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  1. Evaluation Summary:

    The authors present the first digital adult zebrafish brain anatomy atlas that includes ten highly informative markers for different brain regions and cell types. The authors utilize new clearing techniques to enable staining of the whole fixed zebrafish adult brains, and light sheet microscopy fluorescent imaging to obtain high resolution three-dimensional data. Several individual dataset for each marker are registered and aligned to an anatomical model using fluorescent cell nuclei and autofluorescence patterns by state of the art volume registration algorithms. The results appear to be of high quality. The authors also provide a detailed and state-of-the-art anatomical annotation of brain regions. Using ten different antibodies to fluorescently label different neuron types, stem cells and glia, proliferation zones, and some specific anatomical regions, the authors demonstrate the utility of their database. This will be a valuable resource for neurobiology research in zebrafish.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    This paper uses cutting-edge imaging to develop a new 3D map of the zebrafish brain. The use of fixed imaging plus antibody staining with Lightsheet microscopy has developed an excellent high-resolution dataset. The regional imaging data is convincing. The data are well-presented, and the text easy to read.

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  3. Reviewer #2 (Public Review):

    The authors have constructed a brain atlas, AZBA, for adult zebrafish, based on light-sheet imaging to acquire whole-brain image stacks, followed by registration and manual segmentation. Everything from image acquisition to anatomical annotation has been beautifully performed to state of the art standards. The result is an atlas that is more useful than the classic Wullimann book, because sections can be viewed in arbitrary section planes. Even more critically, as a digital atlas, this resource also enables new computational approaches to analyzing adult zebrafish brain imaging data. Thus, AZBA will quickly become a central and widely used resource for zebrafish neurobiological studies. I have no major concerns, as it stands, this work immediately propels work using adult zebrafish.

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