Control of canalization and evolvability by Hsp90

Abstract

Partial reduction of Hsp90 increases expression of morphological novelty in qualitative traits of Drosophila and Arabidopsis, but the extent to which the Hsp90 chaperone also controls smaller and more likely adaptive changes in natural quantitative traits has been unclear. To determine the effect of Hsp90 on quantitative trait variability we deconstructed genetic, stochastic and environmental components of variation in Drosophila wing and bristle traits of genetically matched flies, differing only by Hsp90 loss-of-function or wild-type alleles. Unexpectedly, Hsp90 buffering was remarkably specific to certain normally invariant and highly discrete quantitative traits. Like the qualitative trait phenotypes controlled by Hsp90, highly discrete quantitative traits such as scutellor and thoracic bristle number are threshold traits. When tested across genotypes sampled from a wild population or in laboratory strains, the sensitivity of these traits to many types of variation was coordinately controlled, while continuously variable bristle types and wing size, and critically invariant left-right wing asymmetry, remained relatively unaffected. Although increased environmental variation and developmental noise would impede many types of selection response, in replicate populations in which Hsp90 was specifically impaired, heritability and 'extrinsic evolvability', the expected response to selection, were also markedly increased. However, despite the overall buffering effect of Hsp90 on variation in populations, for any particular individual or genotype in which Hsp90 was impaired, the size and direction of its effects were unpredictable. The trait and genetic-background dependence of Hsp90 effects and its remarkable bias toward invariant or canalized traits support the idea that traits evolve independent and trait-specific mechanisms of canalization and evolvability through their evolution of non-linearity and thresholds. Highly non-linear responses would buffer variation in Hsp90-dependent signaling over a wide range, while over a narrow range of signaling near trait thresholds become more variable with increasing probability of triggering all-or-none developmental responses.

Figure 1. Experimental design partitions genetic and purely-environmental components of variation.

Females heterozygous for P582i (Sam1;Sam2;P582i/Sami; left) were crossed to males from each of 9 highly inbred (95% homozygous) RI line backgrounds (different colors) to create matched “clones” of Hsp90 mutant and control male progeny from the same vial and maternal environments and hybrid for Sam and each RI line background.

Figure 2. Hsp90 controlled phenotypic variation of most invariant quantitative traits.

(A) Effects of P582i (yellow) and Sami (blue) alleles of Hsp90 in the isogenic Sam background on V_P of mutant and control sets of 450 males across the 9 RI line backgrounds. (B and C) Effects of 3^rd chromosomes carrying null (P582), and dominant-negative (9J1) Hsp90 mutations or wild-type Sam alleles introgressed into the Sam background in mutant and control sets of 225 male or 225 female sibs. TM6B contains the dominant Humeral (Hu) mutation , which increases humeral TH bristle numbers. Therefore, for comparison of TH bristles between TM6B and the other genotypes we used TH-HU, indicating that TH was scored only for the remaining 18 non-humeral TH bristle types. All experiments were conducted under temperature, density and humidity controlled conditions. Environmental effects were further controlled by direct comparisons between flies from the same vial and maternal environments. Coefficients of variation (CV = standard deviation/mean) are shown to enable between-trait comparisons. Statistical tests of the significance of Hsp90 effects on phenotypic variability and P-values are found in Table 1 (for A) and Table 2 (for B and C).

Figure 3. Hsp90 controlled environmental variation specific to previously invariant or canalized traits.

Comparison of mean-normalized components of purely-environmental variation (z-axis) across the RI line backgrounds (x-axis). Developmental stability was calculated as the averaged (unsigned) deviations of left and right from the mean, i.e. (L+R)/2, within each individual (V_e within) or on the averaged (unsigned) deviations of each individual (L+R) from their clone means for each RI line genotype (V_e among). There was generally no effect of Hsp90 allele (Sami, blue or P582i, yellow) on the variable traits, and a highly significant effect on either measure of V_e for canalized bristle traits but not wing area, which was highly canalized independent of Hsp90 (Table 4).

Figure 4. Hsp90-buffered variation contributed to predicted selection responses.

Fold-increase in the predicted response to truncation, directional or stabilizing selection of the ‘populations’ of 9 RI line genotypes in the P582i mutant relative to equivalent ‘populations’ of genotypes in the Sami control flies (Table 5).

Figure 5. Upper and lower thresholds control SC bristle number.

A threshold trait model was applied the effect of Hsp90 on signalling pathways underlying SC bristle development with assumptions based on current understanding of bristle development and Hsp90 function , . The positions of upper and lower thresholds were estimated for P582i and Sami populations, which differed by the presence of wild-type (Sami) or heterozygous mutant (P582i) levels of Hsp90 in otherwise nearly identical genetic backgrounds (N = 450 flies each). A consistent, but arbitrary, standard deviation (SD) scale was inferred from the frequency of abnormal flies in the P582i background, which had the highest frequencies of abnormal flies. SC was canalized at 4 over a range of from 5 1/2 to 6 1/2 SD. On the same scale, we estimate that the P582i mutation decreased the strength of proneural and inhibitory bristle development pathways, shifting signalling across the population >2.5 SD to the left relative to fixed upper and lower thresholds.

Figure 6. Model for biological control of canalization and evolvability. In any individual the strength of signaling through Hsp90 target pathways is directly proportional to the level of Hsp90 function (top panel), but the phenotypic effects of changes in signaling strength are often highly nonlinear, creating thresholds (the inflection point, second from top).

A simple consequence of steep non-linearity is that sensitivity to all kinds of perturbation and stochastic effects is expected to be highest at the inflection point (second panel from bottom). This simple relationship between sensitivity to variation and the steepness of the signaling-phenotype relationship at the inflection point, the inverse of canalization (bottom panel), suggests why all variation increases in concert for individuals and/or populations near trait thresholds.