Dissecting cellular diversity of cortical GABAergic cells across multiple modalities: A turning point in neuronal taxonomy

Decoding the complexity of the brain requires an understanding of the architecture, function, and development of its neuronal circuits. Neuronal classifications that group neurons based on specific features/behaviors have become essential to further analyze the different subtypes in a systematic and reproducible way. A comprehensive taxonomic framework, accounting for multiple defining and quantitative features, will provide the reference to infer generalized rules for cells ascribed to the same neuronal type, and eventually predict cellular behaviors, even in the absence of experimental measures. Technologies that enable cell-type classification in the nervous system are rapidly evolving in scalability and resolution. While these approaches depict astonishing diversity in neuronal morphology, electrophysiology, and gene expression, a robust metric of the coherence between different profiling modalities leading to a unified classification is still largely missing. Focusing on GABAergic neurons of the cerebral cortex, Gouwens et al.1 pioneered the first integrated cell-type classification based on the simultaneous analysis of the transcriptional networks, the recording of intrinsic electrophysiological properties, and the reconstruction of 3D morphologies of the same cell. Their comprehensive and high-quality data provide a new framework to shed light on what may be considered a "neuronal cell type.

Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI

Transcription factors with gradients of expression in neocortical progenitors give rise to distinct motor and sensory cortical areas by controlling the area-specific differentiation of distinct neuronal subtypes. However, the molecular mechanisms underlying this area-restricted control are still unclear. Here, we show that COUP-TFI controls the timing of birth and specification of corticospinal motor neurons (CSMN) in somatosensory cortex via repression of a CSMN differentiation program. Loss of COUP-TFI function causes an area-specific premature generation of neurons with cardinal features of CSMN, which project to subcerebral structures, including the spinal cord. Concurrently, genuine CSMN differentiate imprecisely and do not project beyond the pons, together resulting in impaired skilled motor function in adult mice with cortical COUP-TFI loss-of-function. Our findings indicate that COUP-TFI exerts critical areal and temporal control over the precise differentiation of CSMN during corticogenesis, thereby enabling the area-specific functional features of motor and sensory areas to arise.Stem Cell and Regenerative Biolog

Monitoring the December 2015 summit eruptions of Mt. Etna (Italy): Implications on eruptive dynamics

A lengthy period of eruptive activity fromthe summit craters ofMt. Etna started in January 2011. It culminated in early December 2015 with a spectacular sequence of intense eruptive events involving all four summit craters (Voragine, Bocca Nuova,NewSoutheast Crater, and Northeast Crater). The activity consisted of high eruption columns, Strombolian explosions, lava flows andwidespread ash falls that repeatedly interferedwith air traffic. The most powerful episode occurred on 3 December 2015 from the Voragine. After three further potent episodes fromthe Voragine, activity shifted to the NewSoutheast Crater on 6 December 2015, where Strombolian activity and lava flow emission lasted for two days and were fed by the most primitive magma of the study period. Activity once more shifted to the Northeast Crater, where ash emission and weak Strombolian activity took place for several days. Sporadic ash emissions from all craters continued until 18 December, when all activity ceased. Although resembling the summit eruptions of 1998–1999, which also involved all four summit craters, thismultifaceted eruptive sequence occurred in an exceptionally short time window of less than three days, unprecedented in the recent activity of Mt. Etna. It also produced important morphostructural changes of the summit area with the coalescence of Voragine and Bocca Nuova in a single large crater, the “Central Crater”, reproducing themorphological setting of the summit cone before the formation of Bocca Nuova in 1968. The December 2015 volcanic crisis was followed closely by the staff of the Etna Observatory to monitor the on-going activity and forecast its evolution, in accordance with protocols agreed with the Italian Civil Protection Department.Published53-695V. Dinamica dei processi eruttivi e post-eruttiviJCR Journa

Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.Harvard University. Society of FellowsNational Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170)United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445)United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.)Wyss Institute of Biologically Inspired EngineeringNational Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01))European School of Molecular Medicine (predoctoral fellowship

Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype–specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity

Molecular development and laminar distribution of GABAergic interneurons of the cerebral cortex

GABAergic interneurons of the cerebral cortex represent one of the most diversified populations of cells of the Central Nervous System. It is well established that different subtypes of interneurons populate and integrate into the cortical layers, where they critically modulate the firing activity of their excitatory projection neuron partners. The developmental origin of interneurons has been extensively studied, and elegant prior work has demonstrated that in rodents, the vast majority of interneurons are born in the ventral telencephalon within the medial and caudal ganglionic eminences (MGE and CGE). Although great progress has been made over the last decade in understanding the molecular diversity and fate-specification of MGE-derived interneuron subtypes, the mechanisms controlling the birth and diversification of CGE-derived interneurons, as well as their functional roles within cortical circuitry, remain poorly understood. Here, I addressed this question directly, by investigating a potential role over CGE-derived interneurons of COUP-TFI, a transcription factor highly expressed in the embryonic CGE. I generated and studied conditional, null-mutant mice where the COUP-TFI gene was selectively deleted from the ventral telencephalon (COUP-TFIfl;fl x Dlx5/6CRE-IRES-GFP). In this mouse model, all GABAergic interneurons that arise from the subventricular zone of the ventral telencephalon lack COUP-TFI, without loss of this gene from neurons within the cerebral cortex. These conditional mutants are viable and survive to adulthood, thus enabling investigation of the birth and differentiation of interneuronal subtypes through postnatal stages of development, when local cortical microcircuitry is built. I found that conditional loss-of-function of COUP-TFI in subventricular precursors and post-mitotic cells of the basal ganglia led to a decrease within the cortex of late-born, CGE-derived, VIP- and CR-expressing bipolar interneurons, which was compensated by a concurrent increase of early-born, MGE-derived, PV-expressing interneurons. Strikingly, COUP-TFI mutants were more resistant to pharmacologically induced seizures, a phenotype that we found is dependent on GABAergic signaling. Together, the data support a model by which COUP-TFI regulates the delicate balance between MGE- and CGE-derived interneurons that reach the cortex, likely by influencing intermediate progenitor cell division in the CGE. Upon fate-specification in the ventral telencephalon, interneurons travel long distances to reach and enter the cerebral cortex. Here, they precisely distribute into cortical layer and contact projection neuron partners. The developmental events governing the integration of excitatory projection neurons and inhibitory interneurons into balanced local circuitries are still poorly understood. In order to investigate the role of projection neurons in cortical interneuron lamination, I analyzed the cortex of Fezf2-/- mice, a unique mutant model in which a single population of projection neurons, subcerebral projection neurons, is absent and replaced by callosal projection neurons, without any effect on interneuron cell-autonomous fate specification and differentiation. Using this model, I found that replacement of one projection neuron type with another was sufficient to cause distinct abnormalities of interneuron lamination and altered GABAergic inhibition. The data indicate that different subtypes of projection neurons uniquely and differentially determine the laminar distribution of interneurons in the cerebral cortex. In agreement, in parallel gain-of-function experiments, I found that distinct populations of projection neurons (i.e. corticofugal projection neurons or upper layer II/III callosal projection neurons) that were experimentally generated below the cortex could recruit cortical interneurons to these ectopic locations. Strikingly, the identity of the projection neurons generated, rather than strictly their birthdate, determined the specific types of interneurons recruited. These data demonstrate that in the neocortex individual populations of projection neurons cell-extrinsically control the laminar fate of interneurons and the assembly of local inhibitory circuitry. Together, the work identifies a new transcriptional control over cell-autonomous development of CGE-derived interneurons, and provides a first demonstration that neuronal subtype-specific interactions among excitatory and inhibitory neurons is critically necessary for the building of balanced local microcircuitry in the cerebral cortex

Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription.

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells