Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis.

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9-Brn2 and Isl1-Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

Chx10 Consolidates V2a Interneuron Identity through Two Distinct Gene Repression Modes.

During development, two cell types born from closely related progenitor pools often express identical transcriptional regulators despite their completely distinct characteristics. This phenomenon implies the need for a mechanism that operates to segregate the identities of the two cell types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). We demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing the MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate the V2a and MN pathways. Our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.

Detection and monitoring of microRNA expression in developing mouse brain and fixed brain cryosections.

MicroRNAs (miRNAs) are 20-25 nucleotide long, noncoding, and single-strand RNAs that have been found in almost all organisms and shown to exert essential roles by regulating the stability and translation of target mRNAs. In mammals most miRNAs show tissue specific and developmentally regulated expression. Approximately 70 % of all miRNAs are expressed in the brain and a growing number of studies have shown that miRNAs can modulate both brain development function and dysfunction. Moreover, miRNAs have been involved in a variety of human pathologies, including cancer and diabetes and are rapidly emerging as new potential drug targets. In order to further characterize miRNA functions, it is therefore crucial to develop techniques enabling their detection in tissues (both fixed and in vivo) with single-cell resolution. Here, we describe methods for the detection/monitoring of miRNA expression, that can be applied in both developing embryos and fixed samples, which we and others have applied to the investigation of both embryonal and postnatal neurogenesis in mice, but also in zebrafish, and cell cultures.

Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex.

In the absence of external stimuli, the mammalian neocortex shows intrinsic network oscillations. These dynamics are characterized by translaminar assemblies of neurons whose activity synchronizes rhythmically in space and time. How different cortical layers influence the formation of these spontaneous cellular assemblies is poorly understood. We found that excitatory neurons in supragranular and infragranular layers have distinct roles in the regulation of intrinsic low-frequency oscillations in mice in vivo. Optogenetic activation of infragranular neurons generated network activity that resembled spontaneous events, whereas photoinhibition of these same neurons substantially attenuated slow ongoing dynamics. In contrast, light activation and inhibition of supragranular cells had modest effects on spontaneous slow activity. This study represents, to the best of our knowledge, the first causal demonstration that excitatory circuits located in distinct cortical layers differentially control spontaneous low-frequency dynamics.

Convergent repression of Foxp2 3'UTR by miR-9 and miR-132 in embryonic mouse neocortex: implications for radial migration of neurons.

MicroRNAs (miRNAs) are rapidly emerging as a new layer of regulation of mammalian brain development. However, most of the miRNA target genes remain unidentified. Here, we explore gene expression profiling upon miRNA depletion and in vivo target validation as a strategy to identify novel miRNA targets in embryonic mouse neocortex. By this means, we find that Foxp2, a transcription factor associated with speech and language development and evolution, is a novel miRNA target. In particular, we find that miR-9 and miR-132 are able to repress ectopic expression of Foxp2 protein by targeting its 3' untranslated region (3'UTR) in vivo. Interestingly, ectopic expression of Foxp2 in cortical projection neurons (a scenario that mimics the absence of miRNA-mediated silencing of Foxp2 expression) delays neurite outgrowth in vitro and impairs their radial migration in embryonic mouse neocortex in vivo. Our results uncover a new layer of control of Foxp2 expression that may be required for proper neuronal maturation.