Primer sequences for RT-qPCR analyses.

Germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomic analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

胚层特化是多能细胞获得谱系特异性命运的关键发育转变。本研究鉴定出母源转录因子Foxi2与Sox3为非洲爪蟾外胚层胚层特化的关键主调控因子。在预定内胚层组织中异位共表达Foxi2与Sox3，可诱导外胚层标记基因的表达，并抑制中内胚层标记基因的表达。转录组分析显示，Foxi2与Sox3可协同且独立调控数百个外胚层靶基因。在早期卵裂阶段，Foxi2与Sox3预先结合至关键顺式调控模块（cis-regulatory modules, CRMs），标记后续招募Ep300并促进组蛋白H3赖氨酸27乙酰化（histone H3 lysine 27 acetylation, H3K27ac）沉积的位点，从而塑造外胚层基因组的表观遗传图谱。上述CRMs在外胚层特异性超级增强子（super-enhancers, SEs）中高度富集。本研究结果凸显了外胚层超级增强子相关CRMs在精准且稳健的外胚层基因激活中的关键作用，确立了Foxi2与Sox3作为外胚层谱系特化的核心调控架构师。