Genetic mechanisms control the linear scaling between related cortical primary and higher order sensory areas.

In mammals, the neocortical layout consists of few modality-specific primary sensory areas and a multitude of higher order ones. Abnormal layout of cortical areas may disrupt sensory function and behavior. Developmental genetic mechanisms specify primary areas, but mechanisms influencing higher order area properties are unknown. By exploiting gain-of and loss-of function mouse models of the transcription factor Emx2, we have generated bi-directional changes in primary visual cortex size in vivo and have used it as a model to show a novel and prominent function for genetic mechanisms regulating primary visual area size and also proportionally dictating the sizes of surrounding higher order visual areas. This finding redefines the role for intrinsic genetic mechanisms to concomitantly specify and scale primary and related higher order sensory areas in a linear fashion.

Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies. First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results. The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Geniculocortical input drives genetic distinctions between primary and higher-order visual areas.

Studies of area patterning of the neocortex have focused on primary areas, concluding that the primary visual area, V1, is specified by transcription factors (TFs) expressed by progenitors. Mechanisms that determine higher-order visual areas (V(HO)) and distinguish them from V1 are unknown. We demonstrated a requirement for thalamocortical axon (TCA) input by genetically deleting geniculocortical TCAs and showed that they drive differentiation of patterned gene expression that distinguishes V1 and V(HO). Our findings suggest a multistage process for area patterning: TFs expressed by progenitors specify an occipital visual cortical field that differentiates into V1 and V(HO); this latter phase requires geniculocortical TCA input to the nascent V1 that determines genetic distinctions between V1 and V(HO) for all layers and ultimately determines their area-specific functional properties.

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.

Cortical area size dictates performance at modality-specific behaviors.

The mammalian neocortex is organized into unique areas that serve functions such as sensory perception and modality-specific behaviors. The sizes of primary cortical areas vary across species, and also within a species, raising the question of whether area size dictates behavioral performance. We show that adult mice genetically engineered to overexpress the transcription factor EMX2 in embryonic cortical progenitor cells, resulting in reductions in sizes of somatosensory and motor areas, exhibit significant deficiencies at tactile and motor behaviors. Even increasing the size of sensorimotor areas by decreasing cortical EMX2 levels can lead to diminished sensorimotor behaviors. Genetic crosses that retain ectopic Emx2 transgene expression subcortically but restore cortical Emx2 expression to wild-type levels also restore cortical areas to wild-type sizes and in parallel restore tactile and motor behaviors to wild-type performance. These findings show that area size can dictate performance at modality-specific behaviors and suggest that areas have an optimal size, influenced by parameters of its neural system, for maximum behavioral performance. This study underscores the importance of establishing during embryonic development appropriate levels of regulatory proteins that determine area sizes, thereby influencing behavior later in life.

Regulation of laminar and area patterning of mammalian neocortex and behavioural implications.

We will focus on describing our recent studies on the laminar and area patterning of the mammalian neocortex. We describe a novel IgCAM, MDGA1, that is a unique laminar and area specific marker, and functional studies showing its influence on radial migration. We also describe time-lapse imaging studies showing that the pre-plate and its derivative, the subplate, is a cellular protomap of the cortical ventricular zone, and the implications of this finding for mechanisms of arealization and development of area-specific TCA projections. We will summarize studies of each of the four transcription factors, Emx2, Pax6, Couptfl and Sp8, expressed by cortical progenitors and involved in specifying area patterning. Finally, we will describe studies showing that area size dictates performance at modality-specific behaviours.

EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors.

Genetic studies of neocortical area patterning are limited, because mice deficient for candidate regulatory genes die before areas emerge and have other complicating issues. To define roles for the homeodomain transcription factor EMX2, we engineered nestin-Emx2 transgenic mice that overexpress Emx2 in cortical progenitors coincident with expression of endogenous Emx2 and survive postnatally. Cortical size, lamination, thalamus, and thalamocortical pathfinding are normal in homozygous nestin-Emx2 mice. However, primary sensory and motor areas are disproportionately altered in size and shift rostrolaterally. Heterozygous transgenics have similar but smaller changes. Opposite changes are found in heterozygous Emx2 knockout mice. Fgf8 expression in the commissural plate of nestin-Emx2 mice is indistinguishable from wild-type, but Pax6 expression is downregulated in rostral cortical progenitors, suggesting that EMX2 repression of PAX6 specification of rostral identities contributes to reduced rostral areas. We conclude that EMX2 levels in cortical progenitors disproportionately specify sizes and positions of primary cortical areas.

Cloning and cortical expression of rat Emx2 and adenovirus-mediated overexpression to assess its regulation of area-specific targeting of thalamocortical axons.

A goal of this study was to use recombinant adenovirus (AdV) to ectopically express Emx2 in the embryonic neocortex as a gain-of-function approach to study its role in the area-specific targeting of thalamocortical axons (TCAs), using the rat as a model. First, we cloned the cDNA for the full-length coding region of rat Emx2, a homologue of Drosophila empty spiracles. We also used this sequence to define the full-length coding region of mouse Emx2 from genomic DNA. Our analysis of Emx2 expression shows that in rat, as reported in mouse, Emx2 is expressed in high caudal to low rostral, and high medial to low lateral, gradients across the cortex throughout cortical neurogenesis, and expression is primarily restricted to progenitors in the neuroepithelium. We also carried out an analysis of the distribution of cells infected with a replication defective recombinant type 5 adenovirus (AdV) containing a CAG/LacZ expression construct, following an injection into the lateral ventricle of the cerebral hemisphere at different stages of embryonic cortical neurogenesis. AdV-infected cells are broadly distributed tangentially, but their laminar distribution is differentially restricted and reflects the temporal sequence of generation of cortical neurons. This finding indicates that the AdV predominantly infects progenitors in the ventricular zone, which leads to a preferential labeling of their immediate progeny, and infects cells that have recently become postmitotic and have yet to move far from the ventricular surface. We then show that AdV-mediated ectopic Emx2 expression results in aberrant intracortical pathfinding and areal targeting of TCAs from the dorsal lateral geniculate nucleus. These findings indicate that EMX2 imparts positional information normally associated with caudal cortical areas, such as the primary visual area, that influences the area-specific targeting of TCAs. These results are consistent with a role for EMX2 in areal specification of the neocortex as suggested by recent analyses of Emx2 null mutants.