UNC-6/Netrin promotes both adhesion and directed growth within a single axon

Reviewer #1 (Public Review):

Summary:

This paper investigates the mechanism of axon growth directed by the conserved guidance cue UNC-6/Netrin. Experiments were designed to distinguish between alternative models in which UNC-6/Netrin functions as either a short-range (haptotactic) cue or a diffusible (chemotactic) signal that steers axons to their final destinations. In each case, axonal growth cones execute ventrally directed outgrowth toward a proximal source of UNC-6/Netrin. This work concludes that UNC-6/Netrin functions as both a haptotactic and chemotactic cue to polarize the UNC-40/DCC receptor on the growth cone membrane facing the direction of growth. Ventrally directed axons initially contact a minor longitudinal nerve tract (vSLNC) at which UNC-6/Netrin appears to be concentrated before proceeding in the direction of the ventral nerve cord (VNC) from which UNC-6/Netrin is secreted. Time-lapse imaging revealed that growth cones appear to pause at the vSLNC before actively extending ventrally directed filopodia that eventually contact the VNC. Growth cone contacts with the vSLNC were unstable in unc-6 mutants but were restored by the expression of a membrane-tethered UNC-6 in vSLNC neurons. In addition, the expression of membrane-tethered UNC-6/Netrin in the VNC was not sufficient to rescue initial ventral outgrowth in an unc-6 mutant. Finally, dual expression of membrane-tethered UNC-6/Netrin in both vSLNC and VNC partially rescued the unc-6 mutant axon guidance defect, thus suggesting that diffusible UNC-6 is also required. This work is important because it potentially resolves the controversial question of how UNC-6/Netrin directs axon guidance by proposing a model in which both of the competing mechanisms, e.g., haptotaxis vs chemotaxis, are successively employed. The impact of this work is bolstered by its use of powerful imaging and genetic methods to test models of UNC-6/Netrin function in vivo thereby obviating potential artifacts arising from in vitro analysis.

Strengths:

A strength of this approach is the adoption of the model organism C. elegans to exploit its ready accessibility to live cell imaging and powerful methods for genetic analysis.

Weaknesses:

A membrane-tethered version of UNC-6/Netrin was constructed to test its haptotactic role, but its neuron-specific expression and membrane localization are not directly determined although this should be technically feasible. Time-lapse imaging is a key strength of multiple experiments but only one movie is provided for readers to review.

Reviewer #2 (Public Review):

Nichols et al studied the role of axon guidance molecules and their receptors and how these work as long-range and/or local cues, using in-vivo time-lapse imaging in C. elegans. They found that the Netrin axon guidance system works in different modes when acting as a long-range (chemotaxis) cue vs local cue (haptotaxis). As an initial context, they take advantage of the postembryonic-born neuron, PDE, to understand how its axon grows and then is guided into its target. They found that this process occurs in various discrete steps, during which the growth cone migrates and pauses at specific structures, such as the vSLNC. The role of the UNC-6/Netrin and UNC-40/DCC axon guidance ligand-receptor pair was then looked at in terms of its requirement for
(1) initial axon outgrowth direction
(2) stabilization at the intermediate target
(3) directional branching from the sublateral region or
(4) ventral growth from the intermediate target to the VNC.

They found that each step is disrupted in the unc-6/Netrin and unc-40/DCC mutants and observed how the localization of these proteins changed during the process of axon guidance in wild-type and mutant contexts. These observations were further supported by analysis of a mutant important for the regulation of Netrin signaling, the E3 ubiquitin ligase madd-2/Trim9/Trim67. Remarkably, the authors identified that this mutant affected axonal adhesion and stabilization, but not directional growth. Using membrane-tethered UNC-6 to specific localities, they then found this to be a consequence of the availability of UNC-6 at specific localities within the axon growth path. Altogether, this data and in-vivo analysis provide compelling evidence of the mechanistic foundation of Netrin-mediated axon guidance and how it works step by step.

The conclusions are well-supported, with both imaging and quantification of each step of axon guidance and localization of UNC-6 and UNC-40. Using a different type of neuron to validate their findings further supports their conclusions and strengthens their model. It's not yet known whether this model holds true for other ligand-receptor pairs, but the current work sets the stage for future analysis of other axon guidance molecules using time-lapse in-vivo imaging. There are still two outstanding questions that are important to address to support the authors' model and conclusions.

(1) The results of UNC-6-TM expression at different locations are clear and support the conclusions but need to consider that there's no diffusible UNC-6 available. What would happen if UNC-6 is tethered to the membrane in an otherwise completely 'normal' UNC-6 gradient. Does the axon guidance ensue normally or does it get stuck in the respective site of the membrane tethered-UNC-6 and doesn't continue to outgrow properly? This is an important control (expression of the UNC-6-TM at the vSLNC or VNC in the wild type background) that would help clarify this question and gain a better insight into the separability of both axon guidance steps and the ability to manipulate these.

(2) Axon guidance systems do not work in a vacuum and are generally competing against each other. For example, the SLT-1/Slit and SAX-3/ROBO axon guidance ligand-receptor pair is also required for PDE, and other post-embryonic neurons, axon guidance. It would be interesting to test mutants for these genes with the membrane tethered-UNC-6 to determine if the different steps of axon guidance are disrupted and if so, to what degree these are disrupted.

Reviewer #3 (Public Review):

Summary:

This manuscript from Nichols, Lee, and Shen tackles an important question of how unc6/netrin promotes axon guidance: i.e. haptotaxis vs chemotaxis. This has recently been a large topic of investigation and discussion in the axon guidance field. Using live cell imaging of unc6/netrin and unc40/DCC in several neurons that extend axons ventrally during development, as well as TM localized mutants of Unc6, they suggest that unc6 promotes first haptotaxis of the emerging growth cone followed by chemotaxis of the growth cone. This is timely, as a recent preprint from the Lundquist group, using a similar strategy to make only a TM anchored unc6 similarly found that this could rescue only the haptotaxis-like growth of the PDE neuron, but not the second phase of growth. However, their conclusions were quite different based on the overexpression of unc6 everywhere rescuing the second phase, and thus they conclude that a gradient is not present.

Strengths:

As this has been quite a controversy in both the invertebrate and vertebrate field, one strength of this paper is that they use an unc6-neon green to demonstrate unc6 localization, and show a gradient of localization.

Weaknesses:

This is important, although it could be strengthened by first showing a more zoomed-out image of unc6 in the animal, and second demonstrating the localization of the transmembrane anchored unc6 mutants, to help define what may be the "diffusible Unc6". I suggest two additional experimental or analysis suggestions: First, the authors clarify the phenotype of ventral emergence of the growth cone. Though the manuscript images suggest that no matter the mutant there is ventral emergence of the growth cone, but then later defects, yet they claim ventral emergence defects with the UNC6 tethered mutants, but there is no comparison of rose plots. This is confusing and needs to be addressed. Second, I have concerns that the analysis of unc40 polarization may be misleading in some cases when there appears to indeed be accumulation in the growth cone, but since the only analysis shown is relative to the rest of the cell, that can be lost.