Novel regulatory mechanisms of cell growth in Drosophila development

Abstract

Developmental cues regulate cell growth in coordinated with environmental conditions such as nutrients. However, the mechanisms for how nutrient-regulated signaling pathways are coordinated during development are largely unknown. Target of rapamycin complex I (TORC1) has been extensively investigated as a central regulator of cell growth in response to nutrients. As a first step for investigating how TORC1 is regulated during development of multicellular organisms, I generated the antibody for phosphorylated ribosomal protein S6 (p-S6). The specificity of p-S6 antibody was confirmed by immunostaining Drosophila tissues containing S6K, TOR and Raptor mutant cells. Immunostaining with p-S6 antibody revealed that TORC1 was selectively activated in S phase cells in the second mitotic wave (SMW) of Drosophila eye disc. When insulin signaling, well-known TORC1 upstream regulator, was disrupted, TORC1 activity was abolished, indicating that it is required for TORC1 activity. In contrast to TORC1, insulin signaling showed constant activity regardless of the location in wild type eye disc. Although endogenous Akt was active in the outside of the SMW, it did not lead to TORC1 activation. Moreover, activation of Akt induced TORC1 activation in the SMW, but not in the outside of the SMW. However, TSC1 mutation resulted in dramatic increase of TORC1 activity in the entire region of eye disc. These results indicated that Akt is not upstream of TSC1 in Drosophila eye disc. To confirm this possibility, I generated double mutant clones of Akt and TSC1. Expectedly, these clones abolished TORC1 activity in the entire region of eye disc including the SMW, indicating that TSC1 mutation requires Akt to induce TORC1 and that Akt acts on TORC1 in parallel with TSC and Rheb. Therefore, in the outside of the SMW, TSC1 inhibits the activation of TORC1 regardless of insulin signaling activity and undefined factors activate TORC1 at the upstream of or in parallel with TSC in the SMW. Hedgehog (Hh) signaling regulates furrow movement, neuronal cell fate determination and cell cycle progression in the eye disc. I investigated whether Hh signaling regulates TORC1 in the SMW. Interestingly, Smo mutant abolished TORC1 activity. In the absence of Hh signaling, the repressive Ci (CiR) is generated by protein kinase A (PKA)-mediated modifications and inhibits Hh target gene expression. Surprisingly, expression of dominant negative PKA restored the TORC1 activity in Smo mutant cells. In addition, knockdown of Ci mRNA also rescued the defective TORC1 activity in Smo mutant cells. Taken together, Smo activates TORC1 by inhibiting the formation of CiR via PKA in the SMW. Further epistatic analysis revealed that Smo is at the upstream of or in parallel with Rheb to activate TORC1. Subsequently, I also observed that TOR mutant clones exhibited delayed G1/S transition which was rescued by overexpression of cyclin A, indicating that TORC1 controls G1/S transition through cyclin A. Interestingly, overexpression of Rheb partially rescued the defective G1/S transition of Smo mutant cells, suggesting that Hh signaling regulates G1/S transition at the upstream of or in parallel with Rheb. Based on these results, I could propose novel mechanisms for the spatial regulation of TORC1 by insulin, TSC and Hh signaling in Drosophila eye development.

During fly development, various signaling pathways are converged to regulate cell growth. Discovery of novel regulators for cell growth is essential for understanding animal developmental regulation. In this study, I identified LTV1, a novel cell growth regulator in Drosophila. To investigate LTV1 in vivo function, I generated and characterized null mutant of LTV1. LTV1 mutant larvae exhibited developmental delay and lethality at second larval stage. I purified ribosomal protein S3 (RpS3) as a physical interactor of LTV1 by mass spectrometry analysis, suggesting LTV1 has a role in ribosomal function. Subsequent biochemical studies demonstrated that LTV1 is critical for 40S ribosomal subunit synthesis and pre-ribosomal RNA processing. In wing disc, decreasing LTV1 mRNA by RNAi induced cell death, indicating that LTV1 is also required for cell survival. As an upstream regulator of LTV1, I characterized Myc, an oncogenic transcription factor. Myc regulated LTV1 at the level of mRNA. Ectopic expression of Myc induced cell growth in wing disc and salivary gland. Interestingly, knockdown of LTV1 mRNA suppressed cell growth stimulated by Myc in those tissues. Furthermore, the increased amount of ribosomes and rough endoplasmic reticulum in Myc expressing salivary glands was suppressed by co-expression of LTV1 RNAi, suggesting that LTV1 is critical for Myc-induced cell growth and ribosome biogenesis in Drosophila. Taken together, I identified and characterized LTV1, a crucial regulator for cell growth and ribosome biogenesis at the downstream of Myc. My studies for the in vivo regulation of cell growth will be of help to understand how various cell signals interplay for cell growth and animal development.