Balancing growth and repulsion: Slit proteolysis generates diverse signals in axon guidance
Authors
Kellermeyer, Riley
Issue Date
2021
Type
Dissertation
Language
Keywords
axon guidance , Slit , Slit cleavage , SLIT2
Alternative Title
Abstract
The nervous system gathers and interprets information from the environment and can generate a physiological response. Interpreting environmental stimuli requires communication between the surface of the body and the brain via neural networks. Correct formation of the central and peripheral nervous systems (CNS and PNS, respectively) require precise connections to allow localization of stimuli and movement. Neurons extend long projections called axons to connect with distant targets, a process controlled by axon guidance. Axons are guided to the correct location by extracellular axon guidance cues. Key regulators of axon guidance are large secreted proteins called Slits. Slits were first identified as repellents of CNS axons at the midline, the medial separation of the left and right sides of the body. In the absence of Slits, all CNS axons collapse onto the midline. The repulsion conducted by Slits is essential to maintain separation of axons on the left and right sides of the body, conducting coordinated movement, and survival. Slits are proteolytically processed in the extracellular space into N-terminal (Slit-N) and C-terminal (Slit-C) fragments. While Slits are essential repellents, several early studies indicated that Slit fragments, particularly Slit-N, can have non repellent functions in axon guidance, by promoting axon growth, branching, and fasciculation. Slit-N appears to only act as a repellent at higher than endogenous concentrations. Despite these early findings, the majority of subsequent studies have used Slit-FL and Slit-N as interchangeable repellents. In Chapter 2, we clarify the roles of individual Slit fragments and full-length Slit (Slit-FL) and identified the protease responsible for cleaving Slits. We found that the Slit protease in Drosophila is the zinc metalloprotease Tolkin (Tok). Tok is the sole protease for Slit in Drosophila embryonic development. We confirm that Slit cleavage is not required for midline repulsion, but is required for longitudinal axon fasciculation. Defasciculation and guidance errors in tok mutants are fully rescued by expression of Slit-N, but not Slit-C, suggesting that Slit is the primary substrate for Tok at the CNS midline. We determine that neither Slit-N nor Slit-C are capable of conducting midline repulsion. These results confirm that Slit-FL and Slit fragments are both required in early embryonic development to conduct different functions. Our identification of Tok as the Slit protease will aid in future evaluation of the distinct functions of Slit fragments. In Chapter 3, we identified a conserved mechanism of Slit processing in vertebrates. The vertebrate homolog of Slit, SLIT2, is proteolytically processed and has similar function in CNS midline repulsion. SLIT2 also acts as a repellent for sensory neurons from dorsal root ganglia (DRG) that infiltrate the spinal cord and branch to grow longitudinally. DRGs cultured ex vivo are repelled by an uncleaved form of SLIT2 (SLIT2-UC) but promoted to grow and branch in response to SLIT2-N. DRGs are an excellent system to evaluate the potential conserved function of Slit proteolysis and Slit fragments. We find that the vertebrate homolog of Tok, Tolloid-like1 (TLL1), is a primary protease responsible for SLIT2 cleavage in mice. In vivo, the full-length SLIT2 produced by a SLIT2-UC allele is sufficient to repel DRG axons from the CNS midline but is not capable of rescuing longitudinal axon fasciculation or errors in DRG branching as they enter the spinal cord. We find that SLIT2-N and cleavable SLIT2-FL are capable of stimulating DRG fasciculation, but no obvious function for SLIT2-C. This is the first identification of dual functions of SLIT2 fragments in vivo, with SLIT2-UC repelling axons and SLIT2-N instead providing a fasciculation cue. These findings are essential for applying the proteolytic interaction between TLL1 and SLIT2 to other biological systems. In Chapter 4, we review the current literature for evidence of individual Slit fragments functions. From a thorough assessment of Slit fragments across development and disease biology, we find that Slit fragments are inappropriately identified or mischaracterized in most experimental systems. Because SLIT2-FL and SLIT2-N are frequently interchanged, we find that Slits have dual functions in axon guidance, angiogenesis, metastasis, and cell migration and adhesion, some of which can be clearly attributed to Slit processing. These findings corroborate that other biological systems would benefit from careful testing of separate SLIT2 fragments in future analysis.
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Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 United States