Differential adhesion regulates neurite placement via a retrograde zippering mechanism
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Evaluation Summary:
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript includes some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos. The surprising finding here is that neurite position is not solely dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shifts direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil , and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.
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Author Response:
Reviewer #1 (Public Review):
In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and …
Author Response:
Reviewer #1 (Public Review):
In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and zippering onto the second neighborhood. Importantly, axon outgrowth and neighborhood choice are continuously visualized during embryogenesis, an impressive experiment typically constrained by lack of cell-specific reporters during early development as well as the struggle of imaging embryos.
The authors posit that zippering is mediated by temporally regulated differential adhesive forces between AIB's neighboring pre- and post-synaptic neurons. How this differs from differential adhesion in classic fasciculating neurons is described but could be made much clearer. They proceed to identify the immunoglobulin syg-1/syg-2 receptor-ligand pair to be necessary and sufficient for AIB's axon switch; in syg-1/syg-2 mutants, AIB is not able to position itself in the second neighborhood and remains fasciculated with the first one, suggesting that adhesive forces are dampened in syg-1/syg-2 mutants. Lastly, the authors show that pre-synapse assembly follows zippering, linking AIB axon placement with synaptogenesis, and that this is also compromised in syg mutants.
The pipeline used to study axon outgrowth at a single-cell level in the embryo at relevant time points is commendable and will be useful to people studying C. elegans nervous system establishment. Although the overall manuscript and data are well-presented, we think the mechanism of retrograde zippering could be better described. Also, syg-1/syg-2 expression needs to be delineated to support the notion of differential adhesion between neighborhoods.
We have further clarified the novelty of the zippering mechanism, contrasting it with tip-directed outgrowth. We have also performed a thorough analysis of syg-1 expression.
Reviewer #2 (Public Review):
A large amount of data is presented in this paper. The experiments are carefully documented and support the conclusions. Of particular importance is the live imaging of the outgrowth of the AIB neurite in the embryo. This is challenging and required the development of a new marker for labelling and the adaptation of a new type of microscope. This enabled the initial and surprising observation that part of the neurite relocates after outgrowth. I'm not sure that the mathematical modeling adds much. The main conclusion is that the modeling is consistent with a "net increase of adhesive forces in the anterior neighbourhood", which is to be expected. The authors then try to identify the relevant adhesion molecules and find that a pair of IgCAMs (syg-1 and syg-2), which are known to act as receptor-ligand pair, are involved. A series of experiments establishes that syg-2 act in the AIB neurons, whereas syg-1 does not. The neurite positioning defects in syg-1 and syg-2 mutants are partially penetrant, suggesting that other adhesion molecules must be involved. While a large percentage of mutant animals show defects, the defects within an individual animal are surprisingly low with only 21.5% +/- 4% of the neurite detached. This would suggest that syg-1/syg-2 aren't even the major adhesion molecules involved here. In further studies, where the authors ablate the RIM neurons (which express syg-1), the authors use a different measure to quantify the defects (minimal distance between neurite segments, Suppl Figure 7). This makes it difficult to compare the results to those of the syg-1 mutants. For the ectopic expression experiments with syg-1 the authors only report the percentage of animal with defects and not the extent of the defects (how much of the neurite was in an abnormal position).
Overall, this is a very detailed study describing an important novel mechanism for neurite positioning within an nerve bundle.
We have added in this revised version additional quantifications, including ‘the minimal distance between neurite segments’ measure (the one used for the RIM ablation experiments) in Figure 4-figure supplement 1 for the placement defects in syg-1(ky652) and syg-2(ky671) mutants, allowing direct comparisons between the phenotypes. We have also added a measure for the percentage of the distal neurite that is mispositioned in the ectopic expression experiments (Figure 6-figure supplement 1A). We do not claim that SYG-1/SYG-2 are the only adhesion molecules involved in AIB neurite placement, but that they are required for complete and proper placement of the neurite. We clarify this in the text.
Reviewer #3 (Public Review):
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript included some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos which are very hard to do. The surprising finding here is that neurite position is not sole dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shift direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2. Overall, I think this is a strong candidate for eLife. I have one main point and a few minor points.
My main point is about the relationship between synapse formation and neurite zippering. In my opinion, this is an interesting point because it would tell us if the zippering behavior is a consequence of synapse formation or it is a distinct specificity step before synapse formation. From the time course that was described in the paper, it seems that the accumulation of RAB-3 only starts after the zippering has completed. I would suggest the authors to examine at least another synaptic marker like SNB-1 or SYD-2. We have created cell specific endogenous labeling of several active zone markers that can be used for these experiments. If the results hold, then, I think the authors should make it clear in the text that the zippering takes place before synapse formation and serves as a distinct step in achieving the neighborhood specificity.
We thank the reviewer for generously sending us strains for cell-specific endogenous active zone protein labeling (McDonald et al., 2021) using the SapTrap method (Schwartz and Jorgensen, 2016). We made constructs expressing FLP recombinase downstream of the inx-1 and unc-42 promoters for cell-specific labeling of these active zone proteins in AIB and injected them into the strains. Although we observed cell-specific synaptic signal in larvae with both inx-1p and unc-42p-driven FLP, we were not able to observe signal during embryogenesis, probably due to cell-specific synaptic protein expression levels being low.
Therefore and to address the reviewer’s question about the temporal order of zippering and synapse formation, we have cell-specifically expressed two active zone proteins in AIB (CLA-1 and SYD-2) and measured their intensities over time in AIB in embryos (Figure 7, Figure 7-figure supplement 1A-F). We find that similar to RAB-3, synaptic signal is not visible until after the end of zippering, and progressively increases over time following zippering. These observations suggest that synapses do not initiate retrograde zippering. We added the time-course of active zone protein localization in AIB in the context of the time course of retrograde zippering (Figure 7J). Consistent with these observations, in a syd-2(ola341) allele identified in our screen, we find that although synapses are mislocalized, AIB neurite placement is unaffected, consistent with the idea that synapse formation is not upstream of zippering-mediated placement (Figure 7-figure supplement 1G-K).
We acknowledge, however, that our studies are limited by detection of the synaptic proteins, and that, while it does not appear that synaptogenesis leads to zippering, a cooperative and synergistic relationship might exist between the process of zippering and synaptogenesis to hold the neurite position in place. We have added text to better discuss this relationship between zippering and synaptogenesis in light of these findings.
Minor points
- The schematic diagram is somewhat misleading because in the axial view, the anterior and posterior segment of the nerve ring should appear on top of each other. The lateral view is the right view to show the anterior and posterior segments.
This is a valid point - if we look at the worm directly head-on, the two segments of the neurite would be on top of one another. A slight tilt of the worm head enables visualization of the parts of the neurite in the two neighborhoods. We have now clarified this in the figure legends.
- Describe the screen that led to the mutant alleles of syg-1 and syg-2 better. Any other mutants?
We have described the screen further in Methods and now include another allele, corresponding to syd-2 (Figure 7-figure supplement 1), isolated from the screen.
- "Consistent with the importance of adhesion-based mechanisms in the observed phenotypes, ectopic expression of the SYG-1 endodomain in the posterior neighborhood did not result in mislocalization of AIB (Figure 6-figure supplement 1A,B). " This statement is wrong. I suspect the authors meant in syg-2 mutants.
The statement might have been confusing, but it is not wrong. We have found that ectopic expression of the SYG-1 endodomain, which lacks SYG-1’s extracellular domains, does not cause ectopic placement of the AIB neurite, which is what we described in that statement. We have edited to make it more clear.
- For Fig. 7-figure supplement 1, please quantify this phenotype.
We have now included quantifications for this in Figure 7-figure supplement 3.
References
MCDONALD, N. A., FETTER, R. D. & SHEN, K. 2021. Author Correction: Assembly of synaptic active zones requires phase separation of scaffold molecules. Nature, 595, E35. SCHWARTZ, M. L. & JORGENSEN, E. M. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics, 202, 1277-88.
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Evaluation Summary:
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript includes some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos. The surprising finding here is that neurite position is not solely dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shifts direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2.
(This preprint has been reviewed by …
Evaluation Summary:
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript includes some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos. The surprising finding here is that neurite position is not solely dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shifts direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and zippering onto the …
Reviewer #1 (Public Review):
In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and zippering onto the second neighborhood. Importantly, axon outgrowth and neighborhood choice are continuously visualized during embryogenesis, an impressive experiment typically constrained by lack of cell-specific reporters during early development as well as the struggle of imaging embryos.
The authors posit that zippering is mediated by temporally regulated differential adhesive forces between AIB's neighboring pre- and post-synaptic neurons. How this differs from differential adhesion in classic fasciculating neurons is described but could be made much clearer. They proceed to identify the immunoglobulin syg-1/syg-2 receptor-ligand pair to be necessary and sufficient for AIB's axon switch; in syg-1/syg-2 mutants, AIB is not able to position itself in the second neighborhood and remains fasciculated with the first one, suggesting that adhesive forces are dampened in syg-1/syg-2 mutants. Lastly, the authors show that pre-synapse assembly follows zippering, linking AIB axon placement with synaptogenesis, and that this is also compromised in syg mutants.
The pipeline used to study axon outgrowth at a single-cell level in the embryo at relevant time points is commendable and will be useful to people studying C. elegans nervous system establishment. Although the overall manuscript and data are well-presented, we think the mechanism of retrograde zippering could be better described. Also, syg-1/syg-2 expression needs to be delineated to support the notion of differential adhesion between neighborhoods. -
Reviewer #2 (Public Review):
A large amount of data is presented in this paper. The experiments are carefully documented and support the conclusions. Of particular importance is the live imaging of the outgrowth of the AIB neurite in the embryo. This is challenging and required the development of a new marker for labelling and the adaptation of a new type of microscope. This enabled the initial and surprising observation that part of the neurite relocates after outgrowth. I'm not sure that the mathematical modeling adds much. The main conclusion is that the modeling is consistent with a "net increase of adhesive forces in the anterior neighbourhood", which is to be expected. The authors then try to identify the relevant adhesion molecules and find that a pair of IgCAMs (syg-1 and syg-2), which are known to act as receptor-ligand pair, …
Reviewer #2 (Public Review):
A large amount of data is presented in this paper. The experiments are carefully documented and support the conclusions. Of particular importance is the live imaging of the outgrowth of the AIB neurite in the embryo. This is challenging and required the development of a new marker for labelling and the adaptation of a new type of microscope. This enabled the initial and surprising observation that part of the neurite relocates after outgrowth. I'm not sure that the mathematical modeling adds much. The main conclusion is that the modeling is consistent with a "net increase of adhesive forces in the anterior neighbourhood", which is to be expected. The authors then try to identify the relevant adhesion molecules and find that a pair of IgCAMs (syg-1 and syg-2), which are known to act as receptor-ligand pair, are involved. A series of experiments establishes that syg-2 act in the AIB neurons, whereas syg-1 does not. The neurite positioning defects in syg-1 and syg-2 mutants are partially penetrant, suggesting that other adhesion molecules must be involved. While a large percentage of mutant animals show defects, the defects within an individual animal are surprisingly low with only 21.5% +/- 4% of the neurite detached. This would suggest that syg-1/syg-2 aren't even the major adhesion molecules involved here. In further studies, where the authors ablate the RIM neurons (which express syg-1), the authors use a different measure to quantify the defects (minimal distance between neurite segments, Suppl Figure 7). This makes it difficult to compare the results to those of the syg-1 mutants. For the ectopic expression experiments with syg-1 the authors only report the percentage of animal with defects and not the extent of the defects (how much of the neurite was in an abnormal position).
Overall, this is a very detailed study describing an important novel mechanism for neurite positioning within an nerve bundle. -
Reviewer #3 (Public Review):
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript included some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos which are very hard to do. The surprising finding here is that neurite position is not sole dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shift direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2. …
Reviewer #3 (Public Review):
This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript included some technically difficult experiments of imaging neurite outgrowth in C. elegans embryos which are very hard to do. The surprising finding here is that neurite position is not sole dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shift direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2. Overall, I think this is a strong candidate for eLife. I have one main point and a few minor points.
My main point is about the relationship between synapse formation and neurite zippering. In my opinion, this is an interesting point because it would tell us if the zippering behavior is a consequence of synapse formation or it is a distinct specificity step before synapse formation. From the time course that was described in the paper, it seems that the accumulation of RAB-3 only starts after the zippering has completed. I would suggest the authors to examine at least another synaptic marker like SNB-1 or SYD-2. We have created cell specific endogenous labeling of several active zone markers that can be used for these experiments. If the results hold, then, I think the authors should make it clear in the text that the zippering takes place before synapse formation and serves as a distinct step in achieving the neighborhood specificity.
Minor points
1. The schematic diagram is somewhat misleading because in the axial view, the anterior and posterior segment of the nerve ring should appear on top of each other. The lateral view is the right view to show the anterior and posterior segments.
2. Describe the screen that led to the mutant alleles of syg-1 and syg-2 better. Any other mutants?
3. "Consistent with the importance of adhesion-based mechanisms in the observed phenotypes, ectopic expression of the SYG-1 endodomain in the posterior neighborhood did not result in mislocalization of AIB (Figure 6-figure supplement 1A,B). " This statement is wrong. I suspect the authors meant in syg-2 mutants.
4. For Fig. 7-figure supplement 1, please quantify this phenotype. -
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