Interpreting Human Genetic Variation With In Vivo Zebrafish Assays
Material below is adapted from the SfN Short Course Interpreting Human Genetic Variation With In Vivo Zebrafish Assays, by Erica E. Davis, PhD, Stephan Frangakis, and Nicholas Katsanis, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.
Amidst the accelerating pace of gene discovery lies the realization that each individual’s genome contain a sobering degree variation containing hundreds of mutations and variants — many of them rare. Accurately interpreting the biological relevance of this variation poses considerable challenges to traditional methods of studying human genetic disorders.
Researchers have learned a great deal studying human genes inserted into mouse models. However, the onslaught of genetic discoveries combined with the time and costs required to screen each variant in mouse models make such methods unworkable.
Faster, cheaper screening can be achieved with other model organisms such as the roundworm (Caenorhabditis elegans) and fruit fly (Drosophila melanogaster). While both are easy to manipulate genetically, they are a lot less like human beings. Zebrafish (Danio rerio) models are beginning to bridge the gap between species.
Researchers began using zebrafish about 20 years ago in a quest to understand vertebrate development. The common freshwater aquarium species has generation times of just three to four months, and each mating pair produces several hundred transparent embryos that develop rapidly and synchronously, making their development easy to observe.
Like humans, zebrafish are vertebrates with many homologous structures — 70 percent of zebrafish genes have a human ortholog. Decades of developmental study has spawned a vast catalog of mutants and a public, annotated genome all stored in ZFIN, the Zebrafish Model Organism Database, along with anatomical atlases and useful molecular tools.
Those tools include several methods developed over the past 15 years that precisely target candidate genes and alleles. Morpholinos, antisense oligonucleotides, can knock down expression of specific genes. Scientists can create germline mutations using retroviruses, transposons, and a technique called TILLING that induces local, targeted genomic lesions.
Newer genetic tools allow very precise editing of the genome using specific nucleases and molecular guides. These include zinc finger nucleases to target sequences with Fokl endonuclease that guide DNA cleavage and repair at target sites, TALENs (transcription activator-like TAL effector nucleases), and CRISPRs (clustered, regularly interspaced, short palindromic repeats), which guide RNAs to direct site-specific DNA cleavage via the Cas9 endonuclease.
Zebrafish models have contributed to our understanding of several human disorders. For one, Timothy syndrome, a disorder characterized by irregular heartbeat, webbed fingers and toes, and abnormal development of the face and jaw, is caused by a mutation in the gene for a voltage-gated calcium channel. Expression of the gene variant CACNA1C in zebrafish revealed a gain of function and a new role for the channel in non-excitable cells of the developing jaw.
Similarly, a zebrafish model revealed that the gene KCTD13 drove the neurocognitive defects associated with human genetic disorder copy number variant 16p11.2, a duplication and deletion on chromosome 16. And a stable, transgenic zebrafish model carrying the human 4repeat Tau gene accumulates Tau protein within neurons resembling the neurofibrillary tangles found in Alzheimer’s disease.
The zebrafish model has drawbacks. The most serious is that their genomic structure, like that of all teleost fishes, differs notably from other vertebrates. A teleost-specific genome duplication disrupts the correspondence between their genome and ours, with a preponderance of many-to-one and one-to-many rather than one-to-one relationships among orthologous genes.
Still the genetic tractability, ease of breeding and care, and early success in some human diseases point to the potential for many contributions to our understanding of how human genetic variety contributes to health.