第412次SKLBE 学术论坛

以下为第412次SKLBE 学术论坛信息，请阅。

第412次SKLBE 学术论坛——“斑马鱼与疾病模型研究”系列报告

时间：2018-4-24  9:00-15:00

地点：实验十八楼315室

主持人：刘琴教授

报告内容：

9:30-10:00 主题报告（1）

报告题目：Hemodynamic-mediated endocardial signaling regulates ventricle regeneration

报告人：张瑞霖研究员复旦大学生命科学学院

10:00-10:30主题报告（2）

报告题目：基于斑马鱼研究Irf8调控髓系细胞发育及髓系白血病发生的分子机理

报告人：李礼教授西南大学生命科学学院

10:30-11:00 主题报告（3）

报告题目：细胞代谢成像新技术与药物发现

报告人：赵玉政特聘研究员华东理工大学

11:00-11:30主题报告（4）

报告题目：雷帕霉素靶蛋白（mTOR）通路对适应性免疫的调控•从哺乳动物到硬骨鱼

报告人：杨嘉龙研究员华东师范大学生命科学学院

13:30-14:00 主题报告（5）

报告题目：Reprogramming of lipid metabolism in transgenic zebrafish models of hepatic tumorigenesis

报告人：王旭青年研究员复旦大学基础医学院

14:00-14:30 主题报告（6）

报告题目：斑马鱼NOD like receptor在巨噬细胞发育及分枝杆菌感染中的作用

报告人：晏博副研究员上海市公共卫生临床中心

14:30-15:00主题报告（7）

报告题目：Unlocking the non-canonical inflammasome activation in zebrafish

报告人：阳大海博士华东理工大学

Hemodynamic-mediated endocardial signaling regulates ventricle regeneration

Ruilin Zhang  (张瑞霖)

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University

E-mail: zhangruilin@fudan.edu.cn

Unlike the human heart, the zebrafish heart possesses a strong regenerative capacity in response to myocardial injury.  However, how cardiac injury initiates signaling pathways that control this crucial regenerative process remains to be defined.  Previously we have observed that Notch signaling becomes activated in the atrial endocardium following ventricular injury and this activation is required to promote ventricular myocardial regeneration through reprogramming of atrial cardiomyocytes.  Here, we utilize a combination of dynamic biophysical analysis and in vivo imaging to reveal that altered hemodynamic forces due to cardiac injury activate a sequential endocardial-myocardial signaling cascade to direct cardiomyocyte reprogramming and heart regeneration.  Overall, our finding not only reveals how the heart senses environmental changes due to cardiac injury to adaptively respond but also provides insight into new signaling mechanisms that may be manipulated to stimulate cardiomyocyte reprogramming and regeneration.

基于斑马鱼研究Irf8调控髓系细胞发育及髓系白血病发生的分子机理

李礼

西南大学生命科学学院

E-mail: swu_lili@126.com

Interferon regulatory factor (IRF)-8 is a critical transcription factor involved in the pathogenesis of myeloid neoplasia. However, the underlying mechanisms in vivo are not well known. Investigation of irf8 mutant zebrafish in this study indicated that Irf8 is evolutionarily conserved as an essential neoplastic suppressor through tight control of the proliferation and longevity of myeloid cells. Surviving irf8 mutants quickly developed a myeloproliferative neoplasm (MPN)-like disease with enhanced output of the myeloid precursors, which recurred after transplantation. Multiple molecules presented notable alteration and Mertk signaling was aberrantly activated in the hematopoietic cells in irf8 mutants. Transgenic mertk overexpression in Tg(coro1a:mertk) zebrafish recapitulated the myeloid neoplasia-like syndrome in irf8 mutants. Moreover, functional interference with Mertk, via morpholino knockdown or genetic disruption, attenuated the myeloid expansion phenotype caused by Irf8 deficiency. Therefore, Mertk signaling is a critical downstream player intheIrf8-mediatedregulation of the progression of myeloid neoplasia. Our study extends the understanding of the mechanisms underlying leukemogenesis.

细胞代谢成像新技术与药物发现

赵玉政

生物反应器工程国家重点实验室，华东理工大学

E-mail：yuzhengzhao@ecust.edu.cn

原位、高时空分辨地检测细胞代谢状态是生命科学研究的一个瓶颈问题和迫切需求。依赖于细胞裂解的传统生化方法难以实时追踪代谢物变化，更难以应用于高通量筛选。针对细胞内核心代谢物NADH和NADPH，我们开发了系列遗传编码的荧光探针，实现了在活体、活细胞及亚细胞中对细胞代谢的高时空分辨检测与成像，并建立了高通量代谢分析技术，解决了细胞代谢研究中两个关键技术瓶颈问题。这些细胞代谢荧光探针不仅可应用于代谢途径与通路分析，也可用于衰老及相关疾病创新药物的发现。目前全球已有来自哈佛大学、麻省理工学院、牛津大学、剑桥大学等200多个国内外一流机构实验室使用相关检测技术。

主要参考文献：

1. Zhao, Y.*, Zhang, Z., Zou, Y., Yang, Y.* Visualization of nicotine adenine dinucleotide redox homeostasis with genetically encoded fluorescent sensors. Antioxidants & Redox Signaling, 2018, 28(3), 213-229.

2. Hu, H., et al. Monitoring cellular redox state under hypoxia using a fluorescent sensor based on eel fluorescent protein. FreeRadicalBiology&Medicine, 2018, online.

3. Tao, R.#, Zhao, Y.#, Chu, H.#, et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. (#Co-first authors). Nature Methods, 2017, 14(7), 720-728.

4. Zhao, Y., et al. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state. Nature Protocols, 2016, 11(8), 1345-1359. (Cover story).

5. Zhao, Y., et al. SoNar, a highly responsive NAD (+) /NADH sensor, allows high-throughput metabolic screening of anti-tumor agents. Cell Metabolism, 2015, 21(5), 777-789.

6. Zhao, Y., et al. Genetically encoded fluorescent sensors for intracellular NADH detection. Cell Metabolism, 2011, 14(4), 555-566.

7. Zhao, Y., and Yang, Y. Profiling metabolic states with genetically encoded fluorescent biosensors for NADH. Current Opinion in Biotechnology, 2015, 31, 86-92.

8. Zhao, Y.*, and Yang, Y*. Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD+/NADH sensors. FreeRadicalBiology&Medicine, 2016, 100, 43-52.

雷帕霉素靶蛋白（mTOR）通路对适应性免疫的调控——从哺乳动物到硬骨鱼

杨嘉龙

华东师范大学生命科学学院

E-mail: jlyang@bio.ecnu.edu.cn

雷帕霉素靶蛋白（mTOR）通路对适应性免疫的调控是生命科学研究中蓬勃发展的前沿领域。本研究分别以小鼠和尼罗罗非鱼为对象，探讨了mTOR通路对哺乳动物和硬骨鱼（T）淋巴细胞介导适应性免疫的调控机制。在哺乳动物的研究中发现，T细胞中敲除mTOR造成组成型和诱导型滤泡辅助性T（Tfh）细胞和GC-B细胞数量的降低，并进而影响抗体的产生；mTOR通路的两个蛋白复合体mTORC1和mTORC2通过截然不同的机制调控了Tfh细胞的分化过程；mTORC1通过细胞增殖的途径、而mTORC2通过AKT/GSK3β/β-catenin通路促进转录调控因子TCF1的表达，分别调控了Tfh细胞的分化。在硬骨鱼的研究中发现，尼罗罗非鱼具有保守的mTOR通路，且该通路调控了淋巴细胞的激活、增殖、和其介导的感染清除等过程。虽然相关研究仍在继续，已有的证据表明硬骨鱼可能使用了与哺乳动物类似的mTOR通路调控了淋巴细胞介导的适应性免疫应答。研究结果为从全新的视角揭示硬骨鱼适应性免疫的调控机制，和总结适应性免疫系统的进化提供重要的证据和参考。

Reprogramming of lipid metabolism in transgenic zebrafish models of hepatic tumorigenesis

Xu Wang (王旭)

Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University,

E-mail: wangxu2013@fudan.edu.cn

1) There is limited understanding of the effects of major oncogenic pathways on lipid metabolism in hepatocellular carcinoma (HCC). Here, we observed a negative correlation of Wnt/Myc activity with steatosis in human HCC and performed in vivo functional exploration using zebrafish. Transgenic zebrafish larvae conditionally expressing human CTNNB1mt and zebrafish tcf7l2 or murine Myc in hepatocytes led to hepatomegaly and a significantly attenuated accumulation of lipid droplets triggered by krasv12 expression. Lipidomics studies further revealed that Wnt/Myc activity promotes triacylglycerol to phospholipid transformation in a Ras-dependent manner, while also increasing unsaturated fatty acyl groups in phospholipids. Additionally, middle-scale screenings suggest that supplementation of certain free fatty acids (FA) and/or inhibition of FA desaturation represses zebrafish hepatic hyperplasia and proliferation of human HCC cells.  2) At whole organism level, transgenic zebrafish overexpressing murine Myc and zebrafish krasv12 in hepatocytes developed fast hepatocellular carcinoma (HCC) and anorexia/cachexia-like phenotypes with extensively increased leptin expression. Genetic knockout of either lepr or mc4r improved the orexigenic neuroendocrine axis, and partially restored the appetite. Homozygous lepr mutation or Stat3 inhibition, but not mc4r mutation significantly rescued the wasting of skeletal muscle and white adipose tissue around mesentery. Further investigation using both zebrafish and mammalian cell lines indicated that high dosage of leptin directly induced insulin resistance via Socs3 in normal peripheral tissues, but not significantly in the tumorized hepatocytes, while the inhibition of Stat3 partially recovered the insulin sensitivities in muscle and adipose tissues. Together, our studies provide in vivo evidence that classical oncogenic pathways play a dual role in modulating the nutritional metabolism of both the tumor and non-tumor tissues in zebrafish HCC models, demonstrating zebrafish as a powerful and convenient platform for studying HCC-CACS conditions.

斑马鱼NOD like receptor在巨噬细胞发育及分枝杆菌感染中的作用

晏博

上海市公共卫生临床中心，新发与再现传染病研究所

E-mail: bo.yan@shphc.org.cn

小胶质细胞作为常驻脑部的巨噬细胞可以吞噬程序性死亡的神经细胞和外源性病原微生物，被证明在脑发育和脑部感染中起重要的作用。我们的前期工作中针对斑马鱼脑部巨噬细胞缺陷做了大规模正向遗传学筛选，发现了一个温敏型的脑部巨噬细胞缺失突变体，并成功定位其基因突变位于一个NOD like receptor中。通过活体实时成像观察，该突变体中的小胶质细胞缺失被证明是由于突变体小胶质细胞在发育过程中会有更多的细胞死亡，并且在形态学呈现出细胞焦亡的典型表型。进一步遗传学分析，我们发现该突变体表型可以通过阻断 caspase-1通路而被得到部分回补。同时为了探究该基因在感染性疾病中的作用，我们用海分枝杆菌感染该基因缺失的斑马鱼，发现细菌体内的增殖被抑制。我们拟利用活体实时成像，基因敲除等技术进一步验证其机制是否是由于在分枝杆菌感染状态下该基因的缺失可以诱导巨噬细胞焦亡从而起到控制感染的作用。

Unlocking the non-canonical inflammasome activation in zebrafish

Dahai Yang (阳大海)

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China 200237;

Shanghai Engineering Research Center of Marine Cultured Animal Vaccines, Shanghai, China 200237

E-mail: dahaiyang@ecust.edu.cn

The non-canonical inflammasome is critical for cytosolic sensing of gram-negative pathogens in the innate immune responses. In the present study, we demonstrated that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates significant pyroptosis via caspase-5 activity. Zebrafish caspy2 binds directly to lipopolysaccharide via its N-terminal pyrin death domain, resulting in caspy2 oligomerization, which correlates with the catalytic activity for pyroptosis. Furthermore, we revealed that caspy2 is highly expressed in the zebrafish gut and is significantly activated during infection. Essentially, knockdown of caspy2 expression impaired the ability of zebrafish to restrict bacterial invasion in vivo, and also protected larvae from lethal sepsis. Collectively, our results offer a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular LPS-sensing pathway in innate immunity, and also highlight the double-sided roles of the non-canonical inflammasome in modulating infection in lower vertebrates.