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The 3D atlas builder: a dynamic and expandable 3D tool for monitoring the changes in the neuronal differentiation domain during hindbrain morphogenesis

Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a protocol and provide experimental proof for building a digital 3D atlas (here, for zebrafish hindbrain) that integrates spatial and temporal data for neuronal differentiation and brain morphogenesis, through a combination of in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a reference 3D hindbrain from several imaged specimens and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain vs. the progenitor domain in the whole hindbrain. Third, we used in vivo Kaede-photoconversion experiments to generate a temporal heatmap of the neuronal growth in the whole hindbrain, revealing the spatiotemporal dynamics of neuronal differentiation upon morphogenesis. Last, as proof-of-concept, we assessed the birthdate order of GABAergic neurons using our temporal registration map. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction.Brain morphogenesis studies can use our atlas-builder tool, which precisely maps cell populations in the embryonic brain over time and can be easily shared.