We have discovered a requirement for a novel connexin gene (frm-cx) in specification of the anterior neural plate in the invertebrate chordate Ciona. (The connexin proteins make gap junctions.) In embryos from the frimousse (frm) mutant line, the anterior neural plate is transfated to epidermis. The anterior neural plate of Ciona gives rise both to brain and to two other non-neural tissues, the mouth and adhesive palp. The initial FGF-dependent induction of the anterior neural plate appears to be normal in the mutant, as judged by expression of early response genes. However, the expression of these genes is quickly lost, and the anterior neural plate derivatives become incorporated into the epidermis and express epidermal genes. We hypothesize that the frm-cx gene product is required for maintaining neural plate identity by allowing for intercellular communication between the newly induced anterior neural plate cells. We also hypothesize that that frm-cx expression is an early response to anterior neural induction. Experiments in the first two Specific Aims of this proposal will test these hypotheses. Because some connexins have biological activities distinct from their channel forming role, in the first Specific Aim we will investigate the necessity of gap junction communication within the newly-induced neural plate. In further experiments to determine whether the anterior neural plate makes a distinct communication compartment within the embryo, we will assess the cellular connectivity of cells using a photoactivatable gap junction-permeable fluorescent dye. Additional experiments will investigate whether over expression of frm-cx is sufficient to cause expansion of the neural plate. In the second Specific Aim we will place frm-cx within the regulatory context of anterior neural induction by determining its epigenetic relationship to other known anterior neural transcription factors. To test for the necessity of these factors we will isolate a minimal regulatory element from the frm-cx gene. In this element we will identify and then mutate potential transcription factor binding sites. The final Specific Aim will characterize a new mutant line, bugeye (bug) that disrupts the same set of cells affected in the frm mutant. In the bug mutant neural tube folding and closure (neurulation), but not specification, are disrupted. Specific experiments will fully characterize the mechanical defects in neurulation in bug mutants by confocal imaging and 3-dimensional modeling. The final goal of this Specific Aim is to postionally clone the bug mutant using established methods.
The formation of the central nervous system is a critical component of human development. This proposal will examine the cellular and molecular mechanisms of early events in brain development using an animal that models human brain development.
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