Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila

Abstract

The NF-kappaB-related transcription factor, Dorsal, forms a nuclear concentration gradient in the early Drosophila embryo, patterning the dorsal-ventral (DV) axis to specify mesoderm, neurogenic ectoderm, and dorsal ectoderm cell fates. The concentration of nuclear Dorsal is thought to determine these patterning events; however, the levels of nuclear Dorsal have not been quantified previously. Furthermore, existing models of Dorsal-dependent germ layer specification and patterning consider steady-state levels of Dorsal relative to target gene expression patterns, yet both Dorsal gradient formation and gene expression are dynamic. We devised a quantitative imaging method to measure the Dorsal nuclear gradient while simultaneously examining Dorsal target gene expression along the DV axis. Unlike observations from other insects such as Tribolium, we find the Dorsal gradient maintains a constant bell-shaped distribution during embryogenesis. We also find that some classical Dorsal target genes are located outside the region of graded Dorsal nuclear localization, raising the question of whether these genes are direct Dorsal targets. Additionally, we show that Dorsal levels change in time during embryogenesis such that a steady state is not reached. These results suggest that the multiple gene expression outputs observed along the DV axis do not simply reflect a steady-state Dorsal nuclear gradient. Instead, we propose that the Dorsal gradient supplies positional information throughout nuclear cycles 10-14, providing additional evidence for the idea that compensatory combinatorial interactions between Dorsal and other factors effect differential gene expression along the DV axis.

Fig. 1.

Cross-sections and whole mount in situ hybridizations and antibody staining. (A) Dorsal antibody staining visualized by manual cross-section. (B) mRNA in situ hybridization of genes expressed along the DV axis. (C) Three-dimensional whole mount in situ hybridization of sog gene expression in a single embryo, shown in blue, detected using a riboprobe made to the sog transcript, co-labeled using antibodies for Dorsal protein (green) and Histone H3 (red). (D) Computational unrolling of 3D images of whole mount embryo from C allows for protein and mRNA expression to be analyzed in 2D. This technique was used to generate the quantitative data for each of the following figures. [A and B reproduced with permission from Reeves GT, Stathopoulos A (Graded Dorsal and differential gene regulation in the Drosophila embryo. Perspectives on Generation and Interpretation of Morphogen Gradients, eds Briscoe J, Lawrence P, Vincent J.-P. (Copyright 2009, Cold Spring Harbor Lab Press, Plainview, NY).]

Fig. 2.

Dorsal quantification and target mRNA expression in individual wt embryos shows the location of germ layer-specific target gene expression at nc 14. (A–C) Single embryo measurements of fluorescent intensity values of Dorsal within the nuclei (gray dots) fit by best-fit Gaussian curves (black curve) in raw fluorescent intensity units (left axis). Colored curves represent normalized intensity of gene expression (right axis). Numbers on the x axis represent distance from the ventral midline. dl¹/dl¹ mutants were used to determine background fluorescence in the absence of Dorsal protein (horizontal black line; standard deviation shown as thickness of line). (A) vnd expression (red trace) starts within the steepest part of the Dorsal gradient and ends at the dorsal border of Dorsal nuclear localization. (B) sog expression (green trace) spans from the ventral region of vnd expression to lateral regions of the embryo where Dorsal levels are uniform. (C) ind expression (blue trace) lies largely outside the Dorsal gradient. Note that the amplitudes of Dorsal concentration vary among the three embryos shown; this variability is seen even when the embryos are all at the same nuclear cycle (see Fig. 3). (D) Overlay of all three gene expression outputs (averages of multiple embryos; n = 12 for vnd, 7 for sog, and 8 for ind) onto a single plot with averaged Dorsal gradient in black (n = 35 nc 14 embryos).

Fig. 3.

Developmental time course of wt Dorsal gradient shows no change in the width of the gradient centered at the ventral midline. (A) Whole mount quantification of Dorsal levels in computationally staged embryos from nc 10–14, color-coded by stage (n = 56). The top 15% of Dorsal nuclear levels at each nuclear cycle is shown in the inset. (B) Box-and-whisker plot of Dorsal levels in ventral-most nuclei correspond to the peak amplitude at each nuclear cycle (blue). Basal levels represent Dorsal levels in lateral and dorsal regions of the embryo outside of the graded distribution of Dorsal (orange). Median intensity is shown as a horizontal bar in the box; box denotes data bounded by interquartile range. Whiskers show the distribution of data. Asterisks denote outliers. (Inset) Cartoon of Amplitude and Basal portion of signal. (C) When the peaks of each of the curves in A are normalized to 1, all curves fall along the same Gaussian curve with minor variation in curve width. (D) Box-and-whisker plots show Dorsal nuclear gradient widths remain constant throughout embryogenesis. (Inset) Width parameters correspond to 2× width of best-fit curve at 60% maximal. For nc 10–14, n = 7, 3, 3, 7, and 35, respectively.

Fig. 4.

Dorsal nuclear localization in wt and mutant embryos reveals a wide range of nuclear concentrations. Dual fluorescent in situ and antibody staining: vnd, ind, and/or sna riboprobes and anti-Dorsal antibody were used. (A) Dorsal nuclear localization in wt (green) with wt expression domains of vnd (red) and ind (blue) transcripts. (B) sna expression (purple) in Toll10b mutant embryos is ubiquitous except for repression in posterior of the embryo. (C and D) Tollrm9/Tollrm10 mutant embryos with variable expression of ind (blue) and vnd (red) transcripts. A temporal change in the patterns is observed: (C) early nc 14, and (D) late nc 14. (E) Nuclear localization of Dorsal in nc 14 embryos of Toll^10B mutants (n = 15) (green) and Toll^rm9/Toll^rm10 mutants (n = 17) (blue) and the top 15% of all nc 14 wt embryos (n = 6) (red).

Fig. 5.

Mutant embryos with genetically manipulated Dorsal produce similar gene expression outputs. (A) dl¹/+ heterozygous embryos (black) have a flattened plateau of Dorsal concentration instead of the peak in ventral regions seen in wt embryos, top 15% of nc 14 (n = 6) (red). dl-gfp embryos contain an additional copy of dorsal and have significantly wider and higher Dorsal gradients (green). dl¹/+; dl-gfp/+ embryos (cyan) are wider than wt, yet not higher. (B) Average nc 14 Dorsal gradients from wt (solid red), dl¹/+ (dashed black, n = 16), dl-gfp/+ embryos (dotted green, n = 8), and dl¹/+; dl-gfp/+ embryos (dot-dashed cyan, n = 5). From these average gradients, the trends from A are clearly seen. Furthermore, note that gradients from wt and dl¹/+ embryos have close overlap in ventral-lateral regions. Also shown: sog mRNA expression patterns. While sog expression in dl¹/+ and dl¹/+; dl-gfp/+ embryos is indistinguishable from wt, dl-gfp/+ embryos exhibit a widened expression domain extending into more dorsal regions of the embryo (SI Text, section 13). (C and D) Box plots of gradient amplitudes (C) and widths (D) of each of the genotypes described here.

Fig. 6.

Proposed mechanism of Dorsal-mediated patterning. (A) A combinatorial model for DV patterning. Dorsal and EGFR may function together to specify ind, and other genes, in the presumptive neurogenic ectoderm. Additionally, repression by an unknown factor (X) may serve to limit the dorsal-extent of these genes. (B) Gene expression in the midst of dynamic Dorsal nuclear concentration. Dorsal levels fluctuate during and between nuclear cycles (red curve). When Dorsal surpasses a minimally sufficient level (dashed line) of protein in the nucleus, and the requisite additional factors are present, transcription of a given target gene occurs. Transcripts (green curve) accumulate during the time when Dorsal is above a given threshold and then diminish when Dorsal falls below that threshold. The bar at the top of the simulated plot demarcates interphase (white) and mitosis (black).