Hyams Yosef, Panov Julia, Rosner Amalia, Brodsky Leonid, Rinkevich Yuval, Rinkevich Baruch
Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 9753, Tel Shikmona, Haifa, 3109701, Israel; Marine Biology Department, Leon H. Charney School of Marine Sciences, University of Haifa, Haifa, 31905, Israel.
Tauber Bioinformatics Research Center, University of Haifa, Haifa, 31905, Israel; Sagol Department of Neurobiology, University of Haifa, Haifa, 3498838, Israel.
Dev Biol. 2022 Oct;490:22-36. doi: 10.1016/j.ydbio.2022.06.005. Epub 2022 Jul 7.
Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.
恶劣环境会促使生物表现出行为、形态、生理和生殖方面的反应，包括蛰伏。在此，我们通过全生物体透射电子显微镜（TEM）和光学显微镜观察、RNA测序、对选定基因的实时聚合酶链反应（qPCR）定量以及WNT、SMAD和SOX2基因表达的免疫定位，研究群体尾索动物拟柱囊菌属近缘种leachii蛰伏原型中的形态、细胞和分子变化。在形态学层面，蛰伏始于所有游动孢子和芽体的逐渐退化，群体以浓缩的脉管系统残余形式存活，这些残余在环境变化时可能会“苏醒”，以再生出功能完全的群体。同时，我们观察到血淋巴细胞类型的分布发生了变化。吞噬细胞数量翻倍，而桑椹细胞数量减少了一半。此外，还观察到两种新的循环细胞类型，即多核细胞和含细菌细胞。RNA测序技术揭示了不同生物体区室和状态（活跃的游动孢子和壶腹，以及蛰伏中期与未处理群体、未处理与蛰伏群体之间）的基因表达存在显著差异。基因本体术语富集分析进一步显示了不同的生物学过程。在蛰伏群体中，我们总共观察到233个上调基因。这些基因包括NR4A2、EGR1、MUC5AC、HMCN2等。此外，在蛰伏群体中有27个转录因子上调，包括ELK1、HDAC3、RBMX、MAZ、STAT1、STAT4和STAT6。有趣的是，参与发育过程的基因，如SPIRE1、RHOA、SOX11、WNT5A和SNX18在蛰伏群体中也上调。我们通过qPCR进一步验证了蛰伏期间22个基因的失调。对来自三个信号通路的代表性基因进行免疫组织化学分析，结果显示这些基因在蛰伏期间的循环细胞中高表达。给予WNT激动剂后，80%的蛰伏群体提前从蛰伏状态苏醒，而在活跃群体中，WNT激动剂则引发了蛰伏状态。因此，上述结果表明与拟柱囊菌属leachii蛰伏相关的独特转录组景观。
