Anatomy and Neuroscience - Theses
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ItemCloning and characterisation of gripe: a novel interacting partner of e12 during brain development( 2002-10)The mammalian cerebral cortex is a remarkable product of brain evolution, and is the structure that most distinctively delineates the human species from others (Northcutt and Kaas, 1995; Rakic, 1988). Neurons in the adult brain are organised into cytoarchitectonic areas, defined by distinct biochemical, morphological and physiological characteristics (Rakic 1988). Remarkably, this complex structure is generated from a simple neuroepithelium. What are the signalling mechanisms that direct neuron formation and subsequent functional-parcellation of the cerebral cortex? Key to the study of this process is an understanding of neuronal fate determination. Available evidence demonstrates an intrinsic programming potential by neuronal progenitors within subdomains of the developing cerebral cortex that is instructive for proper corticogenesis. These regional domains are demarcated by expression of certain transcription factors, including members of the Helix-Loop-Helix (HLH) family of proteins. The HLH family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and haematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. While studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD (Lee et al., 1995; Miyata et al., 1999; Liu et al., 2000) and Mash1 (Casarosa et al., 1999) are intimately involved in neuronal fate determination and terminal differentiation, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. Originally discovered as an important regulator of lymphopoiesis, expression studies revealed its widespread expression in proliferative zones of multiple nascent organs of the embryo, including the developing cerebral cortex; implying a role for E12 in development of the nervous system. Since the function of E12 is, in part, coded by its capacity for protein dimerisation, a search was undertaken for binding partners in developing mouse brain, and using a yeast 2-hybrid assay. Yeast 2-hybrid prey libraries were constructed using complementary DNA (cDNA) isolated from embryonic mouse forebrain tissue at early (embryonic day e11.5) and peak (e15.5) stages of neurogenesis. Screening of these libraries for binding partners to an E12 bait resulted in cloning of HLH factors, such as Mash1, NSCL and Id2. Importantly, a novel binding partner, named GRIPE, was cloned as a novel GAP Related Interacting Protein to E12. GRIPE binds to the HLH region of E12, and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidently detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were reconfirmed through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein whose dimerisation with E12 has important consequences for cells undergoing neuronal differentiation. A model is proposed to suggest how neurogenic HLH proteins that dimerise to E12 may promote signalling cascades driving early neuroblast differentiation.