ABSTRACT

1. A morphogenetic framework for analyzing gene expression in Drosophila melanogaster blastoderms. Soile V.E. Keränen1, Cris L. Luengo Hendriks1, Charless Fowlkes2, Gunther H. Weber3, Oliver Rübel4, Min-Yu Huang3, Lisa Simirenko1, Damir Sudar1, Bernd Hamann3, Jitendra Malik2, Michael Eisen1, Mark D. Biggin1, David W. Knowles1, Berkeley Drosophila Transcription Network Project. Lawrence Berkeley National Laboratory, Berkeley, CA. 2) Department ofComputer Science, UC Berkeley, Berkeley, CA. 3) Computer and Data Visualization Department, UC Davis, Davis, CA 4) University Kaiserslautern, Germany ABSTRACT To fully understand and be able to computationally model the spatial complexity of developmental regulatory networks, it is critical to measure gene expression patterns at the resolution of individual cells. To this end, we have developed image analysis techniques for extracting 3D embryo morphology and quantifying gene expression at cellular (i.e., nuclear) resolution in Drosophila melanogaster blastoderm embryos (see also Luengo et al., Fowlkes et al., Knowles et al.). Using these methods on data from blastoderms of whole fixed embryos, we have discovered that well before gastrulation there are complex a/p and d/v nuclear density changes around the embryonic blastoderm. These changing densities correspond to nuclear movements whose directions and magnitudes we have estimated using both time-lapse images of live, histone2A-GFP embryos and pointcloud data from fixed embryos. Because the scale of nuclear movements are significant relative to the x,y,z locations of different genes’ expression patterns in the embryo, our data indicate that blastoderm pattern formation needs to be analyzed in a morphodynamic, rather than a morphostatic environment. As an example, we describe the movement of eve and ftz pair-rule stripes during stage 5 and compare these to the predicted nuclear flow. O2-permeable membrane embryo coverslip Density maps Live cell data shows that the local density differences are real and result from pregastrula nuclear movements Nuclear density changes through time in stage 5 Drosophila melanogaster blastoderm If we want to accurately map the temporal expression profiles of each individual cell, these changes need to be included in the spatio-temporal analysis of expression patterns and gene regulation. Corresponding unrolled view showing eve and sna expression Live embryo density maps early late D V D D V D eve (green), sna (red), isodensity (white) 0 20 40 60 80 100 % egg length 0 20 40 60 80 100 % egg length Anterior Posterior Dorsal Nuclear flow field map Arrows indicate direction, color intensity the strength of data and color hue (red-yellow-blue) the total magnitude of movement. These three maps are composites of 16 time-lapse images of GFP-H2A embryos at different dorso-ventral orientations. Density changes through time Ventral Dorsal a/p and d/v patterning mutants have altered nuclear density patterns The d/v differences in ftz stripe positions depend on d/v axial patterning of morphogenesis Comparing ftz patterns in late bcd-mutant vs late wild type embryos is difficult because the a/p pattern in general is very disturbed in bcd-mutant embryos (A). Toll10B has been classified as a fully ventralized phenotype. In Toll10B mutant embryos, however, the late ftz stripes similar to wild type ventrally but not as close to each other in dorsal side as in the late wild type embryos (B). gd7 has been classified as a fully dorsalizing mutation. In gd7-mutants, the dorsal sides of the stripes resemble the wild type stripes, but the ventral sides of the ftz-stripes draw together like dorsal wild type stripes (C). Although the effects in Toll10B might result from disturbances in cell adhesion (3), gd7 results suggest that d/v signals regulate also a/p pattern elements. Because Toll10B and gd7 mutants also show aberrant density patterns (left), the abnormal stripe movement is likely to result, at least in part, from the d/v regulation of morphogenetic movements. Conclusions: 3D nuclear density patterns change rapidly during stage 5 in Drosophila blastoderm. This involves pre-gastrula morphogenetic movements of blastoderm nuclei. Cellular resolution expression analysis of Drosphila blastoderm requires multiple morphological maps and positional correspondence tables for each nucleus between the maps of different stages. Because a/p expression borders of different elements and different genes shift differently in d/v axis, 3D analyses of expression patterns are essential for modeling regulatory interactions. Comparison between mutant and wild type embryos shows that d/v and a/p density patterns are connected, as are d/v elements of a/p pattern changes. Novel models are required to analyse: which part of the expression change is caused by direct regulatory interactions between cells (induction/inhibition) and which part of the change is due to the shifting cell positions how the a/p and d/v patterning axes interact with each other wild type gd7 Toll10B bcd9 Sytox image markers nuclei cells normals surface shell DNA cytoplasm atten. corr. density map Cy3 image pointcloudfile Cou image Changes in d/v patterning system cause changes in d/v density patterns. In gd7 mutants the denser dorsal patch in wild type embryos extends ventrally and laterally. In Toll10B mutants, the development of denser patches is disturbed. Changes in a/p patterning system cause changes also in d/v density patterns. In bcd9 a/p patterning mutants the posterior denser patch is duplicated and the anterior density decrease is disturbed, but also dorsal denser patch is less developed. References: Jaeger et al. (2004) Nature 40(6997):368-371. Carroll et al. (1987) Development 99(3):327-332. Keith and Gay (1990) EMBO Journal 9(13):4299-4306 Nuclear density changes were observed in living embryos To check that the observed morphological differences were not caused by fixation and other treatment artefacts, nuclear densities and movements were studied in living histone 2A - GFP embryos mounted in halocarbon oil under air-permeable membrane (materials and methods a kind gift from E. Wieschauss) and the nuclei in the top 1/3 of the embryo were time-lapse imaged in confocal microscope. From confocal images to computer analyzable, cellular resolution spatial expression data The xyz-positions of the blastoderm nuclei and the expression intensity of two genes around each nucleus are extracted from a confocal image (A) using an algorithm (B) and converted into computer readable data table (C). This data can be transformed into other forms, like an unrolled view of ~6000 blastoderm nuclei showing normalized gene expression levels (D). The principle of live cell imaging A B Early A time-lapse view of dorsal development through stage 5. As the density in the mid-dorsal region increases, cells flow in from the anterior and posterior ends and from lateral. This indicates that the local density patterns in fixed material are an in vivo phenomenon and that the changes in stage 5 density patterns result from morphogenetic movements. anterior posterior C id, x, y, z, Nx, Ny, Nz, Vn, Vc, Sytox, Cy3_n, Cy3_a, Cy3_b, Cy3_g, Cou_n, Cou_a, Cou_b, Cou_g 1, 102.36, 142.14, 112.00,-0.396, 0.851, 0.344, 207.96, 605.36, 52.18, 23.55, 18.76, 22.55, 22.10, 11.95, 8.13, 28.01, 12.04 2, 264.63, 172.01, 79.36, 0.103, 0.972,-0.208, 281.73, 599.90, 82.12, 31.67, 34.97, 15.95, 31.93, 21.06, 12.56, 41.40, 19.12 3, 225.91, 174.99, 88.65,-0.030, 0.999,-0.015, 185.79, 418.35, 85.32, 35.63, 31.27, 14.77, 34.00, 19.59, 20.53, 38.80, 21.35 4, 318.42, 48.34, 138.91, 0.095,-0.744, 0.660, 182.46, 464.19, 37.61, 19.31, 15.15, 12.47, 17.55, 21.01, 13.78, 26.87, 17.53 5, 110.18, 34.40, 109.65,-0.186,-0.913, 0.362, 127.81, 432.01, 55.78, 24.12, 23.53, 12.19, 19.71, 13.81, 7.57, 28.16, 12.40 6, 340.48, 73.79, 37.548, 0.205,-0.299,-0.931, 208.26, 607.49, 80.23, 33.04, 26.75, 21.24, 28.91, 31.48, 20.69, 50.45, 26.96 . . . Unrolling the embryo D Late Pattern shift The a/p expression patterns can develop differently on dorsal and ventral sides of the embryo early border position: late border position: border position projected from cell movement: As known, the expression borders of gap genes gt, hb and kr shift anteriorly (1). Also the expression borders of the posterior-middle stripes of pair-rule genes shift anteriorly, anterior stripes posteriorly. However, dorsal parts of the stripes often move differently from ventral parts of the stripes. This phenomenon has been explained by differential a/p induction dynamics caused by d/v signal(s) (2). Further modeling that includes the cell movements and density changes is required to determine what fraction of pattern movement is caused by differential induction and what fraction follows from the cell flow.