Repurposing the Hippo pathway for cell fate specification
AuthorPojer, Jonathan Meir
AffiliationSir Peter MacCallum Department of Oncology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-02-17. This item is currently available to University of Melbourne staff and students only, login required.
© 2019 Jonathan Meir Pojer
The Hippo pathway is an important regulator of organ growth during development where it ensures correct scaling of tissues and organs. It is well-conserved in animals and deregulation of the pathway has been found to drive development of a range of cancers. The Hippo pathway was first identified in the fruit fly, Drosophila melanogaster, and has been extensively studied in a number of different tissues. As well as controlling organ growth in Drosophila, the Hippo pathway has been repurposed to control the binary cell fate choice of the R8 photoreceptor cell in the Drosophila eye. The R8 photoreceptor cell is one of eight photoreceptor cell types in the Drosophila eye and is responsible for much of colour vision in Drosophila. There are two main subtypes of R8 cells – around 30% are the 'pale' (p) subtype and express Rhodopsin 5 (Rh5; responsive to blue light); the remaining 70% are the 'yellow' (y) subtype and express Rh6 (responsive to green light). The Hippo pathway acts in a bistable feedback loop to both specify and maintain R8 subtype specification. This bistable feedback loop is composed of the Hippo pathway kinase, Warts, the downstream transcriptional coactivator, Yorkie, and the Pleckstrin-homology domain containing protein, Melted. Yorkie and Melted both promote pR8 cell fate, while Warts represses pR8 cell fate and promotes yR8 cell fate. Although we have begun to study the role of the Hippo pathway in R8 cell fate choices, we still lack a clear understanding how the Hippo pathway functions in these cells. I sought to further our understanding of the Hippo pathway in R8 cells by answering three key questions: (1) Which Hippo pathway proteins control R8 cell fate? (2) What is the subcellular localisation of Hippo pathway components in R8 cells? (3) What are the target genes of the Hippo pathway in each R8 subtype? (1) I used genetic experiments to modulate the expression of Hippo pathway genes and found that Crumbs and Cka promote pR8 cell fate, while the apical spectrin cytoskeleton and the 14-3-3 proteins promote yR8 cell fate. Modulating other Hippo pathway regulators did not obviously alter the ratio of R8 subtypes, suggesting that not all Hippo pathway proteins function in R8 cell fate choices. (2) Using confocal and multiphoton microscopy, I showed that only Warts differed in expression or localisation between R8 subtypes, suggesting that regulation of Warts levels in R8 cells controls the Hippo pathway's role in R8 cell fate. I also showed that the upstream Hippo pathway proteins, Tao, Merlin and alpha-spectrin are relocalised between late pupal and adult photoreceptor cells, hinting that there may be differences in their mechanisms of action between these two stages. (3) Targeted DamID-seq showed that Yorkie and its binding partner Scalloped reside at over a thousand loci in each R8 subtype. Yorkie and Scalloped target genes included known target genes from proliferating Drosophila tissues, as well as genes that are specific to photoreceptor cells. I also investigated the role of a target gene in pR8 cells, ninaB, and found that it promoted pR8 cell fate in male Drosophila retinas. These results provide insight into the role of the Hippo pathway in R8 cells and highlight similarities and differences between the roles of the Hippo pathway in proliferating cells and in R8 cell fate specification.
KeywordsDrosophila melanogaster; Hippo pathway; Cell fate specification; Binary cell fate choice; Eye development; Photoreceptor; R8 cell; DamID
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