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

SummaryElucidating 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. CreERT2 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

Lhx6 Directly Regulates Arx and CXCR7 to Determine Cortical Interneuron Fate and Laminar Position

Cortical GABAergic interneurons have essential roles for information processing and their dysfunction is implicated in neuropsychiatric disorders. Transcriptional codes are elucidating mechanisms of interneuron specification in the MGE (a subcortical progenitor zone), which regulate their migration, integration, and function within cortical circuitry. Lhx6, a LIM-homeodomain transcription factor, is essential for specification of MGE-derived somatostatin and parvalbumin interneurons. Here, we demonstrate that some Lhx6⁻/⁻ MGE cells acquire a CGE-like fate. Using an in vivo MGE complementation/transplantation assay, we show that Lhx6-regulated genes Arx and CXCR7 rescue divergent aspects of Lhx6⁻/⁻ cell-fate and laminar mutant phenotypes and provide insight into a neonatal role for CXCR7 in MGE-derived interneuron lamination. Finally, Lhx6 directly binds in vivo to an Arx enhancer and to an intronic CXCR7 enhancer that remains active in mature interneurons. These data define the molecular identity of Lhx6 mutants and introduce technologies to test mechanisms in GABAergic interneuron differentiation

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

Subpallial Enhancer Transgenic Lines: a Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate.

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment

Subpallial Enhancer Transgenic Lines: a Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate.

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development