Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish

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

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):

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, 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 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). 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. 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. 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.

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 lateral-line 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.
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.
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.
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.