Dissecting successful spinal cord regeneration in adult zebrafish

Adult zebrafish have the amazing capacity to regain swimming function after a complete spinal lesion. In mammals, such lesions lead to complete and permanent paralysis. Successful spinal cord regeneration in zebrafish is a highly complex process involving long processes (axons) of neurons in the brain (which convey movement commands from the brain to the spinal cord), motor neurons in the spinal cord (which grow their axons towards muscle targets and cause their contraction), as well as interneurons in the spinal cord (which coordinate the input from the brain). We have previously shown that severed axons form the brain regrow, that destroyed motor neurons are replaced by new motor neurons and that spinal interneurons may change their connections in order to adapt to altered input from the brain. In order to determine how these different components of regeneration are interconnected, we devised ways to reduce or enhance regeneration of spinal motor neurons. Thus we can ask what the role of spinal motor neuron regeneration is for recovery of swimming, for regrowth of axons from the brain and for connections of spinal interneurons. Moreover, we address the molecular mechanism of motor neuron regeneration from adult spinal stem cells by analysing the function of an important signalling pathway for stem cell differentiation. By elucidating successful spinal cord regeneration in zebrafish, we aim to identify the components necessary for a return of function after a spinal lesion or motor neuron disease from the molecular to the systems level.

Adult zebrafish, in contrast to mammals, functionally regenerate after complete spinal cord transection. This complex process likely involves regrowth of axons from the brainstem, cellular regeneration of spinal cell types, and plasticity of the existing spinal circuitry. We have previously shown that regrowth of axons from the brainstem is necessary for functional recovery and that lost motor neurons regenerate after a spinal lesion. Moreover, spinal interneurons express plasticity-associated genes in the lesioned spinal cord. Here, we intend to characterize which spinal cell types regenerate/undergo plasticity after a lesion using multiple labelling protocols of different cell types in adult fish, in which different interneuron cell types are transgenically labelled. In pilot experiments, we have established pharmacological tools by which we can reduce (cyclopamine) or increase (newly discovered agonist) the number of regenerating motor neurons. Thus, we can ask how motor neuron regeneration influences functional recovery, spinal plasticity, and axon regrowth from the brainstem by manipulating motor neuron numbers during spinal cord regeneration. It has been suggested that mammals are unable to regenerate spinal neurons, because spinal stem/progenitor cells show strong activation of the notch pathway, which keeps these cells in an undifferentiated stage in vitro. We will address the role of the notch pathway for successful motor neuron regeneration in vivo. We have established means to genetically augment (overexpression of activated notch) and pharmacologically inhibit (DAPT) notch pathway activity. With this project we aim to increase our understanding of the complex of contributing factors to functionally successful spinal cord regeneration at the molecular, cellular and systems level. Ultimately, our results may inform future therapies of conditions such as spinal cord injury and motor neuron disease.