Edgar A et al. (2019), Embryo microinjection of the lecithotrophic sea...

ECB-ART-48778

J Biol Methods 2019 Sep 17;63:e119. doi: 10.14440/jbm.2019.292.

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Embryo microinjection of the lecithotrophic sea urchin Heliocidaris erythrogramma.

Edgar A , Byrne M , Wray GA .

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Microinjection is a common embryological technique used for many types of experiments, including lineage tracing, manipulating gene expression, or genome editing. Injectable reagents include mRNA overexpression, mis-expression, or dominant-negative experiments to examine a gene of interest, a morpholino antisense oligo to prevent translation of an mRNA or spliceoform of interest and CRISPR-Cas9 reagents. Thus, the technique is broadly useful for basic embryological studies, constructing gene regulatory networks, and directly testing hypotheses about cis-regulatory and coding sequence changes underlying the evolution of development. However, the methods for microinjection in typical planktotrophic marine invertebrates may not work well in the highly modified eggs and embryos of lecithotrophic species. This protocol is optimized for the lecithotrophic sea urchin Heliocidaris erythrogramma.

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Species referenced: Heliocidaris erythrogramma
Genes referenced: LOC100887844

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References [+] :

Armstrong, The evolution of larval developmental mode: insights from hybrids between species with obligately and facultatively planktotrophic larvae. 2015, Pubmed, Echinobase

	Figure 1. Example of a pulled needleâs tip. The preferred needle size is relatively fine compared to the size of H. erythrogramma eggs.
	Figure 2. Healthy H. erythrogramma eggs. They are positively buoyant and bright orange in color. Left: Spawning female. Right: Washed eggs floating on the surface of FSW.
	Figure 3. Set up of injection dish immediately before injection. A. Allow zygotes to equilibrate in Ficoll-PFSW in the injection dish. B. Draw off Ficoll-PFSW using a fire-polished Pasteur pipette. C. Continue to remove Ficoll-PFSW until the embryos are immobilized against the agarose pad, leaving just enough solution to cover them.
	Figure 4. Microinjection of H. erythrogramma embryos. Top row: photomicrograph of an injection. Middle row: cartoon illustration of focal embryo in top row. Bottom row: Schematic of early of H. erythrogramma development. A and Aâ. The needle dents the fertilization envelope and the embryo itself. B and Bâ. Injection solution in pulsed into the embryo. C and Câ. The needle is withdrawn and the injected solution disperses evenly within the embryo. D. Approximate timing of H. erythrogramma development from fertilization to hatching at 22Â°C. Grey boxes indicate timing windows for injection and sorting. See also Video S1.
	Figure 5. Screen injected embryos. Use a fluorescent dissecting microscope. Pick only evenly injected embryos cleaving normally at 4-cell stage or beyond and transfer into a fresh, low-attachment plate to culture. A. Diagrammatic view of injected embryos under white light. B. Diagrammatic view of injected embryos under fluorescent light; dark embryos are not injected.
	Figure 6. Representative results. A. Control embryo, injected with 1Ã injection mix and fixed at late gastrula stage. B. Experimental embryo, injected with 50 nM Axin2 translation-blocking morpholino and fixed at late gastrula stage.