MOTIVATION: Understanding the mechanisms by which the zebrafish pectoral fin develops is expected to produce insights on how vertebrate limbs grow from a 2D cell layer to a 3D structure. Two mechanisms have been proposed to drive limb morphogenesis in tetrapods: a growth-based morphogenesis with a higher proliferation rate at the distal tip of the limb bud than at the proximal side, and directed cell behaviors that include elongation, division and migration in a non-random manner. Based on quantitative experimental biological data at the level of individual cells in the whole developing organ, we test the conditions for the dynamics of pectoral fin early morphogenesis. RESULTS: We found that during the development of the zebrafish pectoral fin, cells have a preferential elongation axis that gradually aligns along the proximodistal (PD) axis of the organ. Based on these quantitative observations, we build a center-based cell model enhanced with a polarity term and cell proliferation to simulate fin growth. Our simulations resulted in 3D fins similar in shape to the observed ones, suggesting that the existence of a preferential axis of cell polarization is essential to drive fin morphogenesis in zebrafish, as observed in the development of limbs in the mouse, but distal tip-based expansion is not. AVAILABILITYAND IMPLEMENTATION: Upon publication, biological data will be available at http://bioemergences.eu/modelingFin, and source code at https://github.com/guijoe/MaSoFin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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This paper presents a new multiscale method to denoise three-dimensional images of cell nuclei. The specificity of this method is its awareness of the noise distribution and object shapes. It combines a multiscale representation called Isotropic Undecimated Wavelet Transform (IUWT) with a nonlinear transform, a statistical test and a variational method, to retrieve spherical shapes in the image. Beyond extending an existing 2D approach to a 3D problem, our algorithm takes the sampling grid dimensions into account. We compare our method to the two algorithms from which it is derived on a representative image analysis task, and show that it is superior to both of them. It brings a slight improvement in the signal-to-noise ratio and a significant improvement in cell detection.

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.

MOTIVATION: Multi-view microscopy techniques such as Light-Sheet Fluorescence Microscopy (LSFM) are powerful tools for 3D + time studies of live embryos in developmental biology. The sample is imaged from several points of view, acquiring a set of 3D views that are then combined or fused in order to overcome their individual limitations. Views fusion is still an open problem despite recent contributions in the field. RESULTS: We developed a wavelet-based multi-view fusion method that, due to wavelet decomposition properties, is able to combine the complementary directional information from all available views into a single volume. Our method is demonstrated on LSFM acquisitions from live sea urchin and zebrafish embryos. The fusion results show improved overall contrast and details when compared with any of the acquired volumes. The proposed method does not need knowledge of the system's point spread function (PSF) and performs better than other existing PSF independent fusion methods. AVAILABILITY AND IMPLEMENTATION: The described method was implemented in Matlab (The Mathworks, Inc., USA) and a graphic user interface was developed in Java. The software, together with two sample datasets, is available at http://www.die.upm.es/im/software/SPIMFusionGUI.zip A public release, free of charge for non-commercial use, is planned after the publication of this article.

We propose to directly process 3D + t image sequences with mathematical morphology operators using a new classification of the 3D+t structuring elements. Several methods (filtering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebrafish embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebrafish early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it benefits from the intrinsic redundancy of the temporal dimension and minimizes the needs for human intervention in semi-automatic algorithms.

Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis.

We designed a strategy for extracting the shapes of cell membranes and nuclei from time lapse confocal images taken throughout early zebrafish embryogenesis using a partial-differential-equation-based segmentation. This segmentation step is a prerequisite for an accurate quantitative analysis of cell morphodynamics during embryogenesis and it is the basis for an integrated understanding of biological processes. The segmentation of embryonic cells requires live zebrafish embryos fluorescently labeled to highlight sub-cellular structures and designing specific algorithms by adapting classical methods to image features. Our strategy includes the following steps: the signal-to-noise ratio is first improved by an edge-preserving filtering, then the cell shape is reconstructed applying a fully automated algorithm based on a generalized version of the Subjective Surfaces technique. Finally we present a procedure for the algorithm validation either from the accuracy and the robustness perspective.

We propose an extension of the classical morphological filtering based on openings by line segment structuring elements. It consists in filtering a 3D+time image with the opening by all the possible rotations of a segment in the 4D space, given an initial segment and a rotation angle. The method has been applied to remove noise from confocal laser scanning microscopy images of live zebrafish embryos engineered to fluorescently label all their cell membranes.