Analysis of pattern precision shows that Drosophila segmentation develops substantial independence from gradients of maternal gene products

Abstract

We analyze the relation between maternal gradients and segmentation in Drosophila, by quantifying spatial precision in protein patterns. Segmentation is first seen in the striped expression patterns of the pair-rule genes, such as even-skipped (eve). We compare positional precision between Eve and the maternal gradients of Bicoid (Bcd) and Caudal (Cad) proteins, showing that Eve position could be initially specified by the maternal protein concentrations but that these do not have the precision to specify the mature striped pattern of Eve. By using spatial trends, we avoid possible complications in measuring single boundary precision (e.g., gap gene patterns) and can follow how precision changes in time. During nuclear cleavage cycles 13 and 14, we find that Eve becomes increasingly correlated with egg length, whereas Bcd does not. This finding suggests that the change in precision is part of a separation of segmentation from an absolute spatial measure, established by the maternal gradients, to one precise in relative (percent egg length) units.

Fig. 1

The segmentation hierarchy in Drosophila. A: An anterior-high gradient of the maternally derived protein Bicoid (Bcd) is established before zygotic gene expression. Posterior gradients also form: a major regulator of downstream expression is B) Caudal (Cad), graded by translational repression by Bcd. C: Gap genes, such as hunchback (hb) are expressed in response to maternal regulators and cross-interactions. D: The first periodic segmentation patterns are observed in the expression of the pair-rule genes, such as even-skipped (eve). Although Eve expression depends in a complicated way on upstream regulators and cross-interactions, direct comparison of the precision in positioning Eve stripes to Bcd spatial precision shows that passive reading of the maternal gradient is sufficient to initiate Eve, but not for the mature segmentation pattern. A–D are confocal microscope images from embryos stained with fluorescently tagged antibodies to the above proteins (anterior left, posterior right, dorsal up, ventral down). A, B, and D are from the same, triply stained, embryo. Most of the pattern formation occurs in nuclear cleavage cycle 14A, before cellularization (each dot is a nucleus). The embryos shown are from later cycle 14A (T6, see the Experimental Procedures section for temporal classes). E–H: For Bcd, Cad, Hb, and Eve, respectively, fluorescence intensity (on an 8-bit [0–255] scale), at each nucleus, vs. anteroposterior (AP) position (relative, in percent egg length [%EL]; 0% anterior, 100% posterior), with the extracted profile (see the Experimental Procedures section for details) in red.

Fig. 2

Multiple-embryo overlays for Bcd, with summary statistics and precision trends. A: Overlay of Bcd gradients from 61 embryos, early cleavage cycle 14 (T1–2). Fluorescence intensity (proportional to concentration) is on the vertical, anteroposterior (AP) position (percent egg length [%EL]; 0% anterior, 100% posterior) on the horizontal. Each line is the profile extracted (see the Experimental Procedures section) from a single embryo (e.g., red line in Fig. 1E). The broad scatter of positions at which any particular concentration of Bcd is encountered suggests low precision for positional specification. B: Mean positions, for selected intensities, with one standard deviation error bars, for the same embryos. C: Positional error (standard deviation) against AP position. There is a posteriorly rising trend in the positional errors. D: Bcd gradients in time. Each curve is generated from the average exponential parameters of each developmental stage; this is a pictorial representation of the profiles for mean k and C₀ values in Table 1. There appears to be some deterministic drop in Bcd over these stages. However, Bcd’s exponential decay constant (k) does not change over cycle 14A (see text), so spatial dependence of positional errors also remains unchanged.

Fig. 3

Overlay of Cad patterns, with summary statistics and trends. A–C: Cleavage cycle 13 pattern. A: Overlay of anteroposterior (AP) profiles, for 47 embryos. B: Mean positions, at select intensities, with one standard deviation error bars. C: Plot of these standard deviations against AP position. Like Bcd, Cad shows a rise in positional errors toward the posterior. D–F: Early cleavage cycle 14 (T1–2). D: Overlay of AP profiles, for 43 embryos. E: Mean positions, at select intensities, with one standard deviation error bars. F: Plot of these standard deviations against AP position. The posteriorly rising trend in positional errors increases from cycle 13. Bcd is a translational repressor of Cad: the change between C and F may reflect the sustained effects of Bcd’s greater variability toward the posterior.

Fig. 4

Eve patterns, over time, with summary statistics and trends. A–C: cleavage cycle 13. A: Overlay of anteroposterior (AP) profiles for 93 embryos. B: Mean positions, at select intensities, with one standard deviation error bars. C: Plot of these standard deviations against AP position. At this stage, both anterior and posterior error trends are comparable to Bcd (cf. Fig. 2C; Table 1, 2nd row). D–F: Early cycle 14 (T1) Eve pattern. D: Overlay of AP profiles for 101 embryos. E: Mean positions, at select intensities, with one standard deviation error bars. F: Plot of these standard deviations against AP position. The posterior error trend is still comparable to Bcd’s (Fig. 2A–C), but the anterior (20–30 percent egg length [%EL]) of the early Eve peak is becoming much more precise. G–I: Mature (late cycle 14A, T7) Eve segmentation pattern. G: Overlay of AP profiles for 96 embryos. H: Mean positions, for selected intensities, with one standard deviation error bars. I: Plot of these standard deviations against AP position. These errors are much lower than Bcd’s, and no longer have a posteriorly rising trend, pointing toward a divergence of maternal positional specification and zygotic segmentation patterning over cycle 14A. Note: H and I show positional errors calculated with 94, 95, or 96 of the embryos, to show positional errors along all stripe borders. See Supplementary Figure S4 for the same plots with all 96 embryos: not as many positional errors can be calculated, but the trends are the same.

Fig. 5

Spatial scaling. A,B: Eve staining on two embryos in later cycle 14A (T6), on the same spatial scale. A is 468 μm long (white bar, 50 μm), B is 555 μm long. C: The Eve and Bcd patterns in these embryos are shown, in absolute units (μm). D: The same patterns, in relative units (percent egg length [%EL]). Long embryo: Bcd, red; Eve, green. Short embryo: Bcd, blue; Eve, magenta. Bcd patterns are similar (precise) in absolute units, whereas Eve patterns are similar (precise) in relative units. The absolute scale for Bcd likely reflects its establishment by diffusion and degradation, while the relative scale for Eve suggests that zygotic patterning involves feedback with egg size. This is an example for one pair of embryos: see text for statistics on 17 embryos of the same time class.

Fig. 6

Eve position increasingly correlates with egg length (becomes more precise in relative units). A: Cycle 13, Eve peak 1 position vs. egg length (both in μm), R = 0.55. B: Cycle 14, T1, R = 0.81. C: Cycle 14, T2, R = 0.87. Scatterplots later in cycle 14 are comparable to T2.