The role of Bicoid cooperative binding in the patterning of sharp borders in Drosophila melanogaster

Abstract

In Drosophila embryonic development, the Bicoid (Bcd) protein establishes positional information of downstream developmental genes like hunchback (hb), which has a strong anterior expression and a sharp on-off boundary in the mid-embryo. The role of Bcd cooperative binding in the positioning of the Hb pattern has been previously demonstrated. However, there are discrepancies in the reported results about the role of this mechanism in the sharp Hb border. Here, we determined the Hill coefficient (nH) required for Bcd to generate the sharp border of Hb in wild-type (WT) embryos. We found that an n(H) of approximately 6.3 (s.d. 1.4) and 10.8 (s.d. 4.0) is required to account for Hb sharpness at early and late cycle 14A, respectively. Additional mechanisms are possibly required because the high nH is likely unachievable for Bcd binding to the hb promoter. To test this idea, we determined the nH required to pattern the Hb profile of 15 embryos expressing an hb14F allele that is defective in self-activation and found nH to be 3.0 (s.d. 1.0). This result indicates that in WT embryos, the hb self-activation is important for Hb sharpness. Corroborating our results, we also found a progressive increase in the required value of n(H) spanning from 4.0 to 9.2 by determining this coefficient from averaged profiles of eight temporal classes at cycle 14A (T1 to T8). Our results indicate that there is a transition in the mechanisms responsible for the sharp Hb border during cycle 14A: in early stages of this cycle, Bcd cooperative binding is primarily responsible for Hb sharpness; in late cycle 14A, hb self-activation becomes the dominant mechanism.

Fig. 1. Hb and Bcd protein profiles (Lopes et al., 2008)

An embryo at mid-nuclear cleavage cycle 14A immunostained for Bcd (A) and Hb (B). (C) Fluorescence intensities for A and B are shown in green and blue, respectively, as a function of position along the AP axis. The diffusion of Bcd protein, translated from its mRNA localized at the anterior end of the egg, forms an exponential concentration gradient. In A and B, the anterior pole is on left and the dorsal side is on top.

Fig. 2. Estimating the required Hill coefficient to pattern Hb profiles in embryos of different stages or backgrounds (Lopes et al., 2008)

The Hill coefficient is given by the inclination of the straight line (see equation (2) in Material and Methods). (A): Plot from a WT embryo at early cycle 14A exhibiting an n_H of 6.3 that is similar to the mean n_H (6.3, s.d. 1.4) measured from 15 embryos at this stage (inset). (B): The same plot as in (A) but from a WT embryo at late cycle 14A with an n_H of approximately 11.6 that is similar to the mean n_H (10.8, s.d. 4.0) measured from 15 embryos at this stage (inset). (C): Same plot as in (A) but from an hb^14F embryo at cycle 14A with an n_H of approximately 3.0. Inset: 15 embryos at this stage with the mean n_H (3.0, s.d. 1.0). (D): The same plot as above from an embryo expressing the pThb5 lacZ artificial construct at cycle 14A and exhibiting an n_H of 7.0. Inset: 9 embryos with mean n_H (7.0, s.d. 1.5). We assumed early and late cycle 14A embryos corresponded to stages T1-T4 and T5-T6, respectively, according to previous classification (Surkova et al., 2008).

Fig. 3. Estimating the required Hill coefficient to account for Hb sharpness using an alternative plot

(A) The same data from the 15 WT early embryos used for Fig. 2A but plotted similarly to Fig. 4A by Gregor et al. (Gregor et al., 2007a). (B) The same 15 WT late embryos as in Fig. 2B but plotted as in A. In these graphics, the red, green, cyan and black lines show the plots for an n_H of 5, 6, 7 and 10, respectively. It is easy to see that in (A), the n_H choice of 6 fits the data better, while in (B), the best choice is 12. This method (see equation (1) in Material and Methods) does not allow a precise determination of n_H because visual inspection is used. The straight-line fitting, used for Figs. 2 and 4, is more precise and accurate.

Fig. 4. Required Hill coefficients for each temporal class in cycle 14A necessary to account for Hb sharpness as determined from averaged data

Poustelnikova et al., 2004; Surkova et al., 2008)(A) – (H): Plots for time classes T1-T8, which last for approximately 6 minutes each. The Hill coefficients, shown in the insets followed by the r.m.s.d., were determined by the straight-line inclination of these plots. (I): Evolution of the Hill coefficient (red line) from A to H and r.m.s.d. (blue line) as a function of time classes. The increase in r.m.s.d. indicates a reduction in how suitable the Hill approach is for describing this system. The increase in n_H indicates that Bcd cooperative binding cannot account for the sharpness of the Hb pattern after T4. To generate these plots, we used the equation (2) in Material and Methods.