Whole brain mapping of spinal-projecting neurons in larval zebrafish

Reviewer #1 (Public review):

Summary:

The authors have achieved an excellent, thorough anatomical characterization of all spinal projecting neurons in the larval zebrafish. The comprehensive nature of their labeling approach and their quantification will make this work an instant reference benchmark for a wide number of zebrafish researchers. In addition, the scholarly approach in comparisons with other work in and outside of fish makes the manuscript valuable to researchers outside the field who would like to know how to translate between zebrafish and mouse terminologies.

Strengths:

The figures are clear and easy to follow. The literature review is impressive. The authors are careful to note the few limitations of their approach (eg the absence of Mauthner cell labeling and associated large neuron weak label). The manuscript does the whole field a major service.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

Summary:

The vertebrate spinal cord receives inputs from many supraspinal regions. The authors used optical backfilling to trace neurons in the zebrafish larval brain sending axons to the spinal cord. With two-photon microscopy, they managed to render a comprehensive 3D map of these neurons and drew homologs with mammalian brain structures.

Strengths:

The main strength lies in the precise 3D mapping. The fact that most of the previously reported neuron groups have been confirmed by their approach is a solid endorsement of their methodology.

This study provides a comprehensive alternative anatomical reference framework for studying individual groups of supraspinal neurons with projections to the zebrafish spinal cord.

Weaknesses:

The whole approach could be enhanced by counter-staining their preparation to profile brain structures, including many nuclei more precisely.

Also, the backfilling approach does not reveal the full trajectories of axons, which is already available to some degree by ZExplorer Atlas.

Reviewer #3 (Public review):

In this study, the authors aim to provide the most comprehensive and detailed topographic map to date of spinal projection neurons in the larval zebrafish brain. They achieve this by retrogradely photoactivating, in the rostral spinal cord, a photoconvertible GFP expressed pan-neuronally, and by constructing a template larval zebrafish brain atlas to regionalize the location of all labeled somata across the brain. The labeling strategy, together with the chosen animal model, provides strong support for the completeness of the dataset. The generation of a standardized anatomical atlas establishes a rigorous framework for analysis. Molecular and anatomical evidence suggesting evolutionary conservation of selected regions of interest appears solid.

Overall, the authors successfully achieve their aim. By generating this atlas of spinal projection neurons, they provide not only an anatomical framework of the regions involved, but also an important reference for improving the orientation and regionalization of the zebrafish brain, which has historically been difficult to define. This work may serve as a valuable resource for future evolutionary, developmental, and comparative studies of spinally projecting neuronal populations implicated in diverse functions.