Distance measurements via the morphogen gradient of Bicoid in Drosophila embryos

Abstract

Background: Patterning along the anterior-posterior (A-P) axis in Drosophila embryos is instructed by the morphogen gradient of Bicoid (Bcd). Despite extensive studies of this morphogen, how embryo geometry may affect gradient formation and target responses has not been investigated experimentally.

Results: In this report, we systematically compare the Bcd gradient profiles and its target expression patterns on the dorsal and ventral sides of the embryo. Our results support a hypothesis that proper distance measurement and the encoded positional information of the Bcd gradient are along the perimeter of the embryo. Our results also reveal that the dorsal and ventral sides of the embryo have a fundamentally similar relationship between Bcd and its target Hunchback (Hb), suggesting that Hb expression properties on the two sides of the embryo can be directly traced to Bcd gradient properties. Our 3-D simulation studies show that a curvature difference between the two sides of an embryo is sufficient to generate Bcd gradient properties that are consistent with experimental observations.

Conclusions: The findings described in this report provide a first quantitative, experimental evaluation of embryo geometry on Bcd gradient formation and target responses. They demonstrate that the physical features of an embryo, such as its shape, are integral to how pattern is formed.

Figure 1

Dorsal and ventral sides of the embryo have different geometric features. (A) Illustration showing different distances in geometry. (B) Digital image from DAPI channel of a wt (w¹¹¹⁸) embryo to illustrate the parameters used to analyze geometric features in this study. (C) Relative differences (with error bar shown) in contour distances between the dorsal and ventral sides (Δc_embryo/C_D) as a function of fractional embryo length (x/L) from 28 wt embryos at early nuclear cycle 14. All distances along the A-P axis in this report are measured from the anterior pole unless otherwise stated.

Figure 2

Bcd and Hb profiles expressed in two different distance measurements. (A and B) Average raw Bcd intensity profiles on the dorsal (blue) and ventral (red) sides of 28 wt embryos when measured as a function of projected distance x (A) or contour distance c (B) from the anterior. Error bars are shown for the profile from the dorsal side. In each panel, the inset shows a magnified view of the region surrounding the Hb expression boundary positions, which are marked with arrowheads for reference. (C and D) Average normalized Hb intensity profiles on the dorsal (blue) and ventral (red) sides of the same wt embryos when measured as a function of projected distance x from the anterior (C) or contour distance c (D). The Hb expression boundary positions on the two sides are marked with arrowheads.

Figure 3

Similar Bcd-Hb input/output relationship on D and V sides. (A) Scatter plots of normalized Hb intensity values against raw Bcd intensity values on the dorsal (blue) and ventral (red) sides of wt embryos. The effective dissociation constant K_d for Bcd activating Hb expression on the two sides can be extracted from the plots, as illustrated by dashed lines. (B) Average input/output relationship on the dorsal (blue, with error bars) and ventral (red) sides of wt embryos. B and K_d values are background-subtracted. Green profile represents a Hill curve with cooperativity n = 5. (C) Intensity noise of Hb expression in response to the Bcd gradient. B and K_d values are background-subtracted. (D) Intensity noise of Bcd at and around c_Hb on the dorsal (blue) and ventral (red) sides of wt embryos. In this figure (and Fig. S1 H and S2H), noise from measurement and background is not subtracted from the Bcd intensity noise profiles.

Figure 4

Contributions of Bcd gradient behaviors to target expression slanting. (A) Profiles of distance differences between the dorsal and ventral sides of the wt embryos when measured as either projected distance (blue, left scale) or contour distance (red, right scale). The profiles are shown as a function of raw Bcd intensity on log scale. (B) Positions with equal Bcd intensity values are marked on the dorsal and the ventral sides of an average wt embryo frame. Lines that connect the locations on both sides of the embryo can be viewed as iso-concentration contour lines. The embryo shape (blue) is averaged from the 28 wt embryos analyzed in this study, with dorsal side up and anterior to the left. (C) Differences in target boundary positions between the dorsal and ventral sides. The expression boundary positions are measured as either projected distance from the anterior (blue, left scale) or contour distance (red, right scale), and the differences are plotted at their respective projected boundary positions (measured from the dorsal side and expressed as fractional embryo length x/L). From anterior to posterior, each pair of results represents data in the following order: Otd in 30 wt embryos, Hb in 24 1×-bcd embryos, Hb in 28 wt embryos, and Hb in 29 3×-bcd embryos. (D) Target expression boundary locations on both dorsal and ventral sides are marked on the average embryos frames. The average 1×-bcd embryo frame is shown in cyan, wt in blue, and 3×-bcd in green. Both Otd and Hb boundary positions are marked on the average wt embryo frame. Lines connecting the target boundary positions on two sides of the embryos are shown for visual comparison with those shown in Fig. 4B.

Figure 5

Simulating the effect of embryo geometry on Bcd gradient profiles. (A) A heat map of local total Bcd concentration on the midsagittal plane of a simulated embryo at nuclear cycle 14. The shape of the simulated embryo is based on the experimentally observed average wt embryos (Fig. 4B) with an asymmetric geometry on the dorsal and ventral sides (see Methods for details). Color bar represents normalized local total Bcd concentration. (B and C) Heat maps of local total Bcd concentration of the same simulated embryo on transverse planes at x_Otd/L (panel B) and x_Hb/L (panel C). Color bar represents normalized local total Bcd concentration only showing the range for the cortical layer relevant to our discussions. (D and E) Nuclear Bcd concentration profiles from the cortical layer of the dorsal (blue) and ventral (red) sides of the simulated embryo, measured as a function of either projected distance x from the anterior (panel D) or contour distance c (panel E). See text and Methods for further details.