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.

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.

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.