The Role of Transforming Growth Factors Type Beta During Murine Embryogenesis

Millan, Fergus A (1992) The Role of Transforming Growth Factors Type Beta During Murine Embryogenesis. PhD thesis, University of Glasgow.

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Abstract

The role of transforming growth factors type beta (TGF-beta) was investigated during murine embryogenesis. There are three known mammalian TGF-beta genes. The developmental pattern of expression of TGF-beta RNA has previously been elucidated by in situ hybridisation and the localisation of TGF-beta1 protein by immunohistochemistry. Based on this data it has been suggested that TGF-beta1 acts in both a paracrine and autocrine role during development. The expression of both TGF-beta2 and TGF-beta3 RNA has been observed in murine embryos by Northern analysis. In order to understand the role of these two genes, individually, and in relation with each other, the developmental regulation of all three genes was investigated by in situ hybridisation using mouse specific probes on serial mouse embryo sections from 6.5 days p.c. to 16.5 days p.c.. A comparison was made of the data concerning the temporal and spatial expression patterns of TGF-beta1, TGF-beta2 and TGF-beta3 RNA, and the role of each gene discussed. The pattern of expression of each of the TGF-beta RNAs was compared with available protein localisation data. TGF-beta2 RNA was found to be expressed in the early embryo, where It was localised to the myocardium of the developing heart. This pattern of expression persisted, although it was regionally restricted to the myocardium overlying the presumptive heart valves. TGF-beta1 RNA had previously been localised to the endothelial cells of the heart valves. It was suggested that TGF-beta2 was providing the signal which induced the endothelial cells, overlying the myocardial tissue of the presumptive heart valve, to undergo a epithelial/mesenchymal transformation to form valve cushion tissue. TGF-beta2 RNA was also expressed In prechondrogenic blastemae and growth zones of developing long bones. There was also widespread expression of TGF-beta2 RNA in epithelial cells, including many which were undergoing differentiation. These included the nasal, retinal and otic epitheliunn. TGF-p2 RNA was also transiently expressed in ventral horns of the neural chord at a time of motor neurone differentiation. TGF-beta3 RNA had a more restricted pattern of expression than TGF-beta2. TGF-beta3 RNA was expressed in intervertebral discs aniagen, cartilaginous capsules of the ear and nasal septum. TGF-beta3 RNA was also expressed in epithelial tissue. The medial edge epithelium of the developing palate was an abundant source of TGF-beta3 RNA both during and after fusion of the palates. The three TGF-beta genes were often expressed in the same developmental system, but rarely in the same cell or tissue. The lung develops as simple endodermal tubes which are induced to branch distally, the lung expands as rapid growth occurs at the terminal end buds and in the surrounding lung mesenchyme. The epithelial cells that line the respiratory tubes change in morphology at defined points, from a columnar to a simple cuboidal shape. The simple cuboidal epithelium eventually becomes more flattened in shape, forming the alveolar ducts. The change in epithelial morphology is marked by a change in expression from TGF-beta3 RNA seen in the columnar epithelium to TGF-beta2 RNA observed in the simple and flattened epithelium. TGF-beta1 RNA is expressed in the mesenchyme of the lung, and TGF-beta1 protein has been localised underneath the bronchiolar epithelium and at the clefts of the bronchioles. This data suggests there is close co-operation between the three TGF-beta Isoforms during morphogenetic events. The role of the TGF-beta genes remains unclear. The interactions of TGF-beta isoforms with a variety of polypeptides In the ECM has been elucidated from in vitro analysis, this data, combined with RNA expression and protein localisation data, has not clarified the specific roles of the TGF-beta isoforms. To determine a complete picture of the role of TGF-beta isoforms during development, dominant-negative mutations were created and analysed in vitro. The dimeric structure of mature TGF-beta isoforms lends itself to the production of dominant-negative mutations which can bind to wild-type monomers of TGF-beta, resulting in the formation of heterodimers, which reduce biological activity of the selected TGF-beta isoform. Mutations of human TGF-beta2 were generated which were sub-cloned into the eukaryotic expression vector pH3M and analysed in vitro. The first mutant was generated by a deletion of part of the human TGF-beta2 cDNA sequence which encodes the signal peptide sequence at the N-terminal. The second mutation was generated by site-directed mutagenesis of the human TGF-beta2 cDNA sequence which encodes the proteolytic cleavage site. The constructs were transiently transfected into COS-7 cells and the conditioned media assayed. The biological activity of the mutant constructs were compared to biological activity of constructs which contained the wild-type human TGF-beta2 cDNA.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Rosemary Akhurst
Keywords: Developmental biology
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75302
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:16
Last Modified: 19 Nov 2019 21:16
URI: http://theses.gla.ac.uk/id/eprint/75302

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