Studying the biological functions of the TET and TDG proteins during zebrafish embryonic development

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5'-methylcytosine (5mC) is a transcriptionally repressive epigenetic mark, enriched at CpG dinucleotideswithin vertebrates. The TET proteins (TETs 1, 2 and 3) oxidize 5mC to 5'-hydroxymethylcytosine (5hmC) thensubsequently to 5'-formylcytosine (5fC) and 5'-carboxylcytosine (5caC)1. 5fC and 5caC are excised bythymine-DNA glycosylase (TDG)2. This process is known as active DNA demethylation.Active demethylation is essential during embryonic development. It prevents aberrant repression ofpluripotency factor expression and allows for the de-repression of lineage specification factors' expression -essential for regulating stem cell differentiation and organogenesis. Worth considering is that passivedemethylation (linked to cell division) may contribute significantly to embryonic development too; however theimportance relative to active demethylation is unknown.The relationship between TET expression, 5hmC presence and pluripotency varies across vertebrate species- big differences exist between mice and zebrafish (see Figs.1&2)3. This suggests a phylogenetically varieddevelopmental role for the TET proteins. Why these variations exist and the differences in theirdevelopmental functions are unknown.In mouse embryos, the knockout of individual TET genes leads to phenotypes of varying severity, with TET1causing a very mild impact upon development and viability4 and TET3 being lethal post-implantation5. TDGknockout is also lethal6. Combinatorial knockout of TETs 1&2 results in a severe phenotype4. Little reliabledata has been obtained regarding the consequences of TET1 and TET3 knockout upon zebrafish embryonicdevelopment and none for TDG. However available evidence suggests both phenotypic similarities anddifferences exist between mouse4 and zebrafish7 TET-depleted embryos.Generating CRISPR/Cas9-mediated knockout lines for TETs 1,2,3 and TDG in zebrafish is a crucial step inelucidating the functions of both the enzymes and the epigenetic marks which they deposit/process.