Dampened regulates the activating potency of Bicoid and the embryonic patterning outcome in Drosophila

Abstract

The Drosophila morphogen gradient of Bicoid (Bcd) initiates anterior-posterior (AP) patterning; however, it is poorly understood how its ability to activate a target gene may have an impact on this process. Here we report an F-box protein, Dampened (Dmpd) as a nuclear cofactor of Bcd that can enhance its activating potency. We establish a quantitative platform to specifically investigate two parameters of a Bcd target gene response, expression amplitude and boundary position. We show that embryos lacking Dmpd have a reduced amplitude of Bcd-activated hunchback (hb) expression at a critical time of development. This is because of a reduced Bcd-dependent transcribing probability. This defect is faithfully propagated further downstream of the AP-patterning network to alter the spatial characteristics of even-skipped (eve) stripes. Thus, unlike another Bcd-interacting F-box protein Fate-shifted (Fsd), which controls AP patterning through regulating the Bcd gradient profile, Dmpd achieves its patterning role through regulating the activating potency of Bcd.

Figure 1. Dmpd is a nuclear factor that stimulates Bcd-dependent reporter activity in cells

(a and b) FLAG-Dmpd interacts with HA-Bcd. Coimmunopreciptiation (co-IP) experiments were performed to detect the interaction between FLAG-Dmpd and HA-Bcd. Here, extracts from HEK293T cells expressing the indicated fusion proteins were subject to co-IP by the indicated antibodies, followed by Western blot detections by the indicated antibodies. Lanes 5 and 6 in these panels document the specificity of the antibodies used in our study. (c) Bcd activity is stimulated by Dmpd. Reporter assays were performed in Drosophila S2 cells to evaluate the impact of Dmpd expression on CAT reporter activity. For each set of experiments, CAT activities were normalized to those obtained from cells without Dmpd expression (set as 100). Data shown are from three independent sets of experiments, with standard deviation (sd) shown for Dmpd-expressing cells. (d-f) Nuclear localization of Dmpd. A plasmid expressing the HA-tagged Dmpd protein (HA-Dmpd) was transfected into S2 cells. Immunocytochemistry experiments were then performed to evaluate its sub-cellular localization through the use of the anti-HA antibody (e). To-pro-3 was used to counterstain the nucleus (d). Panel f is a merge of images shown in panels d and e. Scale bar is 10 μm.

Figure 2. Establishing a platform for quantifying the amplitude of hb transcription

(a) Shown are the mean profiles (with sd shown) of un-normalized hb mRNA FISH intensities (in arbitrary units) from embryos that have one, two, or four copies of the hb gene. The mean and sd of x_hb expressed as fractional embryo length are: 0.435 ± 0.013, 0.445 ± 0.013 and 0.454 ± 0.019 for 1×, 2× and 4×hb embryos, respectively (n = 16, 20 and 17). These results show that x_hb remains largely insensitive to changes in hb copy number in our experimental system; a relatively minor shift might be related to the limitations inherent to experimental detections . (b) Shown are hb_plat values (in arbitrary units) in individual embryos with different copies of the hb gene. The mean and sd are also shown in the figure (n = 16, 20 and 17 for 1×, 2× and 4×hb embryos, respectively). The linear fit between hb_plat and hb gene copy number is shown as a red line in the figure. (c) Shown are the mean profiles of un-normalized hb mRNA FISH intensities in embryos from mothers with one, two, or three copies of bcd. The mean and sd of x_hb expressed as fractional embryo length are: 0.373 ± 0.013, 0.446 ± 0.009, and 0.510 ± 0.010 for 1×, 2× and 3×bcd embryos, respectively (n = 11, 9, and 13). (d) Shown are hb_plat values in individual embryos from mothers with different copies of bcd. The mean and sd of hb_plat are: 23.93 ± 6.75, 24.51 ± 5.13, and 24.94 ± 4.42 for 1×, 2× and 3×bcd embryos, respectively (n = 11, 9, and 13). p values (obtained from Student’s t-tests and shown in the figure) reveal no significant differences.

Figure 3. hb mRNA level is reduced in dmpd embryos

(a) A schematic diagram showing the deletion caused by the dmpd^6-3 allele. The boxes represent exons with the annotated open reading frame in cyan and the un-translated regions in grey. The blue arrow shows the P-element insertion site for dmpd^EY08992. (b) Shown are the mean profiles of un-normalized hb mRNA FISH intensities in wt and dmpd embryos (n = 22 and 18, respectively). Note that the level of the Bcd-independent posterior hb expression domain remains unaffected in dmpd embryos (p = 0.93, Student’s t-test); see text and panel c legend for further details). The mean and sd of x_hb expressed as fractional embryo length are: 0.433 ± 0.012 and 0.462 ± 0.018 for wt and dmpd embryos, respectively (p = 4.8 × 10⁻⁷, Student’s t-test). (c) Shown are hb_plat values in individual wt and dmpd embryos (n = 22 and 18, respectively). Unlike the significant amplitude reduction for the Bcd-activated anterior hb expression domain shown in this figure, the amplitude of the Bcd-independent posterior hb expression domain, hb_post (defined as the average FISH intensities at the three peak positions of the posterior domain), is insensitive to dmpd mutation.

Figure 4. The spacing between eve stripes 3 and 4 is sensitive to the dmpd mutation

(a) Shown are the normalized mean intensity profiles of eve in wt and dmpd embryos (n = 16 and 13, respectively). (b) Shown is the calculated distance between eve stripes 3 and 4 in each individual wt and dmpd embryos (n = 16 and 13, respectively). The distance values shown are expressed as percentage of embryo length. Also shown are mean and sd for these two groups, along with the p value obtained from Student’s t-test.

Figure 5. The transcribing probability near Bcd-responsive promoter of hb is decreased in dmpd embryos

(a) Shown is an image of hb intron staining from a cycle 14 embryo. Nuclear envelope is shown in red and intron dots in green. Scale bar, 50μm. A magnified view of a section of the expression domain is shown on the right side of this panel. (b) Shown is a computer-processed image of panel a. Here each of the identified nuclei is shown in a color according to the number of intron dots detected inside. White, blue and green represent zero, one and two intron dots detected, respectively. (c) Shown is the plot of ρ as a function of x/L based on panel b. (d-i) Shown are the mean ρ profiles from wt and dmpd embryos at time classes t1-t6. The mean and sd of ρ_plat for t1-t6 time classes are: 0.50 ± 0.07; 0.84 ± 0.04; 1.03 ± 0.08; 0.71 ± 0.14; 0.52 ± 0.10; 0.34 ± 0.03 for wt embryos (n = 3, 3, 7, 7, 5 and 4, respectively) and 0.51 ± 0.11; 0.86 ± 0.25; 0.79 ± 0.21; 0.54 ± 0.06; 0.38 ± 0.12; 0.23 ± 0.05 for dmpd embryos (n = 3, 4, 5, 5, 5 and 4), respectively. The p values (from Student’s t-tests) between wt and dmpd embryos for t1-t6 time classes are: 0.93; 0.92; 0.016; 0.028; 0.078; 0.0087, respectively. The mean ρ at x/L ~0.15 (which is located outside of the plateau domain for calculating ρ_plat) appears higher in dmpd embryos than in wt embryos at t6 (panel i); this is likely caused by an “outlier” dmpd embryo that has a very high level of ρ at this location (see Fig. S8f).

Figure 6. Similar length constant of the Bcd gradient profiles in wt and dmpd embryos

(a) Shown are mean Bcd gradient profiles from wt and dmpd embryos plotted as ln(B/B_max) against AP position x/L. Here both B and B_max are background-adjusted intensities without any further adjustments. The solid lines representing linear fits (y = −6.78x + 0.46, R squared for linear regression; R² = 0.999 for wt embryos; y = −6.77x + 0.58; R squared for linear regression, R² = 0.998 for dmpd embryos). (b) Shown are calculated λ values of Bcd gradient profiles from individual wt and dmpd embryos (n =15 and 13, respectively). These values were obtained from fitting the individual Bcd intensity profiles to an exponential function. Shown are mean and sd for these two groups, along with the p value obtained from Student’s t-test.