Hsp90 Selectively Modulates Phenotype in Vertebrate Development

Abstract

Compromised heat shock protein 90 (Hsp90) function reveals cryptic phenotypes in flies and plants. These observations were interpreted to suggest that this molecular stress-response chaperone has a capacity to buffer underlying genetic variation. Conversely, the protective role of Hsp90 could account for the variable penetrance or severity of some heritable developmental malformations in vertebrates. Using zebrafish as a model, we defined Hsp90 inhibitor levels that did not induce a heat shock response or perturb phenotype in wild-type strains. Under these conditions the severity of the recessive eye phenotype in sunrise, caused by a pax6b mutation, was increased, while in dreumes, caused by a sufu mutation, it was decreased. In another strain, a previously unobserved spectrum of severe structural eye malformations, reminiscent of anophthalmia, microphthalmia, and nanophthalmia complex in humans, was uncovered by this limited inhibition of Hsp90 function. Inbreeding of offspring from selected unaffected carrier parents led to significantly elevated malformation frequencies and revealed the oligogenic nature of this phenotype. Unlike in Drosophila, Hsp90 inhibition can decrease developmental stability in zebrafish, as indicated by increased asymmetric presentation of anophthalmia, microphthalmia, and nanophthalmia and sunrise phenotypes. Analysis of the sunrise pax6b mutation suggests a molecular mechanism for the buffering of mutations by Hsp90. The zebrafish studies imply that mild perturbation of Hsp90 function at critical developmental stages may underpin the variable penetrance and expressivity of many developmental anomalies where the interaction between genotype and environment plays a major role. Genetic variation is not always expressed as a single consistent phenotype even in familial diseases. Unilateral malformations in paired organs, such as the failure of an eye to develop on one side only, also remind us that gene function is often modified by environmental factors. Following observations by others in fruit flies, we explored the underlying mechanisms for such phenotypic fluctuation, using zebrafish as a vertebrate model. Earlier work suggested involvement of chaperone proteins like Hsp90, which assist with normal protein folding during development and also work overtime to keep proteins functional in response to environmental stress. Using specific drugs at defined times in early development for the limited reduction of Hsp90 activity, we showed that different cryptic genetic variants could be revealed consistently in genetically distinct fish strains. Once uncovered, the frequency of these variants was increased by inbreeding, confirming the role of underlying genetic factors. Similarly, we could modify the phenotypic severity of some—but not all—known gene variants, worsening some and improving others. It emerged that the most susceptible variants were those carrying amino acid alterations, in which assisted protein folding may either restore near normal function or facilitate malfunction, thus worsening phenotype. This insight may allow us to prevent recurrent malformations by minimizing or perhaps even counteracting the effects of exposure to environmental stress during development.