2018 - NECC
RACER Pre-proposal Proposal

Name: Mr. Benjamin L King
Institution/Firm: MDIBL
Address: Mount Desert Island Biological Laboratory
PO Box 35
Salsbury Cove, ME 04672
Email: bking@mdibl.org
Telephone: (207) 288-9880 Extension: 139
Project Title: Discovery of microRNAs Expressed in Response to Hypoxia in the Estuarine Fish, Fundulus heteroclitus
Abstract: The ability for animals to maintain cellular homeostasis under a spectrum of environmental stressors, such as low dissolved oxygen concentrations, is critical for survival. After low oxygen concentrations are sensed, a hypoxic stress response is induced in attempt to restore homeostasis. This response is relevant to several human diseases such as myocardial infarction and neurological dysfunction. In cancer, regions of proliferating tumors continue to thrive in hypoxic conditions as they outgrow blood supply. Goals of hypoxia studies in model organisms include determining how cells sense and survive with low oxygen concentrations in order to design human intervention strategies. Hypoxia has been widely studied in many model organisms, but only a relatively few genes such as hypoxia induced factor (HIF-1a) have been characterized. Beyond protein coding genes, microRNAs mediate stress responses by rapidly repressing expressed transcripts of target genes[1]. The role of microRNAs in hypoxia are largely limited to cancer cell line studies[2] and not organismal responses. To address this gap, we propose to characterize microRNA expression levels in the estuarine fish, killifish (Fundulus heteroclitus), that are the most hypoxia tolerant among other abundant estuarine fishes and can survive in dissolved oxygen levels as low as 1 mg/L[3]. These fish tolerate a variety of environmental stressors due to the dynamic conditions they encounter in estuaries that make them a powerful model to understand genetic-environment interactions including anthropogenic environmental change[3]. Not only does poor coastal water quality contribute to hypoxic conditions for these fish, but exposure to particular pollutants, such as PCBs, blocks the upregulation of glycolytic genes normally initiated by hypoxia[4]. As the first study of microRNAs in killifish to date, we will test the hypothesis that microRNAs are part of the hypoxic stress response in killifish using high-throughput sequencing. Specifically, we will characterize microRNAs expressed in gills of fish kept in normoxic versus hypoxic environments and compare the response to that of other organisms. This experiment will broaden our understanding hypoxic responses as killifish can tolerate extremely hypoxic environments and be the first characterization of microRNAs in killifish. This project is the first of many envisioned experiments to characterize a variety of environmental stressors in killifish that we plan to encapsulate into a database to compare and contrast responses.


1. Leung AK, Sharp PA. (2010) MicroRNA functions in stress responses. Mol Cell. 40(2):205-15.

2. Pocock R. (2011) Invited review: decoding the microRNA response to hypoxia. Pflugers Arch. 461(3):307-15.

3. Burnett KG, Bain LJ, Baldwin WS, Callard GV, Cohen S, Di Giulio RT, Evans DH, Gomez-Chiarri M, Hahn ME, Hoover CA, Karchner SI, Katoh F, Maclatchy DL, Marshall WS, Meyer JN, Nacci DE, Oleksiak MF, Rees BB, Singer TD, Stegeman JJ, Towle DW, Van Veld PA, Vogelbein WK, Whitehead A, Winn RN, Crawford DL. (2007) Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics. Comp Biochem Physiol D (Genomics Proteomics). 2(4):257-86.

4. Kraemer, L.D., Schulte, P.M. (2004) Prior PCB exposure suppresses hypoxia-induced up-regulation of glycolytic enzymes in Fundulus heteroclitus. Comp Biochem Physiol C (Toxicol.). 139, 23-29.
Collaborative Project Details: Mr. King (MDIBL, Maine) will be the Principal Investigator and work closely with collaborators to accomplish project goals. In preparation of this proposal, two key collaborations have been established. First, Mr. Bruce Kingham (Delaware Biotechnology Institute, Delaware) is willing to prepare the small RNA sequencing libraries and conduct the sequencing on a fee-for-service basis. Second, Dr. Kevin Peterson (Dartmouth College, New Hampshire) will work with Mr. King on bioinformatics data analysis to find known and novel microRNAs in high-throughput sequence data. We plan to utilize the NECC Shared Data Center to share data between the three states.

We are actively seeking additional collaborators at NECC institutions that have interests in hypoxia, environmental stress responses or that use killifish as a model organism.
Project Overview - include goals, general expertise *: The objective of this proposal is to characterize microRNAs that are differentially expressed under hypoxic conditions in killifish, Fundulus heteroclitus.

The proposed project will involve three major steps. The first is to obtain RNA samples from gill tissue of killifish exposed to normoxic or hypoxic conditions. MDIBL has the infrastructure and expertise to house locally caught killifish, expose the fish to the appropriate conditions and isolate RNA samples. In the second step, RNA samples will be sent to Mr. Bruce Kingham (Delaware Biotechnology Institute, Delaware) where he will prepare a small RNA sequencing library for each sample and then sequence each library. In the last step, the sequence data will be transferred to Maine and New Hampshire to be analyzed using bioinformatics data analysis tools. Specifically, we will identify known and novel microRNAs and then calculate whether they are significantly differentially expressed. Mr. King has valuable experience identifying microRNAs in previously uncharacterized species using high-throughput sequencing and bioinformatics data analysis. We performed the first characterization of microRNAs in the bichir (P. senegalis) and skate (L. erinacea) while profiling their expression profiles during sequential stages of limb regeneration in these two species along with axolotls (A. mexicanum). We plan to use Dr. Kevin Peterson's (Dartmouth College, New Hampshire) miRminer software to identify known and novel microRNAs from the high-throughput sequence data. Dr. Peterson is an expert in bioinformatics analysis of finding novel microRNAs using his miRminer software[5-8].


5. Wheeler BM, Heimberg AM, Moy VN, Sperling EA, Holstein TW, Heber S, Peterson KJ. (2009) The deep evolution of metazoan microRNAs. Evol Dev. 11(1):50-68.

6. Sperling EA, Vinther J, Moy VN, Wheeler BM, Semon M, Briggs DE, Peterson KJ. (2009) MicroRNAs resolve an apparent conflict between annelid systematics and their fossil record. Proc Biol Sci. 276(1677):4315-22.

7. Campo-Paysaa F, Semon M, Cameron RA, Peterson KJ, Schubert M. (2011) microRNA complements in deuterostomes: origin and evolution of microRNAs. Evol Dev. 13(1):15-27.

8. Philippe H, Brinkmann H, Copley RR, Moroz LL, Nakano H, Poustka AJ, Wallberg A, Peterson KJ, Telford MJ. (2011) Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature. 470(7333):255-8.