Unipolar (Dendritic) Brush Cells Are Morphologically Complex and Require Tbr2 for Differentiation and Migration.

Previous studies demonstrated specific expression of transcription factor Tbr2 in unipolar brush cells (UBCs) of the cerebellum during development and adulthood. To further study UBCs and the role of Tbr2 in their development we examined UBC morphology in transgenic mouse lines (reporter and lineage tracer) and also examined the effects of Tbr2 deficiency in Tbr2 (MGI: Eomes) conditional knock-out (cKO) mice. In Tbr2 reporter and lineage tracer cerebellum, UBCs exhibited more complex morphologies than previously reported including multiple dendrites, bifurcating dendrites, and up to four dendritic brushes. We propose that "dendritic brush cells" (DBCs) may be a more apt nomenclature. In Tbr2 cKO cerebellum, mature UBCs were completely absent. Migration of UBC precursors from rhombic lip to cerebellar cortex and other nuclei was impaired in Tbr2 cKO mice. Our results indicate that UBC migration and differentiation are sensitive to Tbr2 deficiency. To investigate whether UBCs develop similarly in humans as in rodents, we studied Tbr2 expression in mid-gestational human cerebellum. Remarkably, Tbr2+ UBC precursors migrate along the same pathways in humans as in rodent cerebellum and disperse to create the same "fountain-like" appearance characteristic of UBCs exiting the rhombic lip.

Low availability of choline in utero disrupts development and function of the retina.

Adequate supply of choline, an essential nutrient, is necessary to support proper brain development. Whether prenatal choline availability plays a role in development of the visual system is currently unknown. In this study, we addressed the role of in utero choline supply for the development and later function of the retina in a mouse model. We lowered choline availability in the maternal diet during pregnancy and assessed proliferative and differentiation properties of retinal progenitor cells (RPCs) in the developing prenatal retina, as well as visual function in adult offspring. We report that low choline availability during retinogenesis leads to persistent retinal cytoarchitectural defects, ranging from focal lesions with displacement of retinal neurons into subretinal space to severe hypocellularity and ultrastructural defects in photoreceptor organization. We further show that low choline availability impairs timely differentiation of retinal neuronal cells, such that the densities of early-born retinal ganglion cells, amacrine and horizontal cells, as well as cone photoreceptor precursors, are reduced in low choline embryonic d 17.5 retinas. Maintenance of higher proportions of RPCs that fail to exit the cell cycle underlies aberrant neuronal differentiation in low choline embryos. Increased RPC cell cycle length, and associated reduction in neurofibromin 2/Merlin protein, an upstream regulator of the Hippo signaling pathway, at least in part, explain aberrant neurogenesis in low choline retinas. Furthermore, we find that animals exposed to low choline diet in utero exhibit a significant degree of intraindividual variation in vision, characterized by marked functional discrepancy between the 2 eyes in individual animals. Together, our findings demonstrate, for the first time, that choline availability plays an essential role in the regulation of temporal progression of retinogenesis and provide evidence for the importance of adequate supply of choline for proper development of the visual system.-Trujillo-Gonzalez, I., Friday, W. B., Munson, C. A., Bachleda, A., Weiss, E. R., Alam, N. M., Sha, W., Zeisel, S. H., Surzenko, N. Low availability of choline in utero disrupts development and function of the retina.

Reduced phosphoCREB in Müller glia during retinal degeneration in rd10 mice.

PURPOSE: The mechanisms that trigger retinal degeneration are not well understood, despite the availability of several animal models with different mutations. In the present report, the rd10 mouse, a model for retinitis pigmentosa (RP) that contains a mutation in the gene for PDE6ß (Pde6b), is used to evaluate gliosis, as a marker for retinal stress, and cyclic AMP response element binding protein (CREB) phosphorylation, which may be important early in retinal degeneration. METHODS: Wild-type C57Bl6J and rd10 mice raised under cyclic light were examined for changes in gliosis and CREB phosphorylation for approximately 3 weeks beginning at P14 to P17 using immunocytochemistry. Mice raised under normal cyclic light and in complete darkness were also compared for changes in CREB phosphorylation. RESULTS: Gliosis in rd10 mice raised under cyclic light was apparent at P17, before extensive degeneration of the photoreceptor layer is visible, and increased over time. Phosphorylation of CREB at Ser133 (pCREB) was detected in Müller glia (MG) in the wild-type and rd10 mice. However, at all phases of photoreceptor degeneration, the pCREB levels were lower in the rd10 mice. We also observed extensive migration of MG cell bodies to the outer nuclear layer (ONL) during degeneration. In contrast to the mice raised under cyclic light, the rd10 mice raised in the dark exhibited slower rates of degeneration. When the dark-reared mice were exposed to cyclic light, the photoreceptor layer degenerated within 4 days to approximately one to two rows of nuclei. Interestingly, the pCREB levels in the MG also decreased during this 4-day cyclic light exposure compared to the levels in the rd10 mice raised continuously in the dark. CONCLUSIONS: The results of these studies suggest that photoreceptors communicate directly or indirectly with MG at early stages, inducing gliosis before extensive retinal degeneration is apparent in rd10 mice. Surprisingly, phosphorylation of CREB is downregulated in the MG. These results raise the interesting possibility that Müller glia undergo CREB-mediated transcriptional changes that influence photoreceptor degeneration either positively or negatively. Unlike canine models of RP, no increase in pCREB was observed in photoreceptor cells during this period suggesting possible mechanistic differences in the role of CREB in photoreceptors between these species.

Sox2-Deficient Müller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina.

PURPOSE: Müller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Müller glia has yet to be determined. METHODS: We use inducible, MG-specific genetic ablation of Sox2 in vivo at the peak of MG genesis to analyze its function in the maturation of murine MG and effects on other cells in the retina. Histologic and functional analysis of the Sox2-deficient retinas is conducted at key points in postnatal development. RESULTS: Ablation of Sox2 in the postnatal retina results in disorganization of MG processes in the inner plexiform layer and mislocalized cell bodies in the nuclear layers. This disorganization is concurrent with a thinning of the neural retina and disruption of neuronal processes in the inner and outer plexiform layers. Functional analysis by electroretinography reveals a decrease in the b-wave amplitude. Disruption of MG maturation due to Sox2 ablation therefore negatively affected the function of the retina. CONCLUSIONS: These results demonstrate a novel role for SOX2 in glial process outgrowth and adhesion, and provide new insights into the essential role Müller glia play in the development of retinal cytoarchitecture. Prior to this work, SOX2 was known to have a primary role in determining cell fate. Our experiments bypass cell fate conversion to establish a new role for SOX2 in a committed cell lineage.

SOX2 maintains the quiescent progenitor cell state of postnatal retinal Muller glia.

Within discrete regions of the developing mammalian central nervous system, small subsets of glia become specialized to function as neural stem cells. As a result of their self-renewal and neurogenic capacity, these cells later serve to replenish neurons and glia during persistent or injury-induced adult neurogenesis. SOX2, an HMG box transcription factor, plays an essential role in the maintenance of both embryonic and adult neural progenitors. It is unclear, however, which biological mechanisms regulated by SOX2 are required for neural stem cell maintenance. In this study, we address this question through genetic analysis of SOX2 function in differentiating postnatal Müller glia, a cell type that maintains neurogenic capacity in the adult retina. By utilizing molecular analysis and real-time imaging, we show that two progenitor characteristics of nascent Müller glia - their radial morphology and cell cycle quiescence - are disrupted following conditional genetic ablation of Sox2 in the mouse postnatal retina, leading to Müller cell depletion and retinal degeneration. Moreover, we demonstrate that genetic induction of the Notch signaling pathway restores Müller glial cell identity to Sox2 mutant cells, but does not secure their quiescent state. Collectively, these results uncouple the roles of SOX2 and the Notch signaling pathway in the postnatal retina, and uncover a novel role for SOX2 in preventing the depletion of postnatal Müller glia through terminal cell division.

Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex.

Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia, we show that migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron's migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may in part underlie the neurological defects in Joubert syndrome patients.

Expression of histone deacetylases in cellular compartments of the mouse brain and the effects of ischemia.

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1-3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell-and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen-glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner.

Histone deacetylase inhibitors preserve white matter structure and function during ischemia by conserving ATP and reducing excitotoxicity.

The importance of white matter (WM) injury to stroke pathology has been underestimated in experimental animal models and this may have contributed to the failure to translate potential therapeutics into the stroke clinic. Histone deacetylase (HDAC) inhibitors are neuroprotective and also promote neurogenesis. These properties make them ideal candidates for stroke therapy. In a pure WM tract (isolated mouse optic nerve), we show that pan- and class I-specific HDAC inhibitors, administered before or after a period of oxygen and glucose deprivation (OGD), promote functional recovery of axons and preserve WM cellular architecture. This protection correlates with the upregulation of an astrocyte glutamate transporter, delayed and reduced glutamate accumulation during OGD, preservation of axonal mitochondria and oligodendrocytes, and maintenance of ATP levels. Interestingly, the expression of HDACs 1, 2, and 3 is localized to astrocytes, suggesting that changes in glial cell gene transcription and/or protein acetylation may confer protection to axons. Our findings suggest that a therapeutic opportunity exists for the use of HDAC inhibitors, targeting mitochondrial energy regulation and excitotoxicity in ischemic WM injury.

Role of interstitial branching in the development of visual corticocortical connections: a time-lapse and fixed-tissue analysis.

We combined fixed-tissue and time-lapse analyses to investigate the axonal branching phenomena underlying the development of topographically organized ipsilateral projections from area 17 to area 18a in the rat. These complementary approaches allowed us to relate static, large-scale information provided by traditional fixed-tissue analysis to highly dynamic, local, small-scale branching phenomena observed with two-photon time-lapse microscopy in acute slices of visual cortex. Our fixed-tissue data revealed that labeled area 17 fibers invaded area 18a gray matter at topographically restricted sites, reaching superficial layers in significant numbers by postnatal day 6 (P6). Moreover, most parental axons gave rise to only one or occasionally a small number of closely spaced interstitial branches beneath 18a. Our time-lapse data showed that many filopodium-like branches emerged along parental axons in white matter or deep layers in area 18a. Most of these filopodial branches were transient, often disappearing after several minutes to hours of exploratory extension and retraction. These dynamic behaviors decreased significantly from P4, when the projection is first forming, through the second postnatal week, suggesting that the expression of, or sensitivity to, cortical cues promoting new branch addition in the white matter is developmentally down-regulated coincident with gray matter innervation. Together, these data demonstrate that the development of topographically organized corticocortical projections in rats involves extensive exploratory branching along parental axons and invasion of cortex by only a small number of interstitial branches, rather than the widespread innervation of superficial cortical layers by an initially exuberant population of branches.