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.
발생과정 동안 발생 신호는 영양분과 같은 환경에 따라 세포 성장을 조절한다. 그러나, 영양분에 의해 조절 받는 신호전달계가 발생과정 중에 어떻게 조절 받는지에 대해 잘 알려지지 않았다. TOR complex 1 (TORC1) 신호전달계는 영양분에 반응하여 세포 성장을 조절하는 신호전달계로서 많은 연구가 수행되어왔다. 초파리에서 TORC1의 조절 기작을 규명하기 위해, 리보솜 단백질 S6의 인산화를 인지하는 항체를 제작하였다. 흥미롭게도, 초파리 눈 성충판에서 second mitotic wave (SMW) 의 S phase에 들어가있는 세포들에서 TORC1이 특이적으로 활성을 갖는다는 것을 확인하였다. 이어서, TORC1의 상위 조절자로 알려진 인슐린 신호전달계이 SMW에서 TORC1 활성에 필요하다는 것을 밝혀내었다. 또한 TORC1과 다르게 인슐린 신호전달계는 눈 성충판에서 위치와 상관없이 균일한 활성을 갖으며, SMW가 아닌 위치에서 Akt는 활성화 되어 있지만, 이는 TORC1을 활성화 시키지 못하는 것을 확인하였다. 반면, TSC complex의 돌연변이는 눈 성충판 모든 지역에서 TORC1을 활성화시켰으며, 여기에는 Akt가 필요한 것으로 보아, Akt와 TSC가 평행하게 TORC1을 조절함을 알 수 있었다. Hedgehog (Hh) 신호전달계는 morphogenetic furrow의 생성과 이동, 세포 운명의 결정, SMW에서의 세포 분열 등을 조절하는 인자로 연구되어왔다. 유전학적 방법을 이용하여, Smoothened (Smo) 는 SMW에서 억제인자 형태의 Ci가 형성되지 못하게 함으로서, TORC1의 활성에 필요한 것을 밝혀내었고, Smo가 Rheb의 상위에서 또는 Rheb와 평행하게 TORC1을 조절함을 확인하였다. 또한, TOR 돌연변이 세포에서 G1/S phase 변환이 지연되는 것을 관찰하였다. Smo 돌연변이 세포에서 G1/S phase 변환이 억제되어 있는데, Rheb를 과발현시킨 경우 이러한 결점이 부분적으로 회복되는 것을 확인하였다. 본 연구를 통해 초파리 눈 발생에서 인슐린, TSC 그리고 Hh 신호전달계에 의한 공간적인 TORC1 조절 기작을 제시하고자 한다.
초파리 발생에서는 다양한 신호전달계가 상호 협동하여 세포성장을 조절한다. 새로운 세포 성장 조절인자의 발굴은 발생의 이해에 필수적이다. 본 연구에서, 새로운 세포 성장 조절인자인 LTV1이 초파리의 지방세포에서 세포 성장에 필요하다는 것을 확인하였다. 생화학적 방법을 통해 리보솜 단백질 S3가 LTV1과 결합함을 확인하였다. 이어서, LTV1이 40S 리보솜 소단위의 합성에 필요하며, 리보솜 RNA 전구체의 변형 과정에 필요함을 밝혀내었다. 또한 초파리 날개 성충판에서 LTV1을 제거하였을 때, 세포 사멸이 일어나는 것을 확인하였다. 발암성 전사인자인 Myc이 LTV1의 mRNA 양을 조절하며, Myc에 의한 세포 성장과 리보솜 생합성의 증가에 LTV1이 필요하다는 것을 밝혀내었다. 종합적으로, 본 연구는 생체내에서 다양한 신호전달계가 어떻게 상호작용하여 세포 성장과 발생을 조절 기작을 이해하는데 도움을 줄 수 있을 것이다.
