Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on 1xed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface (https://github.com/hci-unihd/plant-seg).

A fundamental question in biology is how morphogenesis integrates the multitude of processes that act at different scales, ranging from the molecular control of gene expression to cellular coordination in a tissue. Using machine-learning-based digital image analysis, we generated a three-dimensional atlas of ovule development in Arabidopsis thaliana, enabling the quantitative spatio-temporal analysis of cellular and gene expression patterns with cell and tissue resolution. We discovered novel morphological manifestations of ovule polarity, a new mode of cell layer formation, and previously unrecognized subepidermal cell populations that initiate ovule curvature. The data suggest an irregular cellular build-up of WUSCHEL expression in the primordium and new functions for INNER NO OUTER in restricting nucellar cell proliferation and the organization of the interior chalaza. Our work demonstrates the analytical power of a three-dimensional digital representation when studying the morphogenesis of an organ of complex architecture that eventually consists of 1900 cells.

BackgroundA salient topic in developmental biology relates to the molecular and genetic mechanisms that underlie tissue morphogenesis. Modern quantitative approaches to this central question frequently involve digital cellular models of the organ or tissue under study. The ovules of the model species Arabidopsis thaliana have long been established as a model system for the study of organogenesis in plants. While ovule development in Arabidopsis can be followed by a variety of different imaging techniques, no experimental strategy presently exists that enables an easy and straightforward investigation of the morphology of internal tissues of the ovule with cellular resolution.ResultsWe developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide. We further improved the microscopy by employing a homogenous immersion system aimed at minimizing refractive index differences. The method allows complete inspection of the cellular architecture even deep within the ovule. Using the new protocol we were able to generate digital three-dimensional models of ovules of various stages.ConclusionsThe protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages. From the imaging data three-dimensional digital ovule models with cellular resolution can be rapidly generated using image analysis software, for example MorphographX. Such digital models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species.

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, and acquisition settings. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.Recently an approach combining the output of two neural networks and watershed to detect individual cells showed promising results on segmentation of cells in 2D [19]. Although this method can in principle be generalized to 3D images, the necessity to train two separate networks poses additional difficulty for non-experts.While deep learning-based methods define the state-of-the-art for all image segmentation problems, only a handful of software packages strives to make them accessible to non-expert users in biology (reviewed in [20]). Notably, the U-Net segmentation plugin for ImageJ [21] conveniently exposes U-Net predictions and computes the final segmentation from simple thresholding of the probability maps. CDeep3M [22] and DeepCell [23] enable, via the command-line, the thresholding of the probability maps given by the network, and DeepCell allows instance segmentation as described in [19]. More advanced post-processing methods are provided by the ilastik Multicut workflow [24], however, these are not integrated with CNN-based prediction.Here, we present PlantSeg, a deep learning-based pipeline for volumetric instance segmentation of dense plant tissues at single-cell resolution. PlantSeg processes the output from the microscope with a CNN to produce an accurate prediction of cell boundaries. Building on the insights from previous work on cell segmentation in electron microscopy volumes of neural tissue [13,15], the second step of the pipeline delivers an accurate segmentation by solving a graph partitioning problem. We trained PlantSeg on 3D confocal images of fixed Arabidopsis thaliana ovules and 3D+t light sheet microscope images of developing lateral roots, two standard imaging modalities in the studies of plant morphogenesis. We investigated a range of network architectures and graph partitioning algorithms and selected the ones which performed best with regard to extensive manually annotated groundtruth. We benchmarked PlantSeg on a variety of datasets covering a range of plant organs and image resolutions. Overall, PlantSeg delivers excellent results on ...

Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially-coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here we present recent advances in the MorphoGraphX software (Barbier de Reuille et al., 2015)⁠ that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

A fundamental question in biology is how morphogenesis integrates the multitude of distinct processes that act at different scales, ranging from the molecular control of gene expression to cellular coordination in a tissue. Investigating morphogenesis of complex organs strongly benefits from three-dimensional representations of the organ under study. Here, we present a digital analysis of ovule development from Arabidopsis thaliana as a paradigm for a complex morphogenetic process. Using machine-learning-based image analysis we generated a three-dimensional atlas of ovule development with cellular resolution. It allows quantitative stage- and tissue-specific analysis of cellular patterns. Exploiting a fluorescent reporter enabled precise spatial determination of gene expression patterns, revealing subepidermal expression of WUSCHEL. Underlying the power of our approach, we found that primordium outgrowth progresses evenly, discovered a novel mode of forming a new cell layer, and detected a new function of INNER NO OUTER in restricting cell proliferation in the nucellus. Moreover, we identified two distinct subepidermal cell populations that make crucial contributions to ovule curvature. Our work demonstrates the expedience of a three-dimensional digital representation when studying the morphogenesis of an organ of complex cellular architecture and shape that eventually consists of 1,900 cells.

Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially-coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here we present recent advances in the MorphoGraphX software (Barbier de Reuille et al. eLife 2015;4:e05864) that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

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