Newt Lens Regeneration: Role of Oct-4 in Newt Regenerating Tissue and Proteome Analysis of Regeneration Competent Vs. Regeneration Incompetent Cells

Newts possess remarkable ability for regenerating various body organs and parts.Lens regeneration in newts is achieved by transdifferentiation of dorsal iris to lens cells.This ability of transdifferentiation is exhibited only from the dorsal iris and not by the ventral iris.In order to study the process of transdifferentiation during lens regeneration and the characteristics that allow lens regeneration only from the dorsal iris, we studied the role of reprogramming factor; oct-4 during newt lens regeneration and analyzed the proteome of dorsal iris cells vs. ventral iris cells respectively.During transdifferentiation, the dorsal iris has to change its cellular identity in order to achieve identity of lens cells.Such mechanisms are also observed during generation of iPS cells and previous studies have shown that these two processes share some similarities.It was observed that one of the crucial reprogramming factor oct-4 was absent in the regenerating tissues of newt.Thus, !iv! we hypothesize that absence of oct-4 during newt lens regeneration restricts the IPE cells to only a particular fate (lens cells).We over expressed oct-4 in the newt IPE cells and studied its effect on transdifferentiation potential of these cells.The results showed oct-4 inhibits the process of transdifferentiation in newt dorsal IPE cells by interfering with sox-2 and pax-6, factors important for synthesis of crystallin during lens formation.Thus, oct-4 absence during newt lens regeneration allows the dorsal iris to change its cellular identity to regenerate the lost lens.Further, we also examined the proteome of cultured regeneration competent dorsal IPE cells and regeneration incompetent ventral iris cells with a goal to identify any molecular marker specific to either dorsal or ventral IPE cells.The proteome was studied using newt's de novo assembled transcriptome as a reference library.The results did not reveal any specific markers for either of the cell population.However, on comparison of in vitro proteome to in vivo proteome of 0, 4 and 8 day regenerating iris identified some factors related to gene regulation and retinoic acid synthesis with higher expression in the dorsal IPE cells compared to the ventral IPE cells.!