamen) and ventral striatum (nucleus accumbens and olfactory tubercle). Progenitors of these neurons arise in the lateral ganglionic eminence (LGE). Using conditional deletion, we show that mice lacking the transcription factor genes Sp8 and Sp9 lose virtually all D2 MSNs as a result of reduced neurogenesis in the LGE, whereas D1 MSNs are largely unaffected. SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants. Finally, ChIP-Seq reveals that SP9 directly binds to the promoter and a putative enhancer of Six3. Thus, this study defines components of a transcription pathway in a regionally restricted LGE progenitor domain that selectively drives the generation of D2 MSNs. Summary: Conditional deletion of Sp8, Sp9 and Six3 reveals a regionally specific transcription pathway that selectively drives the generation of striatal DRD2-expressing medium spiny neurons in mouse striatum.