Properly functioning muscle is vital for the wellbeing of most animals (humans included) and diseases affecting muscle, such as muscular dystrophy, can be completely devastating. Whilst we have a fairly good understanding of how muscle works, our knowledge of how muscle function develops during embryogenesis is scanty. To help address this lack of understanding, in this study we investigated an immotile zebrafish mutant called sloth which has a defect in its muscle.
Sloth mutant embryos are completely immotile (see movie) and they die at around 6-7 days of development probably because they cannot feed. The mutation is recessive, meaning that adult fish can carry the mutation and remain healthy but 1/4 of the offspring of male and female carriers will be immotile. The paper describes the characterisation of the mutant phenotype, that is an investigation into the biological cause of the immotility. The study also identifies the gene, that when mutated leads to the muscle defect. In principle, the immotility could have had three possible causes: 1. A defect in the nervous system (brain and nerves) that controls movement; 2. A defect in the way the nervous system connects to the muscle; or 3. It could have been a defect in the muscle itself. We eliminated the possibility of defects in the nervous system or its connection to the muscle and found that there was a problem with the muscle of the embryos.
The defect we found was in the fine structure of the muscle. In order to contract (shorten), to move a limb for example, muscles use millions of tiny repetitive units called sarcomeres lined up in parallel. These sarcomeres are only about two thousanths of a millimetre long but when supplied with energy and nervous impulse they can contract, shortening their length. The way muscles are set up is exquisitely ordered so that when instructed by the nervous system, many millions of the sarcomeres contract simutaneously and the tiny shortening of the individual sarcomeres is amplified to produce a shortening of the whole muscle and a movement of the limb, or fin in the case of the zebrafish. Sarcomeres are made from alternating bands of thick and thin filaments that slide over each other to lengthen and shorten the sarcomere. When we looked closely at the sarcomeres in the muscle of sloth mutants, we found that the thick filaments were missing (see picture). Without the thick filaments the sarcomeres cannot change length and thus the muscle cannot contract and the embryo cannot move. This is the cause of the immotility.
As mentioned above, the sloth mutation is recessive and is therefore the result of a change in a single gene. Our next step was to identify this gene and explain why it caused the absence of thick filaments in the muscle. By careful pairing of specific carrier pairs of fish and the collection and examination of the DNA - the genetic material of the embryos, we were able to find which gene was changed in sloth mutants. We discovered that the affected gene in sloth mutants is a well known gene called Heat-shock protien 90a or Hsp90a for short. The Hsp90a protein is part of a family of proteins called chaperones. Chaperones are important because they help or chaperone other proteins to form the correct shapes and structures, and they also help groups of proteins to come together correctly in multi-protein complexes. The thick filaments of the muscle sarcomeres are an example of such a complex of proteins. With an additional simple experiment we showed that the sloth mutation causes Hsp90a to be completely ineffective as a chaperone protein.
Given our two main findings: The lack of thick filaments and the ineffective Hsp90a chaperone protein, we can conclude that chaperoning activity by the Hsp90a protein is essential for assembly of thick filaments and the construction of fully-functional muscles.
Why is this study important for the wider scientific community and society in general? Recently, a number of papers have indicated the importance of chaperones in muscle development. Our study adds to this and will help direct the search for new drug targets to treat muscle diseases, particularly drugs which may encourage regeneration of muscles through the recapitulation of development. Another way in which the sloth mutation may prove to be important is because the affected gene (Hsp90) is a known target for cancer drugs as it is thought to aid the survival of cancerous cells. A mutant in this gene is likely to be a useful tool in the investigation of the role of Hsp90 in cancer.
If you have any more questions about this work, please contact Tom (email@example.com) or Steve (firstname.lastname@example.org)
Our work on this project was supported by the ZF-Models EU integrated project and by the Wellcome Trust.