Maria Armentano

COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas

We used cortex-specific deletion of the transcription factor gene COUP-TFI (also known as Nr2f1) in mice to demonstrate previously unknown fundamental roles for it in patterning mammalian neocortex into areas. The highest COUP-TFI expression is observed in the cortical progenitors and progeny in parietal and occipital cortex that form sensory areas, and the lowest expression was observed in frontal cortex that includes motor areas. Cortical deletion of COUP-TFI resulted in massive expansion of frontal areas, including motor, to occupy most of neocortex, paralleled by marked compression of sensory areas to caudal occipital cortex. These area patterning changes are preceded and paralleled by corresponding changes in molecular markers of area identity and altered axonal projections to maintain patterned area-specific input and output connections. We conclude that COUP-TFI is required for balancing patterning of neocortex into frontal/motor and sensory areas by acting in its expression domain to repress frontal/motor area identities and to specify sensory area identities.

The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain.

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.

COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth

The transcription factor COUP-TFI (NR2F1), an orphan member of the nuclear receptor superfamily, is an important regulator of neurogenesis, cellular differentiation and cell migration. In the forebrain, COUP-TFI controls the connectivity between thalamus and cortex and neuronal tangential migration in the basal telencephalon. Here, we show that COUP-TFI is required for proper axonal growth and guidance of all major forebrain commissures. Fibres of the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants. Moreover, hippocampal neurons lacking COUP-TFI have a defect in neurite outgrowth and show an abnormal axonal morphology. To search for downstream effectors, we used microarray analysis and showed that, in the absence of COUP-TFI, expression of various cytoskeleton molecules involved in neuronal morphogenesis is affected. Diminished protein levels of the microtubule-associated protein MAP1B and increased levels of the GTP-binding protein RND2 were confirmed in the developing cortex in vivo and in primary hippocampal neurons in vitro. Therefore, based on morphological studies, gene expression profiling and primary cultured neurons, the present data uncover a previously unappreciated intrinsic role for COUP-TFI in axonal growth in vivo and supply one of the premises for COUP-TFI coordination of neuronal morphogenesis in the developing forebrain.

Loss of COUP-TFI Alters the Balance between Caudal Ganglionic Eminence- and Medial Ganglionic Eminence-Derived Cortical Interneurons and Results in Resistance to Epilepsy

In rodents, cortical interneurons originate from the medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) according to precise temporal schedules. The mechanisms controlling the specification of CGE-derived interneurons and their role in cortical circuitry are still unknown. Here, we show that COUP-TFI expression becomes restricted to the dorsal MGE and CGE at embryonic day 13.5 in the basal telencephalon. Conditional loss of function of COUP-TFI in subventricular precursors and postmitotic cells leads to a decrease of late-born, CGE-derived, VIP (vasoactive intestinal peptide)- and CR (calretinin)-expressing bipolar cortical neurons, compensated by the concurrent increase of early-born MGE-derived, PV (parvalbumin)-expressing interneurons. Strikingly, COUP-TFI mutants are more resistant to pharmacologically induced seizures, a phenotype that is dependent on GABAergic signaling. Together, our data indicate that COUP-TFI controls the delicate balance between MGE- and CGE-derived cortical interneurons by regulating intermediate progenitor divisions and ultimately affecting the activity of the cortical inhibitory circuitry.

Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons

In the adult brain, active stem cells are a subset of astrocytes residing in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Whether quiescent neuronal progenitors occur in other brain regions is unclear. Here, we describe a novel neurogenic system in the external capsule and lateral striatum (EC-LS) of the juvenile guinea pig that is quiescent at birth but becomes active around weaning. Activation of neurogenesis in this region was accompanied by the emergence of a neurogenic-like niche in the ventral EC characterized by chains of neuroblasts, intermediate-like progenitors and glial cells expressing markers of immature astrocytes. Like neurogenic astrocytes of the SVZ and DG, these latter cells showed a slow rate of proliferation and retained BrdU labeling for up to 65 days, suggesting that they are the primary progenitors of the EC-LS neurogenic system. Injections of GFP-tagged lentiviral vectors into the SVZ and the EC-LS of newborn animals confirmed that new LS neuroblasts originate from the activation of local progenitors and further supported their astroglial nature. Newborn EC-LS neurons existed transiently and did not contribute to neuronal addition or replacement. Nevertheless, they expressed Sp8 and showed strong tropism for white matter tracts, wherein they acquired complex morphologies. For these reasons, we propose that EC-LS neuroblasts represent a novel striatal cell type, possibly related to those populations of transient interneurons that regulate the development of fiber tracts during embryonic life.

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.

Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

Culturing conditions remarkably affect viability and organization of mouse subventricular zone in ex vivo cultured forebrain slices.

Mammalian neurogenic sites are good models for physiological neural cell renewal in the perspective of brain repair. Yet, investigating their stem cell niches is not easy since they are small areas deeply hidden in the brain hemispheres. Organotypic slices could be a useful tool since they substantially retain the three-dimensional tissue organization. The postnatal forebrain subventricular zone (SVZ), as a dynamic structure endowed with proliferation and migration, might undergo striking cellular changes in culture. Literature concerning this ex vivo approach applied to SVZ neurogenic activity and response to damage is scarce and heterogeneous, not considering the fine cellular composition of the slice and not taking into account the modifications occurring as a consequence of the culture conditions. Our aim was to describe in detail what happens in the SVZ when establishing an ex vivo model. We addressed the changes occurring in five day-old, postnatal mice forebrain organotypic slices cultured for several days in vitro, by using confocal and ultrastructural analyses. We found that during the first two days in vitro the slices undergo progressive structural disaggregation accompanied by remarkable increase in cell proliferation and death with respect to basal levels. In addition, these facts occur in parallel with strong activation of astrocytic cells and microglia. Our results highlight technical limits in the use of forebrain organotypic slices for studying the activity of SVZ neurogenic niche, indicating that they can be reliable for a very short time (1-2 days) and could be misleading when addressing lesion-induced responses.

Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15−/− mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.