A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development [ATAC-seq]

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that decrease neural crest formation, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits PI3K/Akt signaling specifically in FGF-stimulated cells, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest development. Overall design: To evaluate the effect of CAPE on chromatin accessibility in zebrafish neural crest cells, we treated sox10:GFP transgenic embryos with 10 micromolar CAPE or DMSO control at 2 ss, then sorted GFP+ cells at 17 or 20 ss for analysis by ATAC-seq.

神经嵴（neural crest）是一类动态的祖细胞群体，起源于神经外胚层与非神经外胚层的边界区域。成纤维细胞生长因子（FGF）、Wnt以及骨形态发生蛋白（BMP）在神经板边界的诱导作用已得到充分证实，但后续神经嵴发育所需的信号通路仍未得到充分阐明。
本研究针对原代斑马鱼胚胎培养体系开展化学筛选，以crestin:EGFP的表达作为检测指标，筛选能够抑制神经嵴形成的化合物。
研究发现，天然产物咖啡酸苯乙酯（CAPE）可通过降低Sox10的活性，干扰神经嵴的基因表达、细胞迁移以及黑素细胞分化过程。
CAPE仅在经FGF刺激的细胞中抑制磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路；经CAPE处理的胚胎所出现的神经嵴发育缺陷，可通过组成型激活的Akt1得到挽救。
通过组成型激活的张力蛋白同源物（PTEN）抑制Akt活性，同样会降低crestin的表达水平以及Sox10的活性。
本研究鉴定出Akt是神经嵴发育所必需的全新细胞内信号通路。
总体实验设计：为评估CAPE对斑马鱼神经嵴细胞染色质开放状态的影响，我们将sox10:GFP转基因胚胎在2体节期（somite stage, ss）分别用10 μmol/L的CAPE或二甲基亚砜（DMSO）对照处理，随后在17体节期或20体节期分选GFP阳性细胞，通过转座酶可及性测序（ATAC-seq）进行分析。