Sato and Siomi Genome Biology 2010, 11:109
http://genomebiology.com/2010/11/3/109

RESEARCH HIGHLIGHT

Is canalization more than just a beautiful idea?
Kaoru Sato and Haruhiko Siomi*

Abstract
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 transposonmediated mutagenesis.

In the 1940s, the developmental biologist and geneticist
CH Waddington coined the concept of ‘developmental
stability’, or the robustness of the phenotype against
genetic and environmental perturbations [1,2]. It has
been claimed that this robustness, termed ‘canalization’,has evolved under natural selection to stabilize pheno­
types and decrease their variability. This is achieved bybuffering the expression of traits, holding them near theiroptimal states despite genetic and environmentalperturbations. Canalization also allows the accumulationof ‘cryptic genetic variation’ caused by mutations that do
not affect the phenotype. Canalized traits are pheno­typically expressed only in particular environments orgenetic backgrounds and become available for naturalselection, a mechanism that can lead to the assimilationof novel traits.
It was found some years ago that reduction in thefunction of the Hsp90 protein in Drosophila (whether bymutation or by specific inhibitors) apparently uncovered
previously silent genetic variation, which led to anincrease in morphological variation [3]. Hsp90 is achaperone and heat-shock protein, which in Drosophila
is encoded by the Hsp83 gene. The morphologicalchanges could become fixed and stably transmitted even
if wild-type Hsp90 function were restored in subsequent
generations. These findings implied that functional Hsp90
is a capacitor (that accumulates cryptic genetic variation
and releases it under certain circumstances) that masks
the effect of hidden or pre-existing genetic variation(Figure 1).
*Correspondence: awa403@sc.itc.keio.ac.jp
Department of Molecular Biology, Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan© 2010 BioMed Central Ltd© 2010 BioMed Central Ltd

The Hsp90 story in flies has become very complicated,however. Recent studies have shown that the buffering by
Hsp90 is limited to specific morphological traits and does
not affect others. This supports the idea that numerous
mechanisms are involved in developmental buffering,and that Hsp90 is just one of many capacitors for genetic
variation [1,2]. In addition, Hsp90 is a very abundant
protein, in some cells accounting for up to 2% of the total
protein content, and a reduction in Hsp90 activity affects
the expression levels of numerous genes. A new study
that implicates Hsp90 in the repression of transposonmediated mutagenesis now further complicates the story.
In work recently published in Nature, Specchia et al. [4]show that biogenesis of the small PIWI-interacting RNA(piRNA) in Drosophila depends on the activity of Hsp90.
These results are of interest not only for the insights they
provide into the molecular pathways of piRNA produc­tion, but also because they imply that Hsp90 prevents
phenotypic variation by suppressing de novo mutation
caused by the activity of transposons in the germline, oneof the known roles of the piRNAs in Drosophila. Thiscalls for current ideas on the buffering role of Hsp90 inflies to be revisited.
piRNAs are one class of the numerous small RNAs(around 20 to 30 nucleotides long) that are expressed byeukaryotic cells and that trigger sequence-specific genesilencing called RNA silencing [5,6]. By base pairing withtarget mRNAs, the small RNAs guide inhibitory
complexes based on members of the Argonaute class ofproteins (which includes the PIWI proteins) to themRNAs, resulting in mRNA destruction or the inhibitionof translation. RNA silencing is thought to have evolved
as a form of nucleic-acid-based immunity to inactivate
parasitic and pathogenic invaders such as viruses andtransposable elements (transposons) [7]. In Drosophila,
the endogenous small interfering RNA (esiRNA) pathway
of RNA silencing restrains the expression of transposons
in somatic cells, whereas the piRNA pathway represses
transposon activity in germline cells.
Transposons are generally considered as ‘selfish DNA’elements usually hidden from sight. They can movearound the genome, transposing into new sites andcausing insertion mutations that are frequently deleteri­ous. Thus, host genomes have evolved multiple mecha­
nisms for regulating transposons, including RNA silencing.

Sato and Siomi Genome Biology 2010, 11:109
http://genomebiology.com/2010/11/3/109(a)Page 2 of 4(b)Normal HSP90 function

Reduced HSP90 function

Genetic signal intensity

Genetic signal intensity

Vanation in phenotype

Vanation inphenotype

Genetic variation

Genetic variation

Figure 1. A model for the buffering role of Hsp90 in canalization. Hsp90 conceals cryptic genetic mutations. (a) When Hsp90 is normal,
underlying genetic variation (gray peaks) is hidden and genetic signal inputs (black peaks) are outputted normally, resulting in a phenotype thatvaries within normal limits. (b) When Hsp90 is impaired, hidden genetic variation is revealed, resulting in altered genetic signal inputs (black peaks)
that are abnormally outputted. These altered outputs may lead to an abnormal phenotypic variation.

Transposition is also potentially adaptive by occasionallyproviding a source of genetic diversity [8]. Thus, a trans­posable element is often defined as a natural, endogenous,
genetic toolbox for mutagenesis. In addition, transposon
defense mechanisms have recently been shown to be coopted or borrowed to provide additional regulatorycomplexity for host genes [7-9].
The production of esiRNAs from their longer precursor
transcripts requires the processing activity of theribonuclease Dicer. By contrast, the production ofpiRNAs is independent of Dicer. Drosophila has three
distinct PIWI proteins, AGO3, Aubergine, and Piwi, allof which exhibit the small RNA-guided ribonuclease(‘Slicer’) activity. Deep sequencing and bioinformatic
analyses of Drosophila piRNAs suggest a model for
piRNA biogenesis in which PIWI subfamily proteins
guide the 5’ end formation of piRNAs by reciprocallycleaving or slicing long sense and antisense transcripts oftransposons. Thus, in this amplification loop, which iscalled the ping-pong cycle, transposons are both a sourceof piRNAs and a target of piRNA-mediated silencing.
However, classification of piRNAs according to their
origins indicated that piRNAs derived from a particularpiRNA cluster locus are exclusively loaded onto one of
the PIWI proteins, Piwi, indicating that those piRNAs areproduced by a pathway independent of the ping-pongcycle. This pathway is called the primary processing path­way [5,6]. The mechanism of their production, however,has been largely unclear.

During spermatogenesis in Drosophila males, antisense
piRNAs derived from the repetitive Suppressor of Stellate[Su(Ste)] locus on the Y chromosome silence the X-linked
Stellate locus. In Su(Ste) and piRNA pathway mutants,
piRNAs targeting Stellate are lost, causing crystals of
Stellate protein to form in primary spermatocytes [6].
Specchia et al. [4] found that mutations in the Hsp83
gene encoding Hsp90, or treatment with the specific
Hsp90 inhibitor geldanamycin also caused the accumu­
lation of crystalline aggregates in primary spermatocytes,
suggesting that Hsp90 is involved in a piRNA-mediatedmechanism that silences the expression of repetitive
sequences and transposons. Consistent with this, theauthors found that Hsp90 mutations result in a marked
reduction in the accumulation of piRNAs corresponding
to Su(Ste) and various transposon sequences. Conversely,
the expression of various types of transposons wasupregulated in both the ovaries and the testes of Hsp90
mutants. These results showed that Hsp90 represses theexpression of transposons through piRNA-mediatedmechanisms (Figure 2a).
Specchia et al. [4] examined the effect of Hsp90
mutations on transposon mobility in individual flies andfound that in homozygous Hsp90 null mutants, severaltransposons had jumped into new sites within thegenome. They further showed that approximately 1% of
Hsp90 mutants screened (30 out of 3,220 flies) exhibitedmorphological abnormalities. Together, these findings
suggested that the phenotypic variation observed among

Sato and Siomi Genome Biology 2010, 11:109
http://genomebiology.com/2010/11/3/109

Page 3 of 4(a)(b)

Reduced HSP90 function
Decrease of piRNAs
Transposon activation

Transposon

Transposon insertional mutagenesis

Transposon silencing
HSP90

piRNA

(Piwi proteins)
Insertional mutagenesis

Phenotypic variation

Figure 2. Hsp90 functions in the suppression of transposon-mediated ‘canonical’ mutagenesis via the piRNA pathway. (a) Activatedtransposons can move from one site in a genome to another by the process of transposition and so have the potential to induce de novo genemutations by insertion. Hsp90 could be involved in the control of transposition by affecting piRNA biogenesis. (b) Reduced Hsp90 function leads totransposon-mediated insertional mutagenesis and the induction of morphological variability, such as variation in eye and body color.

Hsp90 mutants could be due to de novo mutations
produced by activated transposable elements rather thanto the buffering of pre-existing cryptic genetic variation.
For example, among the abnormalities observed bySpecchia et al. [4] among their Hsp90 mutants was a fly
resembling the Scutoid phenotype (in which there is aloss of bristles from the head and thorax of the adult),which is caused by a mutation in the noc gene. Theauthors demonstrated that the coding sequence of thenoc gene in this fly was indeed interrupted by an Ielement-like transposon sequence. This indicates that theScutoid phenotype found in the screen was caused by ade novo mutation and not by the expression of a preexisting genetic variation (Figure 2b).
As well as suggesting that a reinterpretation of thebuffering role of Hsp90 [3] might be needed, these newfindings also provide evidence supporting a model inwhich Hsp90 is involved in the control of transposon
activity in germ cells by affecting piRNA biogenesis.
piRNAs in Drosophila are produced almost exclusively ingerm cells from intergenic repetitive genes, transposable
elements and piRNA clusters by two pathways: theprimary processing pathway, and the amplification ‘pingpong’ loop [5,6]. Mature piRNAs are loaded onto thePIWI subfamily of Argonaute proteins, and the amplifi­
cation loop is known to be independent of Dicer but
dependent on the Slicer activity of PIWI proteins.
However, the mechanisms of primary piRNA processing
remain elusive. How does Hsp90 function in piRNAbiogenesis and which of the two piRNA production
pathways is it involved in? Hsp90 can, for example, be copurified with the Slicer activity of Ago2, one of themammalian Argonaute proteins [10].
Hsp90 could play a role in the biogenesis of small
silencing RNAs either as a chaperone for the correct
folding of the Argonaute proteins or by providing an
assembly platform for components of the small RNA
biogenetic machinery to promote the loading of small
RNAs onto the Argonaute proteins. It will be important
to ascertain whether Hsp90 interacts with the PIWI
proteins in flies and has a role in their function, such asensuring their correct cellular localization, and also
whether mutations in Hsp90 affect either or both of thetwo piRNA biogenesis pathways. It will also be interesting
to examine whether Hsp90 is required for the esiRNA
pathway that silences transposable elements in somaticcells. Further investigation should reveal the role ofHsp90 in RNA silencing and help expand ourunderstanding of transposon regulation by RNAsilencing pathways.
Published: 16 March 2010
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doi:10.1186/gb-2010-11-3-109
Cite this article as: Sato K, Siomi H: Is canalization more than just a beautiful
idea? Genome Biology 2010, 11:109.