A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development - PubMed (original) (raw)
A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development
Mohit Dave et al. Front Physiol. 2022.
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% O2. 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).
Copyright © 2022 Dave, Levin, Ruffins, Sato, Fraser, Lansford and Kawahara.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
FIGURE 1
Hyperoxia (60% O2) improves embryo survival for long-term culture in the egg-in-cube system. (A) Captured frames from live imaging of an EGK-X wild-type quail embryo. The images shown here are acquired using an Android Motorola E5 phone camera taken from the dorsal aspect of the embryo in the cube at 5 min intervals. Images were acquired from EGK-X (E0) to day 4 of development, the lighting and cube were readjusted, and imaging was continued until the embryo started to deteriorate by day 11.25. Raw JPEG files were acquired from the phone and processed into TIFF files using ImageJ. Time frames at approximately 24 h intervals were isolated from these TIFFs to make a representative figure here. The original video file can be seen in Supplementary Video S1. Scale bar = 5 mm. (B) Kaplan Meier survival curve plotted between embryos incubated in Air (20.9% O2, blue line, n = 21 embryos) and 60% O2 (red line, n = 15 embryos). Percent embryos survival plotted on the Y-axis vs. Time of incubation in the cube (in days) on the X-Axis. X denotes the death of embryos and filled squares denote the survival endpoint of embryos is unknown/embryos were censored from analysis. Mantel-Haenszel Log-rank test, χ21 = 7.16, p < 0.0074.
FIGURE 2
Imaging gastrulation using an upright fluorescent stereoscope. Captured frames from live imaging of an EGK-X [Tg (hUbC.membrane.GFP)] quail embryo. The images shown here are acquired using the upright Olympus stereomicroscope taken from the dorsal aspect of the embryo in the cube at 10 min intervals. Images were acquired from EGK-X (E0) to HH3+ (9.8 h of incubation). Time frames at 1–2 h intervals were isolated from the original TIFF file. The original video file can be seen as Supplementary Video S2 (EGK-X to HH3+). Scale bar = 700 µm.
FIGURE 3
Using fluorescent bead injections to study blood flow dynamics in embryos using the cube on an upright fluorescent stereoscope. (A) Captured frame from live imaging of an E3 wild-type quail embryo in the cube. The embryo was microinjected with a small bolus of fluorescent microspheres/beads in the lateral left vitelline vein, the cube was covered with the high transparency membrane and mounted into the custom incubator for imaging. The images shown here are acquired using the upright Olympus stereomicroscope taken from the dorsal aspect of the embryo in the cube at 50 ms intervals. Panel (A) shows the representative region of interest (ROI) used for bead tracking analysis. (B) Captured frames from the ROI (white square) from (A). The encircled bead moves through the small capillary and is tracked through its path in the vessel here. Representative images from four time frames 50 ms, 450 ms, 900 ms, and 1500 ms from the start of the time-lapse show the bead moving through the vessel (labeled 1–4 in order of frame sequence). (C) The first frame and last frame are shown in (B) and are used for tracking the displacement of the microsphere through the circulation (Measurement tool in Imaris). The original video file can be seen as Supplementary Video S3A [original time-lapse of region shown in Panel 3A] and Supplementary Video S3B [Zoomed in time-lapse of region shown in Panel 3B].
FIGURE 4
Cardiac neural crest cell migration imaged by photoconversion of an HH10 [Tg(hUbC:H2B-Cerulean-2A-Dendra2)] embryo using the egg-in-cube system. (A) Confocal images of the native green form of Dendra2 (referred to as Dendra2green here), photoconverted red form of Dendra2, and Cerulean channels in an HH10 [Tg(hUbC:H2B-Cerulean-2A-Dendra2)] embryo before and after photoconversion (PC). The neural tube cells between the otic placode and the 3rd somite expressing the Dendra2green are photoconverted using the 405 nm UV laser to induce the activated red form of Dendra2 (referred to as Dendra2red here) expression. The Dendra2 green fluorescence intensity decreases with its photoconversion into its red form whereas the fluorescent intensity of the Cerulean channel remains unchanged. (B) Confocal images of the photoconverted red form of Dendra and Cerulean channels at different time points were sampled from the time-lapse data at 0 h, 3 h, and 7 h acquired using the upright confocal microscope. Photoconverted cardiac neural crest cells migrate laterally out of the neural tube towards the somites. The original time-lapse files can be seen in Supplementary Video S5.
FIGURE 5
The egg-in-cube system enables dynamic imaging of early embryonic development. Captured frames from live confocal microscope imaging of an EGK-X [Tg(hUbC.membrane.EGFP)] quail embryo. The 5x images, with ×0.6 optical zoom, are maximum intensity projections of 11 optical slices (100 µm each) taken at 10 min intervals taken from the dorsal aspect of the embryo on the inverted confocal microscope. (A) Frames are shown at different time points highlighting key features of early avian development through the first 24 h of development. (B) Frames shown at different time points highlight the expansion of the blastodisc in 3D following its natural curvature on the yolk. Time-lapse images were acquired using the Zen 2011 (black) software, the acquisition was halted momentarily to readjust the focus and resumed several times. Acquired images were converted to a maximum intensity projection and stitched together in time to present the development as a continuous time-lapse. Images shown in (B) were taken from the first 9.1 h of development in 4D and presented as an orthogonal slice view using Imaris. The video files can be seen in Supplementary Video S5A (Panel 5A) and Supplementary Video S5B (Panel 5B).
FIGURE 6
The Egg-in-cube system enables imaging of hypoblast migration. (A) Panels show the putative hypoblast region of interest in the anterior germinal crescent of an HH2 [Tg(hUbC:H2B-Cerulean-2A-Dendra2)] quail embryo before and after photoconversion. The 405 nm UV laser is used to photoconvert a region of interest using the “Regions” and “bleaching” function in Zen Black. Nuclei are labeled by Cerulean fluorescent protein (Cyan). Photoconverted cells are labeled by Dendra2red fluorescent protein (red). Scale: 100 µm. (B) The migration of cells (red cells, region bounded by a white dotted line) in the photoconverted region of interest is shown over different time points (see Supplementary Video S6). Captured frames from live confocal microscope imaging from the dorsal side of an HH2 [Tg(hUbC:H2B-Cerulean-2A-Dendra2)] quail embryo. Images are maximum intensity projections of ×20 tiled images (24 slices of 6 µm) with ×0.6 optical zoom acquired every 10 min. Scale: 100 µm. (C) The last frame of the time-lapse (Dendra2red, upper left panel, 3 h) is compared to the Dendra2red channel (Lower left panel) to demonstrate the position of the hypoblast layer (region bounded by a white dotted line in all panels) before and after ApoA1 HCR staining. Images are maximum intensity projections of ×20 confocal Z stacks (20 slices of 8 mm) from whole-mount in situ hybridization for hypoblast specific ApoA1 (green) staining within an HH2 [Tg(hUbC:H2B-Cerulean-2A-Dendra2)]. DAPI stain in the nuclei is shown as blue. Scale: 150 µm.
FIGURE 7
The egg-in-cube system enables electroporation of the neural tube with fluorescent mRNA. The images in the top panel show a representative midbrain region of the [Tg(PGK1-H2B-mCherry)] embryo (red nuclei) electroporated with membrane-eGFP mRNA (green) and the bottom panel shows a region caudal to the midbrain (closer to the hindbrain and otic placode). These images show a small proportion of H2B-mCherry expressing cells colocalizing with membrane eGFP post electroporation. Scale: 70 µm.
References
- Bohr C., Hasselbalch K. A. (1903). Ueber die Wärmeproduction und den Stoffwechsel des Embryos1. Skand. Arch. Physiol. 14, 398–429. 10.1111/j.1748-1716.1903.tb00541.x -DOI
- Byerly T. C. (1930). Time of Occurrence and Probable Cause of Mortality in Chick Embryos. Proc. World's Poult. Congr. 4, 178–186.
LinkOut - more resources
Full Text Sources