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The expression and regulation of Prod 1 in salamanders during regeneration

Shaikh, N.; (2011) The expression and regulation of Prod 1 in salamanders during regeneration. Doctoral thesis, UCL (University College London).

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Abstract

Urodele amphibians have a remarkable ability to regenerate and are the only adult vertebrates capable of regenerating their limbs. Mammals have a limited ability to regenerate, with urodeles and premetamorphic frogs being the only vertebrates known to regenerate this complex appendage. After amputation of a salamander limb, mature differentiated cells at the cut site de-differentiate to produce a heterogeneous population of proliferating cells which re-differentiate to form the lost tissues of the regenerate. The cells at the amputation site possess a property called positional identity such that they regenerate only the structures distal to their level of origin. This property of distal cells can be respecified by treatment with retinoic acid (RA) and synthetic retinoids. The urodele limb is a powerful model in which to study positional identity and pattern formation in a regenerating limb. Positional information is apparently encoded in part by a small glycolipid-anchored protein called Prod 1, which is expressed in a graded manner on the proximodistal axis. If the level of expression of Prod 1 in distal cells is raised they now behave like proximal cells. This activity is also shown by the Meis homeoproteins which are implicated as determinants of proximodistal identity in limb development in drosophila, chick and mouse, as well as in limb regeneration in the axolotl. This raises the question of whether Meis proteins regulate Prod1 expression. Altering the expression of Prod 1 at the site of injury alters the positional information encoded by the cells at the site. Over-expression of Prod 1 leads to proximalisation of the limb elements; distal blastemas grafted to a proximal amputation site leads to intercalary regeneration. The current hypothesis for is, the cells detect a disparity in the level of Prod 1 leading to limb regeneration from the proximal portion of the limb stump and the distal grafted blastema giving rise only to the distal structures. The work in this thesis has investigated the regulation of Prod 1 gene at the promoter level, firstly by use of RT-PCR to assess where in the limb Prod 1 is expressed and how RA affects Prod 1 expression in the skin and mesenchyme. These experiments revealed that Prod 1 expression was predominantly in the dermal layer of the skin. The promoters for Prod 1 were isolated from newt and axolotl genomic DNA, and a dual luciferase reporter assay was used to measure normal and mutant promoter activity after transfection in cell culture and in the animal. Meis transcription factor binding sites were found in both newt and axolotl Prod 1 promoter sequences and the effects of mutating the sites in the axolotl case was analysed in cultured salamander cells, and after transfection of axolotl limb blastemas. Intact Meis binding sites are required for normal promoter activity in cultured cells and in proximal blastemal cells.

Type:Thesis (Doctoral)
Title:The expression and regulation of Prod 1 in salamanders during regeneration
Language:English
Additional information:Permission for digitisation not received
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology

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