Fishing for treatments for muscle diseases
Jane Patrick, a PhD student at the Wellcome Trust Sanger Institute, studies zebrafish to learn more about muscle diseases such as muscular dystrophy. She explains her work in her commended entry for the 2014 Max Perutz Science Writing Award.
Which muscles are you using right now? Perhaps you’re absent-mindedly shaking a leg or munching on food? At the very least, I expect you’re breathing. The chances are you haven’t even noticed your muscles working. Most of us take our muscles for granted, but for a child born with an inherited muscle disease, such as myopathy or muscular dystrophy, it isn’t that simple.
These children have a faulty copy of a gene meaning their muscle doesn’t develop or work properly, so they have weak or degenerating muscles from birth or a very young age, and often developmental problems too. The problem is there are a vast number of different genes that can be affected, some unique to one patient, which gives a huge range of symptoms and makes it difficult to find an effective treatment.
Recently scientists have used DNA sequencing technology to identify new genes linked to inherited muscle disease by looking for the faulty genes in affected children. The question is, how can we use this list of genes to help to find a treatment?
In my PhD research I’m trying to do this by using genomics, the study of all of the genes in an organism, along with zebrafish. You might be thinking that fish are nothing like humans, but actually zebrafish are extremely useful for research in genetics and ideal for investigating muscle.
Unlike with mammals, the fish eggs are laid before fertilisation and develop entirely outside the mother. They have a transparent ‘eggshell’ meaning you can observe all stages of development without interfering, almost like having a window into a womb.
Usefully, the muscle is very obvious during development. It forms evenly spaced chevron shapes down the embryo, like those lines on the motorway that tell you how big a gap to leave behind the car in front. When zebrafish muscle doesn’t form properly this arrangement is disrupted and the pattern is often very obviously disorganised.
In my research, I’m trying to disrupt the same genes in zebrafish as those that are affected in children born with muscle diseases. Since the Human Genome Project sequenced, or ‘read’, the entire DNA sequence of humans, many other organisms have had their DNA sequenced too, including the zebrafish.
Most genes that cause muscle problems in humans have a partner in zebrafish and we can manipulate them to understand their function. I’m using a technique called CRISPR (pronounced “crisper”), which scientists developed from a mechanism found in bacteria. It involves targeting an enzyme to cut the DNA at a chosen position to prevent a specific gene from working.
I inject the instructions to make this enzyme, along with directions to the correct DNA section, into zebrafish embryos just after fertilisation. At this point just one single cell has formed, so my injection mix is taken into the cell and passed on to subsequent cells as they divide to form the embryo. I use this to make zebrafish with the same faulty gene as human patients and then look at how their muscle develops, to model the human disease.
Zebrafish with muscle defects have moving problems as they develop, which you can see by poking their tail and watching how well they swim away. I also look at the arrangement of different muscle components using antibodies, which can attach to a specific protein and then fluoresce under a microscope, to highlight the arrangement of the protein within the muscle and show any differences from a normal healthy embryo.
As well as looking at the muscle structure I’m trying to probe deeper into the molecular changes in my fish by using the rapidly advancing techniques of genomics. I measure how many products of every single gene are being made in my disease model fish and compare these to healthy fish, to look for differences that could be caused by the faulty gene.
I’m hoping to identify genes that are used more or less by the embryo as the muscle development goes wrong. Some of these may be the same across a number of the model fish despite them having different faulty genes originally, which could reveal common features amongst the genetic spectrum of diseases. They could provide exciting new targets for treatment that would be effective for many patients, despite differences in the exact genetic cause of their diseases.
So why do I think my research is important? Inherited muscle diseases are rare compared to diseases such as cancer, heart disease and diabetes, but for the children and families affected by them the numbers don’t matter.
What matters is the difference between walking and being confined to a wheelchair, between breathing effortlessly like you or me and struggling with each breath, perhaps the difference between life and death. It’s the difference between that absent-minded leg shake and a lifetime of struggling to move.