Petiot, Anita; (2001) Fibroblast growth factor receptors and skeletogenesis. Doctoral thesis (Ph.D.), University College London (United Kingdom).

Text Fibroblast_growth_factor_recep.pdf Download (29MB)

Abstract

Mutations causing craniosynostosis, a condition characterised by premature fusion of the cranial sutures, as well as dwarfism syndromes have been mapped to the fibroblast growth factor receptors (FGFR) genes. Most of the mutations result in constitutive activation of these tyrosine kinase receptors. To gain further insight into the functional consequences of these mutations during skeletogenesis, biological in vitro assays were developed. The craniofacial skeleton is derived mainly from cranial neural crest (NC) cells. Previous studies showed that, with 10 ng/ml FGF2, cultured primary quail NC cells differentiate into cartilage in vitro and, after several weeks, into bone. In contrast, 1 ng/ml FGF2 leads only to cell proliferation. To further investigate these findings the expression of specific marker genes such as collagen type II, a marker of cartilage differentiation, and osteopontin (a bone marker) was assessed using RT-PCR on NC cells in the presence of either 1 or 10 ng/ml FGF2. To draw a parallel with different stages of cell maturity expression analysis of these genes was also carried out on an osteogenic cell line, MC3T3-E1, which has a fibroblastic appearance when sub-confluent, then condenses and differentiates into bone. The molecular findings were consistent with precocious bone differentiation upon FGF2 exposure. Mutant forms of the human FGFRs genes were transfected into NC cells using a lipid-mediated integrin targeted system. A few cultures transfected with the FGFR1 or FGFR2 mutant differentiated into cartilage, in the presence of a low concentration of FGF2, as characterised by alcian blue and RT-PCR analysis. However, due to a lack of reproducibility of this finding, the method of electroporation in ovo was used as an alternative means of transfecting NC cells. Cultures established from these cells showed an effect of mutant FGFR expression in increasing the incidence of differentiation and expression of osteopontin. In parallel, the MC3T3-E1 cell line was stably transfected with the mutant FGFRs and revealed an earlier onset of osteogenesis in the absence of FGF2. These results correlate with the clinical situation and demonstrate the activation of FGFRs in a biological system.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item