Coiled coil protein 1, a novel gene downstream of FGF2 expressed in the developing brain

Pellicano, Francesca (2006) Coiled coil protein 1, a novel gene downstream of FGF2 expressed in the developing brain. PhD thesis, University of Glasgow.

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Fibroblast Growth Factor 2 (FGF2) plays an important role in modulating cell proliferation and differentiation in the developing central nervous system. It has been shown that FGF2 affects the transcription of several genes. In this study, a previously uncharacterised gene downstream of FGF2, named 'coiled coil protein 1' (ccpl), was identified and characterised. The expression at the tissue levels, particularly, in the brain, and cellular localization of ccp1 was investigated. In addition, functional analysis was carried out by overexpression and RNAi approaches. Ccp1 was identified by previous microarray analysis of primary cortical neuron culture derived from mouse embryonic brains stimulated by FGF2. Microarray data showed that ccp1 was up-regulated by 2.3-fold upon FGF2 stimulation. The study in this thesis showed that FGF2 up-regulated ccp1 both at the transcript and protein levels. Ccp1 cDNA is 1697 nucleotides long and the protein size is predicted to be 180 amino acids. Bioinformatic analysis revealed ccp1 homologues in various species, including human, rat, zebrafish, and drosophila. The gene is located in mouse chromosome 1,1B and its human homolog in chromosome 2q21.2. Next, in order to gain insight into the physiological role of ccp1 in mouse forebrains, its expression pattern was further analysed using in situ RNA hybridisation. Ccp1 was highly expressed early in the brain development in a ventrolateral area of the cortex and in the specific layer of the dorsal cortex. The pattern of ccp1 expression reflects some of the aspects of the tangential and radial migration that occurs at the early stages of brain development. A number of conserved domains are contained within the amino acid sequence of ccp1, including two coiled coil regions, a leucine zipper and two nuclear exporting signals (NES). These domains are related to specific protein functions and they may provide information in determining a role of ccp1 in the cell and in animal development. Analysis of enhanced green fluorescent protein (EGFP)-tagged ccp1 expressed in cells demonstrated that ccp1 was localised to the cytoplasm in a punctate pattern. Finally, ccp1 function was investigated using a retroviral overexpression system and RNAi in vitro. Ccp1 overexpression induced cell proliferation in the absence and presence of serum. Silencing of ccp1 expression by RNAi decreased cell proliferation, which provided further evidence for the role of ccp1 in this process. Incorporation of the S phase marker, 5-Bromo 2-Deoxyuridine (BrdU), has indicated that ccp1 enhances G1-S transition. To address the signalling mechanism by which ccp1 induces proliferation, involvement of 'Mitogen-Activated Protein Kinase' (MAPK) pathway was investigated. The addition of the 'MAPK or ERK Kinase' (MEK) inhibitor, U0126, blocked cell proliferation in cells overexpressing ccp1 in the presence of serum. The data suggested that MAPK pathway may be at least partially responsible for the ccp1-induced cell proliferation. The data presented in this thesis form the bases for functional study of ccp1 in vivo in the future, and provides novel insights into the mechanism of cell proliferation by growth factors.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Tomoko Iwata
Keywords: Neurosciences, developmental biology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-74074
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Aug 2019 10:36
Last Modified: 07 Aug 2019 10:39
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