Understanding intestinal development at the molecular level is critical for developing future methods of promoting organ regeneration and treatments for intestinal failure. Genetic screens in the zebrafish have identified numerous genes required for intestinal development that are also known to be involved in protein biosynthesis. A common feature of many of these protein biosynthesis genes is their regulation by the target of rapamycin (TOR). That mutations in genes controlled by TOR are selective to the digestive organs suggests that an additional common mechanism of control exists that provides organ specificity to the phenotype of these defects. It is our hypothesis that TOR is the leading candidate for mediating this specificity given its central role in regulating these anabolic pathways and their gene products. ? ? Testing this hypothesis in a future R01 proposal will require addressing experimentally several important questions: Does TOR activity vary in time and space during development? Does TOR activity correlate with particular morphological transitions? Are certain organs are more reliant on TOR activity than others? Are there subsets of cells in developing organs with high levels of TOR activity? Most importantly, what is the functional significance of TOR signaling during development? At present, there is no experimental system for addressing these questions. Accordingly, in this R21 exploratory proposal we aim to develop a novel tool we term the """"""""TORfish"""""""" that will allow us to address these questions experimentally by permitting the functional imaging of TOR activity in living vertebrate embryos. ? ? Specific Aim 1: We will create a novel transgenic zebrafish reporter line for functional imaging of TOR signaling activity in vivo (TORfish): In particular, we will generate dicistronic constructs containing a TOR-dependent reporter fused to a TOR-independent reporter to normalize for transcriptional variation, screen reporter constructs for rapamycin sensitive translation; and create transgenic lines with germline transmission of the reporter construct. ? ? Specific Aim 2: We will validate the TORfish and assess the role of TOR signaling during digestive organ development using the TORfish: In particular, we will (a) perform time-lapse fluorescence imaging of TORfish embryos during normal development, (b) phamacologically inhibit TOR with rapamycin or its analogs CCI-779 (Wyeth) and RAD-001 (Novartis), and (c) employ antisense morpholinos to knockdown TOR, its positive downstream effectors (p70S6 kinase, eIF4E), and its antagonists (PTEN, TSC1/TSC2, and 4E-BP). Successful development and validation of the TORfish will allow us to experimentally test our hypothesis that TOR signaling plays an important role in organ-specific organogenesis in a future RO1 proposal. The development of the TORfish in this R21 exploratory proposal therefore represents an important risk-reward hurdle that must be overcome in order to pursue future hypothesis-driven research in this system. ? ? ? ?
| Marshall, Kathryn E; Tomasini, Amber J; Makky, Khadijah et al. (2010) Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition. Dev Dyn 239:3000-12 |
| Makky, Khadijah; Tekiela, Jackie; Mayer, Alan N (2007) Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate intestine. Dev Biol 303:501-13 |
