TRAINING and EDUCATION
Demystifying the Lab
Reverse Genetics : Discovering New Functions of Known Genes Now that the human and zebrafish genomes have been completely sequenced , researchers are using reverse genetic approaches to directly investigate the functions of genes and pathways of interest and to validate genetic discoveries in patients . 3 , 4
“ As with many areas of biology , we can only answer questions when we have the appropriate technology , and sometimes we have to wait for the technology to catch up ,” said Seth Corey , MD , MPH , professor and chief of the Division of Hematology and Oncology and the Children ’ s Hospital Foundation Endowed Chair in Pediatric Cancer Research at the Children ’ s Hospital of Richmond at Virginia Commonwealth University . “ We proposed using fish to model Shwachman-Diamond Syndrome 10 years ago , when all we had were morpholinos to knock down gene expression . We were only successful when new gene-editing technologies came about . The use of CRISPR / Cas9 editing has revolutionized the way mutant strains can be developed .”
Dr . Berman agreed : “ CRISPR / Cas9-based genome editing has greatly facilitated the ability to knock out and knock in genes in the zebrafish genome to more accurately model human diseases .” A gene knock-out is when the gene of interest is inactivated to disrupt protein expression and analyze loss of function ; a knock-in involves the insertion or substitution of DNA at a specific locus in the genome to create a disease model and observe new phenotypes . Such manipulations of zebrafish genes can provide useful information about the function or associated phenotypes of similar human genes . 1 , 2
Dr . Berman explained that these approaches are now well established for germline mutations , and recent efforts have applied this technology to induce somatic mutations , such as those found in sporadic cancers .
Animal models are not completely accurate representations of what occurs in humans , but some models can provide novel insights into human disease , Dr . Corey added . “ It depends on the gene or pathway you ’ re interested in ,” he said . “ When there is high conservation between humans and fish , then the model is likely to be more accurate .”
Chemical Screens In addition to characterizing the mechanisms behind human diseases , zebrafish models can be used to identify and test new drugs for the treatment of these diseases using chemical screens .
“ We can bathe fish in thousands of different chemicals , and then find ones that affect something like stem cells or cancer cells inside the animal ,” Dr . White said . “ There is no other vertebrate animal that is capable of doing this .”
Dr . Zon , who uses zebrafish to study the developmental biology of hematopoiesis and cancer , agreed . “ Zebrafish is the best animal model for chemical genetics ,” he said . “ With embryos , drugs can be added to the water in well plates , and this allows high-throughput chemical screening of a whole vertebrate live animal .”
Adult zebrafish can undergo a similar process : Chemicals are placed in the water or injected intravenously or intraperitoneally . “ Since marrow transplants can be done on a large scale [ in zebrafish ], it is possible to do ex vivo chemical treatments and look for effects in vivo ,” Dr . Zon said . “ About 75 percent of chemicals from humans work on the zebrafish , and vice versa .”
“ We can bathe fish in thousands of different chemicals , and then find ones that affect ... stem cells or cancer cells inside the animal .”
— RICHARD WHITE , MD , PhD
High-throughput chemical screens “ can reveal new potential therapeutic avenues that can be prioritized and subsequently validated in other model systems for their ultimate translation back to the clinic ,” added Dr . Berman .
Zebrafish and Hematology The researchers who spoke with ASH Clinical News agreed that zebrafish provide a powerful tool in hematology / oncology research . “ Many laboratories have shown that zebrafish have conservation of all types of blood cells , including erythrocytes , neutrophils , lymphocytes , monocytes , mast cells , and HSCs ,” Dr . Berman said . The ability to track the fish ’ s development in real time “ has shed light on the origin and biology of these different hematologic cell populations .”
Highlighting a “ success story ” of zebrafish in hematologic research , Dr . Zon described how his group identified genes responsible for congenital sideroblastic anemia via large-scale forward genetic screens in mutant fish . The investigators also found patients with these same genetic mutations , “ so we discovered five human diseases as a result of our fish ,” he said . Similarly , “ ferroportin was first found ( and named ) in the zebrafish .” This iron transporter was subsequently shown to be mutated in patients with iron overload disorders . 8
Dr . Zon ’ s laboratory also used a zebrafish chemical screen to identify a drug that could enhance hematopoietic cell transplantation engraftment . 9 “ We found a drug , prostaglandin E2 , that can stimulate production of blood stem cells in the fish , and later showed that it worked to enhance stem cell numbers in the mouse ,” Dr . Zon said . “ The compound is now in its fourth clinical trial , looking at patients receiving cord blood units or mobilized peripheral blood stem cells for leukemia .”
More recently , “ transgenic zebrafish models of both acute lymphoid leukemia and acute myeloid leukemia have been used to identify a number of promising therapeutic compounds ,” noted Dr . Berman , including cyclooxygenase inhibitors in myeloid disease and phosphoinositide 3-kinase inhibitors in lymphoid malignancies .” 10
Transplantation of human cells into zebrafish , a technique known as xenotransplantation , also is being used to model human blood disorders and cancers and to screen human cells for drug susceptibility in vivo . 11 “ Transplanted larvae are bathed in the compound of interest , and the anti-proliferative response to the drug can be determined within the clinically-relevant timespan of a week ,” Dr . Berman explained .
“ Zebrafish have also been used to identify safer ways of providing chemotherapy ,” he added . “ By applying chemical screening to zebrafish larvae transplanted with human leukemia cells , compounds that protect against the cardiac damage caused by anthracyclines were identified , which importantly did not compromise the anti-leukemic effects .” 12
With an expanding repertoire of tools available to researchers who use zebrafish as a model organism , many more new discoveries are sure to follow . “ What fish offer is the ability to find new and unexpected things that would be tough to find in other models of cancer ,” remarked Dr . White . “ We validate what we find in the fish using human samples and tissues , making it a really exciting opportunity for discovery .” — By Amy Dear , PhD ●
REFERENCES
1 . Burke E . “ Why Use Zebrafish to Study Human Diseases ?” Accessed February 8 , 2018 , from https :// irp . nih . gov / blog / post / 2016 / 08 / why-use-zebrafish-tostudy-human-diseases .
2 . Clark KJ , Ekker SC . How zebrafish genetics informs human biology . Nature Education . 2015 ; 8:3 .
3 . Lawson ND , Wolfe SA . Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish . Dev Cell . 2011 ; 21:48-64 .
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7 . White RM , Sessa A , Burke C , et al . Transparent adult zebrafish as a tool for in vivo transplantation analysis . Cell Stem Cell . 2008 ; 2:183-9 .
8 . North TE , Zon LI . Modeling human hematopoietic and cardiovascular diseases in zebrafish . Dev Dyn . 2003 ; 228:568-83 .
9 . North TE , Goessling W , Walkley CR , et al . Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis . Nature . 2007 ; 447:1007-11 .
10 . Liu W , Wu M , Huang Z , et al . c-myb hyperactivity leads to myeloid and lymphoid malignancies in zebrafish . Leukemia . 2017 ; 31:222-33 .
11 . Deveau AP , Bentley VL , Berman JN . Using zebrafish models of leukemia to streamline drug screening and discovery . Exp Hematol . 2017 ; 45:1-9 .
12 . Liu Y , Asnani A , Zou L , et al . Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase . Sci Transl Med . 2014 ; 6:266ra170 .
50 ASH Clinical News March 2018