Microinjection to deliver protein, mRNA, and DNA into zygotes of the cnidarian endosymbiosis model Aiptasia sp.

Victor A S Jones,Annika Guse,Elizabeth A Hambleton,Madeline Bucher

doi:10.1038/s41598-018-34773-1

Abstract

Reef-building corals depend on an intracellular symbiosis with photosynthetic dinoflagellates for their survival in nutrient-poor oceans. Symbionts are phagocytosed by coral larvae from the environment and transfer essential nutrients to their hosts. Aiptasia, a small tropical marine sea anemone, is emerging as a tractable model system for coral symbiosis; however, to date functional tools and genetic transformation are lacking. Here we have established an efficient workflow to collect Aiptasia eggs for in vitro fertilization and microinjection as the basis for experimental manipulations in the developing embryo and larvae. We demonstrate that protein, mRNA, and DNA can successfully be injected into live Aiptasia zygotes to label actin with recombinant Lifeact-eGFP protein; to label nuclei and cell membranes with NLS-eGFP and farnesylated mCherry translated from injected mRNA; and to transiently drive transgene expression from an Aiptasia-specific promoter, respectively, in embryos and larvae. These proof-of-concept approaches pave the way for future functional studies of development and symbiosis establishment in Aiptasia, a powerful model to unravel the molecular mechanisms underlying intracellular coral-algal symbiosis.

Highlights

Progress towards a better understanding of symbiosis establishment has been slow, primarily due to the historical lack of tools and workable laboratory systems: reef-building corals grow slowly, are sensitive to environmental conditions, and typically sexually reproduce to spawn larvae only once annually[7,8,9]
Here we show the establishment of microinjection in the Aiptasia model system in a simple and robust workflow to introduce exogenous material into embryos for subsequent analysis
We establish a workflow to successfully introduce exogenous protein, mRNA, and DNA via microinjection into Aiptasia zygotes and, critically, we demonstrate that such manipulation has no significant effects on development or symbiosis establishment in Aiptasia larvae

Summary

Introduction

Progress towards a better understanding of symbiosis establishment has been slow, primarily due to the historical lack of tools and workable laboratory systems: reef-building corals grow slowly, are sensitive to environmental conditions, and typically sexually reproduce to spawn larvae only once annually[7,8,9]. Would be especially useful to study how Aiptasia, and by extension reef-building corals, establish symbiosis anew in the developing larval stage To this end, microinjection of material into embryos has proven an efficient method of genetic engineering in many models, while making possible the direct production of F0 manipulated larvae and juveniles for immediate phenotypic analysis. It would open the door to myriad observational and functional studies, propelling the symbiosis field forward and allowing both broad approaches as well as specific hypothesis testing based on candidate genes To this end, here we show the establishment of microinjection in the Aiptasia model system in a simple and robust workflow to introduce exogenous material into embryos for subsequent analysis. Introduction of such exogenous material appears to have no significant effects on either development or symbiosis establishment, demonstrating the utility of these tools to study fundamental questions of development and symbiosis establishment in the Aiptasia system

Methods

Results

Conclusion