Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution II: dynamics

Abstract

Background: To accurately describe gene expression and computationally model animal transcriptional networks, it is essential to determine the changing locations of cells in developing embryos.

Results: Using 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.

Conclusion: Our 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.

Figure 1

Changing local nuclear density patterns during stage 5. Average local nuclear densities on the blastoderm surface were computed for PointCloud data derived from embryos for six consecutive time intervals spanning stage 5. (a-f) Cylindrical projections of the average for each of these temporal cohorts. The range of membrane invagination for embryos in each temporal cohort is shown above each panel (for example, 5:0-3%). Isodensity contours were plotted over a color map representing local average densities from 0.025 nuclei/μm² (dark blue) to 0.05 nuclei/μm² (dark red). The position on the y-axis of the dorsal midline (D), ventral midline (V), and left (L) and right (R) lateral midlines are indicated. On the x-axis, anterior is to the left, posterior is to the right, and the distance along the a/p axis is given as a percent egg length. The number of embryos in each cohort (n) and the standard deviation of nuclear density values (SD) are also shown. It can be seen that over time the local nuclear densities increased dorsally, decreased at the poles, and changed little ventrally.

Figure 2

Nuclear density patterns and movements in living Histone2A-GFP embryos. (a,b) Cylindrical projections of average nuclear density maps derived from portions of 22 living embryos expressing Histone2A-GFP. Density maps for this set of embryos are shown at the start of stage 5 (a) and at the end of stage 5 (b). The axis and isodensity contours are labeled as in Figure 1. The density patterns seen are remarkably similar to those derived from fixed material (Figure 1). (c) Orthographic projections of the average distance and direction of nuclear movement in two dimensions for n = 1 embryo in the dorsal orientation, n = 8 embryos in the lateral orientation, and n = 4 embryos in the ventral orientation. These arrows represent a local average movement within each embryo calculated from time-lapse series as well as an averaging over the various embryos in similar orientations. As expected from the changes in nuclear densities, a net flow of nuclei from the poles towards the mid-dorsal region was observed.

Figure 3

Synthetic density maps are similar to measured density maps. Cylindrical projections of nuclear density patterns in PointClouds from: (a) fixed early embryos (stage 5:0-3%); (b) an early synthetic embryo modeled to have shapes and nuclear density patterns of stage 5:0-3% fixed embryos; (c) fixed late embryos (stage 5:75-100%); and (d) a late synthetic embryo modeled to have shapes and density patterns of stage 5:75-100% fixed embryos according to the model described in the text. All other information and scales are as used in Figure 1.

Figure 4

Predicted nuclear flow in the stage 5 blastoderm. The movement of nuclei in three dimensions was estimated using PointCloud data derived from fixed embryo material. Three orthographic projections of this model are shown, illustrating movement dorsally (top), laterally (center), and ventrally (bottom). The length and direction of arrows indicate the direction and distance of nuclear movement. The position on the y-axis of the dorsal midline (D), ventral midline (V), and left (L) and right (R) lateral midlines are indicated. On the x-axis, anterior is to the left and posterior to the right. The scale is in μm from the embryo center of mass. The predicted movements broadly agree with those seen in the live embryos, being greater at the poles and dorsally than ventrally. Note that the apparent movement towards the center of each view results from the basal movement of nuclei inward.

Figure 5

Basal movement of nuclei during stage 5 in living Histone2A-GFP embryos. Two optical slices through the middle of a living embryo are superimposed. The red image was taken at the beginning of stage 5, whereas the green image was taken at the end. The bright line is the water-oil interface at the vitelline membrane, and was used to align the two images. All nuclei move inwards and elongate during stage 5. Anterior is to the left and dorsal is up.

Figure 6

Movement of gap and pair rule stripe borders. Lateral orthographic projections of the mean positions of the anterior borders of eve and ftz stripes from early (4-8%) (blue), mid (26-50%) (green) and late (76-100%) (red) stage 5 cohorts, and of selected borders of gt, hb and Kr stripes from early (0-3%) (blue), mid (9-25%) (green), and late (51-75%) (red) stage 5 cohorts. The stages chosen for gap gene analysis were earlier than those for pair rule genes because, unlike pair rule mRNA, gap mRNA is rapidly down-regulated towards the end of stage 5, whereas pair rule expression increases throughout stage 5. The error boxes at each measurement point represent 95% confidence intervals for the mean in a/p and d/v directions. Anterior is to the left, dorsal is to the top. The x- and y-axes show the distance in μm from the center of embryo mass. It can be seen that most stripe borders changed spatial location during stage 5. The silhouettes of PointClouds were smaller for later stage embryos because of basal nuclear movements. Note that, for each eve and ftz stripe, the posterior stripe border shows a broadly similar movement to the anterior border, indicating that the movements observed are not principally due to the narrowing of stripes (data not shown).

Figure 7

The relative contributions of nuclear flow and expression flow to pattern flow. Orthographic projections of the locations of ftz, hb, eve, gt, and Kr stripe borders in early stage 5 (blue lines) and late stage 5 (red lines) embryos. The stripe locations are taken from the earliest and latest applicable embryo cohort (5:4-8% to 5:75-100% for ftz and eve; 5:0-3% to 5:51-75% for hb, gt and Kr). The axes are labeled as in Figure 4. Our model of nuclear flow was used to predict the location of stripe borders in late embryos in the absence of changing expression levels (dotted black lines). The left panels compare the measured locations of the early and late stripe borders, and thus show the pattern flow. The center panels show the movement predicted to be due only to nuclear flow. The right panels show the residual movement (expression flow) that can be attributed to zones of up/down-regulation along stripe boundaries.

Figure 8

bcd, gd, and Tl regulate nuclear density patterns along both major body axes. Cylindrical projections of nuclear density patterns in (a) wild-type, (b) bcd¹² mutant, (c) gd⁷ mutant, and (d) Tl^10B mutant embryos. To reduce noise, information from the left and right sides of each embryo was averaged. All embryos were from stages 5:25-100%. Axes and isodensity contours are as described in Figure 1. All three mutants exhibit changes in the pattern of density along both body axes. Note that while it appears that the total number of nuclei in Tl^10B mutants is less than in the wild-type embryos, this reflects a difference between fly strains and not an effect of the Tl gene as there is no statistically significant difference between the average number of nuclei in Tl^10B mutants versus their wild-type-like siblings, which are derived from Tl^10B hetrozygous mothers.

Figure 9

gd and Tl regulate ftz stripe location. Quantitative comparison of ftz expression (a) between mutant embryos derived from gd⁷ homozygous mothers and wild-type-like embryos derived from gd⁷ heterozygous mothers and (b) between mutant embryos derived from Tl^10B homozygous mothers and wild-type-like embryos derived from Tl^10B heterozygous mothers; both show lateral orthographic projections indicating the position of each of the seven stripes in wild-type-like embryos (blue stripes) and mutant embryos (red stripes). All embryos were from stages 5:25-75%. The confidence intervals, embryo orientation, and scales are as described in Figure 4. Shifts in the ftz expression boundaries are consistent with dorsalized (gd) and ventralized (Tl) nuclear flow, respectively. (c) The effects of disrupting the d/v system on stripe curvature and placement in single embryo images, shown in a lateral view. The stripes in the mutant embryos (right) clearly differ from those in the wild-type-like embryos (left), but because of small differences in embryo orientation and shape it is difficult to draw a precise understanding of how stripe locations have changed from such raw image data.

Figure 10

gd and Tl regulate the levels of ftz stripe expression in the direction of the d/v axis. Plotted are averaged expression intensities of gene stripes for ftz in wild-type-like embryos derived from (a) gd⁷ heterozygous mothers, (b) dorsalized mutant embryos from gd⁷ homozygous mothers, (c) wild-type-like embryos from Tl^10B heterozygous mothers, and (d) ventralized mutant embryos derived from Tl^10B homozygous mothers. Expression intensity (y-axis) is plotted against the location along the stripe in the direction of the d/v axis as described in [12]. Each of the seven ftz stripes is shown in a different color and the error bars give the 95% confidence intervals for the means. The differences in expression seen in wild-type embryos in the direction of the d/v axis are not seen in the mutant embryos.

Figure 11

The distance and direction of nuclear movement in an individual living Histone2A-GFP embryo. Nuclei were tracked throughout stage 5, and the vector of their total motion was plotted on top of the image of the embryo at the beginning of stage 5. The color of the arrows is given by their length, short arrows being blue and long ones red. Vectors that are very different from nearby vectors were not used when generating the averaged plots in Figure 2. Anterior is to the left and dorsal is up.