The control of organ size is a long-standing puzzle in developmental biology. Classic embryological studies suggest that many organs possess intrinsic information about their final size. For example, when two-thirds of a mouse liver is surgically removed, the remaining one-third regenerates its original mass within 7–10 days and then stops growing. Similarly, when imaginal discs from newly hatched larvae are transplanted into adult flies, they grow to a final size characteristic of that seen in situ. The molecular mechanisms that stop organ growth at the appropriate point during development or regeneration remain largely unknown.
My laboratory uses Drosophila and mice as model systems to investigate size-control mechanisms in normal development and their pathological roles in cancer. Our general approach is to use Drosophila as a genetic tool to discover size-control genes. We then use a combination of genetics and biochemistry to place these genes into signaling networks. Finally, we use mouse genetics to investigate how the size-control mechanisms we have uncovered in Drosophila regulate tissue homeostasis in mammals. With these concerted efforts, we aim to decipher the general mechanisms underlying control of organ size in animals.
To discover novel size-control genes, we conducted genetic screens in Drosophila for mutations that result in overgrowth of adult structures. These overgrowth mutants can be broadly divided into two classes: those associated with an increase in cell size and those associated with an increase in cell number. Earlier studies from my laboratory focused on the cell-size mutants, which led to the discovery of a cell size-controlling pathway that involves the tuberous sclerosis tumor suppressors Tsc1 and Tsc2, the small GTPase Rheb, and the protein kinase TOR. The functional link between Tsc1 and Tsc2 and TOR uncovered in Drosophila paved the way for the clinical development of mTOR inhibitor everolimus in the treatment of subependymal giant cell astrocytoma associated with tuberous sclerosis.
Much of our recent work focused on the overgrowth mutants associated with an increase in cell number. These studies led us to elucidate a novel kinase cascade, the Hippo pathway, which plays a critical role in stopping organ growth by simultaneously promoting cell death and cell cycle exit as cells enter the differentiation phase of organogenesis. In Drosophila, the Ste20-like kinase Hippo (Hpo) phosphorylates and activates the NDR family kinase Warts (Wts). Wts, in turn, phosphorylates and inactivates the oncoprotein Yorkie (Yki) by excluding it from the nucleus, where it normally functions as a coactivator for the DNA-binding transcription factor Scalloped (Sd). Building on insights from Drosophila, we further delineated a mammalian Hippo pathway that links the mammalian homologues of Hpo (Mst1/2), Wts (Lats1/2), Yki (YAP), and Sd (TEAD/TEF family members) in an analogous signaling cascade.
Our current efforts focus on several outstanding issues in Hippo signaling.
Hippo Signaling in Drosophila: Composition, Mechanism, and Regulation
Studies in Drosophila suggest that the Hippo kinase cascade functions as a signaling module that integrates multiple upstream inputs, many of which are poorly defined. We are conducting sensitized genetic screens, cell-based RNAi screens, and protein interaction screens to discover the missing components of this emerging pathway. Through these unbiased approaches, we have identified several upstream regulators of Hippo signaling, including the apically localized transmembrane protein Crumbs, the cytoplasmic adaptor protein Kibra, and the cytoskeletal protein spectrin. Kibra is part of an apical cortex–associated protein complex that includes two additional tumor suppressors (Merlin and Expanded), and Crumbs is a transmembrane protein required for the apical localization of Expanded in epithelial cells. These proteins act in concert to target the Hippo kinase cassette to apical plasma membrane for activation. In contrast, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These findings support an intriguing link between tissue architecture and Hippo signaling, although exactly how these parameters of tissue architecture such as apical-basal polarity and cytoskeleton tension are modulated spatially and temporally to achieve proper organ size in development remains unclear.
Moving downstream of the Hippo pathway, we are investigating the molecular mechanism by which the Sd-Yki complex regulates target gene transcription. We discovered that Sd has a dual role in gene transcription: it functions by default as a transcriptional repressor in the absence of Yki by engaging a co-repressor called Tgi, and Yki regulates normal tissue growth by antagonizing Sd’s repressor function in a “relief-of-repression” manner. We further showed that Yki activates gene transcription by directly recruiting a specific H3K4 methyltransferase complex to Hippo target genes. Our ultimate goal is to define a complete Hippo signaling network that relays information from tissue architecture to nuclear gene transcription.
Hippo Signaling in Mammalian Development, Regeneration, and Tumorigenesis
Insights learned from Drosophila allow us to formulate specific hypotheses regarding the composition and physiological function of Hippo signaling in mammals, which can be tested using mouse genetics. Using a liver-specific tetracycline-inducible YAP transgenic model, we showed that YAP induction results in a robust and uniform expansion of liver size to five times normal, providing the first functional evidence that the Hippo pathway is a conserved regulator of organ size in mammals. Using knockout mouse models, we demonstrated that Merlin and Kibra play conserved roles in Hippo-mediated organ size control in mammals. These findings not only illustrate the predictive power of fly genetics but also help to clarify the ill-defined molecular functions of these proteins in mammalian physiology. Besides normal development and homeostasis, we are also interested in tissue regeneration and have uncovered a critical role for Hippo signaling in preventing excessive compensatory proliferation in a mouse model of intestinal regeneration.
An ongoing project in the lab is to develop inhibitors of the YAP oncoprotein, which is overexpressed/activated in a wide spectrum of human cancers. We have identified verteporfin (a drug currently used to treat macular degeneration) as a chemical that inhibits YAP function by disrupting the TEAD–YAP transcription factor complex, providing a proof of principle that targeting the TEAD–YAP complex is a pharmacologically viable strategy against the YAP oncoprotein. The discovery of Sd co-repressors and Yki-recruited H3K4 methyltransferase suggests additional modalities for targeting the YAP oncoprotein.
Hippo Signaling in a Unicellular Organism
Given its prominent role in organ size control, it was widely assumed that the Hippo pathway is relevant only in multicellular organisms. Through phylogenetic analysis coupled with functional validation, we recently refuted this assumption by showing that a biochemically conserved Hippo pathway is present in unicellular relatives of metazoa such as the ameboid Capsaspora. Remarkably, the Sd and Yki homologues from the unicellular Capsaspora form a functional transcription factor complex capable of driving massive tissue overgrowth in Drosophila; conversely, overexpression of the Capsaspora Hpo homologue in Drosophila decreases organ size. We are interested in understanding the ancestral function of Hippo signaling in this unicellular organism, which will shed light on the evolution of the Hippo pathway and potentially the genetic mechanisms underlying the transition from unicellular to multicellular life. From the reductionist point of view, unicellular organisms like Capsaspora offer a remarkably simple model for mechanistic understanding of Hippo pathway composition and regulation.
This research has been supported by grants from the National Institutes of Health and the Department of Defense.
As of February 25, 2016