Stark, David Robert; (1997) A molecular analysis of amphibian limb regeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The regeneration of the urodele limb is a striking phenomenon. The molecular mechanisms which regulate growth and pattern formation during limb regeneration, however, are poorly understood. The aim of this thesis was to isolate genes and gene products that might play key roles in patterning during regeneration. A protocol was developed using avidin-biotin technology and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualise the cell-surface proteins expressed in the regenerate, and was used to investigate the potential role of the cell surface in the morphogenesis of regeneration. In a separate study, a gene was characterised, whose function during Drosophila development suggested that it might be involved in pattern formation during regeneration. The Notch gene was chosen because it was a cell-surface signal receptor, it could function as an adhesion molecule, and it was known to mediate cell fate determination in the Drosophila neuroectoderm. The expression pattern of newt Notch did not support a role for the gene in regulating pattern formation. However, the surprising result that the gene was expressed in differentiated cultured newt cells prompted a study of newt Notch function using a constitutively active Notch construct. Cultured myogenic newt cells remained able to differentiate in the presence of Notch signalling. Since Notch is not known to be expressed in the differentiated cells of other organisms, its expression in differentiated newt cells may be related to the capability of these cells to dedifferentiate. During the period of my study, vertebrate homologues of the Drosophila hedgehog gene were shown to play a key role in many developmental processes including limb development. I have therefore investigated whether members of this family are expressed during limb regeneration and are regulated by retinoic acid (RA), since this derivative induces proximodistal (PD) duplications in regenerating limbs. A homologue of Xenopus banded hedgehog and avian and murine Indian hedgehog was uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration, and is up-regulated by RA. In addition, the newt hedgehog gene (N- bhh) was uniformly expressed in the early limb bud of the embryo. Since neither bhh nor Ihh has been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles. The expression of this urodele hedgehog in newts suggests a significant difference between the development of the limb in urodeles and other vertebrates.

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