Phillip Cleaves, Stanford University

Moss Landing Marine Labs Seminar Series - September 13th, 2018

Hosted by the Invertebrate Zoology & Molecular Ecology Lab

MLML Seminar Room, 4pm

Open to the public

Using Transcriptomics and Reverse Genetics to Understand Mechanisms of Cnidarian-dinoflagellate Symbiosis

Phillip A. Cleves¹, Cory J. Krediet¹, Erik M. Lehnert¹, Benjamin M. Mason¹, Marie Strader², Mikhail Matz², and John R. Pringle¹

¹Department of Genetics, Stanford University, Stanford, CA, USA

²Integrative Biology, The University of Texas at Austin, Austin, TX, USA

 

The endosymbiosis between corals and dinoflagellate algae (genus Symbiodinium) is essential to the energetic requirements of coral-reef ecosystems.  However, coral reefs are in danger due to elevated ocean temperatures and other stresses that lead to the breakdown of this symbiosis and consequent coral "bleaching".  Despite its importance, the molecular basis of how corals establish and maintain a healthy symbiosis is poorly understood, in part because of the lack of a tractable genetic model organism. The small anemone Aiptasia is symbiotic with Symbiodinium strains like those in reef-building corals but has many experimental advantages over corals, making it an attractive laboratory model for cnidarian symbiosis.  To explore the possible transcriptional basis of heat-induced bleaching, we used RNA-Seq to identify genes that are differentially expressed during a time course of thermally stressed symbiotic and aposymbiotic Aiptasia strains. We observed a strong upregulation of hundreds of early stress response genes at time points long before bleaching begins in symbiotic anemones. The putative promoters of these early stress response genes are enriched for NFKB and HSP1 transcription factor binding sites suggesting that many of these stress response genes share core transcriptional inputs. The overall expression patterns were similar between the symbiotic anemones and the aposymbiotic anemones, indicating that many of the expression changes are not specific to the presence of the algae. However, blocking protein synthesis or HSP1 DNA binding with pharmacological inhibitors during this up-regulation results in more severe bleaching suggesting this symbiont-independent early stress response is protective against thermal stress and bleaching.

Genetic tools are needed to allow rigorous functional testing of the roles in symbiosis of candidate genes and pathways. As a first step in developing transgenic methods for Aiptasia, we have successfully expressed the photoconvertible Kaede fluorescent protein in larvae by microinjection of capped mRNA into 1-cell zygotes obtained by spawning in the laboratory.  This technique should allow both expression of tagged proteins for localization studies and the overexpression of candidate genes to analyze gain-of-function phenotypes.  In addition, we have promising results for two different methods for analyzing loss-of-function phenotypes in Aiptasia.  First, we have microinjected zygotes with translation-blocking morpholinos targeting the FGF1a gene, which has been shown to be required for apical-tuft formation in another anthozoan.  Preliminary results show apparent loss of apical-tuft formation in successfully injected larvae, suggesting that the Fgf1a protein was effectively knocked down.  Meanwhile, we have successfully used the CRISPR-Cas9 technology to create genetic changes in embryos of the coral Acropora millepora.  Through the establishment of both gain-of-function and loss-of-function methods in both Aiptasia and corals, Aiptasia will be a uniquely powerful genetic model organism (with year-round spawning) for the study of cnidarian-Symbiodinium symbiosis, and the discoveries made can be validated using similar technologies in corals.

 

We thank the Gordon and Betty Moore Foundation and the Simons Foundation for support.