Growing Pains: The Loss of Plasticity to Extreme Anoxia in Developing Embryos of Austrofundulus limnaeus

Author

• Patrick Ryan Clouser, Portland State UniversityFollow

Sponsor

Portland State University. Department of Biology

First Advisor

Jason Podrabsky

Term of Graduation

Spring 2026

Date of Publication

6-4-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.) in Biology

Department

Biology

Language

English

Subjects

anoxia, diapause, RNA

Physical Description

1 online resource (xi, 231 pages)

Abstract

Oxygen plays a pivotal role for most animal life on Earth, so much so that even short exposures to no oxygen, anoxia, can have severe effects on the animal's wellbeing. Conversely, aerobic recovery after anoxic insult can be just as detrimental. Some animals are the exception to this rule and demonstrate survival through anoxia and recovery. There has been a great deal of research around anoxia tolerant animals, but what remains unclear are the genetic underpinnings that determine the anoxia tolerant phenotype. RNA sequencing allows for the quantification of RNA transcripts within a sample, which can indicate if a gene is being actively expressed and at what level. Regulation of gene expression is controlled by many factors, and cellular stress can induce major changes in gene expression. To date, the annual killifish, Austrofundulus limnaeus, is the most anoxia tolerant vertebrate observed on the planet, making it a great model to investigate the genetic underpinnings of anoxia tolerance and recovery. Anoxia tolerance is associated with the ability to enter a state of developmental and metabolic arrest called diapause. During diapause, embryos exhibit an incredible anoxia tolerance with survival being observed for over 100 days. Shortly after exiting diapause, Wourms' stage (WS) 36 embryos exhibit an even greater anoxia tolerance making them the most anoxia tolerant vertebrate identified to date. However, as development continues anoxia tolerance is reduced to just hours by the time the embryo is ready to hatch. I hypothesize that investigation of the transcriptomic changes during anoxia and recovery across stages of development in embryos of A. limnaeus will uncover cellular pathways and genetic programs needed to support anoxia tolerance and successful recovery from anoxic stress.

Bulk RNA sequencing was performed on 4 developmental timepoints that demonstrate a gradient of anoxia tolerance, each timepoint underwent a time course of anoxia and recovery with 5 total exposure conditions sampled, making for 20 different samples. Within each developmental stage, differential expression analysis was performed to see which transcripts changed in abundance across the experiment, and pathway enrichment analysis was used to highlight biological processes that were enriched through anoxia and recovery. Finally, multiple weighted gene coexpression network analysis (multiWGCNA) was performed to identify networks of transcripts that work may together in response to anoxia but were lost through development.

Results illustrate that embryos of A. limnaeus mount a transcriptomic response to anoxia at every stage of development, but the magnitude of the response falls as embryos develop towards hatching and lose their anoxia tolerance. Enrichment of RNAs related to the unfolded protein response, MAPK signaling, and the upregulation of FOS and JUN were common responses to anoxia across all developmental stage. Additional anoxia experiments with the use of drug inhibitors found that Wourms' stage 36 anoxia tolerance is significantly reduced by inhibition of HSF1 and AP-1 transcription factors. Wourms' stage 40 individuals' anoxia tolerance was also sensitive to HSF1 and p38 inhibition. MultiWGCNA uncovered a network of developmentally conserved transcripts highly associated with anoxia and recovery from anoxia whose discoordination through development coincides with the loss of anoxia tolerance in A. limnaeus embryos. The work presented here provides a basis for understanding the transcriptomic response to anoxic stress in a profoundly anoxia tolerant vertebrate, adding insight to the important genetic underpinnings of this phenotype that can be applied to other non-tolerant species.

Rights

© 2026 Patrick Ryan Clouser

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Persistent Identifier

https://archives.pdx.edu/ds/psu/44979

Recommended Citation

Clouser, Patrick Ryan, "Growing Pains: The Loss of Plasticity to Extreme Anoxia in Developing Embryos of Austrofundulus limnaeus" (2026). Dissertations and Theses. Paper 7154.