In the race to understand the genetics of amyotrophic lateral sclerosis (ALS), the lowly zebrafish just might swim off with a share of the prize. At the Fondation André-Delambre’s annual meeting, held September 24-25 in Québec City, Canada, Pierre Drapeau of the Université de Montréal presented his work on genetic interactions between the top ALS-linked genes superoxide dismutase 1 (SOD1), TAR DNA Binding Protein 43 (TDP-43), and Fused in Sarcoma (FUS). By mixing knockdowns of those genes with overexpression of both mutant and wild-type versions in zebrafish embryos, Drapeau is building a data chart, or “matrix,” that shows which genes interact with each other. He concluded that TDP-43 and FUS work together in a pathway distinct from SOD1.

Why zebrafish? These finned vertebrates have much in common with humans. They share the same basic organ and tissue layout, and their proteins have 50-80 percent amino acid identify with those of people. Zebrafish TDP-43, for one, is 73 percent identical with the human protein. Conveniently for researchers, zebrafish embryos develop quickly; their first day is roughly equivalent to the first trimester for mammals. And when poked, they dart away, making it easy for researchers to see motor defects (Drapeau et al., 2002). They are also useful thanks to their simple neural physiology; each somite, or segment, possesses three motor neurons with long axons, again making it easy to spot motor neuron pathology. (Reviewed in Kabashi et al., 2010) and Best and Alderton, 2009.

Scientists have tools to knock down native fish genes or overexpress them, or even alter expression of multiple genes in the same embryo. By targeting ALS-linked genes in this fashion, Drapeau has created zebrafish that have motor and axon defects. “I am not saying this is ALS in the zebrafish,” Drapeau said, but “it recapitulates some features.”

However, there is a caveat to zebrafish studies, Drapeau conceded, whereby researchers may trade speed and convenience for direct disease relevance. “We have to keep in mind that we are dealing, potentially, with developmental defects, rather than neurodegenerative changes,” wrote Jean-Pierre Julien of Université Laval, who organized the symposium, in an e-mail to ARF.

In a recent study (Kabashi et al., 2010), Drapeau and colleagues examined TDP-43 function by overexpressing the human version of the protein. They used wild-type as well as three mutations that cause ALS in people: A315T, G348C, and A382T. They injected the knockdown or overexpression constructs into embryos one day post-fertilization, and analyzed them upon hatching a day later. The mutant genes caused short, overly branched motor neurons and hampered the fishes’ swimming; overexpressing the wild-type human protein caused similar but milder pathology.

When the researchers knocked down the fish’s normal TDP-43 expression, they observed the same axonal and swimming problems, indicating that both too much or too little TDP-43 is bad for embryonic development. Introducing human wild-type TDP-43, but not the mutants, into embryos was able to rescue the phenotype. The authors suggest that both loss of normal function and gain of toxic function may be at work in people with TDP-43 mutations.

In Québec, Drapeau reported on similar experiments using FUS genes. He used the wild-type as well as disease-associated mutations: R521C, R521H and a deletion after S57. Wild-type FUS was harmful only if expressed at high levels. The embryos were malformed and died. The mutant FUS transgenes impaired swimming at lower expression levels. R521H gave the most serious phenotype including shortened, over-branched axons, which was similar to that of the TDP-43-overexpressing fish.

When the scientists knocked down zebrafish FUS, they observed the same excessively branched axons and poor swimming. Over-expressing wild-type FUS rescued the knockdown embryos, but mutant FUS did not.

TDP-43 and FUS are both RNA-binding proteins (see ARF News story on Kwiatkowski et al., 2009 and Vance et al., 2009), and could conceivably be involved in similar processes. To analyze their relationship, Drapeau mixed the FUS and TDP-43 treatments together to start crafting his matrix. He found that human FUS rescued the motor neuron phenotype in TDP-43 knockdown embryos. However, human TDP-43 overexpression failed to rescue the FUS knockdown deficiencies. Drapeau concluded that the two function in the same processes. Since the presence of TDP-43 negates the need for FUS, FUS must be downstream of TDP-43. “They are working in one pathway,” he said. “It does not matter where you hit it, you will interfere with it.”


Genotype + WT hTDP-43 + mutant hTDP-43 + WT hFUS + mutant hFUS
TDP-43 knockdown Rescue No rescue Rescue  
FUS knockdown No rescue   Rescue No rescue

Pierre Drapeau invited attendees to “enter the ALS matrix” with him as he detailed genetic interactions between TDP-43 and FUS.

In human ALS, TDP-43 pathology is common except in people who carry SOD1 mutations. When Drapeau studied SOD1 and TDP-43 or FUS mutations in the same animals, he found no genetic interaction between SOD1 and TDP-43 or between SOD1 and FUS. This confirms that SOD1 acts independently from the TDP-43 and FUS pathway, he said.

Next, Drapeau plans to use zebrafish with inducible TDP-43 expression to screen for drugs that rescue the phenotype (zebrafish drug screens reviewed in (Zon and Peterson, 2005). Since researchers have to check each fish for its swimming prowess, the process won’t be “high-throughput,” he joked. It’s more like “through-putt-putt.”—Amber Dance.

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News Citations

  1. New Gene for ALS: RNA Regulation May Be Common Culprit

Paper Citations

  1. . Development of the locomotor network in zebrafish. Prog Neurobiol. 2002 Oct;68(2):85-111. PubMed.
  2. . In the swim of things: recent insights to neurogenetic disorders from zebrafish. Trends Genet. 2010 Aug;26(8):373-81. PubMed.
  3. . Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009 Feb 27;323(5918):1205-8. PubMed.
  4. . Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. PubMed.
  5. . In vivo drug discovery in the zebrafish. Nat Rev Drug Discov. 2005 Jan;4(1):35-44. PubMed.

Other Citations

  1. download .pdf of series

Further Reading


  1. . Amyloid precursor protein is required for convergent-extension movements during Zebrafish development. Dev Biol. 2009 Nov 1;335(1):1-11. PubMed.
  2. . Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish. Hum Mol Genet. 2007 Oct 1;16(19):2359-65. PubMed.
  3. . A zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and drug evaluation. J Clin Invest. 2009 May;119(5):1382-95. PubMed.
  4. . Decreased BDNF levels are a major contributor to the embryonic phenotype of huntingtin knockdown zebrafish. J Neurosci. 2009 Feb 4;29(5):1343-9. PubMed.
  5. . Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 2008 May;4(5):295-305. PubMed.
  6. . Zebrafish (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production and requires a critical aspartate residue for its function in amyloidogenesis. Biochemistry. 1999 Oct 12;38(41):13602-9. PubMed.