Opposite Feedbacks in the Hippo Pathway for Growth Control and Neural Fate

Introduction: A finite number of signaling pathways are repurposed during animal development to regulate an extraordinary array of cellular decisions. Elucidating context-specific mechanisms is crucial for understanding how cellular diversity is generated and for defining potential avenues of pathway misregulation during disease. The Hippo tumor suppressor pathway has been primarily studied in growth control where it inhibits the oncogenic transcriptional coactivator Yorkie (Yki) (YAP/TAZ in vertebrates). The Hippo pathway also functions in nongrowth contexts such as postmitotic fate specification. In the Drosophila visual system, R8 photoreceptor neurons terminally differentiate into one of two alternative subtypes that express either blue-light–sensitive Rhodopsin5 (Rh5) or green-light–sensitive Rhodopsin6 (Rh6). These mutually exclusive cell fates are established by the Hippo pathway kinase warts and the growth regulator gene melted, which repress each other’s expression. However, the mechanisms underlying the context-specific use of the Hippo pathway in postmitotic fate decisions remain unclear. Context-specific regulation by the Hippo signaling in postmitotic photoreceptors. The Hippo pathway uses negative feedback through its transcriptional effector Yki for homeostatic control of proliferation. In Drosophila eyes, two alternative fates of blue- versus green-sensitive R8 photoreceptors are regulated by antagonism between the growth regulator Melted and the Hippo pathway. Contrary to the growth mechanism, Yki positive feedback and a cell-type–restricted transcription factor network promote repurposing of the Hippo pathway for binary fate decisions. Methods: To define the regulatory mechanisms of Hippo-dependent cell fate decisions in Drosophila photoreceptor neurons, we used a combination of genetic epistasis analyses, in vivo cis-regulatory studies, a candidate gene RNA interference screen, and cell culture–based transcription assays Results: We show that the transcriptional output of the Hippo pathway in photoreceptor differentiation, as in cell proliferation, is mediated through the factors Yki and Scalloped. In contrast to growth control, where Yki limits its own activity by negative feedback, we identify two Yki positive-feedback mechanisms: In blue-sensitive Rh5 photoreceptors, Yki represses its own negative regulator warts, downstream of melted; Yki also promotes melted expression, which subsequently represses warts to further promote Yki function. Yki cooperates with the transcription factors Orthodenticle (Otd) and Traffic Jam (Tj) to promote melted expression and Rh5 photoreceptor fate. Otd and Tj, othologs of the mammalian OTX/CRX and MAF/NRL transcription factors, form an evolutionarily conserved transcriptional module for generating photoreceptor subtype diversity. We also show that the transcription factors Senseless and Pph13 create a permissive environment for Warts/Hippo signaling to promote the alternative “default” green-sensitive Rh6 fate. Hence, Hippo pathway function integrates with four cell-type–restricted transcription factors, each promoting genetically different aspects of R8 subtypes, such that Yki activity ultimately coordinates the binary fate decision between blue- and green-sensitive photoreceptors. Discussion: This work illustrates how molecular signaling pathways can adopt context-specific regulation. Yki positive feedback in the photoreceptor fate decision is opposite to the negative feedback found in Hippo growth control. These distinct network-level feedback mechanisms provide context-appropriate functions: homeostasis to fine-tune growth regulation and an all-or-nothing fate decision to ensure robust differentiation of sensory neuron subtypes. Altering network-level systems properties, such as positive or negative feedback, within biochemically conserved pathways may be broadly used to co-opt signaling networks for use in cellular contexts as distinct as proliferation and terminal differentiation. Complexity and Diversity Complex organisms must produce and maintain an extraordinary diversity of cell and tissue types with a limited number of genes and molecular pathways. Cells accomplish this by reusing the same signaling networks at different times, in different tissues, and for different purposes, yet how this context-specificity is achieved is poorly understood. Jukam et al. (1238016, published online 29 August) show how a set of genes that function in cell and tissue growth can be used again in nondividing fly photoreceptor neurons to ensure that flies develop appropriate sensitivity to both blue and green light. The Hippo pathway undergoes a regulatory change—from negative to positive feedback—that requires a tissue-specific transcription factor network. This network uses evolutionarily conserved regulatory factors whose mutations in humans result in degenerative retinal diseases. The context-appropriate positive feedback in flies ensures an all-or-nothing fate decision necessary to establish a functional visual system. Hippo directs cell differentiation and fate through context- and tissue-specific feedback and transcription networks. Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network—including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module—that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.