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  1. Evaluation Summary:

    This paper very nicely tackles a methodological problem in aligning different types of datasets (EM and light microscopy) to image embryonic nervous system development in the nematode C.elegans. The paper is important not just from a methodological standpoint, but also provides novel insights into nervous system development that will be of general interest to the reader. The latter deserves more attention in the manuscript.

    (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 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Santella describes methods to align fluorescence microscopy and EM datasets in the C. elegans embryo. The major use of this will be to help analyze the initial development of the embryonic nervous system, including process outgrowth, etc, which so far has been described at the fluorescence but not EM level. The work will be of interest to the community of researchers involved in developmental connectomics and annotation of serial section EM datasets.

    The study uses FM and EM datasets for iterative refinement of cell identities at the tissues and single-cell levels. Some new information is provided about early C. elegans nervous system development e.g. entry points of pioneer axons into the nerve ring, or cell contacts during sensilla development; however, the main focus is the methodology. This is overall technically sound and represents a significant investment of effort.

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  3. Reviewer #2 (Public Review):

    This study provides a new approach to image alignment between electron microscopy (EM) and time-lapse fluorescence microscopy (FM). It uses previously developed methods of EM sampling (FIB-SEM and array-tomography), imaging and software approaches of nuclei lineaging. It combines these together with a new landmark-based co-optimization algorithm, suggested to contribute to high accuracy, for cross-modality image alignment. The study seeks to combine the spatial resolution by EM and the temporal resolution of FM to characterize cell morphologies in C. elegans embryos, specifically its central nervous system neuropil, the major sensory organ, and excretory canal. The work presents the timing of axon growth of specific neurons of the neuropil and specific appositions of neurons and glia in the analyzed sensory organ. The authors present a way towards cross-modality analysis in C. elegans and suggest their combined approach can be modified for future use in alignment and annotation of other complex images.

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  4. Reviewer #3 (Public Review):

    Neuron visualization in a developing animal embryo gives important insights into the earliest stages of neurite outgrowth and nervous system assembly, but existing imaging methods are confounded in one way or another by a multitude of challenges, including temporal and spatial resolution, cell-specific labelling, cell identity and annotation. In this manuscript, Santella et al. cleverly integrate two contrasting methods of visualization, electron microscopy (EM) and fluorescence microscopy (FM) to cross-compensate for some of these shortfalls. Through this method, the authors achieve spatial and temporal resolution in visualizing developing neurons in the C. elegans embryo across a time series. This cross-modality analysis revealed important insights into early formation of the nerve ring as well as the amphid sensory organ.

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