Hsp90 inhibition and the expression of phenotypic variability in the rainforest species Drosophila birchii

Recently a heat shock protein (Hsp90) has been implicated as controlling the expression of cryptic genetic variation through buffering developmental processes. The release of variability in canalized characters following Hsp90 inhibition has been established in model species including Drosophila melanogaster and Arabidopsis thaliana , but has not yet been examined in species with limited distributions. To test if Hsp90 has a role in releasing phenotypic variation in rainforest Drosophila species, developing larvae from a large (> 1000 individuals) outbred population of Drosophila birchii were treated with the Hsp90 inhibitors geldanamycin and radicicol, and morphological traits, desiccation resistance, and life-history traits were measured. The means of all traits were influenced by inhibition. Although only the phenotypic variances of two canalized bristle traits were affected consistently, variability for two of the continuously varying traits (fecundity and development time) were also affected, albeit inconsistently. There was also no effect of Hsp90 inhibition on the developmental stability of the morphological traits as measured by fluctuating asymmetry. Hsp90 inhibition did not increase phenotypic variability in desiccation resistance, a trait previously shown to represent an evolutionary limit in this species. These results question the extent to which Hsp90 buffers variation for both quantitative and discrete traits, and highlight the need for further empirical studies to determine the involvement of Hsp90 in canalization and developmental stability. Nevertheless the results demonstrated increased variability in canalized traits, consistent with observations in model systems. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 457–465.     

Novel genetic capacitors and potentiators for the natural genetic variation of sensory bristles and their trait specificity in Drosophila melanogaster

Cryptic genetic variation (CGV) is defined as the genetic variation that has little effect on phenotypic variation under a normal condition, but contributes to heritable variation under environmental or genetic perturbations. Genetic buffering systems that suppress the expression of CGV and store it in a population are called genetic capacitors, and the opposite systems are called genetic potentiators. One of the best‐known candidates for a genetic capacitor and potentiator is the molecular chaperone protein, HSP90, and one of its characteristics is that it affects the genetic variation in various morphological traits. However, it remains unclear whether the wide‐ranging effects of HSP90 on a broad range of traits are a general feature of genetic capacitors and potentiators. In the current study, I searched for novel genetic capacitors and potentiators for quantitative bristle traits of Drosophila melanogaster and then investigated the trait specificity of their genetic buffering effect. Three bristle traits of D. melanogaster were used as the target traits, and the genomic regions with genetic buffering effects were screened using the 61 genomic deficiencies examined previously for genetic buffering effects in wing shape. As a result, four and six deficiencies with significant effects on increasing and decreasing the broad‐sense heritability of the bristle traits were identified, respectively. Of the 18 deficiencies with significant effects detected in the current study and/or by the previous study, 14 showed trait‐specific effects, and four affected the genetic buffering of both bristle traits and wing shape. This suggests that most genetic capacitors and potentiators exert trait‐specific effects, but that general capacitors and potentiators with effects on multiple traits also exist.     

The Hsp90 Capacitor,Developmental Remodeling,and Evolution: The Robustness of Gene Networks and the Curious Evolvability of Metamorphosis

ABSTRACT

Genetic capacitors moderate expression of heritable variation and provide a novel mechanism for rapid evolution. The prototypic genetic capacitor, Hsp90, interfaces stress responses, developmental networks, trait thresholds and expression of wide-ranging morphological changes in Drosophila and other organisms. The Hsp90 capacitor hypothesis, that stress-sensitive storage and release of genetic variation through Hsp90 facilitates adaptive evolution in unpredictable environments, has been challenged by the belief that Hsp90-buffered variation is unconditionally deleterious. Here we review recent results supporting the Hsp90 capacitor hypothesis, highlighting the heritability, selectability, and potential evolvability of Hsp90-buffered traits. Despite a surprising bias toward morphological novelty and typically invariable quantitative traits, Hsp90-buffered changes are remarkably modular, and can be selected to high frequency independent of the expected negative side-effects or obvious correlated changes in other, unselected traits. Recent dissection of cryptic signal transduction variation involved in one Hsp90-buffered trait reveals potentially dozens of normally silent polymorphisms embedded in cell cycle, differentiation and growth control networks. Reduced function of Hsp90 substrates during environmental stress would destabilize robust developmental processes, relieve developmental constraints and plausibly enables genetic network remodeling by abundant cryptic alleles. We speculate that morphological transitions controlled by Hsp90 may fuel the incredible evolutionary lability of metazoan life-cycles.     

Stress,genomes, and evolution

Evolutionary change, whether in populations of organisms or malignant tumor cells, is contingent on the availability of inherited variation for natural selection to act upon. It is becoming clear that the Hsp90 chaperone, which normally functions to buffer client proteins against the effects of genetic variation, plays a central role in this process. Severe environmental stress can overwhelm the chaperone's buffering capacity, causing previously cryptic genetic variation to be expressed. Recent studies now indicate that in addition to exposing existing variation, Hsp90 can induce novel epigenetic and genetic changes. We discuss key findings that suggest a rich set of pathways by which Hsp90 can mediate the influences of the environment on the genome.     

The Hsp90 capacitor, developmental remodeling, and evolution: the robustness of gene networks and the curious evolvability of metamorphosis

Genetic capacitors moderate expression of heritable variation and provide a novel mechanism for rapid evolution. The prototypic genetic capacitor, Hsp90, interfaces stress responses, developmental networks, trait thresholds and expression of wide-ranging morphological changes in Drosophila and other organisms. The Hsp90 capacitor hypothesis, that stress-sensitive storage and release of genetic variation through Hsp90 facilitates adaptive evolution in unpredictable environments, has been challenged by the belief that Hsp90-buffered variation is unconditionally deleterious. Here we review recent results supporting the Hsp90 capacitor hypothesis, highlighting the heritability, selectability, and potential evolvability of Hsp90-buffered traits. Despite a surprising bias toward morphological novelty and typically invariable quantitative traits, Hsp90-buffered changes are remarkably modular, and can be selected to high frequency independent of the expected negative side-effects or obvious correlated changes in other, unselected traits. Recent dissection of cryptic signal transduction variation involved in one Hsp90-buffered trait reveals potentially dozens of normally silent polymorphisms embedded in cell cycle, differentiation and growth control networks. Reduced function of Hsp90 substrates during environmental stress would destabilize robust developmental processes, relieve developmental constraints and plausibly enables genetic network remodeling by abundant cryptic alleles. We speculate that morphological transitions controlled by Hsp90 may fuel the incredible evolutionary lability of metazoan life-cycles.     

Is canalization more than just a beautiful idea?

The heat-shock protein 90 (Hsp90) is currently thought to buffer eukaryotic cells against perturbations caused by pre-existing cryptic genetic variation. A new study suggests that the buffering function of Hsp90 could instead be due to its repression of de novo transposon-mediated mutagenesis.     

Hsp90 depletion goes wild

Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to revealing cryptic genetic variation.See research article http://wwww.biomedcentral.com/1471-2148/12/25     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.     

Hsp90 selectively modulates phenotype in vertebrate development

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.     

Hsp90 and the quantitative variation of wing shape in Drosophila melanogaster

The molecular chaperone protein Hsp90 has been widely discussed as a candidate gene for developmental buffering. We used the methods of geometric morphometrics to analyze its effects on the variation among individuals and fluctuating asymmetry of wing shape in Drosophila melanogaster. Three different experimental approaches were used to reduce Hsp90 activity. In the first experiment, developing larvae were reared in food containing a specific inhibitor of Hsp90, geldanamycin, but neither individual variation nor fluctuating asymmetry was altered. Two further experiments generated lines of genetically identical flies carrying mutations of Hsp83, the gene encoding the Hsp90 protein, in heterozygous condition in nine different genetic backgrounds. The first of these, introducing entire chromosomes carrying either of two Hsp83 mutations, did not increase shape variation or asymmetry over a wild-type control in any of the nine genetic backgrounds. In contrast, the third experiment, in which one of these Hsp83 alleles was introgressed into the wild-type background that served as the control, induced an increase in both individual variation and fluctuating asymmetry within each of the nine genetic backgrounds. No effect of Hsp90 on the difference among lines was detected, pro,iding no evidence for cryptic genetic variation of wing shape. Overall, these results suggest that Hsp90 contributes to, but is not controlling, the buffering of phenotypic variation in wing shape.     

Little effect of HSP90 inhibition on the quantitative wing traits variation in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Drosophila wings have been a model system to study the effect of HSP90 on quantitative trait variation. The effect of HSP90 inhibition on environmental buffering of wing morphology varies among studies while the genetic buffering effect of it was examined in only one study and was not detected. Variable results so far might show that the genetic background influences the environmental and genetic buffering effect of HSP90. In the previous studies, the number of the genetic backgrounds used is limited. To examine the effect of HSP90 inhibition with a larger number of genetic backgrounds than the previous studies, 20 wild-type strains of Drosophila melanogaster were used in this study. Here I investigated the effect of HSP90 inhibition on the environmental buffering of wing shape and size by assessing within-individual and among-individual variations, and as a result, I found little or very weak effects on environmental and genetic buffering. The current results suggest that the role of HSP90 as a global regulator of environmental and genetic buffering is limited at least in quantitative traits.     

Hsp90 modulates CAG repeat instability in human cells

The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.     

Associations between environmental stress, selection history, and quantitative genetic variation in Drosophila melanogaster

Stressful environments may increase quantitative genetic variation in populations by promoting the expression of genetic variation that has not previously been eliminated or canalized by natural selection. This “selection history” hypothesis predicts that novel stressors will increase quantitative genetic variation, and that the magnitude of this effect will decrease following continued stress exposure. We tested these predictions using Drosophila melanogaster and sternopleural bristle number as a model system. In particular, we examined the effect of high temperature stress (31°C) on quantitative genetic variation before and after our study population had been reared at 31°C for 15 generations. High temperature stress was found to increase both additive genetic variance and heritability, but contrary to the selection history hypothesis prediction, the magnitude of this effect significantly increased after the study population had been reared for 15 generations under high temperature stress. These results demonstrate that high temperature stress increases quantitative genetic variation for bristle number, but do not support the selection history hypothesis as an explanation for this effect.     

Diet and hormonal manipulation reveal cryptic genetic variation: implications for the evolution of novel feeding strategies

When experiencing resource competition or abrupt environmental change, animals often must transition rapidly from an ancestral diet to a novel, derived diet. Yet, little is known about the proximate mechanisms that mediate such rapid evolutionary transitions. Here, we investigated the role of diet-induced, cryptic genetic variation in facilitating the evolution of novel resource-use traits that are associated with a new feeding strategy—carnivory—in tadpoles of spadefoot toads (genus Spea). We specifically asked whether such variation in trophic morphology and fitness is present in Scaphiopus couchii, a species that serves as a proxy for ancestral Spea. We also asked whether corticosterone, a vertebrate hormone produced in response to environmental signals, mediates the expression of this variation. Specifically, we compared broad-sense heritabilities of tadpoles fed different diets or treated with exogenous corticosterone, and found that novel diets can expose cryptic genetic variation to selection, and that diet-induced hormones may play a role in revealing this variation. Our results therefore suggest that cryptic genetic variation may have enabled the evolutionary transition to carnivory in Spea tadpoles, and that such variation might generally facilitate rapid evolutionary transitions to novel diets.     

Effect of E(sev) and Su(Raf) Hsp83 mutants and trans-heterozygotes on bristle trait means and variation in Drosophila melanogaster

The Hsp90 protein encoded by the Hsp83 gene is required for the development of many traits in Drosophila. Hsp83 is also thought to play a role in the expression of phenotypic and genetic variability for subsequent selection and evolutionary change. Here we examine the impact of different E(sev) and Su(Raf) Hsp83 mutants on means and phenotypic variances of invariant and variable bristle traits. One of the mutants influenced the normally invariant thoracic bristle number, while none affected invariant scutellar bristle number. E(sev) alleles consistently influenced variable bristle traits while there were fewer effects of the Su(Raf) alleles. For the variable traits, none of the Hsp83 alleles had any effect on phenotypic variance, environmental variance, or developmental stability of the bristle traits. When alleles were combined in trans-heterozygotes, there were both cumulative and complementary effects on thoracic and variable bristle trait numbers, depending on the allelic combination. Overall, the results suggest that Hsp83 mutants do not have detectable effects on the phenotypic or environmental variance of bristle traits and that complementation of E(sev) and Su(Raf) Hsp83 mutants can extend to thoracic bristles as well as previously reported effects on viability. Some allelic combinations lead to more severe effects on variable bristle trait means than do single Hsp83 mutations.     

Environmental stress-dependent effects of deletions encompassing Hsp70Ba on canalization and quantitative trait asymmetry in Drosophila melanogaster

Hsp70 genes may influence the expression of wing abnormalities in Drosophila melanogaster but their effects on variability in quantitative characters and developmental instability are unclear. In this study, we focused on one of the six Hsp70 genes, Hsp70Ba, and investigated its effects on within- and among-individual variability in orbital bristle number, sternopleural bristle number, wing size and wing shape under different environmental conditions. To do this, we studied a newly constructed deletion, Df(3R)ED5579, which encompasses Hsp70Ba and nine non-Hsp genes, in the heterozygous condition and another, Hsp70Ba(304), which deletes only Hsp70Ba, in the homozygous condition. We found no significant effect of both deletions on within-individual variation quantified by fluctuating asymmetry (FA) of morphological traits. On the other hand, the Hsp70Ba(304)/Hsp70Ba(304) genotype significantly increased among-individual variation quantified by coefficient of variation (CV) of bristle number and wing size in female, while the Df(3R)ED5579 heterozygote showed no significant effect. The expression level of Hsp70Ba in the deletion heterozygote was 6 to 20 times higher than in control homozygotes, suggesting that the overexpression of Hsp70Ba did not influence developmental stability or canalization significantly. These findings suggest that the absence of expression of Hsp70Ba increases CV of some morphological traits and that HSP70Ba may buffer against environmental perturbations on some quantitative traits.     

基于形态及分子标记的濒危植物夏蜡梅自然居群的遗传变异研究

比较了濒危植物夏蜡梅(Sinocalycanthus chinensis)大明山、大雷山、龙须山3个居群的果实与种子的形态变异,采用ISSR分子标记技术分析DNA序列的变异,综合评价夏蜡梅3个居群的遗传变异。结果如下:果实性状中,果柄长、果实长、果实重、每果种子数等指标均以大明山居群最大、大雷山居群次之、龙须山居群最小,它们之间差异显著(P<0.05)。种子重、种子长、种子宽、种子厚等指标均以大雷山居群最大,与大明山居群和龙须山居群差异显著(P<0.05)。基于果实形态特征的表型分化系数(VST)在0.5518—0.9750之间,平均为0.8930,所有果实形态指标的变异大部分存在于居群间。基于种子形态特征的VST在0.1669—0.8678之间,平均为0.6240,除种子长外,其他种子形态指标的变异也大部分存在于居群间。ISSR分析表明,3个居群的多态位点百分率、Shannon信息指数、Nei基因多样性均是大明山居群最高、龙须山居群次之、大雷山居群最低。居群间的遗传分化系数(GST)为0.6050。基于ISSR分子标记数据的聚类结果显示大明山居群先与龙须山居群聚在一起,再与大雷山居群相聚,这与基于种子性状特征的聚类结果相似,而与基于果实性状特征的聚类结果不同。3个居群的种子形态特征变异系数与遗传多样性水平具有显著的正相关,且基于果实形态特征估算的VST要高于GST,而基于种子形态特征估算的VST仅略高于GST,表明夏蜡梅不同居群间的种子形态变异主要由遗传变异所造成的,而环境因子在果实形态变异中起了重要作用。Mantel检验显示3个居群基于分子标记数据的遗传距离矩阵和基于果实及种子形态特征的欧氏距离矩阵之间的相关性不显著,表明果实与种子在居群间出现的表型分化除了受遗传因素影响外,还受到其它环境因子的强烈影响。     

Phylogenetic analysis of nuclear and mitochondrial DNA reveals a complex of cryptic species in Crassicutis cichlasomae (Digenea: Apocreadiidae), a parasite of Middle-American cichlids

We obtained nuclear ITS-1 and mitochondrial cox1 sequences from 225 Crassicutis cichlasomae adults collected in 12 species of cichlids from 32 localities to prospect for the presence of cryptic species. This trematode is commonly found in species of cichlids over a wide geographic range in Middle-America. Population-level phylogenetic analyses of ITS-1 and cox1, assessments of genetic and haplotype diversity, and morphological observations revealed that C. cichlasomae represents a complex of seven cryptic species for which no morphological diagnostic characters have been discovered thus far. Bayesian and Maximum Likelihood analyses of concatenated datasets (906 bp) recovered eight lineages of C. cichlasomae, all with high posterior probabilities and bootstrap branch support. Values of genetic divergence between clades ranged from 1.0% to 5.2% for ITS-1, and from 7.2% to 30.0% for cox1. Morphological study of more than 300 individuals did not reveal structural diagnostic traits for the species defined using molecular evidence. These observations indicate that some traditional morphological characters (e.g., testes position) have substantial intra-specific variation, and should be used with caution when classifying C. cichlasomae and their sister taxa. Additionally, phylogenetic analyses did not reveal a strict correlation between these cryptic species and their host species or geographic distribution, however it appears that genetic distinctiveness of these cryptic species was influenced by the diversification and biogeographical history of Middle-American cichlids.     

Dissecting adaptive clinal variation: markers, inversions and size/stress associations in Drosophila melanogaster from a central field population

Many organisms show latitudinal variation for quantitative traits that is assumed to be due to climatic adaptation. These clines provide an opportunity to study the genetics of the adaptive process both at the phenotypic and the underlying molecular levels. Yet researchers rarely try to link variation in quantitative traits to their underlying molecular genetic basis. We describe a novel approach for exploring the genetic basis for clinal variation in size and stress traits in Drosophila melanogaster. We look for associations between genetic markers and traits that exhibit clinal patterns on the east coast of Australia using a single, geographically central population. There are strong associations between markers found within In(3R)Payne and variation in size, suggesting that this inversion explains much of the clinal variation in this trait. We also find that development time is associated with the Adh allozyme locus, cold resistance is negatively associated with the In(3L)Payne inversion and a genetic marker for Hsp70, a heat‐shock protein, is associated with heat resistance. Finally we discuss the importance of inversions in clinal variation for quantitative traits and for identifying quantitative trait loci.