Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish

Reviewer #1 (Public Review):

Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure. However, the connection between different isoforms of Sema7a and the axon arborization needs to be substantiated. Furthermore, the effect of loss of Sema7a on the presynaptic cell is not ruled out as a contributing factor to the synaptic and axon structure phenotypes. These issues weaken the claims made by the authors including the statement that they have identified dual roles for the GPI-anchored verses secreted forms of Sema7a on synapse formation and as a chemoattractant for axon arborization respectively.

Reviewer #2 (Public Review):

In this work, Dasgupta et al. investigates the role of Sema7a in the formation of peripheral sensory circuit in the lateral line system of zebrafish. They show that Sema7a protein is present during neuromast maturation and localized, in part, to the base of hair cells (HCs). This would be consistent with pre-synaptic Sema7a mediating formation and/or stabilization of the synapse. They use sema7a loss-of-function strain to show that lateral line sensory terminals display abnormal arborization. They provide highly quantitative analysis of the lateral line terminal arborization to show that a number of specific topological parameters are affected in mutants. Next, they ectopically express a secreted form of Sema7a to show that lateral line terminals can be ectopically attracted to the source. Finally, they also demonstrate that the synaptic assembly is impaired in the sema7a mutant. Overall, the data are of high quality and properly controlled. The availability of Sema7a antibody is a big plus, as it allows to address the endogenous protein localization as well to show the signal absence in the sema7a mutant. The quantification of the arbor topology should be useful to people in the field who are looking at the lateral line as well as other axonal terminals. I think some results are overinterpreted though. The authors state: "Our findings demonstrate that Sema7A functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development." However, they have not actually demonstrated which isoform functions in HCs (also see comments below). In addition, they have to be careful in interpreting their topology analysis, as they cannot separate individual axons. Thus, such analysis can generate artifacts. They can perform additional experiments to address these issues or adjust their interpretations.

Reviewer #3 (Public Review):

The data reported here demonstrate that Sema7a defines the local behavior of growing axons in the developing zebrafish lateral line. The analysis is sophisticated and convincingly demonstrates effects on axon growth and synapse architecture. Collectively, the findings point to the idea that the diffusible form of sema7a may influence how axons grow within the neuromast and that the GPI-linked form of sema7a may subsequently impact how synapses form, though additional work is needed to strongly link each form to its' proposed effect on circuit assembly.

Comments on revised submission:

The revised manuscript is significantly improved. The authors comprehensively and appropriately addressed most of the reviewers' concerns. In particular, they added evidence that hair cells express both Sema7A isoforms, showed that membrane bound Sema7A does not have long range effects on guidance, demonstrated how axons behave close to ectopic Sema7A, and analyzed other features of the hair cells that revealed no strong phenotypes. The authors also softened the language in many, but not all places. Overall, I am satisfied with the study as a whole.

Reviewer #4 (Public Review):

This study provides direct evidence showing that Sema7a plays a role in the axon growth during the formation of peripheral sensory circuits in the lateral-line system of zebrafish. This is a valuable finding because the molecules for axon growth in hair-cell sensory systems are not well understood. The majority of the experimental evidence is convincing, and the analysis is rigorous. The evidence supporting Sema7a's juxtracrine vs. secreted role and involvement in synapse formation in hair cells is less conclusive. The study will be of interest to cell, molecular and developmental biologists, and sensory neuroscientists.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This study presents valuable findings on the roles of the axon growth regulator Sema7a in the formation of peripheral sensory circuits in the lateral line system of zebrafish. The evidence supporting the claims of the authors is solid, although further work directly testing the roles of different sema7a isoforms would strengthen the analysis. The work will be of interest to developmental neuroscientists studying circuit formation.

Public Reviews:

Reviewer #1 (Public Review):

In this work, Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure. However, the connection between different isoforms of Sema7a and the axon arborization needs to be substantiated. Furthermore, an autocrine role for Sema7a on the presynaptic cell is not ruled out as a contributing factor to the synaptic and axon structure phenotypes.

Finally, critical controls are absent from the overexpression paradigm.

Comments: Thank you for your valuable comments. We have analyzed the hair cell scRNA transcriptome data of zebrafish neuromasts from published works and have not identified known expression of receptors of the Sema7A protein, particularly PlexinC1 and Integrin β1 molecules (reference 4 and 15) in hair cells. This result suggests that the Sema7A protein molecule, either secreted or membrane-bound, does not possess its cognate receptor to elicit an autocrine function on the hair cells. Moreover, the GPI-anchored Sema7A lacks a cytosolic domain. So it is unlikely that Sema7A signaling directly induces the formation of presynaptic ribbons. We propose that the decrease in average number and area of synaptic aggregates likely reflects decreased stability of the synaptic structures owing to lack of contact between the sensory axons and the hair cells, which has been identified in zebrafish neuromasts (reference 38).

Thank you for pointing missing critical control experiments. Additional control experiments (lines 333-346) with a new figure (Figure 5) have been added.

These issues weaken the claims made by the authors including the statement that they have identified differential roles for the GPI-anchored verses secreted forms of Sema7a on synapse formation and as a chemoattractant for axon arborization respectively.

Comments: We have rephrased our statement and argue in lines 428-430 that our experiments “suggest a potential mechanism for hair cell innervation in which a local Sema7Asec diffusive cue likely consolidates the sensory arbors at the hair cell cluster and the membrane-anchored Sema7A-GPI molecule guides microcircuit topology and synapse assembly.”

The manuscript itself would benefit from the inclusion of details in the text to help the reader interpret the figures, tools, data, and analysis.

Comments: We have made significant revisions to the text and figures to improve clarity and consistency of the manuscript.

Reviewer #2 (Public Review):

In this work, Dasgupta et al. investigates the role of Sema7a in the formation of peripheral sensory circuit in the lateral line system of zebrafish. They show that Sema7a protein is present during neuromast maturation and localized, in part, to the base of hair cells (HCs). This would be consistent with pre-synaptic Sema7a mediating formation and/or stabilization of the synapse. They use sema7a loss-of-function strain to show that lateral line sensory terminals display abnormal arborization. They provide highly quantitative analysis of the lateral line terminal arborization to show that a number of specific topological parameters are affected in mutants. Next, they ectopically express a secreted form of Sema7a to show that lateral line terminals can be ectopically attracted to the source. Finally, they also demonstrate that the synaptic assembly is impaired in the sema7a mutant. Overall, the data are of high quality and properly controlled. The availability of Sema7a antibody is a big plus, as it allows to address the endogenous protein localization as well to show the signal absence in the sema7a mutant. The quantification of the arbor topology should be useful to people in the field who are looking at the lateral line as well as other axonal terminals. I think some results are overinterpreted though. The authors state: "Our findings demonstrate that Sema7A functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development." However, they have not actually demonstrated which isoform functions in HCs (also see comments below).

Comments: Thank you for making this point. To investigate the presence of both sema7a transcripts in the hair cells of the lateral-line neuromasts, we used the Tg(myo6b:actb1EGFP) transgenic fish to capture the labeled hair cells by fluorescence-activated cell sorting (FACS) and isolated total RNA. Using transcript specific DNA oligonucleotide primers, we have identified the presence of both sema7a transcript variants in the hair cell of the neuromast. Even though we have not developed transcript specific knockout animals, we speculate that the presence of both transcript variants in the hair cell implies that they function in distinct fashion. We have changed our interpretation in lines 32-34 to “Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.”

In future we will utilize the CRISPR/Cas9 technique to target the unique C-terminal domain of the GPI-anchored sema7a transcript variant. We believe that this will only perturb the formation of the full-length Sema7A protein and help us determine the role of the membrane-bound Sema7AGPI molecule as well as the Sema7Asec in sensory arborization and synaptic assembly.

In addition, they have to be careful in interpreting their topology analysis, as they cannot separate individual axons. Thus, such analysis can generate artifacts. They can perform additional experiments to address these issues or adjust their interpretations.

Comments: Thank you for this insightful comment. In a previous eLife publication from our laboratory, we utilized the serial blockface scanning electron micrograph (SBFSEM) technique to characterize the connectome of the neuromast microcircuit where patterns of innervation of all the individual axons can be delineated in five-days-old larvae (reference 8). However, the collective behavior of all the sensory axons that build the innervation network remained enigmatic, especially in a living animal during development. In this paper we addressed how the sensory-axon collective behaves around the clustered hair cells and build the innervation network in living animals during diverse developmental stages. Our analyses have not only identified how the axons associates with the hair cell cluster as the organ matures, but also discovered distinct topological features in the arbor network that emerges during organ maturation, which may influence assembly of postsynaptic aggregates (lines 384-403, Figure 6G-I). We believe that our quantitative approach to capture collective axonal behaviors and their topological attributes during circuit formation have highlighted the importance of understanding network assembly during sensory organ development.

Reviewer #3 (Public Review):

Summary:

This study demonstrates that the axon guidance molecule Sema7a patterns the innervation of hair cells in the neuromasts of the zebrafish lateral line, as revealed by quantifying gain- and loss-of function effects on the three-dimensional topology of sensory axon arbors over developmental time. Alternative splicing can produce either a diffusible or membrane-bound form of Sema7a, which is increasingly localized to the basolateral pole of hair cells as they develop (Figure 1). In sema7a mutant zebrafish, sensory axon arbors still grow to the neuromast, but they do not form the same arborization patterns as in controls, with many arbors overextending, curving less, and forming fewer loops even as they lengthen (Figure 2,3). These phenotypes only become significant later in development, indicating that Sema7a functions to pattern local microcircuitry, not the gross wiring pattern. Further, upon ectopic expression of the diffusible form of Sema7a, sensory axons grow towards the Sema7a source (Figure 4). The data also show changes in the synapses that form when mutant terminals contact hair cells, evidenced by significantly smaller pre- and post-synaptic punctae (Figure 5). Finally, by replotting single cell RNA-sequencing data (Figure 6), the authors show that several other potential cues are also produced by hair cells and might explain why the sema7a phenotype does not reflect a change in growth towards the neuromast. In summary, the data strongly indicate that Sema7a plays a role in shaping connectivity within the neuromast.

Strengths:

The main strength of this study is the sophisticated analysis that was used to demonstrate fine-level effects on connectivity. Rather than asking "did the axon reach its target?", the authors asked "how does the axon behave within the target?". This type of deep analysis is much more powerful than what is typical for the field and should be done more often. The breadth of analysis is also impressive, in that axon arborization patterns and synaptic connectivity were examined at 3 stages of development and in three-dimensions.

Weaknesses:

The main weakness is that the data do not cleanly distinguish between activities for the secreted and membrane-bound forms of Sema7a, which the authors speculate may influence axon growth and synapse formation respectively. The authors do not overstate the claims, but it would have been nice to see some additional experimentation along these lines, such as the effects of overexpressing the membrane-bound form,

Comments: We have accepted this useful suggestion. In lines 333-346 and in Figure 5 we have demonstrated the impact of overexpressing the membrane-bound transcript variant on arborization pattern of the sensory axons.

Some analysis of the distance over which the "diffusible" form of Sema7a might act (many secreted ligands are not in fact all that diffusible), or

Comments: We have reported this in lines 311-317 and in Figure 4F,G.

Some live-imaging of axons before they reach the target (predicted to be the same in control and mutants) and then within the target (predicted to be different).

Comments: We have accepted this useful suggestion. We demonstrate the dynamics of the sensory arbors that are attracted to an ectopic Sema7Asec source in lines 325-332, Figure 4I,J; Figure 4—figure supplement 2A, and Videos 13-16.

Clearly, although the gain-of-function studies show that Sema7a can act at a distance, other cues are sufficient. Although the lack of a phenotype could be due to compensation, it is also possible that Sema7a does not actually act in a diffusible manner within its natural context. Overall, the data support the authors' carefully worded conclusions. While certain ideas are put forward as possibilities, the authors recognize that more work is needed. The main shortcoming is that the study does not actually distinguish between the effects of the two forms of Sema7a, which are predicted but not actually shown to be either diffusible or membrane linked (the membrane linkage can be cleaved). Although the study starts by presenting the splice forms, there is no description of when and where each splice form is transcribed.

Comments: We have utilized the HCR™ RNA-FISH Technology to generate transcript specific probes. To generate transcript-specific HCR probes to distinctly detect the sema7aGPI (NM_001328508) and the sema7asec (NM_001114885) transcripts, Molecular Instruments could design only 11 probes against the sema7aGPI transcript and only one probe against the sema7asec transcript (personal correspondence with Mike Liu, PhD, Head of Operations and Product Development Lead Molecular Instruments, Inc.). The HCR probe against the sema7aGPI transcript showed a very faint signal. Unfortunately, the HCR probe against the sema7asec transcript failed to detect the presence of any transcript. For robust detection of transcripts, the protocol demands a minimum of 20 probes. We believe that the very low number of probes against our transcripts is the primary reason for the absence of a signal.

We therefore utilized fluorescence-activated cell sorting (FACS) to capture the labeled hair cells and isolated total RNA to perform RT-PCR using transcript specific DNA oligonucleotide primers. We identified the presence of both the secreted and the membrane-bound transcripts at four-days-old neuromasts (lines 80-84, Figure 1B-D).

Additionally, since the mutants are predicted to disrupt both forms, it is a bit difficult to disentangle the synaptic phenotype from the earlier changes in circuit topology - perhaps the change at the level of the synapse is secondary to the change in topology.

Comments: Thank you for the insightful suggestion. We have analyzed the relationship between the sensory arbor network topology and the distribution of postsynaptic structures (lines 384-403, Figure 6G-I). We identified that the distribution of the postsynaptic aggregates is closely associated with the topological attributes of the sensory circuit. We further clarify the potential origin of disrupted synaptic assemblies in sema7a-/- mutants in lines 380-382 and lines 417-420.

Further, the authors do not provide any data supporting the idea that the membrane bound form of Sema7a acts only locally. Without these kinds of data, the authors are unable to attribute activities to either form.

Comments: We have accepted this useful suggestion and have prepared the Figure 5 with the necessary details.

The main impact on the field will be the nature of the analysis. The field of axon guidance benefits from this kind of robust quantification of growing axon trajectories, versus their ability to actually reach a target. This study highlights the value of more careful analysis and as a result, makes the point that circuit assembly is not just a matter of painting out paths using chemoattractants and repellants, but is also about how axons respond to local cues. The study also points to the likely importance of alternative splice forms and to the complex functions that can be achieved using different forms of the same ligand.

Reviewer #4 (Public Review):

Summary:

The work by Dasgupta et al identifies Sema7a as a novel guidance molecule in hair cell sensory systems. The authors use the both genetic and imaging power of the zebrafish lateralline system for their research. Based on expression data and immunohistochemistry experiments, the authors demonstrate that Sema7a is present in lateral line hair cells. The authors then examine a sema7a mutant. In this mutant, Sema7a proteins levels are nearly eliminated. Importantly, the authors show that when Sema7a is absent, afferent terminals show aberrant projections and fewer contacts with hair cells. Lastly the authors show that ectopic expression of the secreted form of Sema7a is sufficient to recruit aberrant terminals to non-hair cell targets. The sema7a innervation defects are well quantified. Overall, the paper is extremely well written and easy to follow.

Strengths:

(1) The axon guidance phenotypes in sema7a mutants are novel, striking and thoroughly quantified.

(2) By combining both loss of function sema7a mutants and ectopic expression of the secreted form of Sema7a the authors demonstrate the Sema7a is both necessary and sufficient to guide sensory axons

Weaknesses:

(1) Control. There should be an uninjected heatshock control to ensure that heatshock itself does not cause sensory afferents to form aberrant arbors. This control would help support the hypothesis that exogenously expressed Sema7a (via a heatshock driven promoter) is sufficient to attract afferent arbors.

Comments: Thank you for the suggestion. We have added the uninjected heatshock control experiment in Figure 5 and described experimental details in the text, lines 343-345.

(2) Synapse labeling. The numbers obtained for postsynaptic labeling in controls do not match up with the published literature - they are quite low. Although there are clear differences in postsynaptic counts between sema7a mutants and controls, it is worrying that the numbers are so low in controls. In addition, the authors do not stain for complete synapses (pre- and post-synapses together). This staining is critical to understand how Sema7a impacts synapse formation.

Comments: Thank you for raising this issue. We believe the low average numbers of the postsynaptic punctae in control neuromasts arise from lack of formation of postsynaptic aggregates beneath the immature hair cells, which are abundant in early stages of neuromast maturation. We have performed exhaustive analysis on the formation of pre- and postsynaptic structures and have identified how their distribution changes along neuromast development in control larvae. We have further analyzed how such distribution is perturbed in the sema7a-/- mutants. We do not think analyzing the complete synapse structure will add much to our understanding of how Sema7A influence synapse formation and maintenance.

(3) Hair cell counts. The authors need to provide quantification of hair cell counts per neuromast in mutant and control animals. If the counts are different, certain quantification may need to be normalized.

Comments: We have added the raw data with the hair cell counts in both control and sema7a-/- mutants across developmental stages. The homozygous sema7a-/- mutants have slightly less hair cells and we have normalized all our topological analyses by the corresponding hair cell numbers for each neuromast in each experiment (lines 669-675).

(4) Developmental delay. It is possible that loss of Sema7a simply delays development. The latest stage examined was 4 dpf, an age that is not quite mature in control animals. The authors could look at a later age, such as 6 dpf to see if the phenotypes persist or recover.

Comments: The homozygous sema7a-/- mutants are unviable and die at 6 dpf. We therefore restricted our analysis till 4 dpf. The association of the sensory arbors with the clustered hair cells gradually decreases as the neuromasts mature from 2 dpf to 4dpf in the sema7a-/- mutants (lines 174-176, Figure 2I). Moreover, in the sema7a-/- mutants the sensory axons throw long projections that keep getting farther away from the clustered hair cells as the neuromast matures from 2 dpf to 4 dpf (lines 166-168, Figure 2H; Figure 2—figure supplement 1K,L). These observations suggest that if the phenotypes in the sema7a-/- mutants were due to developmental delays, then we should have seen a recovery of disrupted arborization patterns over time. But instead, we observe a further deterioration of the arborization patterns and other architectural assemblies. These findings confirm that the observed phenotypes in the sema7a-/- mutants are not due to delayed development of the larvae, but a specific outcome for the loss of Sema7A protein.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Major concerns:

Issue 1: One of the most interesting conclusions in this manuscript is the function of the GPIanchored vs. secreted form of Sema7a in axon structure and synapse formation. In lines 357360 of the discussion (for example) the authors state that they have shown that the GPIanchored form of Sema7a is responsible for contact-mediated synapse formation while the secreted form functions as a chemoattractant for axon arbor structure. "We have discovered dual modes of Sema7A function in vivo: the chemoattractive diffusible form is sufficient to guide the sensory arbors toward their target, whereas the membrane-attached form likely participates in sculpting accurate neural circuitry to facilitate contact-mediated formation and maintenance of synapses." However, the data do not support this conclusion. Specifically, no analysis is done showing unique expression of either isoform in hair cells and no functional analysis is done to conclusively determine which isoform is important for either phenotype.

Comments: We have shown that both sema7a transcripts are expressed in the hair cells of four-day-old neuromasts (lines 78-84, Figure 1C,D). Ectopic expression of the sema7asec transcript variant robustly attracts the lateral-line sensory arbors toward itself, whereas ectopic expression of the sema7aGPI variant fails to impart sensory guidance from a distance, suggesting that the membrane-bound form likely participates in contact-mediated neural guidance. These experiments decisively show, for the first time in zebrafish, the dual modes of Sema7A function in vivo. However, we agree that the sema7aGPI transcript-specific knockout animal would be essential to conclusively prove that the membrane-attached form is primarily involved in forming accurate neural circuitry and contact-mediated formation and maintenance of synapses. Hence, we have very carefully stated in lines 427-428 that “the membrane-attached form likely participates in sculpting accurate neural circuitry to facilitate contact-mediated formation and maintenance of synapses”. We will follow up on this suggestion in our upcoming manuscript that will incorporate transcript-specific genetic ablations.

Though the authors present RT-PCR analysis of sema7a isoforms, it is not interpretable. The second reverse primer will also recognize the full-length transcript (from what I can gather) so it does not simply show the presence of the secreted form. Is there a unique 3'UTR for the short transcript that can be used? Additionally, for the GPI-anchored version can you use a forward primer that is not present in the short isoform? This would shed some light on the respective levels of both transcripts.

Comments: The C-termini of the two transcript variants are distinct and we have designed distinct primers that will selectively bind to each transcript (lines 503-511). Since, we have not performed quantitative polymerase chain reaction (qPCR), relative levels of each transcript are hard to determine.

Alternatively, and perhaps of more use, in situ hybridization using unique probes for each isoform would allow you to determine which are actually present in hair cells.

Comments: We have tried this approach and explained the point earlier (refer to lines 203212 of this response letter).

To decisively state that these isoforms have unique functions in axon terminal structure and synapse formation, other experiments are also essential. For example, RNA-mediated rescue analyses using both isoforms would tell you which can rescue the axonal structure and synapse size/number phenotypes. Overexpression of the GPI-anchored form, like the secreted form in Figure 4, would allow you to determine if only the secreted form can cause abnormal axon extension phenotypes. Expression of both forms in hair cells (using a myo6b promotor for example) would allow assessment of their role in presynapse formation.

Comments: We have ectopically expressed the sema7aGPI transcript variant near the sensory arbor network and observed that Sema7A-GPI fails to impart sensory axon guidance from a distance.

Thank you for suggesting the rescue experiments. We are in the process of generating CRISPR/Cas9-mediated transcript-specific knockout animals. We are currently preparing another manuscript that incorporates the above-mentioned rescue experiments to dissect the role of each transcript in regulating arbor topology and synapse formation.

For the overexpression experiments, expression of mKate alone (with and without heat shock) is also a critical control to include.

Comments: We have incorporated two control experiments: (1) larvae injected with hsp70:sema7asec-mKate2 plasmid that were not heat shocked and (2) Uninjected larvae that were heatshocked. We think these two controls are sufficient to demonstrate that the abnormal arborization patterns are not artifacts generated due to plasmid injection and heatshocking.

Issue 2: A second concern is the lack of data showing support cell and hair cell formation and function is unaffected. Analysis of support and hair cell number with loss of Sema7a as well as simple analyses of mechanotransduction (FM4-64) would help alleviate concerns that phenotypes are due to disrupted neuromast formation and basic hair cell function rather than a specific role for Sema7a in this process.

Comments: We have measured the hair cell numbers in both control and sema7a-/- mutants across developmental stages. We have added this to our submitted raw data.

We have utilized the styryl fluorophore FM4-64 to test the mechanotransduction function of the hair cells in sema7a-/- mutants. We have detailed our finding in lines 137141 and in Figure 2—figure supplement 1C,D.

Expression analysis of Sema7a receptors would also help strengthen the argument for a specific effect on lateral line afferent axons.

Comments: Thank you for this suggestion. Currently, we do not possess an RNA transcriptome dataset for the lateral line ganglion. This deficit limits a systematic screen for lateral-line sensory neuronal gene expressions either through antibody stains or via HCRmediated in situ techniques. In future we plan to develop an RNA transcriptome for the lateral-line ganglion and identify potential binding partners for Sema7A.

Issue 3: The manuscript could also be improved to include more detail in some areas and less in others. In general, each section has a fairly long lead up but lacks important experimental details that would help the reader interpret the data. For example:

Figure 1: What is the label for the lateral line axons? Is it a specific transgenic? The legend states that 3 asterisks indicate p<0.0001. What about the other asterisk combinations?

Comments: We have clarified these issues in lines 118-121 and in lines 906-907.

Figure 2: For the network analysis, are the traces for all axons that branch to innervate the neuromast?

Comments: Yes, we have traced the entire arbor containing all the axons that branched from the lateral line nerve and extended toward the clustered hair cells. The three-dimensional traces depict a skeletonized representation of the arbor network.

Can the tracing method distinguish individual axons?

Comments: No, our goal is to understand how the axon-collective behave around the clustered hair cells during development.

How do you know where an end is versus continued looping?

Comments: We have categorically defined the topological attributes in lines 187-191 and in Figure 3A.

Also, are all neuromasts similarly affected or is there a divergence based on which organ you are imaging? What neuromast was imaged in this and other figures?

Comments: Yes, all the neuromasts in the trunk and tail regions were affected similarly by the sema7a mutation. We did not observe any region-specific phenotypic outcome. We consistently imaged the trunk neuromasts, particularly the second, third, and fourth neuromasts.

Discussion: The short discussion failed to put these findings into context or to discuss how this unique topological arrangement of axon terminals impacts function.

Comments: We have added a new segment, lines 432-448, in the discussion section which mentions the potential role of the topological features in arranging the distribution pattern of the postsynaptic densities and thereby potentially influencing the network’s ability to gather sensory inputs through properly placed postsynaptic aggregates.

Can you speculate on how the looping structure may alter number of synaptic contacts per axon for instance? For this, it would be useful to know if normally the synapses form on loops versus bare terminals.

Comments: Thank you for this insightful suggestion. We have performed detailed analysis, as mentioned in lines 384-397, to characterize the distribution of the postsynaptic densities between the two topological attributes.

Does this looping facilitate single axons contacting more hair cells of the same polarity? Would that be beneficial?

Comments: Looping behaviors indeed facilitate the contact between the axons and the hair cells. As we have observed, the primary topological attribute that the sensory arbor network underneath the clustered hair cells adopts is a loop. The bare terminals are predominantly projected transverse to the clustered hair cells and lack contact with them. Whether a single axon, being part of a loop, preferentially contacts hair cells of same polarity is yet to be determined. We can address this question by mosaic labeling a single axon in the arbor network and determine its association with the hair cells. We intend to do these experiments in our upcoming manuscript.

Minor concerns:

(1) For the stacked charts quantifying topological features, I found interpreting them challenging. Is it possible to put these into overlapping histograms or line graphs to better compare wild type to mutant directly?

Comments: Thank you for your suggestion. We tried several ways to represent our data and found that the stacked charts optimally signify our analysis and depict the characteristic phenological differences between the control and the sema7a-/- mutants.

(2) There are numerous strong statements throughout not directly supported by the data, e.g. lines 110-113; 206-208; 357-360 and others. These should be tempered.

Comments: For lines 110-113, we have updated this section with new experiments and the new segment is represented in lines 115-126.

For lines 206-208, we have updated the statement to “This result suggests that the stereotypical circuit topology observed in the mature organ may emerge through transition of individual arbors from forming bare terminals to forming closed loops encircling topological holes” in lines 225-227.

Reviewer #2 (Recommendations For The Authors):

The authors should be careful about making any assumptions which form of sema7a is active in NMs. Their RT-PCR demonstrates presence of both isoforms in a whole animal; however, whether they are similarly present in HCs is not investigated here.

Comments: We have addressed this concern and have updated the manuscript with new experiments, detailed in lines 78-84.

Also, there is an issue of translation and trafficking to the membrane with subsequent secretion. An important experiment that would address this question is expressing two sema7a isoforms in mutant HCs and asking whether this can suppress the mutant phenotype.

Comments: Thank you for suggesting the rescue experiments. We are in the process of generating CRISPR/Cas9-mediated transcript-specific knockout animals. We are currently preparing another manuscript that incorporates the above-mentioned rescue experiments to dissect the role of each transcript in regulating arbor topology and synapse formation.

Presumably, sema7a is trafficked to the membrane during HC maturation. This is consistent with the authors' observation that sema7a localization is changing as NM mature. However, actin-sema7a co-labeling does not actually show whether sema7a is on the membrane. Labeling HCs with a membrane marker (transgene) would be much more convincing. Alternatively, can the authors show sema7a localization actually correlates with the presence of sensory axon terminals? They already have immunos that label both. Thus, this should be pretty straightforward.

Comments: Thank you for these suggestions. We have addressed these issues in lines 112114, and in lines 119-126.

Figure 2 should have a control panel, so the reduced sema7a staining can be compared to the control side-by-side.

Comments: We have depicted Sema7A staining in control neuromasts in multiple images, including Figure 1E, Figure 1H, and in Figure 2—figure supplement 1B. We have kept the control panel in the supplementary figure due to space restrictions in Figure 2.

Arborization topology: While I appreciate the very careful characterization of the topology for wild-type and mutant NMs, I think it would be much more informative to mark individual axons and then analyze their topology. The main reason is that the authors cannot really distinguish whether some aspects of topology they describe are really due to the densely packed overlapping terminals of multiple axons or these are really characteristic, higher order organization of individual axons. Because of this, they cannot be certain what is really happening with sema7a mutant terminals. Related to the point above. While it is clear that the overall topology is abnormal in the mutant, the authors should be careful in concluding that sema7a regulates specific aspects of it. The overall structure is probably highly interconnected perturbing one parameter would likely affect all the others.

Comments: Thank you for this comment. In a previous eLife publication from our laboratory, we utilized the serial blockface scanning electron micrograph (SBFSEM) technique to characterize the connectome of the neuromast microcircuit where patterns of innervation of all the individual axons can be delineated in five-days-old larvae (reference number 8). However, the collective behavior of all the sensory axons that build the innervation network remained enigmatic, especially in a living animal during development. In this paper we addressed how the sensory axon-collective behave around the clustered hair cells and build the innervation network in living animals during diverse developmental stages. Our analyses have not only identified how the axon-collective associates itself with the hair cell cluster as the organ matures, but also discovered distinct topological features in the arbor network that emerges during organ maturation, which may influence assembly of postsynaptic aggregates (lines 384-403, Figure 6G-I). We believe that our quantitative approach to capture collective axonal behaviors and their topological attributes during circuit formation have highlighted the importance of understanding network assembly during sensory organ development.

Experiments with the secreted sema7a isoform would be much more informative if they were compared/contrasted to the GPI anchored isoform.

Comments: We added a new section, lines 338-351, and a new Figure 5 to address this issue.

The phenotype of ectopic projections in sema7a overexpression experiments is pretty dramatic, especially given the fact that these were performed in wild-type animals. Does this mean that the phenotype would be even more dramatic in sema7a mutants, as they have more bare axon terminals according to the authors' analysis. Have the authors attempted this type of experiments?

Comments: That is an interesting suggestion. We have not tested that yet. Our guess is that in the sema7a-/- mutants, the abundant bare terminals will be far more sensitive to an ectopic source of Sema7A. But even in the sema7a-/- mutants, other chemotropic cues are still functional, which may impart certain restrictions on how many bare terminals are allowed to leave the neuromast region.

Reviewer #3 (Recommendations For The Authors):

(1) No raw data are shown, such that it is difficult to assess variability across animals or within animals, just the overall trends within the whole dataset. Raw data need to be shown for every measurement, at least in supplemental figures. It would also be useful to reliably show control next to mutant in the same plot, as it is a bit hard to compare across panels, which occurs in several figures.

Comments: We have uploaded all the raw data related to each experiment.

(2) Given the focus on the two possible forms of Sema7a, the authors should use HCR or another form of reliable in situ hybridization to show the spatiotemporal pattern of expression of each isoform.

Comments: We have utilized the HCR™ RNA-FISH Technology to generate transcript specific probes. To generate transcript-specific HCR probes to distinctly detect the sema7aGPI (NM_001328508) and the sema7asec (NM_001114885) transcripts, Molecular Instruments could design only 11 probes against the sema7aGPI transcript and only one probe against the sema7asec transcript (personal correspondence with Mike Liu, PhD, Head of Operations and Product Development Lead Molecular Instruments, Inc.). The HCR probe against the sema7aGPI transcript showed a very faint signal. Unfortunately, the HCR probe against the sema7asec transcript failed to detect the presence of any transcript. For robust detection of transcripts, the protocol demands a minimum of 20 probes. We believe that the very low number of probes against our transcripts is the primary reason for the lack of a signal.

(3) The authors should explain the criteria used to select the 22 embryos used to analyze the effects of expressing diffusible Sema7a.

Comments: We have explained this in lines 291-292. We identified 22 mosaic sema7asecmKate2 integration events, in which a single mosaic ectopic integration had occurred near the network of sensory arbors, from a total of almost 100 integrations. We rejected events where the sema7asec-mKate2 integration occurred either farther away from the sensory arbor network or had happened in multiple neighboring cells.

(4) Although arbors were imaged in live embryos, time is never presented as a variable, so I cannot tell whether axon topology was changing as the images were collected. This needs to be clarified.

Comments: We imaged the trunk neuromasts of both control and sema7a-/- mutant live zebrsfish larvae at 2, 3, and 4 dpf. We imaged the control and the sema7a-/- mutants of each developmental stage in parallel, within a span of two hours, and repeated these experiments multiple times to gather almost a hundred larvae from each genotype. Even though the sensory arbor network is dynamic, we believe imaging both the genotypes in parallel and within a span of two hours, and averaging almost a hundred larvae from each genotype minimize the temporal variability observed in the arbor architecture.

(5) Ideally, the authors should use CRISPR/cas-9 to create a mutation in the C-terminus that would prevent production of the GPI-anchored form and not of the diffusible form. I understand if this is too much work to do in a short time, and would be satisfied with another experiment that could distinguish roles for at least one isoform more clearly. For instance, it would be interesting to see an analysis of how far an axon can be from a source to detect diffusible Sema7a (live imaging would be ideal for this) and then to show that the effect is different when the membrane bound form is expressed.

Comments: Thank you for this comment. We are currently working in generating transcript specific knockout animals.

We have added live timelapse video microscopy data in lines 330-337, Figure 4H-J, Figure 4—figure supplement 2, Video15,16.

We have added a new segment analyzing the membrane-bound transcript variant in lines 338-351.

Reviewer #4 (Recommendations For The Authors):

Feedback to authors

Overall, this is a very important and novel study. Currently the manuscript does need revision.

Major concerns:

(1) Controls. For the ectoptic expression of Sema7a, injection of a construct expressing Sema7a under a heatshock promoter is used to drive ectopic expression. No heatshock (injected) animal are used as a control. In many systems heatshock can impact neuron morphology. And heatshock proteins are required for normal neurite and synapse formation. Please examine sensory axons in uninjected wildtype animals with heatshock.

Comments: We have added this control experiment in a new segment, explained in detail in lines 348-350 and Figure 5.

(2) Synapse staining - regarding Figure 5 and related supplement

Understanding whether guidance defects ultimately impact synapse formation is an important aspect of this paper. Therefore, is necessary to have accurate measurements of the number of complete synapses, and the overall numbers of pre- and postsynaptic components. Currently the data plotted in Figure 5 is extensive, but the way the data is laid out, the relevant comparisons are challenging to make. Perhaps include this quantification in the supplement, and move the data from the supplement to the main figure? The quantifications in the supplement are easier to follow and easier to compare between genotypes.

Comments: We have performed exhaustive analysis on the formation of pre- and postsynaptic structures and have identified how their distribution changes along neuromast development in control larvae. We have further analyzed how such distribution is perturbed in the sema7a-/- mutants. We believe that showing only the average numbers will not reveal the changes in the distribution of the synaptic structures during development and across genotypes.

Looking at the data itself, there seems to be some discrepancies with the synaptic counts compared to published work. While the CTBP numbers seem in order, the Maguk numbers do not. In both mutant and control there are many hair cells without any Maguk puncta/aggregates-leading to 0.75-1 postsynapses per hair cell (Figure 5 supplement H-I). Typically, the numbers should be more comparable to what was obtained for CTBP, 3-4 puncta per cells (Figure 5 supplement B-C), especially by 3-4 dpf. 3-4 CTPB or Maguk puncta per cell is based on previously published immunostaining and EM work.

The Maguk immunostaining, especially at early stages (2-3 dpf) is challenging. To compound a challenging immunostain, around 2019 Neuromab began to outsource the purification of their Maguk antibody. After this outsourcing our lab was no longer able to get reliable label with the Maguk antibody from Neuromab.

Millipore sells the same monoclonal antibody and it works well: https://www.emdmillipore.com/US/en/product/Anti-pan-MAGUK-Antibody-clone-K2886,MM_NF-MABN72

I would recommend this source.

Comments: Thank you for suggesting the new MAGUK antibody. We have utilized this new MAGUK antibody from Millipore and added a new segment in lines 389-408. In future publication we will utilize this antibody to capture the postsynaptic densities in the sensory arbors.

The discrepancies in the postsynaptic punctae number in our control larvae may arise due to the reliability of the Neuromab MAGUK antibody. We have utilized this same antibody to stain the sema7a-/- mutants and have observed a significant decrease in MAGUK punctae number and area. On grounds of keeping parity between the control and the sema7a-/- mutants, we have decided to keep our experimental results in the manuscript.

In addition to a more accurate Maguk label, a combined pre- and post-synaptic label is essential to understand whether synapses pair properly in the sema7a mutants. This can be accomplished using subtype specific antibodies using goat anti-mouse IgG1/Maguk and goat anti-mouse IgG2a/CTBP secondaries.

Comments: Thank you for suggesting this. We are preparing another manuscript in which we will utilize this technique along with other suggestions to tease apart the role of distinct transcript variants in regulating neural guidance and synapse formation.

(3) Does sema7a lesion impact the number of hair cells per neuromast? If hair cell numbers are reduced several of the quantifications could be impacted.

Comments: We have added the raw data with the hair cell counts in both control and sema7a-/- mutants across developmental stages. The homozygous sema7a-/- mutants have slightly less hair cells and we have normalized all our topological analyses by the corresponding hair cell numbers for each neuromast in each experiment (lines 669-675).

(4) Could innervation just be developmentally delayed in sema7a mutants? At 4 dpf the sensory system is just starting to come online and could still be in the process of refinement. Did you look at slightly older ages, after the sensory system is functional behaviorally, for example, 6 dpf? Do the cores phenotypes (synapse defects and excess arbors) persist at 6 dpf in the sema7a mutants?

Comments: The homozygous sema7a-/- mutants are unviable and start to die at 6 dpf. We therefore restricted our analysis until 4 dpf. The association of the sensory arbors with the clustered hair cells gradually decreases as the neuromasts mature from 2 dpf to 4dpf in the sema7a-/- mutants (lines 174-176, Figure 2I). Moreover, in the sema7a-/- mutants the sensory axons throw long projections that keep getting farther away from the clustered hair cells as the neuromast matures from 2 dpf to 4 dpf (lines 166-168, Figure 2H; Figure 2—figure supplement 1K,L). These observations suggests that if the phenotypes in the sema7a-/- mutants were due to developmental delays, then we should have seen a recovery of disrupted arborization patterns over time. But instead, we observe a further deterioration of the arborization patterns and other architectural assemblies. These findings confirm that the observed phenotypes in the sema7a-/- mutants are not due to delayed development of the larvae, but a specific outcome for the loss of Sema7A protein.

Minor comments to address:

Results

Page 4 lines 89-91. For the readers, explain why you examined levels in Sema7a in rostral and caudal hair cells. Also, this sentence is, in general, a little bit misleading-initially reading that there is no difference in Sema7a at 1.5-4 dpf.

Comments: In lines 44-48, we explain that the hair cells in the neuromast contain mechanoreceptive hair cells of opposing polarities that help them detect water currents from opposing directions. In lines 93-106, we tested whether the Sema7A level varies between the two polarities. We observed that the Sema7A level is similar between the two polarities of hair cells, but the average Sema7A intensity increases significantly over the developmental period of 2 dpf to 4 dpf in both rostrally and caudally polarized hair cells.

Page 10-11 Lines 263-270. What was the frequency of these 2 outcomes- out of the 22 cases with ectopic expression?

Comments: We have explained this in lines 291-292. We identified 22 mosaic sema7asecmKate2 integration events, in which a single mosaic ectopic integration had occurred near the network of sensory arbors, from a total of almost 100 integrations. We rejected events where the sema7asec-mKate2 integration occurred either farther away from the sensory arbor network or had happened in multiple neighboring cells.

Discussion

Page 14 Lines 359-360. There is not enough evidence provided in this work to suggest that the membrane attached form of Sema7a is playing a role. Both the secreted and membrane form are gone in the sema7a mutants. If the membrane attached form was specifically lesioned, and resulted in a phenotype, then there would be sufficient evidence. Currently there is strong evidence for a distinct role for the secreted form. Although the authors qualify the outlined statement with the word 'likely', stating this possibility in the discussion take-home is misleading.

Comments: In future we will utilize the CRISPR/Cas9 technique to target the unique Cterminal domain of the GPI-anchored sema7a transcript variant. We believe that this will only perturb the formation of the full-length Sema7A protein and help us differentiate between the roles of the membrane-bound Sema7AGPI molecule and the secreted Sema7Asec in sensory arborization and synaptic assembly.

It might be interesting in either the intro or discussion to reference the role Sema3F in axon guidance in the mouse auditory epithelium. https://elifesciences.org/articles/07830

Comments: We have added this reference in lines 61-64.

Figures

Please indicate on one of your Figures where the mutation is (roughly) in the sema7a mutant (in addition to stating it in the results).

Comments: We have added this information in Figure 2—figure supplement 1A.

Either state or indicate in a Figure where the epitope used to make the Sema7a antibody-to show that the antibody is predicted to recognize both isoforms.

Comments: We have stated the details of the epitope in lines 528-529.

Figure 2-S1 what is the scale in panel A, is it different between mutant and wildtype?

Comments: We have updated the images. New images are depicted in Figure 2—figure supplement 1A.

Methods

What were the methods used to quantify synapse number and area?

Comments: We have added a new section in lines 702-708 to explain the measurement techniques.