Canalization of segmentation and its evolution in Drosophila

Abstract

Segmentation in Drosophila embryogenesis occurs through a hierarchical cascade of regulatory gene expression driven by the establishment of a diffusion-mediated morphogen gradient. Here, we investigate the response of this pattern formation process to genetic variation and evolution in egg size. Specifically, we ask whether spatial localization of gap genes Kruppel (Kr) and giant (gt) and the pair-rule gene even-skipped (eve) during cellularization is robust to genetic variation in embryo length in three Drosophila melanogaster isolines and two closely related species. We identified two wild-derived strains of D. melanogaster whose eggs differ by approximately 25% in length when reared under identical conditions. These two lines, a D. melanogaster laboratory stock (w1118), and offspring from crosses between the lines all exhibit precise scaling in the placement of gap and pair-rule gene expression along the anterior-posterior axis in relation to embryo length. Genetic analysis indicates that this scaling is maternally controlled. Maternal regulation of scaling must be required for consistent localization of segmentation gene expression because embryo size, a genetically variable and adaptive trait, is maternally inherited. We also investigated spatial scaling between these D. melanogaster lines and single lines of Drosophila sechellia and Drosophila simulans, the latter two differing by approximately 25% in egg length. In contrast to the robust scaling we observed within species, localization of gene expression relative to embryo length differs significantly between the three species. Thus, the developmental mechanism that assures robust scaling within a species does not prevent rapid evolution between species.

Fig. 1.

Egg lengths within and among Drosophila species. Average egg lengths (in μm; ±1 SD) for representatives of the D. melanogaster species subgroup, with multiple stocks from D. melanogaster, D. mauritiana (D. maur), D. simulans, and D. sechellia (D. sech). Images are representative eggs photographed at the same magnification and can be directly compared with one another. Measurement error is small compared with the length differences between sampled eggs. All strains were grown under the same conditions, and eggs were collected with the same protocol.

Fig. 2.

eve stripe position varies between species, not within D. melanogaster. (A and B) Anterior (A) and posterior (P) eve stripe boundaries (least-square means ± 1 SE) in stage 14a embryos in three D. melanogaster isofemale lines (Ind, Fra, and w1118) and the closely related species D. simulans and D. sechellia. Stripe boundaries were measured as distances from the anterior pole of the embryo. Means are represented as deviations by line from the mean of all of the lines, with a shift toward positive numbers indicating more posterior localization compared with the grand mean. (A) Means in absolute units (μm), uncorrected for embryo length. Stripes are located further from the anterior pole in the two large-egg strains (Ind and sechellia). (B) Relative measurement means, represented as a percentage of the embryo length. D. sechellia eve stripes remain posterior-shifted when scaled to embryo length, whereas the D. melanogaster Ind eve stripes exhibit the same scaling with length as the other two D. melanogaster lines. (C and D) Statistical significance (negative logarithm of P value) of factors influencing stripe boundary positions based on an ANOVA. Factors are age, line within D. melanogaster (mel lines), species, and the line by age interaction. The dotted line is the Bonferroni-corrected P = 0.05 cutoff. These graphs are a confluence of different ANOVAs (see Materials and Methods), as the effects of line within D. melanogaster and species were modeled separately. The species term has a significant effect on both absolute (C) and relative (D) stripe position, whereas the significance of the within D. melanogaster term (mel lines) disappears when the measurements are corrected for embryo size.

Fig. 3.

Maternal inheritance of eve stripe boundaries. Genetic analysis of D. melanogaster Fra and Ind reciprocal crosses to F₃s. (A) Inheritance of absolute stripe position is consistent with an additive, maternally inherited trait. Line means for absolute measures of stripe positions (μm from anterior pole), shown as the deviation from the grand mean, for each stripe. The designation f (Fra) or i (Ind) after a cross (F1f, F1i, etc.) indicates the maternal lineage. The stripe locations in F1f and F1i lines resemble those of their mothers, Fra and Ind; stripes in F₂ and F₃ lines are localized midway between the parental lines. (B) F₃ embryos exhibit a wide range of embryo lengths (as expected for a segregating trait) but retain nearly perfect linear scaling of eve stripe boundary locations across this length distribution. Relative stripe positions in individual F₃ embryos for the anterior boundaries of each eve stripe are plotted against each embryo's length.

Fig. 4.

Correlations of expression pattern boundary positions within embryos. The Pearson's correlation coefficient for all pairs of expression domain boundaries (all lines and ages, for eve, gt, and Kr) are plotted as a function of average relative distance (%EL) between pairs. Deviations of neighboring stripe boundaries within an individual embryo relative to the mean are highly correlated. The correlation falls off with increasing distance between stripes, suggesting regional influences of stripe positioning.

Fig. 5.

Species differences in eve localization. The relative positioning of eve stripes differs between the three species studied (D. melanogaster, D. simulans, and D. sechellia). Embryos with stripe positions closest to the mean for each species were chosen, and images were scaled to the same length and overlaid.