Investigating the cell type-specific potentials for axon regeneration in zebrafish
- Author(s): Adula, Kadidia Pemba
- Advisor(s): Sagasti, Alvaro
- et al.
Synapses link neuronal populations into circuits, which in turn regulate behavior. Axon damage is a hallmark of traumatic injuries and neurodegenerative diseases such as Alzheimer’s. Its ravages upon individuals and societies alike are well known, yet effective treatment of the consequences remains a challenge to modern medicine. Axon regeneration in humans is quite poor due to the limited regenerative capacities of the peripheral nervous system (PNS) and the inhibitory conditions of the central nervous system (CNS). While axon regeneration is regulated by a fine-tuned balance between intrinsic and extrinsic factors, the contribution of intrinsic factors to this process is not fully understood. The MAP3Ks DLK and LZK are two such intrinsic factors. These axon damage sensors are activated in response to injury and direct diverse, context-specific outcomes to injury. Accounting for the environment around the cell, magnitude of the injury, cell type specificity, and subcellular localization of key actors offers insight into these disparate resolutions. This work designs an experimental approach to investigate the context-specific capacities for axon regeneration and finds that DLK and LZK have cell type- and injury type-specific responses to axon damage. In chapter 1, I describe a new protocol for precise axon injury in single cells using zebrafish. Unlike in mammals, the zebrafish PNS and CNS are permissive to axon regeneration, making them a perfect environment to study the contribution of intrinsic factors. I take advantage of zebrafish genetic tractability and optic clarity to mosaically label neuronal cell types. I then use a precise laser to cut individual axons with minimal damage to the surrounding tissue. Using live imaging, I capture the post-axotomy processes of Wallerian degeneration and the axonal dynamics of regenerating axons. In chapter 2, I describe a new zebrafish model to address the variability in DLK and LZK outcomes following axon injury. DLK and LZK CRISPR mutants, as well as DLK LZK double mutants, are adult viable and fertile. Using the protocol described in chapter 1, I found that DLK and LZK are redundantly required for axon regeneration in motor neurons, but not in touch-sensing neurons. However, DLK and LZK regulate collateral sprouting in non-injured axons of touch-sensing neurons, at both the individual neuron and population levels. In chapter 3, I describe an effort to create lines labeling DLK- and LZK- expressing neurons. In different organisms, RNA studies indicate that DLK and LZK translation is mostly neuronal; however, these studies lack cell-type specificity. Using CRISPR/Cas9 technology, I attempted to insert the signal amplifying GAL4 gene upstream of DLK and LZK start sites. These enhancer traps were characterized using confocal microscopy, and several techniques were used to validate the lines. Although validation revealed incorrect insertion of GAL4, identifying the cell types in which these proteins are expressed under basal and pathological conditions would be a powerful tool in elucidating their multifaceted activation.