Disease Modeling
Disease physiology is complex, involving multiple organs and tissues and often interactions between these tissues. This makes accurate study of these diseases particularly difficult. Traditional mouse models offer low numbers of samples to study, while higher throughput systems such as cell culture can’t capture the full complexity of the disease. One focus of our group is to bypass these issues by modeling human diseases in zebrafish. The rapid development of the zebrafish and its various organs and tissues, combined with their small physical size and large numbers of offspring, makes them an ideal candidate for modeling human diseases. A single female can produce hundreds of eggs while mating, and within a few days the resulting embryos will have already developed fully functioning organs and exhibit many complex behaviors relevant to disease. This provides us with hundreds of larvae with disease relevant symptoms, which can then be used for high throughput drug screening using large chemical libraries to identify small molecules that alter the disease phenotypes. The compounds we discover help us elucidate disease mechanisms and serve as starting points for developing new drug candidates. Compounds discovered in zebrafish screens also have the advantage of having been selected for their ability to be active, efficacious, and well tolerated in animals.
Current disease modeling and drug discovery projects include:
Lysosomal Storage Disorders
Lysosomal storage diseases (LSDs) are a family of over 70 metabolic disorders characterized by loss-of-function mutations in enzymes of lysosomal metabolism, resulting in pervasive lysosomal storage material accumulation and multiple-organ pathologies that contribute to mortality in childhood with limited treatment options. Using CRISPR-Cas9, we have generated 11 zebrafish models of sphingolipidoses, a subcategory of LSDs involving enzymes of sphingolipid catabolism. Zebrafish sphingolipidosis models exhibited marked sphingolipid accumulation and recapitulated major neurological and non-neurological clinical LSD presentations. Current projects focused on these models are as follows: 1) using a Parallel Reaction Monitoring, liquid chromatography–mass spectrometry-based method to reveal structure-specific ceramide regulation in zebrafish early development and in a zebrafish model of Farber disease, 2) uncovering the pathways driving early myelin loss in a zebrafish model of combined saposin deficiency, and 3) developing a high-throughput LC–MS-based chemical screening platform to identify small molecule inhibitors of lipid storage in a zebrafish model of Niemann-Pick disease Type A, B.
Opioid and Addiction
The United States of America are in the midst of an opioid addiction crisis leading to more than 37000 deaths annually. Unfortunately, treatments for substance abuse disorders are not very efficient and have a high risk of relapse. To identify novel therapeutic options, we have pioneered novel assays to study opioid abuse in zebrafish.
Zebrafish represent a unique model to study addiction; the reward pathway is well conserved, and the fish has been shown to exhibit characteristics of addictions to different drugs of abuse. Past research relied on non-contingent assays such as conditioned place-preference, however, the gold standard of substance abuse research are contingent assays such as self-administration paradigms.
We thus decided to develop the first self-administration assay for zebrafish. Using inexpensive electronic, mechanical, and optical components, the Peterson Lab has developed an automated opioid self-administration assay for zebrafish, which enables us to measure drug-seeking behaviors and ultimately gain greater insight into the underlying biological pathways of addiction.
Using this assay, we were able to perform a small-molecule screen to identify novel modulators of opioid self-administration. This led to the identification of finasteride as a potent modulator of intake. Fish treated with finasteride significantly reduce their intake and these observations are conserved in rats.
We are currently investigating the molecular mechanism by which finasteride affects opioid intake.
Sociality and Autism
Sociality is an integral part of human behavior, and its deficits are closely associated with neuropsychiatric disorders such as autism. The Peterson lab has developed large-scale social behavioral assays to identify small molecules that modulate the development and functional regulation of social behavior in zebrafish. Using a scalable social preference assay named Fishbook, we discovered that embryonic exposure to chemical inhibitors of Top2a leads to lasting social deficits in zebrafish and mice. We also applied machine learning to social behavioral analysis. Using an unsupervised deep learning method named ZeChat, we established a social behavioral profile for hundreds of neuroactive compounds and discovered a social stimulative effect in dopamine D3 receptor agonists.
Genome Editing Technology
Zebrafish Genetic Engineering
Zebrafish has proven to be a powerful vertebrate model system for functional genetic screens. A number of features, including high fecundity, rapid development, optical transparency, and genetic tractability make them a good model system. Pioneering large-scale forward genetic screens (where fish are mutagenized and screened for obvious defects) using ENU- and insertional mutagenesis established zebrafish as a model-of-choice for high-throughput in vivo genetic screens. While impressive in scale, forward genetic techniques introduce random mutations and require tremendous time and effort to link a desired phenotype with the genotype.
Our lab has been at the forefront of developing reverse-genetics methods that enable targeted gene disruption in zebrafish.
CRISPR-Cas9
The Peterson lab was one of the first to show that CRISPR-Cas9 technology can successfully be used in zebrafish for targeted gene disruption. More recently, the lab has made clever use of microfluidic technology, DNA barcoding, and CRISPR-Cas9 to enable large-scale reverse-genetic screens in zebrafish.
Transcription activator-like effector nucleases (TALENs) and Zinc-finger nucleases (ZFNs)
TALENs and ZFNs are powerful tools for targeted genome editing. The Peterson lab, in collaboration with Keith Joung’s and Joanna Yeh’s labs, pioneered the use of these technique in zebrafish.
Sander et al., Nat. Biotechnol. (2011) 29:697-8
Cade et al., Nucleic Acids Res. (2012) 40:8001-10
Optogenetics
Optogenetics is a biological technique which involves the use of light to control cells, typically neurons, in living tissue that have been genetically engineered to express certain light-sensitive ion channels. It has become such a powerful and effective research tool because it enables specific and high-resolution optical control of neuronal activity. In the Peterson lab, we've identified a small molecule, known as Optovin, which enables repeated photo-activation of wild-type zebra fish. Miraculously, in animals with severed spinal cords, Optovin treatment activates neuronal pathways which enable control of motor activity in the paralyzed extremities by localized illumination.
Lam et al., J. Am. Chem. Soc. (2020) 142:17457-17468
Counterterrorism
Cyanide, a metabolic poison known for its use as a chemical weapon in World War II and the Jonestown massacre, still presents a chemical threat today in the form of industrial accidents, smoke inhalation, and acts of terror. Cyanide inhibits Complex IV of the electron transport chain, preventing cells from consuming oxygen and producing energy. Cyanide exposure can rapidly result in seizures, cardiac dysfunction, and death. Available antidotes must be delivered intravenously within a narrow time window, meaning modern societies are still ill-equipped to handle mass casualty scenarios.
Our lab has developed a zebrafish model of cyanide toxicity to screen for novel, fast-acting, safe, stable, and potent cyanide antidotes. With collaborators around the nation, we use medicinal chemistry to explore hits from our high-throughput screens, ultimately generating lead compounds for validation in mouse, rabbit, and pig. This powerful pipeline has identified glyoxylate and cisplatin analogs (among others) as compounds of interest that we are continuing to investigate in terms of efficacy and mechanism of action.
Past Research
Disease Modeling and Drug Discovery
Small molecules are powerful tools for studying developmental biology because they provide timing and dosage control over developmental pathways that is difficult to achieve with genetic mutations. Unfortunately, only a handful of developmental pathways can currently be targeted with small molecules. We are discovering novel chemical modifiers of developmental pathways by exposing zebrafish embryos to libraries of structurally diverse small molecules and identifying those that induce specific developmental defects. Using screens of this type, we have discovered dozens of compounds that cause specific defects in hematopoesis, cardiac physiology, embryonic patterning, pigmentation, and morphogenesis of the heart, brain, ear, and eye and germ cell lineage.
Anxiety and Stress Response
Humans and many animals show 'freezing' behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, the Peterson Lab has developed a high-throughput behavioral assay in zebrafish which has allowed us to evaluate approximately 10,000 compounds for their effects on freezing behavior. Remarkably, We found three classes of compounds that switch the threat response from freezing to escape-like behavior.
Behavioral Phenomics Group
Behaviors are accessible readouts of the molecular pathways that control neuronal signaling. Our group develops tools and techniques for comprehensive and high-throughput behavioral phenotyping in the zebrafish. These tools have great potential to improve our understanding of neuronal signaling and may accelerate the pace of neuroactive drug discovery.
Kokel et al. Nat. Chem. Biol. (2010) 6:231-7
Neurodegeneration
Many neurodegenerative diseases involve selective apoptosis of subsets of neurons in the brain. We have developed transgenic zebrafish lines that model key aspects of these neurodegenerative diseases. We have also developed tools for tracking neuron loss in the brains of living zebrafish. Using these reagents, we are screening for small molecules that prevent neuronal cell death in the zebrafish models with the goal of identifying therapeutic leads for neurodegeneration.
Heart failure and diabetes
We have created zebrafish models of diabetes and chemotherapy-induced heart failure and used the models to discover compounds that reverse the disease phenotypes. Ongoing studies include efficacy studies in mouse models, structure activity relationship (SAR) studies, and proteomic-based target identification studies.
Acute myeloid leukemia (AML)
We generated a model of AML by expressing the human AML-causing oncogene AML1-ETO in zebrafish. These zebrafish accumulate granulocytic blast cells that resemble those found in humans with AML. In a robotic expression screen of thousands of small molecules, we discovered that nimesulide can reverse the oncogenic effects of AML1-ETO.
Dorsomorphin
One notable lab success in recent years has been the discovery of dorsomorphin and related BMP receptor antagonists. These small molecules were discovered during a zebrafish screen for compounds that alter development of the embryonic dorsal-ventral axis. As the first compounds to antagonize BMP signaling, the molecules have become powerful tools for studying BMP functions, and the molecules have already been used in hundreds of other studies around the world. In addition, the compounds have proven to be effective in treating animal models of BMP-related disorders, including heterotopic ossification and anemia. The compounds are currently in late stages of preclinical development.
Gridlock suppressors
Zebrafish gridlock mutants exhibit a dysmorphogenesis of the aorta that prevents circulation to the trunk and tail and is a model of human coarctation of the aorta. Gridlock mutants were exposed to thousands of compounds from a diverse small molecule library. Several compounds were identified that completely restore gridlock mutants to normal without causing additional developmental defects. The “gridlock suppressors” identified have revealed fundamental insights into artery formation, and the compounds appear to be effective in promoting new artery formation in mouse models of ischemia.
Peterson et al., Nat. Biotech. (2004) 22:595-9