Investigating mechanisms of tissue growth control during development in D. melanogaster

A remarkable feat of animal development is the precision with which organs grow to a/nconsistent and characteristic size. The lengths of the right and left arm of a human, for/ninstance, match with an accuracy of about 0.2%, and the size of adult mouse brains varies/nby only about 5%. How a developing organ|and the individual cells within|can know/nwhen it is time to stop growing has long-fascinated biologists./nReciprocal organ transplantation experiments from the 1920s were among the rst to/ndemonstrate that, for an individual organ, growth is subject to both extrinsic and intrinsic/nprograms. Working with two closely related but dierent-sized species of salamanders, the/nfaster-growing yet smaller-sized Ambystoma punctatum and the slower-growing yet largersized/nAmbystoma tigrinum, Harrison found that the growth rate of a limb transplanted from/none species to the other matched that of the host, but the overall size of the transplanted/nlimb matched that of the donor. He concluded, therefore, that while some circulating factor,/nperhaps a hormone, dictated the rate of limb growth, the organ's size overall was determined/nby some intrinsic "growth potential."/nNow, close to a century later, we know the identities of many evolutionarily-conserved/nfactors that are critical to achieving a properly-sized organ, but our understanding of what/nultimately determines an organ's size remains far from complete. Much of what we do know/nabout the mechanisms regulating tissue growth comes from studies using the model organism/nDrosophila melanogaster, whose genetic tractability and high reproducibility of organ size/nallow us to easily evaluate the eects of genetic perturbations on tissue growth./nIn Chapter 1, I provide an overview of some of these growth-regulatory mechanisms/nand describe how genetic studies using a well-characterized tissue model of growth|the/nDrosophila wing imaginal disc|have helped to provide some clues into how these mechanisms/nmight operate. I will highlight two signaling pathways|the Hippo pathway and the JNK/npathway|that have very dierent functions but seem to both impact tissue growth in unique/nways. Activities of both of these pathways have been areas of interest for my dissertation/nresearch./nThe Hippo pathway regulates growth in most multicellular organisms and is altered/n(either directly or indirectly) in many human cancers. Since the elucidation of the core/npathway approximately a decade ago, a key aim for researchers has been to identify the/nupstream signals that link this pathway to external cues and understand how these signals/nare sensed. An important upstream regulator of Hippo signaling is the transmembrane/nprotein Fat (Ft), though the mechanistic link between the core pathway and this upstream/nfactor is not fully clear. We identied the Drosophila F-box protein, Fbxl7, as a downstream/neector of Ft activity that is important for regulating the cellular distribution of Dachs, a/nprotein that mediates much of Ft's eect on tissue growth. This work, described in Chapter/n2, provides a more complete understanding of the functional link between Ft signaling and/nthe Hippo pathway./nThe JNK pathway is a well-conserved pathway involved in several morphogenetic processes/nduring development and commonly activated in response to stress. JNK signaling/npromotes apoptosis, yet|quite paradoxically|is also important in promoting tissue proliferation/nduring regeneration and tumorigenesis. The role this pathway plays in regulating/ngrowth during organ development is less established. We found that JNK signaling is active/nat low levels in the developing wing imaginal disc and regulates an enhancer of the/ngene bantam (ban), which encodes a microRNA that promotes growth. This ban enhancer/nactivity is opposed by the transcriptional co-repressor CtBP, which we characterize as a/nnegative growth-regulator in Drosophila. These ndings, described in Chapter 3, support a/nrole for JNK signaling in promoting tissue growth and suggest that CtBP may help to direct/nthis broadly-functioning signaling pathway towards specic eects on growth during normal/ndevelopment./nDuring the course of these studies, I made the surprising observation that a common/ngenetic technique used in Drosophila, namely shRNA-mediated gene knockdown using the/nGal4/UAS system, can lead to an unexpected result: clonal expression of shRNAs causes/nknockdown in cells that do not express Gal4. Chapter 4 describes this phenomenon, which we/nterm "shadow RNAi," and shows how this eect can lead to erroneous conclusions regarding/ncell-autonomous vs. non-autonomous genetic functions. We outline how shadow RNAi can/nbe mitigated, as well as how it can be exploited as an eective lineage-tracing tool./nTaken together, my work oers important clues for solving the mysteries of organ size/ncontrol and explores new applications of standard genetic techniques, while potentially providing/nuseful insights for the development of novel tools in cancer therapy and regenerative/nmedicine.

Advisor: Hariharan, Iswar K