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Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.

During animal development, complex patterns of gene expression provide positional information within the embryo. To better understand the underlying gene regulatory networks, the Berkeley Drosophila Transcription Network Project (BDTNP) has developed methods that support quantitative computational analysis of three-dimensional (3D) gene expression in early Drosophila embryos at cellular resolution. We introduce PointCloudXplore, an interactive visualization tool that supports visual exploration of relationships between different genes’ expression using a combination of established visualization techniques.Two aspects of gene expression are of particular interest: (i) gene expression patterns defined by the spatial locations of cells expressing a gene, and (ii) relationships between the expression levels of multiple genes. PointCloudXplore provides users with two corresponding classes of data views: (i) Physical Views based on the spatial relationships of cells in the embryo, and (ii) Abstract Views that discard spatial information and plot expression levels of multiple genes with respect to each other. Cell Selectors highlight data associated with subsets of embryo cells within a View. Using linking, these selected cells can be viewed in multiple representations. We describe PCX as a 3D gene expression visualization tool and provide examples of how it has been used by BDTNP biologists to generate new hypotheses.

A new spatio-temporal coordinate framework for studying three-dimensional patterns of gene expression in the Drosophila blastoderm is presented that takes account of previously undetected morphological movements.BackgroundTo accurately describe gene expression and computationally model animal transcriptional networks, it is essential to determine the changing locations of cells in developing embryos.ResultsUsing automated image analysis methods, we provide the first quantitative description of temporal changes in morphology and gene expression at cellular resolution in whole embryos, using the Drosophila blastoderm as a model. Analyses based on both fixed and live embryos reveal complex, previously undetected three-dimensional changes in nuclear density patterns caused by nuclear movements prior to gastrulation. Gene expression patterns move, in part, with these changes in morphology, but additional spatial shifts in expression patterns are also seen, supporting a previously proposed model of pattern dynamics based on the induction and inhibition of gene expression. We show that mutations that disrupt either the anterior/posterior (a/p) or the dorsal/ventral (d/v) transcriptional cascades alter morphology and gene expression along both the a/p and d/v axes in a way suggesting that these two patterning systems interact via both transcriptional and morphological mechanisms.ConclusionOur work establishes a new strategy for measuring temporal changes in the locations of cells and gene expression patterns that uses fixed cell material and computational modeling. It also provides a coordinate framework for the blastoderm embryo that will allow increasingly accurate spatio-temporal modeling of both the transcriptional control network and morphogenesis.

A suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology with cellular resolution in whole Drosophila embryos is described.BackgroundTo model and thoroughly understand animal transcription networks, it is essential to derive accurate spatial and temporal descriptions of developing gene expression patterns with cellular resolution.ResultsHere we describe a suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology at cellular resolution in whole embryos. A database containing information derived from 1,282 embryos is released that describes the mRNA expression of 22 genes at multiple time points in the Drosophila blastoderm. We demonstrate that our methods are sufficiently accurate to detect previously undescribed features of morphology and gene expression. The cellular blastoderm is shown to have an intricate morphology of nuclear density patterns and apical/basal displacements that correlate with later well-known morphological features. Pair rule gene expression stripes, generally considered to specify patterning only along the anterior/posterior body axis, are shown to have complex changes in stripe location, stripe curvature, and expression level along the dorsal/ventral axis. Pair rule genes are also found to not always maintain the same register to each other.ConclusionThe application of these quantitative methods to other developmental systems will likely reveal many other previously unknown features and provide a more rigorous understanding of developmental regulatory networks.

Moss et al.(2005) describe, in a recent paper, a filter that they use to detect lines. We noticed that the wavelet on which this filter is based is a difference of uniform filters. This filter is an approximation to the second derivative operator, which is commonly implemented as the Laplace of Gaussian (or Marr-Hildreth) operator (Marr & Hildreth, 1980; Jahne, 2002), Figure 1. We have compared Moss' filter with 1) the Laplace of Gaussian operator, 2) an approximation of the Laplace of Gaussian using uniform filters, and 3) a few common noise reduction filters. The Laplace-like operators detect lines by suppressing image features both larger and smaller than the filter size. The noise reduction filters only suppress image features smaller than the filter size. By estimating the signal to noise ratio (SNR) and mean square difference (MSD) of the filtered results, we found that the filter proposed by Moss et al. does not outperform the Laplace of Gaussian operator. We also found that for images with extreme noise content, line detection filters perform better than the noise reduction filters when trying to enhance line structures. In less extreme cases of noise, the standard noise reduction filters perform significantly better than both the Laplace of Gaussian and Moss' filter.

BackgroundTo accurately describe gene expression and computationally model animal transcriptional networks, it is essential to determine the changing locations of cells in developing embryos.ResultsUsing automated image analysis methods, we provide the first quantitative description of temporal changes in morphology and gene expression at cellular resolution in whole embryos, using the Drosophila blastoderm as a model. Analyses based on both fixed and live embryos reveal complex, previously undetected three-dimensional changes in nuclear density patterns caused by nuclear movements prior to gastrulation. Gene expression patterns move, in part, with these changes in morphology, but additional spatial shifts in expression patterns are also seen, supporting a previously proposed model of pattern dynamics based on the induction and inhibition of gene expression. We show that mutations that disrupt either the anterior/posterior (a/p) or the dorsal/ventral (d/v) transcriptional cascades alter morphology and gene expression along both the a/p and d/v axes in a way suggesting that these two patterning systems interact via both transcriptional and morphological mechanisms.ConclusionOur work establishes a new strategy for measuring temporal changes in the locations of cells and gene expression patterns that uses fixed cell material and computational modeling. It also provides a coordinate framework for the blastoderm embryo that will allow increasingly accurate spatio-temporal modeling of both the transcriptional control network and morphogenesis.

The generalized Radon (or Hough) transform is a well-known tool for detecting parameterized shapes in an image. The Radon transform is a mapping between the image space and a parameter space. The coordinates of a point in the latter correspond to the parameters of a shape in the image. The amplitude at that point corresponds to the amount of evidence for that shape. In this paper we discuss three important aspects of the Radon transform. The first aspect is discretization. Using concepts from sampling theory we derive a set of sampling criteria for the generalized Radon transform. The second aspect is accuracy. For the specific case of the Radon transform for spheres, we examine how well the location of the maxima matches the true parameters. We derive a correction term to reduce the bias in the estimated radii. The third aspect concerns a projection-based algorithm to reduce memory requirements.