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WIREs Dev Biol
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Building quantitative, three‐dimensional atlases of gene expression and morphology at cellular resolution

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Abstract Animals comprise dynamic three‐dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy‐based approaches to establish permanent, quantitative datasets—atlases—that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. WIREs Dev Biol 2013, 2:767–779. doi: 10.1002/wdev.107 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models Technologies > Analysis of Cell, Tissue, and Animal Phenotypes Technologies > Analysis of the Transcriptome

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Examples of three‐dimensional atlases. (a) The FlyCircuit atlas of neuronal connectivity in Drosophila brains. Each color groups members of a neuronal tract. Each panel displays different subsets of tracts. (b) Cell migration during gastrulation in Drosophila embryos. The two left panels show cell movements over time. The two right panels show the net displacement vectors, with mesoderm cells shown in orange and ectoderm cells show in gray. (c) Patterns of mRNA expression of transcription factors in Drosophila blastoderm embryos. The upper view shows a three‐dimensional representation of the embryo. The lower view shows a cylindrical projection in which height indicates the level of expression of each transcription factor in each cell. Both views were generated using the visualization tool PointCloudXplore.

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Visualizing 3D gene expression by parallel coordinates. Each vertical axis shows the mRNA expression levels for one gene in each of the 6000 cells in the Drosophila blastoderm embryo. The lines connect data for the same cells. Blue lines connect cells expressing the anterior most stripe of hunchback (hb), yellow lines the central hb stripe, and pink lines the posterior stripe. The locations of these cells are shown in the physical three‐dimensional view below. In the parallel coordinates, it can be readily seen that the anterior stripe on hb coincides with high slp1 expression, the central hb stripe with high ftz expression, and 50% of the posterior hb stripe with high eve expression. These views were generated using PointCloudXplore, an interactive visualization tool (http://bdtnp.lbl.gov/Fly‐Net/bioimaging.jsp? w=pcx).

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Comparing image quality of SPIM and laser scanning multiphoton microscopy. Multiphoton optical sections are shown for stage‐16 Drosophila embryos stained to label nuclei. (a) The top two images are of a live embryo expressing GFP‐histone. The images were taken using the two‐photon SPIM technique of ‘simultaneous multiview imaging’ (SiMView) and were kindly provided by Philipp J. Keller. (b) The bottom two images are of a fixed emrbyo stained with SYTOX Green and were acquired using standard two‐photon laser scanning microscopy. The embryo's dorsal/ventral direction is shown from top to bottom in each image. Optical sections were selected through the midplane of each three‐dimensional embryo image to show, from left to right, its anterior/posterior (left) and it's left/right (right) directions.

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A pipeline for building and using a three‐dimensional atlas.

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Technologies > Analysis of the Transcriptome
Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models
Technologies > Analysis of Cell, Tissue, and Animal Phenotypes