Quantifying global CG methylation in regenerative Zebrafish caudal fin and Xenopus tail buds.

Abstract

Epigenetics is a control mechanism that creates stable alterations in gene expression thatdoes not require underlying changes in DNA sequence. One of the most iconic and prevalent epigenetic mechanisms involves CG methylation, the erasure of which is a defining characteristic of naïve embryonic stem cells, the most basal stem cells of the mammalian body. Surprisingly, the relationship between developmental potency and global DNA demethylation has not been fully explored in a wide range of vertebrate species. Due to classical experiments analysing DNA methylation in all cytosine contexts, it is often assumed that high levels of CG methylation must exist in a wide range of vertebrate somatic tissue, however, this is yet to be formally proven. Moreover, global erasure of CG methylation in well studied non-mammalian vertebrate models of tissue regeneration (such as Zebrafish and Xenopus) has also not been completed.

The Zebrafish caudal fin is a simple appendage capable of full regeneration. After amputation, a highly proliferative undifferentiated tissue known as ‘blastema’ forms at the injury site, surrounded by a protective apical epithelial cap (AEC). Although somatic tissues are known to contain generally high levels of CG methylation (70-80%), a recent study by Hirose et al., (2013) using dot blot assays and immunohistochemical analysis indicated that Zebrafish blastema undergoes global demethylation during regeneration. However, the methods used by Hirose et al., (2013) could not assess CG methylation in isolation of other cytosine contexts, and even then, the results were semi-quantitative. The larvae of the amphibian Xenopus laevis are also an excellent model of regeneration as they are capable of fully regenerating their tail buds following amputation. Like Zebrafish, Xenopus also produce blastema and have been used in previous studies to identify pathways such as Wntsignalling and shh involved in regeneration, but detailed analysis of global CG methylation dynamics in this system is lacking.

The first aim of this study was to quantify CG methylation from a range of vertebrate species to determine general methylation seen in somatic tissue. The second aim was to test the work of Hirose et al., (2013) by quantifying the levels of CG methylation in regenerating Zebrafish caudal fins using a fully quantitative post-bisulphite adaptor tagging (PBAT) method as developed by Miura et al (2012). The third aim was to quantify Xenopus regenerating tail buds at a range of time points after tail excision to determine the generality of CG methylation in regenerative capability. I did not find any loss of methylation in Zebrafish tail fin as described by Hirose, a surprising result even considering the difficulties of accurately assessing global methylation levels in a heterogeneous stem cell system. A small, but significant methylation loss of 1-2% was seen in Xenopus samples after 24 h regeneration, implying that modest demethylation can occur during regeneration. However, this is not indicative of global epigenetic erasure as occurs in the pluripotent stem cells of mammals.

In order to extend this work, future research should be done on isolated Xenopus blastema using cryosectioning and laser capture. Furthermore, it may be valuable to examine Zebrafish tail fins over a range of regenerative time points after initial clipping to help determine the mechanisms and genes involved during regeneration.