RESUMEN
With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.
Asunto(s)
Analgesia , Dolor Crónico , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Ratas , Macaca , Receptores Opioides , Receptores Opioides mu/genética , TransgenesRESUMEN
FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. Here, we further characterize this phenomenon. We show that tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Factores de Transcripción Forkhead/genética , Mutación/genética , Organogénesis/genética , Animales , Proliferación Celular , Anomalías Congénitas/genética , Drosophila melanogaster/citología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas , Elementos Reguladores de la Transcripción/genética , Especificidad de la Especie , Serina-Treonina Quinasas TOR , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: The atypical Fat cadherin has long been known to control cell proliferation and organ size in Drosophila, but the mechanism by which Fat controls these processes has remained elusive. A newly emerging signaling pathway that controls organ size during development is the Salvador/Warts/Hippo pathway. RESULTS: Here we demonstrate that Fat limits organ size by modulating activity of the Salvador/Warts/Hippo pathway. ft interacts genetically with positive and negative regulators of this pathway, and tissue lacking fat closely phenocopies tissue deficient for genes that normally promote Salvador/Warts/Hippo pathway activity. Cells lacking fat grow and proliferate more quickly than their wild-type counterparts and exhibit delayed cell-cycle exit as a result of elevated expression of Cyclin E. fat mutant cells display partial insensitivity to normal developmental apoptosis cues and express increased levels of the anti-apoptotic DIAP1 protein. Collectively, these defects lead to increased organ size and organism lethality in fat mutant animals. Fat modulates Salvador/Warts/Hippo pathway activity by promoting abundance and localization of Expanded protein at the apical membrane of epithelial tissues. CONCLUSIONS: Fat restricts organ size during Drosophila development via the Salvador/Warts/Hippo pathway. These studies aid our understanding of developmental organ size control and have implications for human hyperproliferative disorders, such as cancers.
