The long term goal of our proposal is to delineate the mechanisms by which Piwi/piRNA pathway normalizes phenotypic variation induced by severe environmental stress and inherent genotype variations. Canalization or developmental robustness provides a framework in which organisms resist displaying phenotypic variation in the face of combined genotype variations and severe environmental stress. Recent research has shown that Hsp90, a molecular chaperone, plays a critical role in canalization. Despite the known role for Hsp90, the lack of a detailed molecular mechanism underlying canalization has provoked great debate for decades. This lab recently validated the existence of 'canalization'and uncovered a major molecular pathway involving Piwi, Hsp90 and Hop. Using a sensitized eye development assay, a reduction in the maternal dosage of Drosophila Piwi and Aubergine was demonstrated, which bind to novel germline-enriched small non-coding RNAs called piRNAs, induces phenotypic variations that can be fixed in a population and stably inherited in later generations. The study also showed that Piwi directly interacts with Hop and Hsp90 and functions in the same pathway as Hsp90 in suppressing phenotypic variations. Further, it was demonstrated for the first time that Piwi becomes phosphorylated in an Hsp90 dependent manner, thus providing insights into how Hsp90 may modulate Piwi's function in canalization. Crucially, this study also revealed two related yet distinct Piwi/piRNA pathway-dependent mechanisms responsible for suppression of phenotypic variation: epigenetic silencing of inherent genotype variations and suppression of transposon-mediated mutagenesis. This work revealed a framework of a pathway that suppresses phenotypic variation. The working hypothesis is that Hsp90 and Hop modulate Piwi function via its phosphorylation which then modulates Piwi function in canalization. By further unraveling the inner workings of each step of this pathway, we aim to shed light on the mechanisms that mediate this very important, yet poorly understood cellular phenomenon.
The aims of the proposal are to- 1) Discover new components that mediate and/or regulate Piwi function in canalization using genetic screens, 2) Unravel the biochemical mechanism of Piwi mediated epigenetic regulation in canalization and finally 3) Understand how Hsp90-Piwi/piRNA pathway counteracts environmental stress induced by industrial pollutants like hexavalent chromium (CrVI). Piwi proteins are present from protozoans to humans with conserved functions in stem cell self-renewal and germ line maintenance. However their role in suppression of phenotypic variation is a novel function that requires further characterization. Unraveling the mechanism by which Piwi functions in canalization using Drosophila as a model system understanding its roles in humans and learning how the dysfunction of this process might affect human will provide the necessary context for development and cause diseases.

Public Health Relevance

Stress induced by toxic pollutants in the environment has deleterious effect on human development. This proposal takes an inter-disciplinary approach to discover new components and fundamental cellular mechanisms that can be used as tools in the future to counteract such environmental stress.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Career Transition Award (K99)
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Special Emphasis Panel (ZES1-LWJ-J (K8))
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Shreffler, Carol K
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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