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A ventral source of UNC-6/Netrin is dispensable for dorsal-ventral axon guidance
Hooper, Kelsey Marie
Hooper, Kelsey Marie
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Abstract
The nervous system is an intricate network of specialized cells that processes and transmits information. It controls both physiological and behavioral responses, regulating everything we think and do. A properly functioning nervous system depends on the ability of neurons to make the correct synaptic connections. Defects in neuronal development can lead to neurodevelopmental disorders such as intellectual disability and ADHD. Neurons establish connections by extending axons that navigate through the developing nervous system to locate their synaptic partners. Axons are guided to their targets by motile structures at their tip that interpret and respond to guidance cues. These structures, referred to as growth cones, are comprised of actin filaments and microtubules. The growth cone body is surrounded by an actin-based lamellipodial veil and dynamic filopodial protrusions that act as sensors for guidance cues. Directed migration is accomplished through the reorganization of the growth cone cytoskeleton to establish a spatial bias of F-actin and stabilized filopodial protrusions, which mark the site of future axonal outgrowth. Growth cones can respond to multiple guidance cues depending on the expression of different receptors. For example, growth cones respond to netrin through interactions with the UNC-40/DCC and UNC-5 receptors. Netrin-mediated axon guidance has been studied in both vertebrates and invertebrates and is classically believed to occur through a secreted gradient that attracts some axons and repels others. Though netrin-mediated axon guidance has been extensively studied, most experiments focusing on uncovering how the growth cone responds to directional gradients of netrin have been done in vitro. Caenorhabditis elegans are small, naturally soil-dwelling nematodes commonly used to study neuronal development due to their relatively simple nervous system and invariant cell lineage. C. elegans are also transparent, allowing for the easy visualization of neurons and the detection of developmental defects through the use of fluorescent proteins. In C. elegans the netrin homolog UNC-6 guides circumferential axons through interactions with UNC-40 and UNC-5. Using this system we can study UNC-6/netrin-mediated axon guidance in vivo.UNC-6/netrin is secreted by ventral tissues and guides multiple axons in C. elegans including the VD/DD axons. The VD/DD axons are dorsally directed axons that extend from the ventral nerve cord to the dorsal nerve cord. The DD axons migrate during embryogenesis, but the VDs migrate during the larval stage allowing us to visualize the growth cone during migration. Our previous work with the VD growth cones revealed a mechanism of UNC-6/netrin-mediated axon guidance that was inconsistent with a directional gradient. Our findings led us to develop a new model, the polarity/protrusion model, which involves first the polarization of the growth cone by UNC-6 and then the regulation of filopodia by UNC-5 and UNC-40 to create a dorsal bias of protrusions. In this work, we focus on expanding our current model and on gaining a deeper understanding of how UNC-6/netrin controls both dorsal and ventral axon guidance.In chapter II, we start by introducing a newly constructed membrane-bound UNC-6 edit. Using this edit we show that secreted UNC-6 is required for the guidance of the ventrally directed AVM, PVM, HSN and PDE axons.In chapter III, we employ our membrane-bound edit to demonstrate that close-range, haptotactic interactions between the growth cone and UNC-6 are sufficient to establish VD growth cone polarity and initiate dorsal axon guidance. In the absence of secreted UNC-6, growth cones eventually lose this polarity. We speculate that continuous interactions with UNC-6 are required to maintain polarity, which is revisited in chapter IV. We also show that the long and short isoforms of UNC-5 have individual roles in determining polarity of the growth cone. The short isoform can establish filopodial polarity as growth cones emerge from the ventral nerve cord in the absence of the long isoforms. We show that the regulation of filopodial polarity by the short isoform does not depend on secreted UNC-6. The long isoforms of UNC-5 alone are also sufficient to establish polarity but, unlike UNC-5 short, the long isoforms can maintain polarity. We find that UNC-40 has no role in regulating filopodial polarity. Finally, in chapter III we demonstrate a new role for the UNC-6 VI domain in VD/DD axon guidance. We show that a missense mutation within this domain has different effects on the short- and long-range functions of UNC-6. This reveals a possible role of the VI domain in regulating the distribution of UNC-6 throughout the extracellular matrix.Chapter IV addresses the role of a ventral source of UNC-6 in axon guidance and VD growth cone polarity. We show secretion of UNC-6 from anterior and dorsal sources rescues the guidance and growth cone polarity defects of both membrane-bound UNC-6 and unc-6 knockouts. Furthermore, we show that ectopic UNC-6 can guide the ventrally directed AVM and PVM axons. This reveals that a ventral source of UNC-6 is dispensable for C. elegans circumferential axon guidance.Chapter V provides insights into the role of glycosylation in UNC-6/netrin-mediated axon guidance. We demonstrate that the loss of N-linked glycosylation sites leads to defects in dorsal axon guidance, but not ventral axon guidance.In summary, this work presents a new framework for thinking about UNC-6/netrin-mediated axon guidance. Our findings suggest that UNC-6/netrin acts as a permissive cue, rather than a directional cue. Additionally, this work builds upon our previous work and underscores the importance of studying axon guidance in vivo.
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Date
2023-01-01
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University of Kansas
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Keywords
Developmental biology, Genetics, Molecular biology, axon guidance, C. elegans, Growth cones, Netrin, UNC-6