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

5‘-甲基胞嘧啶(5mC)是一种转录抑制的表观遗传标记，在脊椎动物中富含CpG二核苷酸。Tet蛋白(Tet 1、Tet 2和Tet 3)将5mC氧化为5‘-羟甲基胞嘧啶(5hmC)，然后是5’-甲酰胞嘧啶(5fC)和5‘-羧基胞嘧啶(5caC)1。5fC和5caC被胸腺嘧啶-DNA糖基酶(TDG)2切除。这一过程被称为活性DNA去甲基化。它防止多能性因子表达的异常抑制，并允许抑制谱系特定因子的表达--这是调节干细胞分化和器官发生所必需的。值得考虑的是，被动去甲基化(与细胞分裂有关)也可能对胚胎发育有重要贡献；然而，相对于主动去甲基化的重要性尚不清楚。Tet表达、5hmC存在和多能性之间的关系在脊椎动物物种中不同-在小鼠和斑马鱼之间存在巨大差异(见图1和2)3。这表明Tet蛋白在系统发育中的不同发育作用。这些变异存在的原因及其发育功能的差异尚不清楚。在小鼠胚胎中，单个Tet基因的敲除会导致不同严重程度的表型，TET1对发育和生存能力造成非常轻微的影响4，TET3在植入后是致命的5。TDG基因敲除也是LETH6。Tet1和Tet2的组合敲除导致了严重的表型4。关于TET1和TET3基因敲除对斑马鱼胚胎发育的影响，以及对TDG的影响，几乎没有可靠的数据。然而，现有的证据表明，小鼠4和斑马鱼7的Tet缺失胚胎在表型上既有相似之处，也有不同之处。在斑马鱼中建立CRISPR/Cas9介导的Tet 1，2，3和TdG基因敲除系是阐明这些酶的功能和它们沉积/加工的表观遗传标记的关键一步。

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.