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Feature Articles : Oct 1, 2013 (Vol. 33, No. 17)

Human Disease Models Move Underwater

  • Nsikan Akpan, Ph.D.

From Ilya Mechnikov discovering phagocytosis in starfish larvae to Hodgkin and Huxley defining the principles of nerve conduction in the giant axons of squid, aquatic animal models have influenced our understanding of human biology for over 100 years.

Yet the power of these marine models extends past the realm of basic biology and into the sphere of translational medicine, offering unique versatility for pathogenic exploration that cannot be found in other animal models.

This facet was on marked display at the “Aquatic Animal Models for Human Disease and Midwest Zebrafish” conference held at the University of Wisconsin-Milwaukee School of Freshwater Sciences in July.

Simplicity and a diversity of tools are the prominent advantages of aquatic models of human disease, according to developmental biologist Anthony De Tomaso, Ph.D., associate professor at the University of California, Santa Barbara.

Such is the case with his study of Botryllus schlosseri, a star-shaped invertebrate whose extracorporeal vasculature—coursing along the surface of its body—allows for direct examination of the interactions between immune cells.

“Basically it is a simplified model for understanding self/nonself recognition or allorecognition,” said Dr. De Tomaso. “Our [human] immune cells are running around inside of our bodies, while in Botryllus, the cells are outside the body. Thus you can touch them or watch them come into contact with each other, all within two sessile cell layers.”

Allorecognition enables genetically compatible Botryllus tunicates to form colonies, but also features in fusion-rejection pathways that functionally mimic how the human body accepts or rejects organ transplants.

In humans, allorecognition is governed by the major histocompatibility complexes, which are the most diverse gene family in vertebrates. In contrast, histocompatibility in Botryllus is governed by single locus—fuhc—that encodes two highly polymorphic genes. Dr. De Tomaso has uncovered that these two inputs let Botryllus’ cells “integrate multiple signaling events from the cell surface and make a decision on a response.”

These pathways have been highly conserved throughout metazoan evolution, suggesting that understanding chordate histocompatibility can provide clinically relevant information on immune processes such as tolerance and education, especially for natural killer cells, according to Dr. De Tomaso.

Another translational feature of Botryllus involves a fierce competition between the stem cells of two tunicates fusing into a new clonal chimera. In some incidents, circulating stem cells from one animal will try to hijack the germline or somatic cells of its new partner.

This cellular parasitism hearkens to the aggressive nature of malignant tumors and cancer stem cells.

“The Botryllus system has a natural situation where variation in stem cell properties—growth, migration, competition—provides a definite correlation to why one stem cell population is better than the next,” said Dr. De Tomaso. “This is the other biomedical aspect of our work. Botryllus could provide clues as to why some cancer stem cells are better at migrating or self-renewing.”

Growing Older with Aplysia and Icefish

While histocompatibility represents an immunological definition of self, people are most cognizant of the identity defined by their mental awareness, especially their memories. This is most pertinent in the clinical realm of Alzheimer’s disease and other forms of dementia.

New dimensions of neurobiological aging are being revealed by research with the seaslug Aplysia californica in the laboratory of Lynne Fieber, Ph.D., associate professor of marine biology and fisheries at the University of Miami. Based across the street from the NIH/University of Miami National Resource for Aplysia Facility, Dr. Fieber’s team of marine biologists has uncovered novel insights into age-related declines in glutamate signaling.

“We’re trying to understand the fundamental differences between sensation and motor activity,” said Dr. Fieber. “When you feel pinch or heat on the tip of your finger, how does that reflex age?”

Over the last two decades it has become clear that changes in sensory and motor function are associated with Alzheimer’s disease, especially at early or presymptomatic stages of the condition. Reductions in glutamate levels within the nervous system are a classic hallmark of aging in both humans and Aplysia.

Aplysia offers a great context for neurobiological aging studies as its entire lifespan is completed within a single year. In addition, its nervous system is rather small (20,000 neurons) in comparison to humans (85 billion neurons) or animal models such as mice (75 million neurons). Thus there is nearly a one-to-one correspondence between sensory neurons, motor neurons, and muscles, which provides better resolution for deciphering neural circuits and electrophysiology.

“The organization of the nervous system is different in invertebrates compared to vertebrates. Nevertheless at a basic level, there are very conserved physiological and neurological phenomena that are present in both kinds of animals,” said Dr. Fieber, who noted that Eric Kandel’s groundbreaking research with Aplysia demonstrated how neurons are able to form and store memories.

Dr. Fieber is capitalizing on the power and simplicity of this model to examine D-aspartate, a chemical in the nervous system that is emerging as a possible endogenous agonist for glutamate receptors. She began the project a few years ago in collaboration with neurobiologist Antimo D’Aniello, Ph.D., of the Stazione Zoologica (Naples, Italy). Dr. D’Aniello has found that free D-aspartate in the brains of humans radically changes with age.

While D-aspartate can substitute for glutamate at certain synapses, it appears that the compound also activates an unidentified receptor in mammals and Aplysia. Dr. Fieber is now conducting a search for this unidentified glutamate receptor.

Cognitive prowess is not the only line of aging research to benefit from aquatic models. John Postlethwait, Ph.D., professor of biology at the University of Oregon, is studying ancient Antarctic icefish to find clues as to why bones lose mineral density over the course of lifetime, a condition known as osteopenia.

Although less severe than osteoporosis, this bone condition is nearly three times as prevalent in people over the age of 50. Nearly four out of five cases are associated with acute pain, and 25% of women with osteopenia will sustain a vertebral compression fracture.

Instead of occurring over developmental time—80–90 years for humans—bone density was lost in Notothenioid fish lineages over the course of 10 million years. As the waters cooled along the coasts of Antartica, the rich fauna near the shore of the island continent began to die off and were replaced by the bottom-feeding—benthic—Notothenioids. However these fish lacked a swim bladder, a lung-like sac that allows fish to regulate their buoyancy. So to complete their upward migration, Notothenioid species either acquired the ability to produce more lipids or lost mass from their bones, which are the most dense organs in the body.

How does evolutionary progression with icefish relate to age-related osteopenia? More buoyant icefish lineages retained their genes for bone mineralization, but changed how they regulate these genes, which is a scenario that mimics bone density loss in humans.

“The two processes, in humans and icefish, are the same,” said Dr. Postlethwait. “We know that most people don’t have mutations that completely block bone mineralization. What they have are mutations that dysregulate the control of bone mineralization genes.”

By comparing the genetic profiles of poorly and robustly mineralized fish, Dr. Postlethwait’s team hopes to isolate regulatory gene regions—enhancers, promoters, and repressors—that are responsible for maintaining bone density.

These findings could be used to improve the resolution of genome-wide association studies (GWAS) of skeletal profiles, which have identified about 60 loci of previously unknown function.

“The problem is many or most of these identified genetic loci are out in the middle of nowhere. They are near genes but not in coding regions,” said Dr. Postlethwait. “If we can identify specific regulatory elements that are responsible for bone mineralization dissonance in the Antarctic icefish, we can then search for similar elements in humans.”

Fish Tools for the Future of Comparative Medicine

Dr. Postlethwait and many other investigators subsequently turn to zebrafish as an in-lab model for examining the functions of potential disease-related genes.

One burgeoning domain for this popular aquarium fish is as an in vivo model for aneuploidy in cancer. Aneuploidy—or an abnormal number of chromosomes—is universally observed in all types of human cancer, yet genetic mouse models of these diseases rarely feature aneuploidy.

This chromosomal phenomenon is relatively easy to replicate in zebrafish, according to Guang Jun Zhang, Ph.D., assistant professor of genetic epidemiology and comparative medicine at Purdue University, whose lab mutates either ribosomal proteins or p53 to develop tumors in zebrafish.

Dr. Zhang has pioneered zebrafish-human oncogenomics, a comparative approach that can reveal novel cancer driver genes. The zebrafish genome shares about 70% identity with the human genome, but through evolutionary synteny, many of the genetic loci have moved to different chromosomal locations.

Oncogenomics allows Dr. Zhang to illuminate patterns in the aneuploidy of human cancers and contrast those with genetic arrangements in zebrafish tumors. Cancer is an automatic evolution process, so the technique reflexively selects for chromosome-related growth advantages.

This investigation has yielded a plethora of gene candidates that may function as oncogenes or tumor suppressors, which Dr. Zhang’s team is now using to build new zebrafish lines that model cancer.

“You cannot explore 100 new genes in mice; it would cost a fortune. But in zebrafish, it is very possible,” said Dr. Zhang, who cites their rapid breeding, cost effectiveness, and broad availability as benefits of the model.

He added that the Sanger Institute’s comprehensive Zebrafish Mutation Project, which aims to create a knockout allele in every protein-coding gene in the animal’s genome, should be completed in the next two years, providing an exceptional resource for comparative medicine studies within the model.

Moreover, recently developed technologies for gene editing—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISP/CAS-9—are rapidly moving into fish models.

“These innovative tools are opening two types of doors,” said molecular physiologist Aron Geurts, Ph.D., of the Medical College of Wisconsin, who specializes in gene targeting with rat models of cardiovascular disease but was invited to attend the conference as a guest lecturer.

“First, gene-editing technology is enabling very elegant experiments that were previously restricted only to the mouse,” remarked Dr. Geurts. “In addition, these tools are empowering precision editing of genomes for the assessment of specific disease-linked alleles.”

This will move animal models away from the realm of simple knockouts and knock-in breeding and will allow future investigators to ask very specific questions about the role of particular single-nucleotide polymorphisms (SNPs) in pathogenic phenotypes.

GWAS and large-scale genetic investigations, like the Encyclopedia of DNA Elements (ENCODE) Consortium and the 1000 Genomes Project, are really driving this research, according to Dr. Geurts, who is a big proponent of using aquatic models in scenarios where biological questions are not easily answered by mammalian systems.

“We’re going to see more people working with genetic engineering in these models, which may be better than mice in some areas of translational medicine,” concluded Dr. Geurts. “That’s where the field is going.”

New Animal Model Introductions

Charles River Labs reports that it will introduce four pain models (rat) for translational disease research this fall.

According to Mike Luther, corporate vp, scientists using a glycolytic inhibitor (monoiodoacetate) will be able to observe behavioral, histological, and biochemical changes that resemble human osteoarthritis and its associated joint pain with the Monoiodoacetate Chronic Joint Pain Model.

Luther also pointed out that the Formalin Model of Spontaneous Behavioral Pain is widely utilized as an acute and rapid in vivo screening assay for evaluating the potential analgesic effects of novel chemical entities.

The Spinal Nerve Ligation (SNL) or Chung Model of Neuropathic Pain is considered one of the most suitable models for analgesia screening against neuropathic pain, noted Luther, adding that the rat paw Complete Freund’s Adjuvant Model of Inflammatory Pain is well characterized in the literature and is routinely used for screening novel compounds targeted for inflammatory pain.

“We have implemented a set of pain models and assays that are robust and reproducible and which provide translational endpoints that allow our clients to make the appropriate decisions regarding progress for their projects and pipelines,” explained Luther.

“These new models are part of our integrated approach to support the lead to candidate process in drug discovery from early PK and PD to efficacy as well as mechanism of action studies, including early safety and toxicology studies for candidate selection.”

Oncology Models

Taconic says it will expand its oncology portfolio later this year with the inclusion of four new mouse models from the Netherlands Cancer Institute.

The Brca1-Associated Breast Cancer Model is a conditional mouse mutant with somatic deletion of Brca1 and Trp53 in several epithelial tissues including mammary epithelium. This model may be helpful in predicting responses of human BRCA1-deficient tumors to therapies, according to Taconic.

The Invasive Lobular Breast Cancer Model was developed to serve as a tissue-specific conditional knockout of Cdh1 (E-cadherin) and Trp53 in mice. It induces metastatic mammary carcinomas that resemble human invasive lobular carcinoma, the second most common type of primary breast cancer. The model is intended to serve as a tool for the development of therapies for the treatment of lobular breast cancer.

The Floxed Ink4a/Arf Mouse contains a targeted mutation of Cdkn2a (Ink4a/Arf), which introduced LoxP sites upstream of exon 2 and downstream of exon 3. It can be crossed with the tissue-specific cre of a researcher’s choice to develop a tumor model.

Floxed p53 Mouse contains a targeted mutation of Trp53, which introduced LoxP sites flanking exons 2 through 10. It can be crossed with a tissue-specific cre to generate a conditional disruption of the Trp53 tumor suppressor gene, the most commonly mutated gene in human cancers. Taconic says it will be useful for studying Trp53 gene function or screening potential cancer intervention therapies.

“Taconic’s newest transgenic oncology mouse models can help researchers accelerate drug discovery by providing better predictability of outcomes in the clinical lab setting and by offering a more reliable screening tool for use in target selection,” said Megan McBride, Ph.D., associate director, scientific marketing. “By using tumor cell lines that correlate well with the tumors they were derived from, these models can also accelerate the target validation process by helping investigators determine early on which targets are best pursued in later-stage drug development efforts for oncology therapeutics.”