Zattara Eduardo E, Turlington Kate W, Bely Alexandra E
Department of Biology, University of Maryland, College Park, MD, 20740, USA.
BMC Dev Biol. 2016 Mar 23;16:6. doi: 10.1186/s12861-016-0104-2.
Time-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional "still-shot" studies and allowing direct examination of tissue and cell dynamics. A major challenge for time-lapse imaging of animals is keeping specimens immobile yet healthy for extended periods of time. Although this is often feasible for embryos, the difficulty of immobilizing typically motile juvenile and adult stages remains a persistent obstacle to time-lapse imaging of post-embryonic development.
Here we describe a new method for long-duration time-lapse imaging of adults of the small freshwater annelid Pristina leidyi and use this method to investigate its regenerative processes. Specimens are immobilized with tetrodotoxin, resulting in irreversible paralysis yet apparently normal regeneration, and mounted in agarose surrounded by culture water or halocarbon oil, to prevent dehydration but allowing gas exchange. Using this method, worms can be imaged continuously and at high spatial-temporal resolution for up to 5 days, spanning the entire regeneration process. We performed a fine-scale analysis of regeneration growth rate and characterized cell migration dynamics during early regeneration. Our studies reveal the migration of several putative cell types, including one strongly resembling published descriptions of annelid neoblasts, a cell type suggested to be migratory based on "still-shot" studies and long hypothesized to be linked to regenerative success in annelids.
Combining neurotoxin-based paralysis, live mounting techniques and a starvation-tolerant study system has allowed us to obtain the most extensive high-resolution longitudinal recordings of full anterior and posterior regeneration in an invertebrate, and to detect and characterize several cell types undergoing extensive migration during this process. We expect the tetrodotoxin paralysis and time-lapse imaging methods presented here to be broadly useful in studying other animals and of particular value for studying post-embryonic development.
延时成像已被证明在研究发育过程中具有极高的价值，能产生比传统“静态拍摄”研究分辨率更高的数据，并可直接观察组织和细胞动态。对动物进行延时成像的一个主要挑战是长时间保持标本静止且健康。虽然这对胚胎通常可行，但固定典型的幼年和成年活动阶段的难度仍然是胚胎后发育延时成像的一个持续障碍。
在此，我们描述了一种对小型淡水环节动物莱氏普里斯蒂纳成虫进行长时间延时成像的新方法，并使用该方法研究其再生过程。用河豚毒素固定标本，导致不可逆麻痹，但再生过程显然正常，然后将其置于琼脂糖中，周围环绕着培养液或卤代烃油，以防止脱水但允许气体交换。使用这种方法，蠕虫可以连续且以高时空分辨率成像长达5天，涵盖整个再生过程。我们对再生生长速率进行了精细分析，并对早期再生过程中的细胞迁移动态进行了表征。我们的研究揭示了几种假定细胞类型的迁移，其中一种与已发表的环节动物新生细胞描述非常相似，这种细胞类型基于“静态拍摄”研究被认为具有迁移性，长期以来一直被假设与环节动物的再生成功有关。
结合基于神经毒素的麻痹、活体固定技术和耐饥饿研究系统，使我们能够获得无脊椎动物完整前后部再生最广泛的高分辨率纵向记录，并检测和表征在此过程中经历广泛迁移的几种细胞类型。我们预计本文介绍的河豚毒素麻痹和延时成像方法在研究其他动物方面将广泛有用，对研究胚胎后发育具有特别价值。
