AbstractWithin the ventral midbrain of the developing vertebrate embryo there is a transient area of conserved gene patterning called the midbrain arcs, this patterning influences the formation and position of nearby nuclei, as well as the position of the MLF (medial longitudinal fascicle) axon tract in the early axon scaffold. A hypothetical regulatory loop has been identified between the genes Nkx1.2 and Emx2 during midbrain arc patterning and this project aimed to identify if a highly conserved non-coding sequence named Nkx1.2.1 acted as an enhancer for the Nkx1.2 gene, and bound directly with the Emx2 protein.
Electroporation of a reporter construct containing the Nkx1.2.1 sequence in the chick embryo identified the element was active specifically in the ventral midbrain during the time of midbrain arc patterning. The Nkx1.2.1 sequence was then analysed for binding affinity with the Emx2 protein using EMSA. The full Emx2 sequence could not be produced in a soluble form, but two artificial sub-forms of the protein were produced. The closest binding affinity identified was a value of 0.2 KdμM, compared to the control experiment using a non-specific sequence of DNA of 2.6 KdμM.
|Date of Award||2016|
|Supervisor||Frank Schubert (Supervisor) & Matt Guille (Supervisor)|