A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish
June 30th, 2021
Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.
3-D zebrafish tail explants let's us exploit transparency of zebrafish embryos to its full extent during somite development and vertebrate segmentation. Fill an empty mating tank with fish system water. Clean the strainer with system water.
Raise the barriers between mating pairs. Selectively move the fish pair into the fresh water tank. Collect embryos in the strainer through water transfers with an empty tank.
Rinse collected embryos with fish system water and flip on a Petri dish. Ethanol-glaze dissection tools. Make two layers of tapes on slides.
Cut the horizontal sides of deep chambers. Complete chamber cuts with vertical sides. Peel the tape off from the chamber surface.
Pour cell culture media in a tube. Mix calcium chloride 2.8 millimolar final concentration. Add antibiotic solution.
Aliquot dissection media in a separate tube. Compliment fetal bovine serum to make tissue culture medium. De-corrinate embryos with needles.
Rinse the embryos in a freshwater dish and collect them for explanting. Stabilize the embryo with needle and start de-yoking. Trim the leftover tissues off.
Transfer the explants to the coverslip. Flatten the tissue and suck excess media out with pipette. Put the coverslip on growth medium containing slide chamber.
Put the explant tissue on a tissue wipe. Apply immersion oil to the objective. Mount the slide chamber on microscope.
Adjust laser channels for image acquisition. Check the z-layer limits of the sample. Set-up time-imaging and z-layers.
3-D tail explant system keeps cell integration to tail bud and excess elongation intact like whole embryos. It is also possible to stop excess elongation by chemically activating the surface with type one collagen. 3-D zebrafish tail explants system lets us to exploit transparency of zebrafish embryos by letting us to take high-resolution and near-objective microscopy images.
Besides that, it lets us to decouple the excess of elongation from morphogen signaling and also control the morphogen signaling sources with simple dissection methods.
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Overview
This article presents a protocol for 3-D tissue culture of the zebrafish posterior body axis, facilitating live observation of vertebrate segmentation. The explant model allows for manipulation of axis elongation and morphogen sources, along with high-resolution tissue-level imaging.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Live Imaging
Background
- Zebrafish embryos are transparent, enabling detailed observation during development.
- Somite development is crucial for understanding vertebrate segmentation.
- 3-D culture techniques enhance the study of tissue dynamics.
- Live imaging provides insights into cellular processes in real-time.
Purpose of Study
- To establish a reliable protocol for 3-D tissue culture of zebrafish.
- To enable live imaging of vertebrate segmentation.
- To manipulate morphogen sources for experimental control.
Methods Used
- Preparation of zebrafish embryos in a controlled environment.
- Utilization of an empty mating tank for embryo collection.
- Application of tissue culture techniques for 3-D explants.
- Live imaging to observe segmentation processes.
Main Results
- Successful establishment of a 3-D tissue culture model.
- Demonstration of live imaging capabilities during segmentation.
- Control over morphogen sources influenced axis elongation.
- Insights into the dynamics of vertebrate development.
Conclusions
- The 3-D zebrafish tail explant model is effective for studying segmentation.
- Live imaging provides valuable data on developmental processes.
- This approach can be applied to further research in vertebrate biology.
