Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

本研究采用数字基因表达（Digital Gene Expression）分析，在无两侧对称动物偏倚的前提下，筛选并鉴定刺胞动物（cnidarian）胚胎模式形成的调控因子。通过对比未受干预的Clytia早期原肠胚胚胎，与经吗啉代反义寡核苷酸（morpholino antisense oligonucleotides，以下简称MO）抑制关键极性调控因子Wnt3活性的胚胎的转录组，我们筛选得到一批显著上调与下调的转录本。这些转录本编码候选Wnt信号通路调控因子、其他转录因子的同源基因、两侧对称动物模型中已被报道的发育调控相关分泌蛋白与跨膜蛋白，或是此前未被表征的蛋白，同时还包含大量刺胞动物特异性蛋白。对比注射靶向Wnt3及其受体Fz1的MO的胚胎样本，我们定义了四类与时空表达谱高度相关的转录本类别：1类与3类转录本分别在发育中的浮浪幼虫的口极与反口极持续表达；2类转录本在原肠胚形成期内陷进入内胚层区域的细胞中表达；而4类基因的表达在早期原肠胚阶段被抑制。Fz1-MO对2类与4类转录本表达的优先调控效应可归因于平面细胞极性（Planar Cell Polarity，以下简称PCP）的破坏，该效应与特异性PCP蛋白Strabismus的吗啉代敲低实验结果高度吻合。本研究据此得出结论：内胚层与原肠胚后特异性基因的表达对PCP破坏尤为敏感，而Wnt/β-连环蛋白信号通路则主导了口-反口轴上的基因调控。通过靶向部分转录本的MO开展表型分析，结果显示这些基因的发育功能与其表达谱相符，既包含保守基因，也包含刺胞动物特异性基因。总体而言，本次无偏筛选实现了区域特异性表达基因的系统性鉴定，为包含已预测与未被探索成员的真后生动物（eumetazoan）发育调控基因集提供了功能支持，同时也证明刺胞动物的轴向模式形成与内胚层形成等核心发育过程，可由新演化（或高度分化）的基因介导。