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  1. Author Response

    Evaluation Summary:

    This manuscript reports advances in the image analysis software package MorphGraphX (MGX). designed to capture the developmental dynamics of growing tissues at cellular resolution. This version, MGX2.0, includes new tools for precise quantitation of cellular behaviors, such as cell division and expansion, within the context of positional information in the growing organs. To illustrate multiple functionalities of MGX2.0, various tissues are analyzed. This presentation style highlights the power and broad applicability of MGX2.0, but leads to a somewhat disjointed narrative, and how it can provide insight into specific biological questions is less clear.

    There has been so much added to MGX since the initial version that is was a bit tough to decide how to present it all. One unifying theme for the work that seemed the most scientifically enabling was the notion of coordinate systems for the annotation and interpretation of spatial data. With this in mind, the story follows the development of the presentation of coordinate systems of increasing complexity, starting from simple gradients to more sophisticated methods such as Beziers, compound systems and deformation maps. Unfortunately, as the reviewers and editor have mentioned, this does make it a bit chaotic from the “biological story” perspective, as the same story may come and go at different parts of the paper when illustrating different tools, or the same dataset may come and go as different techniques are applied to it. To address this problem, we have done as the editor and reviewers have suggested to rearrange some of the text and figures where possible and have tried to provide more context and backup for the biological story and relevance to better demonstrate its utility in specific cases.

    Reviewer #1 (Public Review):

    The work presented here describes the application of a tool (MorphographX 2.0) that opens up possibilities for new image analyses. MorphographX 1.0 is already a valuable tool in the field and the improvements and new functionalities, and approaches presented in this paper allow for the integration and analysis of more positional and temporal information. Specifically, adding positional annotation to analyze the distribution of cell properties across a plant organ will be of great use for the community. The case studies used to showcase MorphographX 2.0's applications highlight the diversity in questions that can be addressed using this tool. As a result, we expect to see MorphographX 2.0 applied in a variety of future plant biology stories. In addition, we believe this tool could also be useful to those outside the plant community. While probably less of use in tissues where there is extensive migration, it can be applied to any system with clearly visible cell membranes.

    We agree that the notion of 2.5D image processing could also prove to be very useful in animal systems, as a great many biological processes happen on layers of cells in animal as well, such as epithelia.

    The examples presented in this story highlight some great applications of the MorphographX 2.0 software. Analyses using more positional, temporal and 3D information will enable new findings across plant tissues and potentially across species. It is however important to be aware that for optimal use this software is designed to analyze high quality, high contrast stacks that can be difficult and time-intensive to acquire. MorphographX 2.0 also requires a powerful computer setup. The presence of both Linux and Windows versions that do not require a nVidia graphics card does open up possibilities. In addition, extensive documentation and the presence of a community forum allow use of the software without intensive training.

    We have added mention of the user forum (forum.image.sc) for MorphoGraphX in the text.

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  2. Evaluation Summary:

    This manuscript reports advances in the image analysis software package MorphGraphX (MGX). designed to capture the developmental dynamics of growing tissues at cellular resolution. This version, MGX2.0, includes new tools for precise quantitation of cellular behaviors, such as cell division and expansion, within the context of positional information in the growing organs. To illustrate multiple functionalities of MGX2.0, various tissues are analyzed. This presentation style highlights the power and broad applicability of MGX2.0, but leads to a somewhat disjointed narrative, and how it can provide insight into specific biological questions is less clear.

    (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 #2 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The work presented here describes the application of a tool (MorphographX 2.0) that opens up possibilities for new image analyses. MorphographX 1.0 is already a valuable tool in the field and the improvements and new functionalities, and approaches presented in this paper allow for the integration and analysis of more positional and temporal information. Specifically, adding positional annotation to analyze the distribution of cell properties across a plant organ will be of great use for the community. The case studies used to showcase MorphographX 2.0's applications highlight the diversity in questions that can be addressed using this tool. As a result, we expect to see MorphographX 2.0 applied in a variety of future plant biology stories. In addition, we believe this tool could also be useful to those outside the plant community. While probably less of use in tissues where there is extensive migration, it can be applied to any system with clearly visible cell membranes.

    The examples presented in this story highlight some great applications of the MorphographX 2.0 software. Analyses using more positional, temporal and 3D information will enable new findings across plant tissues and potentially across species. It is however important to be aware that for optimal use this software is designed to analyze high quality, high contrast stacks that can be difficult and time-intensive to acquire. MorphographX 2.0 also requires a powerful computer setup. The presence of both Linux and Windows versions that do not require a nVidia graphics card does open up possibilities. In addition, extensive documentation and the presence of a community forum allow use of the software without intensive training.

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  4. Reviewer #2 (Public Review):

    The manuscript is of wide interest to the plant development community. It gives an overview of recent extensions to MorphoGraphX, a tool to analyse confocal microscopy images of plant tissues to cell-level. In particular the advance concerns the annotation of cell morphometric and tissue-level topological properties (cell volume, cell shape, cell neighborhood relations, cell lineages) as a function of local coordinate systems in plant organs, such as distance to the root tip along the length of a (potentially curved) root. This allows the end-user to annotate static and time-lapse images in relation to a positional coordinate system close to those likely used by the developmental mechanism itself.

    The useful novel methodology to annotate 3D confocal images of plant organs is illustrated using data of roots, ovules, and leafs. Many of these analysis tools seem to have been developed in the context of research papers cited in the manuscript. The main novelty of the present manuscript seems the bundling and release of these new features in MorphoGraphX 2.0. The software is available from the authors' website allowing users to read it.

    The tools presented in the manuscript will attract the keen interest of plant biologists who (want to) carry out quantitative analyses of developing plant tissues, alongside animal developmental biologists working, e..g, with epithelial tissues that can be well segmented. However, with the manuscript in its present form, it would be very hard for a researcher new to the approach to get started with MorphoGraphX2.0.

    The datasets of the examples are available on the website of MorphoGraphX2.0. It would be very helpful if detailed instructions were given in the manuscript or in the supplements of how the analyses can be reproduced. This would allow the end-user to get started with their own data. Also it would be useful to give more detail on how the data must be generated to make it suitable for use in MorphoGraphX.

    Altogether, the annotation tools presented in the manuscript will be of interest to the community. However, as many of the tools seem to have been presented already elsewhere, the main contribution of the present manuscript would be (apart from 'advertising' MorphoGraphX2.0, which is useful in itself) in a set of instructions and/or directions to help get users started.

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  5. Reviewer #3 (Public Review):

    This manuscript reports the advancement in the image analysis software package MorphGraphX, which was developed about ten years ago. A comprehensive first report of this platform was published five years ago, and now the 2.0 version is being described.

    Included are useful tools to capture the developmental dynamics of growing tissues at cellular resolution. The analyses using this software can provide precise quantitative information of cellular behaviours, such as cell division and expansion, within the context of positional information in the growing organs. The tools developed and reported here are great assets to developmental biologists in general.

    The manuscript is well written in some places, while it reads confusingly in other parts. For example, figures are introduced in the main text in out of order and back-and-forth fashion.

    This article will become a go-to resource for future users of MorphGraphX to learn the power of the software package, with which they can quantify the spatiotemporal dynamics of the growth and development of living organisms.

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