The division of the neocortex into distinct areas is the fundamental way in which neocortical functions and connectivity are organized. How the neocortical area map forms during development is thus a basic question in developmental neurobiology. Mistakes in the map cause behavioral deficits in mice and may underlie human mental health disorders. A major question is when, and how, distinct areas appear in development. Evidence that area-specific signals provide final guidance to thalamocortical afferents (TCAs) suggests distinct areas exist before TCAs enter the NP. The proposed work is designed to test this hypothesis, and potentially resolve a long debate concerning the relative importance of afferent axons versus mechanisms intrinsic to the cortex in setting out the neocortical area map. First I propose to investigate area pattern formation in an unusual mouse mutant brain. Mice that constitutively lack the transcription factor gene, Barhl2, completely lack a thalamus. This mouse represents a striking new resource for investigating the influence of the thalamus on neocortical development. I propose to investigate area boundaries in the Barhl2 null mouse using gene expression that marks out area boundaries in wildtype mice, and by examining cortical connectivity. Determining whether areas develop in the Barhl2 null mouse will provide fresh evidence that the area map does, or does not depend on thalamic innervation for its formation. Prompted by strong evidence that embryonic areas attract appropriate thalamic input, I further propose to search for genes selectively expressed in presumptive areas before TCA entry into neocortex. Sample tissue will be collected from a mouse in which TCAs heading for primary sensory areas express green fluorescent protein (GFP), which marks out primary sensory areas, including primary sensorimotor cortex (S1). In a pilot study, presumptive S1 was detectable at the required age by a thick bundle of GFP-positive axons beneath the cortex proper, waiting to enter S1. Primary motor cortex (M1) is rostral to S1 and markedly lacked GFP- positive TCAs beneath it. Samples of S1 and M1 tissue will be prepared for RNA-Seq and delivered to the Genomics Core Facility at the University of Chicago. Members of the Core will carry out paired-end sequencing at a depth of 80 million reads. This depth will assure detection of transcripts with low expression levels, and RNAs encoding different protein isoforms. I chose this depth to increase sensitivity over previous searches for area-specific genes in developing mouse neocortex. Differentially expressed genes will be validated with qPCR and expression of genes of interest will be examined in both wildtype mice and Barhl2 mutants. Expression in the Barhl2 mutant mouse NP would verify the independence of expression from TCA contact. Identifying genes selectively expressed in presumptive areas would be a major step towards understanding how the area map develops, and set the stage for investigating the functions of these genes, and how their expression is confined to particular areas.
The neocortex controls higher brain functions, such as perception, memory and planning, which are distributed among many distinct areas. Two classic models of how the neocortical area map is patterned in development are once again at odds, and the proposed research is designed to settle these differences. Results will be relevant to human disorders caused by defects in cortical development, including the Autism Spectrum Disorders.
