A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development

Mohit Dave; Joshua Levin; Seth Walter Ruffins; Yuki Sato; Scott Fraser; Rusty Lansford; Tomohiro Kawahara

doi:10.3389/fphys.2022.893736

A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development

Front Physiol. 2022 May 12:13:893736. doi: 10.3389/fphys.2022.893736. eCollection 2022.

Authors

Mohit Dave^{1

2}, Joshua Levin^{1

2}, Seth Walter Ruffins², Yuki Sato³, Scott Fraser^{4

5}, Rusty Lansford^{1

2

6

7}, Tomohiro Kawahara⁸

Affiliations

¹ Department of Radiology and Developmental Neuroscience Program, The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, United States.
² Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.
³ Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
⁴ Translational Imaging Center, University of Southern California, Los Angeles, CA, United States.
⁵ Department of Biological Sciences, Division of Molecular and Computational Biology, University of Southern California, Los Angeles, CA, United States.
⁶ Department of Biological Sciences, Dana and Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA, United States.
⁷ Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.
⁸ Department of Biological Functions Engineering, Kyushu Institute of Technology, Kitakyushu, Japan.

Abstract

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% O₂), which improves to 15 days on switching to a hyperoxic environment of 60% O_2. 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.

Keywords: avian embryo culture; egg-in-cube; embryo development; imaging; long term culture; quail (Coturnix japonica).