Flora M. Vaccarino

Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis

We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.

Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus

The lifelong addition of neurons to the hippocampus is a remarkable form of structural plasticity, yet the molecular controls over proliferation, neuronal fate determination, survival, and maturation are poorly understood. Expression of Notch1 was found to change dynamically depending on the differentiation state of neural precursor cells. Through the use of inducible gain- and loss-of-function of Notch1 mice we show that this membrane receptor is essential to these distinct processes. We found in vivo that activated Notch1 overexpression induces proliferation, whereas γ-secretase inhibition or genetic ablation of Notch1 promotes cell cycle exit, indicating that the level of activated Notch1 regulates the magnitude of neurogenesis from postnatal progenitor cells. Abrogation of Notch signaling in vivo or in vitro leads to a transition from neural stem or precursor cells to transit-amplifying cells or neurons. Further, genetic Notch1 manipulation modulates survival and dendritic morphology of newborn granule cells. These results provide evidence for the expansive prevalence of Notch signaling in hippocampal morphogenesis and plasticity, suggesting that Notch1 could be a target of diverse traumatic and environmental modulators of adult neurogenesis.

Decreased Number of Parvalbumin and Cholinergic Interneurons in the Striatum of Individuals with Tourette Syndrome

Corticobasal ganglia neuronal ensembles bring automatic motor skills into voluntary control and integrate them into ongoing motor behavior. A 5% decrease in caudate (Cd) nucleus volume is the most consistent structural finding in the brain of patients with Tourette syndrome (TS), but the cellular abnormalities that underlie this decrease in volume are unclear. In this study the density of different types of interneurons and medium spiny neurons (MSNs) in the striatum was assessed in the postmortem brains of 5 TS subjects as compared with normal controls (NC) by unbiased stereological analyses. TS patients demonstrated a 50%–60% decrease of both parvalbumin (PV)+ and choline acetyltransferase (ChAT)+ cholinergic interneurons in the Cd and the putamen (Pt). Cholinergic interneurons were decreased in TS patients in the associative and sensorimotor regions but not in the limbic regions of the striatum, such that the normal gradient in density of cholinergic cells (highest in associative regions, intermediate in sensorimotor and lowest in limbic regions) was abolished. No significant difference was present in the densities of medium‐sized calretinin (CR)+ interneurons, MSNs, and total neurons. The selective deficit of PV+ and cholinergic striatal interneurons in TS subjects may result in an impaired cortico/thalamic control of striatal neuron firing in TS. J. Comp. Neurol. 518:277–291, 2010.

Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders

Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Modeling human cortical development in vitro using induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8–10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.

Early Postnatal Astroglial Cells Produce Multilineage Precursors and Neural Stem Cells In Vivo

To identify the fates that astroglial cells can attain in the postnatal brain, we generated mice carrying an inducible Cre recombinase (Cre-ERT2) controlled by the human GFAP promoter (hGFAP). In mice carrying the GCE (hGFAP-Cre-ERT2) transgene, OHT (4-hydroxy-tamoxifen) injections induced Cre recombination in astroglial cells at postnatal day 5 and allowed us to permanently tag these cells with reporter genes. Three days after recombination, reporter-tagged cells were quiescent astroglial cells that expressed the stem cell marker LeX in the subventricular zone (SVZ) and dentate gyrus (DG). After 2–4 weeks, the tagged GFAP lineage included proliferating progenitors expressing the neuronal marker Dcx (Doublecortin) in the SVZ and the DG. After 4 weeks, the GFAP lineage generated mature neurons in the olfactory bulb (OB), DG, and, strikingly, also in the cerebral cortex. A major portion of all neurons in the DG and OB born at the end of the first postnatal week were generated from GFAP+ cells. In addition to neurons, mature oligodendrocytes and astrocytes populating the cerebral cortex and white matter were also the progeny of GFAP+ astroglial ancestors. Thus, genetic fate mapping of postnatal GFAP+ cells reveals that they seed the postnatal brain with neural progenitors/stem cells that in turn give rise to neural precursors and their mature neuronal and oligodendrocytic progeny in many CNS regions, including the cerebral cortex.

Fibroblast Growth Factor 2 Is Required for Maintaining the Neural Stem Cell Pool in the Mouse Brain Subventricular Zone

Cells within the subventricular zone (SVZ) express basic Fgf (Fgf2) and Fgf receptor proteins. We show that the absence of Fgf2 gene products reduces by 50% the dividing progenitor population of the anterior SVZ (SVZa) without changing their cell cycle time. Every 2–3 cell cycles of the SVZa progenitor cell population, 30,000 newly generated neurons capable of long-term survival are added to the glomerular layer of the olfactory bulb. Fgf2 knockout mice have smaller olfactory bulbs due to decreased output of these newly generated cells into the bulbs. A population of slow-dividing neural stem cells (NSCs) residing in the SVZa is identified by its slow cell cycle kinetics (cell cycle approx. 20 days); these cells, called ‘S’ cells, are negative for glial fibrillary acidic protein and occasionally express brain-lipid-binding protein, a molecular marker of radial glia. The number of these dividing NSCs is reduced from about 13,000 in wild-type to 8,500 cells in Fgf2 knockout mice. Thus, FGF2 regulates the number of proliferative cells and olfactory bulb neurogenesis by maintaining a slow-dividing stem cell pool within the SVZa.

Cortical neurogenesis enhanced by chronic perinatal hypoxia

Most regions of the mature mammalian brain, including the cerebral cortex, appear to be unable to support the genesis of new neurons. Here, we report that a low level of neurogenesis occurs in the cerebral cortex of the infant mouse brain and is enhanced by chronic perinatal hypoxia. When mice were reared in a low-oxygen environment from postnatal days 3 to 11, approximately 30% of the cortical neurons were lost after the insult; yet this damage was transient. The loss of cortical neuron number, cortical volume, and brain weight were all reversed during the recovery period. At P18, 7 days after the cessation of hypoxia, there was a marked increase in astroglial cell proliferation within the SVZ, as assessed by 5-bromodeoxyuridine (BrdU) incorporation in S-phase cells. One month after BrdU incorporation, 40% more BrdU-positive cells were found in the cerebral cortex of hypoxic-reared as compared to normoxic control mice. Among these newly generated cortical cells, approximately 45% were oligodendrocytes, 35% were astrocytes, and 10% were neurons in both hypoxic and normoxic mice. However, twice as many BrdU-labeled cells expressed neuronal markers in the neocortex in mice recovering from hypoxia as compared to controls. In both hypoxic-reared and normoxic infant/juvenile mice, putative neuroblasts could be seen detaching from the forebrain subventricular zone, migrating through the subcortical white matter and entering the lower cortical layers, 5 to 11 days after their last mitotic division. We suggest that cortical neurogenesis may play a significant role in repairing neuronal losses after neonatal injury.

Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding

We found that leptin receptors were expressed in hypothalamic astrocytes and that their conditional deletion led to altered glial morphology and synaptic inputs onto hypothalamic neurons involved in feeding control. Leptin-regulated feeding was diminished, whereas feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data reveal an active role of glial cells in hypothalamic synaptic remodeling and control of feeding by leptin.