Schofield, Julian Newby; (1992) In vitro analysis of chick limb development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The limb bud develops as a mesenchymal outgrowth beneath an inductive epithelium known as the apical ectodermal ridge (AER). Cell differentiation occurs in a proximal-distal direction, whilst proliferation and thus generation of new tissues occurs at the distal tip of the bud in a region known as the progress zone. This thesis describes experiments using the high-density micromass culture system to investigate some contemporary issues in limb development. Ectoderm grafted onto micromass cultures inhibits the formation of cartilage. This effect was investigated and found not to be mediated by components of the extracellular matrix. AERs grafted onto micromass cultures result in the formation of a mesenchymal outgrowth. This effect was shown to involve mitogenic stimulation and secretion of an extensive extracellular matrix. The ability of cells from different proximo-distal locations cultured for different periods of time to respond to the effect of a grafted AER was investigated. The influence of different culture media was also analysed. The results suggest that the ability to respond to factors produced by the AER is lost in cells that have progressed too far along the pathway of chondrogenic differentiation. Peptide growth factors have been identified throughout embryogenesis and have marked effects upon differentiation and proliferation of cells in vitro. The effects of several growth factors known to be present during limb development, upon the differentiation and proliferation of limb bud cells in micromass culture has been examined. bFGF stimulated proliferation and cartilage differentiation whilst inhibiting muscle differentiation. TGF-β stimulated cartilage differentiation and blocked the effects of bFGF on proliferation and muscle differentiation. Homeobox-containing genes have been shown to encode positional information during Drosophila embryogenesis and are increasingly implicated in vertebrate development. Expression of the mouse homeobox genes Hox-7.1 and Hox-8.1 in the limb bud is described. Transcripts were retained in vitro in cultured tips and in micromass cultures prepared from mouse limb bud cells. In addition the distribution of the chick homologue of the Hox-7.1 gene in normal limb development and in micromass culture was also investigated. In vitro transcripts were retained in proximal cell cultures but not in distal cell cultures. However transcription of Hox-7.1 was induced in distal cultures beneath a grafted AER. Finally the distribution of the retinoic acid receptor beta during normal limb development has been investigated. A high level of RAR-β transcripts were continually expressed in proximal regions, thus suggesting a role for this receptor in the development of the shoulder. In reciprocal proximal distal grafting experiments RAR-β behaved in a position-dependent manner, with transcripts consistently downregulated at the distal tip of the bud. Expression of RAR-β in micromass culture was consistent with the behaviour of RAR-β in vivo. The results obtained in micromass culture appear to reflect in vivo development, and thus support the use of this culture system as an in vitro model for aspects of early limb development.

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