Disruption of Visc-2, a Brain-Expressed Conserved Long Noncoding RNA, Does Not Elicit an Overt Anatomical or Behavioral Phenotype.

Although long noncoding RNAs (lncRNAs) are proposed to play essential roles in mammalian neurodevelopment, we know little of their functions from their disruption in vivo. Combining evidence for evolutionary constraint and conserved expression data, we previously identified candidate lncRNAs that might play important and conserved roles in brain function. Here, we demonstrate that the sequence and neuronal transcription of lncRNAs transcribed from the previously uncharacterized Visc locus are conserved across diverse mammals. Consequently, one of these lncRNAs, Visc-2, was selected for targeted deletion in the mouse, and knockout animals were subjected to an extremely detailed anatomical and behavioral characterization. Despite a neurodevelopmental expression pattern of Visc-2 that is highly localized to the cortex and sites of neurogenesis, anomalies in neither cytoarchitecture nor neuroproliferation were identified in knockout mice. In addition, no abnormal motor, sensory, anxiety, or cognitive behavioral phenotypes were observed. These results are important because they contribute to a growing body of evidence that lncRNA loci contribute on average far less to brain and biological functions than protein-coding loci. A high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, will be required to establish the distribution of their organismal functions.

Cortical and Clonal Contribution of Tbr2 Expressing Progenitors in the Developing Mouse Brain.

The individual contribution of different progenitor subtypes towards the mature rodent cerebral cortex is not fully understood. Intermediate progenitor cells (IPCs) are key to understanding the regulation of neuronal number during cortical development and evolution, yet their exact contribution is much debated. Intermediate progenitors in the cortical subventricular zone are defined by expression of T-box brain-2 (Tbr2). In this study we demonstrate by using the Tbr2(Cre) mouse line and state-of-the-art cell lineage labeling techniques, that IPC derived cells contribute substantial proportions 67.5% of glutamatergic but not GABAergic or astrocytic cells to all cortical layers including the earliest generated subplate zone. We also describe the laminar dispersion of clonally derived cells from IPCs using a recently described clonal analysis tool (CLoNe) and show that pair-generated cells in different layers cluster closer (142.1 ± 76.8 µm) than unrelated cells (294.9 ± 105.4 µm). The clonal dispersion from individual Tbr2 positive intermediate progenitors contributes to increasing the cortical surface. Our study also describes extracortical contributions from Tbr2+ progenitors to the lateral olfactory tract and ventromedial hypothalamic nucleus.

Examining the relationship between early axon growth and transcription factor expression in the developing cerebral cortex.

The transcription factors Satb2 (special AT-rich sequence binding protein 2) and Ctip2 (COUP-TF interacting protein 2) have been shown to be required for callosal and corticospinal axon growth respectively from subtypes of cerebral cortex projection neurons. In this study we investigated early stages of directed axon growth in the embryonic mouse cerebral cortex, and studied the possible correlation with the expression of Satb2 and Ctip2. Electroporation of an EYFP-expressing plasmid at embryonic day 13.5 to label developing projection neurons revealed that directed axon growth is first seen in radially migrating neurons in the intermediate zone (IZ), prior to migration into the cortical plate, as has been suggested previously. Onset of expression of SATB2 and CTIP2 was also observed in the IZ, correlating well with this stage of migration and initiation of axon growth. Immunohistochemical staining through embryonic and early postnatal development revealed a significant population of Satb2/Ctip2 co-expressing cells, while retrograde axon tracing from the corpus callosum at embryonic day 18.5 back-labelled many neurons with bi-directional axon processes. However, through retrograde tracing and simultaneous immunohistochemical staining we show that these bi-directional processes do not correlate with Satb2/Ctip2 co-expression. Our work shows that although expression of these transcription factors correlates well with the appearance of directed axon growth during cortical development, the transcriptional code underlying the bi-directional axonal projections of early neocortical neurons is not likely to be the result of Satb2/Ctip2 co-expression.

The subventricular zone is the developmental milestone of a 6-layered neocortex: comparisons in metatherian and eutherian mammals.

The major lineages of mammals (Eutheria, Metatheria, and Monotremata) diverged more than 100 million years ago and have undergone independent changes in the neocortex. We found that adult South American gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii) possess a significantly lower number of cerebral cortical neurons compared with the mouse (Mus musculus). To determine whether the difference is reflected in the development of the cortical germinal zones, the location of progenitor cell divisions was examined in opossum, tammar wallaby, and rat. The basic pattern of the cell divisions was conserved, but the emergence of a distinctive band of dividing cells in the subventricular zone (SVZ) occurred relatively later in the opossum (postnatal day [P14]) and the tammar wallaby (P40) than in rodents. The planes of cell divisions in the ventricular zone (VZ) were similar in all species, with comparable mRNA expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in opossum (P12 and P20) and mouse (embryonic day 15 and P0). In conclusion, the marsupial neurodevelopmental program utilizes an organized SVZ, as indicated by the presence of intermediate (or basal) progenitor cell divisions and gene expression patterns, suggesting that the SVZ emerged prior to the Eutherian-Metatherian split.

The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone.

The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.

Evolution of cortical neurogenesis.

The neurons of the mammalian neocortex are organised into six layers. By contrast, the reptilian and avian dorsal cortices only have three layers which are thought to be equivalent to layers I, V and VI of mammals. Increased repertoire of mammalian higher cognitive functions is likely a result of an expanded cortical surface area. The majority of cortical cell proliferation in mammals occurs in the ventricular zone (VZ) and subventricular zone (SVZ), with a small number of scattered divisions outside the germinal zone. Comparative developmental studies suggest that the appearance of SVZ coincides with the laminar expansion of the cortex to six layers, as well as the tangential expansion of the cortical sheet seen within mammals. In spite of great variation and further compartmentalisation in the mitotic compartments, the number of neurons in an arbitrary cortical column appears to be remarkably constant within mammals. The current challenge is to understand how the emergence and elaboration of the SVZ has contributed to increased cortical cell diversity, tangential expansion and gyrus formation of the mammalian neocortex. This review discusses neurogenic processes that are believed to underlie these major changes in cortical dimensions in vertebrates.

Comparative aspects of cortical neurogenesis in vertebrates.

The mammalian neocortex consists of six layers. By contrast, the reptilian and avian cortices have only three, which are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular and subventricular zones, but there are a small number of scattered individual divisions throughout the cortex. Neurogenesis in the cortical subventricular zone is believed to contribute to the supragranular layers. To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stages 18-25) and chick (embryonic days 8-15) brains using phospho-histone H3 (a G2 and M phase marker) immunohistochemistry. In turtle, only few scattered abventricular H3-immunoreactive cells were found outside the ventricular zone; the majority of the H3-immunoreactive cells were located in the ventricular zone throughout the entire turtle brain. Ventricular zone cell proliferation peaks at stages 18 and 20, before an increase of abventricular proliferation at stages 23 and 25. In turtle cortex, however, abventricular proliferation at any given stage never exceeded 17.5+/-2.47% of the total division and the mitotic profiles did not align parallel to the ventricular zone. Phospho-histone H3 immunoreactivity in embryonic chick brains suggests the lack of subventricular zone in the dorsal cortex, but the presence of subventricular zone in the ventral telencephalon. We were able to demonstrate that the avian subventricular zone is present in both pallial and subpallial regions of the ventral telencephalon during embryonic development, and we characterize the spatial and temporal organization of the subventricular zone. Comparative studies suggest that the subventricular zone was involved in the laminar expansion of the cortex to six layers in mammals from the three-layered cortex found in reptiles and birds. Within mammals, the number of neurons in a cortical column appears to be largely constant; nevertheless, there are considerable differences between the germinal zones in mammalian species. It is yet to be determined whether these elaborations of the subventricular zone may have contributed to cell diversity, tangential expansion or gyrus formation of the neocortex and whether it might have been the major driving force behind the evolution of the six-layered neocortex in mammals.

Genes involved in the formation of the earliest cortical circuits.

Building the brain is like erecting a house of cards. The early connections provide the foundation of the adult structure, and disruption of these may be the source of many developmental flaws. Cerebral cortical developmental disorders (including schizophrenia and autism) and perinatal injuries involve cortical neurons with early connectivity. The major hindrance of progress in understanding the early neural circuits during cortical development and disease has been the lack of reliable markers for specific cell populations. Due to the advance of powerful approaches in gene expression analysis and the utility of models with reporter gene expressions in specific cortical cell types, our knowledge of the early cortical circuits is rapidly increasing. With focus on the sub-plate, layer VI and layer V projection neurons, we shall illustrate the progress made in the understanding of their neurochemical properties, physiological characteristics and their integration into the early intracortical and extracortical circuitry. This field benefited from recent developments in mouse genetics in generating models with subtype specific gene expression patterns, powerful cell dissection and separation methods combined with microarray analysis. The emergence of cortical cell type specific biomarkers will not only help neuropathological diagnosis, but will also eventually reveal the causal relations in the pathogenesis of various cortical developmental disorders.

Towards the classification of subpopulations of layer V pyramidal projection neurons.

The nature of cerebral cortical circuitry has been increasingly clarified by markers for the identification of precise cell types with specific morphology, connectivity and distinct physiological properties. Molecular markers are not only helpful in dissecting cortical circuitry, but also give insight into the mechanisms of cortical neuronal specification and differentiation. The two principal neuronal types of the cerebral cortex are the pyramidal and GABAergic cells. Pyramidal cells are excitatory and project to distant targets, while GABAergic neurons are mostly inhibitory non-pyramidal interneurons. Reliable markers for specific subtypes of interneurons are available and have been employed in the classification and functional analysis of cortical circuitry. Until recently, cortical pyramidal neurons have been considered a homogeneous class of cells. This concept is now changing as the powerful tools of molecular biology and genetics identify molecular tags for subtypes of pyramidal cells such as: Otx-1 [Frantz, G.D., Bohner, A.P., Akers, R.M., McConnell, S.K., 1994. Regulation of the POU domain gene SCIP during cerebral cortical development. J. Neurosci. 14, 472-485; Weimann, J.M., Zhang, Y.A., Levin, M.E., Devine, W.P., Brulet, P., McConnell, S.K., 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24, 819-831]; SMI-32, N200 and FNP-7 [Voelker, C.C., Garin, N., Taylor, J.S., Gahwiler, B.H., Hornung, J.P., Molnár, Z., 2004. Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro. Cereb. Cortex 14, 1276-1286]; ER81 [Hevner, R.F., Daza, R.A., Rubenstein, J.L., Stunnenberg, H., Olavarria, J.F., Englund, C., 2003. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev. Neurosci. 25 (2-4), 139-151; Yoneshima, H., Yamasaki, S., Voelker, C., Molnár, Z., Christophe, E., Audinat, E., Takemoto, M., Tsuji, S., Fujita, I., Yamamoto, N., 2006. ER81 is expressed in a subpopulation of layer 5 projection neurons in rodent cerebral cortices. Neuroscience, 137, 401-412]; Lmo4 [Bulchand, S., Subramanian, L., Tole, S., 2003. Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev. Dyn. 226, 460-469; Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; CTIP2 [Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; Fez1 [Molyneaux, B.J., Arlotta, P., Hirata, T., Hibi, M., Macklis, J.D., 2005. Fez1 is required for the birth and specification of corticospinal motor neurons. Neuron 47 (6), 817-831; Chen, B., Schaevitz, L.R., McConnell, S.K., 2005. Fez1 regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102 (47), 17184-17189]. These genes outline the numerous subtypes of pyramidal cells and are increasingly refining our previous classifications. They also indicate specific developmental programs operate in cell fate decisions. This review will describe the progress made on the correlation of these markers to each other within a specific subtype of layer V neurons with identified, stereotypic projections. Further work is needed to link these data with observations on somatodendritic morphology and physiological properties. The integrated molecular, anatomical and physiological characterisation of pyramidal neurons will lead to a much better appreciation of functional cortical circuits.