Identifying conserved mechanisms of cranial muscle morphogenesis using the zebrafish

The human face is capable of a huge range of expressions from joy through to sadness, surprise and confusion. These expressions are caused by the coordinated movement of a large number of muscles, which are also required for talking, eating and looking. Each muscle in the face attaches to a specific location on the skull and this specificity is vital for the correct function of the muscle. Although the formation of heads in animal development has been studied for over 200 years, it is still not clear how the head muscles are positioned and which factors regulate this process. The principal aim of this proposal is to discover how the muscles in heads of animals with backbones (vertebrates) become correctly positioned during development and then attach to the appropriate points on the skull and jaw. The key aims and techniques of this proposal are: 1) describe how the head muscles form in zebrafish. I will observe head muscles as they form during development by labelling them with a coloured fluorescent dye. I can then test if other tissues in the head, such as bone or tendons, are important for positioning the muscles by specifically removing them using a laser. 2) show whether zebrafish head muscles are guided to their correct position by specific factors. To test if a factor is important for head muscle development, I will alter its function during development and show if this causes a change in muscle positioning and attachment to the skull. 3) show which factors are required for head muscle development in all vertebrates. I will show if factors important for positioning of zebrafish head muscles have similar roles in two other representatives of the animal kingdom: a primitive fish (dogfish) and a bird (chick). I use zebrafish embryos as my model system because development of the head is easy to observe in the transparent embryos and it has a fairly simple head organisation. This work will be performed at the Centre for Developmental Neurobiology at Kings College London in collaboration with Professor Susan Guthrie and Professor Anthony Graham. We will bring to bear our combined expertise of head development to provide a model for how the head muscles become properly positioned and attached to the skull in vertebrates. Despite obvious differences between the heads of adult vertebrates, early in development the heads of all vertebrate embryos, including fish, birds and humans, are very similar. This is because all vertebrates have a common ancestor and they have inherited many structures that were present in that ancestor. By comparing these structures such as the skull or jaw, between different animals, we can identify shared features that all vertebrates will be likely to possess. Almost always, these shared features arise in the embryos of different animals by the same process of cell movement and arrangement. These processes in turn, are controlled by the same factors, in different animals. This means that if I can find the factors that control how muscles are correctly positioned in the head of a zebrafish, a dogfish and a chicken, it is likely that they will be the same in all vertebrates, including humans. It is highly possible that all animals use the same factors for positioning of the muscles, BUT vertebrate heads show considerable variation in how the muscles attach, partly because animals have different food sources and lifestyles. How do these differences in muscle attachment and hence different functions of these muscles arise? I want to look at these differences, to show how the muscles attach to different points on the skull in different species. These differences in muscle positioning have lead to dramatic changes in how animals eat and communicate. As a consequence, I hope that my work will eventually lead to an understanding of how we gained the ability to smile and talk to each other.

This proposal aims to identify conserved genes important for vertebrate cranial muscle development. The zebrafish will be used as a model vertebrate system because: gene function can be easily manipulated, head development is relatively simple and cells can be observed in the transparent embryos throughout development. A transgenic line will be made with specific expression of a red fluorescent protein in developing cranial muscles. This line will used to follow development of cranial muscle precursors relative to neural crest cells expressing a green fluorescent protein. Laser ablation of defined populations of neural crest will be used to reveal temporal and spatial requirements for neural crest in cranial muscle morphogenesis. To test for a role of leading muscle cells in driving cranial muscle morphogenesis, specific cranial muscle cells will be laser ablated. A candidate signalling molecule SDF-1 will be tested for positional roles in directing muscle precursors movement by knockdown of gene function and ectopic expression. Genes mediating the response of muscle cells to SDF-1 or to another candidate signalling pathway important for muscle development, GDNF signalling, will be identified by expression profiling of muscle cells lacking active signalling. Candidate genes will then be tested for direct roles in cranial muscle morphogenesis. To show if cranial muscle development occurs by a mechanism conserved between all vertebrates, homologues of genes important for zebrafish cranial muscle morphogenesis will be tested for similar functions in a basal chondricthyan fish (dogfish) and a tetrapod (chick). Specifically, I will test 1) whether cranial muscles arrive at their correct positions by an active migratory behaviour or are moved by the neural crest; 2) how SDF-1 signalling directs muscle positioning; 3) which genes mediate muscle movement; 4) if cranial muscle development occurs by a conserved process.