Dave Mohit, Levin Joshua, Ruffins Seth Walter, Sato Yuki, Fraser Scott, Lansford Rusty, Kawahara Tomohiro
Department of Radiology and Developmental Neuroscience Program, The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, United States.
Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.
Front Physiol. 2022 May 12;13:893736. doi: 10.3389/fphys.2022.893736. eCollection 2022.
The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O), which improves to 15 days on switching to a hyperoxic environment of 60% O Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA "in cubo", followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the "in cubo" electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.
禽蛋是一个封闭系统，可保护发育中的胚胎免受外部因素影响，但阻碍了对胚胎发育的直接观察。文献中存在各种培养系统，用于研究胚胎在短时间孵化（从12小时到最多60小时的蛋孵化）过程中的发育情况。这些培养技术的一个常见缺陷是无法在其天然卵黄上以完整的组织张力培养未孵化的禽胚盘。这项工作的目标是创建一种独特的新型“立方体内的蛋”系统，可用于从未孵化的胚盘阶段开始的鹌鹑胚胎长期培养。“立方体内的蛋”充当一个人工透明蛋壳系统，容纳发育中的胚胎，使其适合显微镜观察。使用“立方体内的蛋”系统，鹌鹑胚胎从未孵化的胚盘开始可培养长达9天（在空气中孵化，氧气含量为20.9%），切换到60%氧气的高氧环境后可延长至15天。在“立方体内的蛋”系统中使用转基因荧光鹌鹑胚胎，利用倒置共聚焦显微镜动态成像未孵化胚盘中的细胞运动，这在其他培养系统中很难实现。除了这些观察结果，本工作还使用直立共聚焦或落射荧光显微镜对转基因荧光鹌鹑胚胎描述了该系统的其他几种成像应用。为了证明“立方体内的蛋”系统在扰动实验中的实用性，将荧光mRNA通过“立方体内电穿孔”导入鹌鹑神经管，然后对电穿孔后的胚胎进行孵化，并在立方体内对电穿孔区域进行显微镜观察。本文所述的“立方体内的蛋”培养系统与“立方体内电穿孔”及动态成像能力相结合，将使研究人员能够利用禽（鹌鹑）胚胎在其天然卵黄上研究早期胚胎发育中的几个基本问题。
