Deficiency in SUMOylation, by injection of Gam1 mRNA into one cell embryos, leads to differential expression of genes in Xenopus. Deficiency in SUMOylation, by injection of Gam1 mRNA into one cell embryos, leads to differential expression of genes in Xenopus

Background: Adenovirus protein Gam1 triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal levels, enabling an examination of the role of this post-translational modification during early vertebrate development. Results: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex as opposed to housekeeping processes. Conclusions: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies. Overall design: Three different time points (early gastrula-9 hours post fertilization, late gastrula- 13.5 hours post fertilization, and early neurula- 16.5 hours post fertilization). Twenty embryos from each time point. Three replicate experiments. Samples from control (injected with water) and experimental (injected with Gam1 mRNA).

背景：腺病毒蛋白Gam1可介导E1型SUMO激活酶（E1 SUMO-activating enzyme）的蛋白水解降解。通过经验确定剂量的Gam1 mRNA显微注射至单细胞期爪蟾（Xenopus）胚胎中，可将SUMO化修饰（SUMOylation）活性降至可检测阈值以下且不会导致胚胎致死，从而得以研究该翻译后修饰在脊椎动物早期发育过程中的功能。
结果：我们发现，SUMO化修饰缺陷型胚胎始终表现出神经管与心脏发育缺陷。我们针对三个发育阶段的对照组胚胎与Gam1 mRNA注射组胚胎开展了基因表达差异分析：早期原肠胚期（合子转录启动后即刻）、晚期原肠胚期（三胚层完全形成时）与早期神经胚期（神经板出现时）。尽管基因表达变化广泛存在且可关联至多种生物学过程，但三条信号通路——非经典Wnt/PCP通路（non-canonical Wnt/PCP pathway）、snail/twist通路与Ets-1通路——对SUMO化活性缺失尤为敏感，且可在很大程度上解释Gam1注射胚胎的主要表型。SUMO化修饰似乎可使同一转录因子产生不同功能亚型池，该翻译后修饰参与调控更为复杂的生物学过程，而非持家基因相关的基础生理过程。
结论：我们已鉴定出SUMO化活性受抑制所导致的神经管与心脏发育表型背后的基因表达变化。值得注意的是，这些发育缺陷与人类中最常见的两种先天性出生缺陷相对应，强烈提示无论是全局SUMO化紊乱还是单一蛋白的SUMO化异常，都可能是此类病理发生的常见诱因。
实验设计：共设置三个不同的采样时间点：早期原肠胚期（受精后9小时）、晚期原肠胚期（受精后13.5小时）与早期神经胚期（受精后16.5小时）。每个时间点收集20枚胚胎，设置3次生物学重复。样品分为对照组（注射无菌水）与实验组（注射Gam1 mRNA）。