近年研究主題

動物早期胚發育之訊息傳導機制

研究室簡介

胚胎發育是一個複雜的動態過程,更是一個迷人的研究課題。單倍體配子成熟後,精卵結合形成雙倍體之合子,合子卵裂形成多細胞胚,內中外三個胚層則在在囊胚期形成。之後依循著前後、背腹及左右體軸進行包括神經及心血管各類器官生成以形成一個全新成熟之個體來繁衍下一代。在這一連串過程中各個細胞基因體之個別基因活性必須在胚發育期間被精準地調控以發育成形態功能正常之個體,任何一個錯誤都可能造成個體之缺陷甚至死亡。我們實驗室即利用分子生物及胚胎學之方法輔以現代體學及顯微鏡顯影技術來研究斑馬魚胚發育過程之基因網絡及訊息傳遞機制。

斑馬魚模式

斑馬魚為新興脊椎動物模式物種,其早期胚快速分裂(每十五分鐘一次)、腔腸化、神經及大部分器官於2-3天內大致發育完成,而其早期胚體透明極適合各類組織學及細胞學之研究。其基因體已被完全定序,包括基因轉殖、突變誘導、基因弱化、 剔除及各類體學生物資訊等方法也都可在斑馬魚進行,更由於其世代間距短(3個月)、體型小(約一吋)可大量飼養、胚數量多(約200顆)且可多次重複產卵,使斑馬魚相較於其他脊椎動物如小鼠在發生學及遺傳學上之研究佔有相對優勢。再者斑馬魚基因與人及其他哺乳類基因相當類似,斑馬魚有至少70%人類疾病相關之同源基因,因此許多種不同疾病相關模式也在斑馬魚被建立,這也墊定其在醫學研究之地位。
adult  zebra fish

腔腸化

斑馬魚胚早期分裂及腔腸化(Gastrulation)主因細胞骨架蛋白質,肌動蛋白(actin)及微管(microtubule)動態變化。肌動蛋白絲及微管變化主要調節因子之一為小三磷酸鳥苷水解脢(small GTPases)即Rho、Rac及Cdc42,及其上下游訊息傳導因子,我們過去一系列之研究利用藥理性抑制及基因弱化方法證明了Rho及其下游因子ROCK、diaphanus 2及肌動蛋白結合蛋白profilin及cofilin均為斑馬魚胚細胞質分裂及卵黃包覆(epiboly)和腔腸化過程中細胞遷徙所必須之因子,後續我們將持續探討Rho, Rac及Cdc42及其上下游訊息傳導因子(如Wnt及FAK)在斑馬魚腔腸化之調控機制。
Gast

胚體軸決定與器官發育調控

胚胎形態發育及後續器官之生成端賴早期體軸適時之正確建立,體軸不正常常導致早期胚死亡或後續之個體缺陷,因此胚體軸決定與發育調控為發生生物學重要之基礎議題。在我們先前的研究中,我們發現弱化水解磷酯酸(lysophosphatidic acid, LPA)的一個受體基因lpar3導致幼魚產生心臟及其他器官左右不對稱之缺陷,進一步之試驗證實LPA可能經由調控庫式泡(Kupffer's Vesicle)來調控胚體軸之左右不對稱性,而影響後續包括心臟等器官之錯置及功能異常。再則我們也發現了另一個LPA受體Lpar1是斑馬魚淋巴管發育所必需,然而LPA在胚發育過程之生成調控卻仍未知,這也是我們未來研究課題之一。

低溫誘導轉錄體及微核醣核酸分析

臺灣冬季寒流侵襲常導致氣溫之急遽下降而造成大量養殖魚類之死亡及經濟上之損失,因此魚類低溫生物學及其應用長久以來一直是台灣水產養殖管理之重要課題。前人之研究已找到幾個冷誘導基因,也利用基因微矩陣之方法瞭解在冷適應下較全面性之基因表現變化。然而冷適應為較緩和之方法,無法全然反應寒流氣溫急遽變化下之反應。因此我們利用次世代定序法來探討在急遽低溫變化下觀察斑馬魚幼魚之生理反應及基因表現變化。本試驗之完成將有助於我們瞭解急遽低溫在斑馬幼魚所導致之生理、基因調控及其影響所及之訊息網路變化。更重要的是,此一系統性之研究未來將可能提供我們建立多個可抗寒害之魚類模式,對未來漁業寒害管理將有重大助益!

斑馬魚人類疾病模式之建立

如前述斑馬魚為最近人類疾病相當重要之模式動物,與台大醫院醫師合作我們已成功建立芳香族L-胺基酸類脫羧基酶缺乏症一種小兒罕見疾病的斑馬魚模式,我們將目前也正利用CRISPR/Cas9之技術建立如人類心臟病及早衰症模式,並以之探討其致病機轉及可能治療方法。
AADC


Research Goals

Embryonic development is a fascinating, complicated and dynamic process. Mature haploid gametes fuse to become a diploid zygote. The zygote undergoes rapid clevages and followed by the formation of blastocoel and three germ layers during gratrulation. Organs develop according to three embryonic axis and thus different systems, including reproductive system, to become a sexually mature adult. During these series of development, differential gene expression in each individual genome of each cell needs to be precisely regulated in correct spatial and temporal orders to shape a normal functional organism. Any mistake may result in organismal defect or even death. Thus, we aim to use contemporary biological tools to investigate signal transduction mechanism during these miracle processes.

Zebrafish Model

Zebrafish is an emergent vertebrate model. The fertilized zebrafish zygote undergoes rapid cleavages at 15 min intervals. After the cleavage period, the embryo has dramatically changes in morphology during gastrulation to form three germ layers. Neurogenesis and most organs develop within 2-3 days. Early zebrafish embryos are transparent that allows cellular and histological analyses. Its genome has been completely assembled. The manipulation of zebrafish genome is also feasible by different methods, including transgenesis, ENU mutagenesis, morpholino knockdown and TALEN/CRISPR knockout. In addition, omic studies are also widely used in zebrafish. Due to its small size (~ an inch) which can be reared in large population, short generation time (3 months), large clutches (~200 embryos per spawning), multiple ovulation (females can lay eggs in 7-10 days)、zebrafish has clear advantages compared that other vertebrates like mice to used in developmental biology and genetic studies. Furthermore, zebrafish genes are highly homologous to mammals including human. It has been estimated that zebrafish genome contains over 70% human disease-related genes that make zebrafish a great candidates for making human disease animal models. In fact many models have been established.
adult  zebra fish

Gastrulation

Dramatic morphological changes occur during cleavage and gastrulation. These changes are mainly due to the dynamic regulation of cytoskeletal actin and microtubule. Small GTPases, Rho, Rac and Cdc42, are known regulators for the dynamic changes of cytoskeletal proteins. In a series of studies, we have explored the Rho-mediated signaling in controlling the progression of cytoplasmic cleavages and cell migration during gastrulation in zebrafish. Using pharmacological and gene knockdown analysis, we have demonstrated how Rho exerts its action via downstream factors like ROCK, diaphanus 2 and actin binding protein profilin and cofilin. We are also investigating upstream or co-regulators of small GTPases like Wnt and FAK for their roles in gastrulation cell migration.
Gast

Embryonic axis formation and organogenesis

During embryonic development, the correct patterning of morphogenesis and organogenesis rely on the early establishment of body axes. Abnormal embryonic axes often cause early lethality or later defects. Therefore, the determination and regulation of embryonic axis are fundamental questions in developmental biology. Previously, we have accidentally observed that knockdown of lysophosphatidic acid (LPA) receptor 3 (lpar3) causes defects in blood circulation and left-right asymmetry. Further experiments demonstrated that LPA may exert its effect on the formation of Kupffer's vesicle, a transient structure equivalent to mammalian node, which is a central regulator of left-right axis. Our earlier work also revealed the necessity of another LPA receptor, Lpar1, in lymphatic vessel formation. It appears that LPA may be an essential signal for embryonic development. However, we are not yet able to demonstrate the existence of LPA during embryonic development. In addition, the regulation of LPA production and metabolism is also topics of interest for our future interest.

Cold-induced transcriptome and micro RNA analyses

In Taiwan, the sudden reduction of temperature often results in massive death of aquacultures species and huge economical loss in winter. Thus, the study of cold biology in fish and its application is an important issue in fisheries. Previously, researchers have found several cold-inducible genes and also used microarray analysis to study cold aclimed genes in fish. However, clod acclimation is a gradual adaptation to environmental changes. It may not reflect the sudden drop in temperature occurs during cold front. Thus, we have used next generation sequencing to analyze changes in transcriptome and miRNA in zebrafish larva treated without or with cold shock. We aim to have a systematic understanding of cold-induced signaling gene and miRNA network for father design of anti-cold strategies in Fishery management.

Establishing zebrafish as a human disease model

Zebrafish is an emergent model for human diseases. In cooperation with NTU hospital, we have established a model for a rare pediatric disease, AADC. One of our goals is to build more disease models to be used for studying diseases mechanisms and therapy. We have collaborated with MDs to use zebrafish for human disease models like AADC, heart and progeria (premature aging) diseases.
AADC


代表著作

Publications

期刊論文 Journal
  1. Lin, M.J., Lee, S.J., 2016. Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae. Sci Rep 6, 36188. Pubmed
  2. Hung, I.C., Hsiao, Y.C., Sun, H.S., Chen, T.M., Lee, S.J., 2016. MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae. BMC genomics 17, 922. Pubmed
  3. Lee, S.J., Tsao, K.C., Cherng, B.W., Liao, Y.H., 2015. Lysophospholipid receptor signaling in zebrafish development. Transl Cancer Res 4, 544-556. Pubmed
  4. Shih, D.F., Chu, C.L., Lee, S.J., 2014. Characterization and expression analysis of stathmin family genes during embryogenesis in zebrafish, Danio rerio. Taiwania 59, 262-280. Pubmed
  5. Han, H.W., Chou, C.M., Chu, C.Y., Cheng, C.H., Yang, C.H., Hung, C.C., Hwang, P.P., Lee, S.J., Liao, Y.F., Huang, C.J., 2014. The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Express Pubmed
  6. Tu, C.F., Tsao, K.C., Lee, S.J., Yang, R.B., 2014. SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development. J Biol Chem 289, 18928-18942. Pubmed
  7. Lee, S.J., 2014. Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly. Biochemical and biophysical research communications 452, 1-7. Pubmed
  8. Hung, I.C., Cherng, B.W., Hsu, W.M., Lee, S.J., 2013. Calnexin is required for zebrafish posterior lateral line development. Int J Dev Biol 57, 427-438. Pubmed
  9. Shih, D.F., Hsiao, C.D., Min, M.Y., Lai, W.S., Yang, C.W., Lee, W.T., Lee, S.J., 2013. Aromatic L-Amino Acid Decarboxylase (AADC) Is Crucial for Brain Development and Motor Functions. PLoS ONE 8, e71741. Pubmed
  10. Tsao, K.C., Tu, C.F., Lee, S.J., Yang, R.B., 2013. Zebrafish scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis. J Biol Chem 288, 5017-5 Pubmed
  11. Moolenaar, W.H., Houben, A.J., Lee, S.J., van Meeteren, L.A., 2013. Autotaxin in embryonic development. Biochimica et biophysica acta 1831, 13-19. Pubmed
  12. Hu, C.W., Tseng, C.W., Chien, C.W., Huang, H.C., Ku, W.C., Lee, S.J., Chen, Y.J., Juan, H.F., 2013. Quantitative Proteomics Reveals Diverse Roles of miR-148a from Gastric Cancer Progression to Neurological Development. Journal of proteome research 12, 399 Pubmed
  13. Lai, S.L., Yao, W.L., Tsao, K.C., Houben, A.J., Albers, H.M., Ovaa, H., Moolenaar, W.H., Lee, S.J., 2012. Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish. Development 139, 4439-4448.Pubmed
  14. Chiang, C.L., Chen, S.S., Lee, S.J., Tsao, K.C., Chu, P.L., Wen, C.H., Hwang, S.M., Yao, C.L., Lee, H., 2011. Lysophosphatidic Acid Induces Erythropoiesis through Activating Lysophosphatidic Acid Receptor 3. Stem Cells 29, 1763-1773.Pubmed
  15. Chen, C.F., Chu, C.Y., Chen, T.H., Lee, S.J., Shen, C.N., Hsiao, C.D., 2011. Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo. PLoS ONE 6, e20654. full text
  16. Yeh, C. M., Liu, Y. C., Chang, C. J., Lai, S. L., Hsiao, C. D., and Lee, S.J.* (2011) Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish.PLoS ONE6(4), e18302. full text.
  17. Tseng, Y. C., Lee, J. R., Lee, S.J., and Hwang, P. P. Functional analysis of the glucose transporters-1, -6, and -13.1 expressed by zebrafish epithelial cells. Am J Physiol Regulatory Integrative Comp Physiol 22, 423-442. Pubmed
  18. Lin, C. W., Yen, S. T., Chang, H. T., Chen, S. J., Lai, S. L., Liu, Y. C., Chan, T. H., Liao, W. L., and Lee, S.J.* (2010) Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish. PLoS ONE 5 (12), e15331. full text
  19. Kapoor, A., Hsu, W. M., Wang, B. J., Wu, G. H., Lin, T. Y., Lee, S.J., Yen, C. T., Liang, S. M., and Liao, Y. F. (2010) Caveolin-1 regulates gamma-secretase-mediated AbetaPP processing by modulating spatial distribution of gamma-secretase in membrane. J Alzheimers Dis 22, 423-442. Pubmed
  20. Lai, S.L., Chan, T.H., Lin, M.J., Huang W.P., Lou S.W. and Lee, S.J.*(2008). Diaphanous-related formin 2 and profilin I are required for gastrulation cell movements. PLoS ONE 3 (10), e3439. full text
  21. Lee, S.J.*, Chan, T.H., Chen, T.C., Liao, B.K., Hwang, P.P., Lee, H. (2008). LPA1 is essential for lymphatic vessel development in zebrafish. FASEB J. 22(10), 3706-3715. Pubmed
  22. Lin, C.I., Chen C.N., Huang M.T., Lee, S.J., Lin, C.H., Chang C.C. and Lee, H. (2008). Lysophosphatidic acid upregulates vascular endothelial growth factor-C and lymphatic marker expressions in human endothelial cells. Cellular and Molecular Life Sciences 65(17), 2740-51. Pubmed
  23. Lin, C.I., Chen C.N., Huang M.T., Lee, S.J., Lin, C.H., Chang C.C. and Lee, H. (2008). Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA1/3, COX-2, and NF-κB activation and EGFR transactivation-dependent mechanisms. Cell Signaling 20 1804-1814. Pubmed
  24. Tseng, U.C. Lee, J.R., Chang, J.C.H., Kuo, C.H., Lee, S.J. and Hwang, P.P. (2008). Regulation of lactate dehydrogenase in tilapia (Oreochromis mossambicus) gills during acclimation to salinity. Zoological Studies 47, 473-480. PDF
  25. Lee, S.J.*, Ju, C.C., Chu, S.L., Chien, M.S., Chan, T.H. and Liao, W.L. (2007). Molecular cloning and expression analysis of parvalbumin in tilapia, Oreochromis mossambicus. J Exp Zool Part A 307, 51-61. Pubmed
  26. Chen, Y. M., Lee, T. H., Lee, S.J., Lin, J. Z., Huang, R. and Chou, H. N. (2006). Potential of a simple solid-phase extraction method coupled to analytical and bioanalytical methods for an improved determination of microcystins in algal samples. J Chromatogr B 844, 134-41. Pubmed
  27. Chen, Y. M., Lee, T. H., Lee, S.J., Huang, H. B., Huang, R. and Chou, H. N. (2006). Comparison of protein phosphatase inhibition activities and mouse toxicities of microcystins. Toxicon 47, 742-6. Pubmed
  28. Chu, S.L., Weng, C.F., Hsiao, C.D., Hwang, P.P., Chen, Y.C., Ho, J.M. and Lee, S.J.*. (2006). Profile analysis of expressed sequence tags derived from the ovary of tilapia, Oreochromis mossambicus. Aquaculture 251, 537-548. Pubmed
  29. A. Robinson, J.M. Fang, P.T. Chou, K.W. Liao, R.M. Chu, and S.J. Lee. Probing Lectin and Sperm Based on Carbohydrate Modified Quantum Dots. 2005. ChemBioChem. 6, 1–8. Pubmed
  30. S.L. Lai, C.N. Chang, P.J. Wang and S.J. Lee*. Rho mediates cytokinesis and epiboly via ROCK in Zebrafish. 2005. Molecular Reproduction and Development. 71, 186-196. Pubmed
  31. P.Z. Wang, M.S. Chien, F.J. Wu , H.N. Chou and S.J. Lee*. Inhibition of embryonic development by micocystin-LR in zebrafish, Danio Rerio. 2005. Toxicon 45, 303-308. Pubmed
  32. S.J. Lee, G. Stapleton, J.H. Greene, and M.B. Hille. Protein kinase C-related kinase 2 (PRK2) phosphorylates protein synthesis initiation factor 4E in starfish oocytes. 2000. Developmental Biology 228, 166-180. Pubmed
  33. Shen, S.-S., Kinsey, W.-H. and Lee, S.-J. (1999) Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg: Development, Growth and Differentiation, 41(3), 345-355. (SCI) Pubmed
  34. Lee, S.-J. and Shen, S.-S. (1998) U73122 blocked the cGMP-induced calcium release in sea urchin eggs: Experience Cell Research, 242, 328-340. Pubmed
  35. Lee, S.-J. and Shen, S.-S. (1998) The calcium transient in sea urchin eggs during fertilization requires the production of inositol 1,4,5-trisphosphate: Developmental Biology, 193, 195-208. Pubmed
  36. Lee, S.J., L. Christenson, T. Martin and S.S. Shen. The cGMP-mediated calcium release pathway in sea urchin eggs is not required for the rise in calcium during fertilization. 1996. Developmental Biology. 180, 324-335. Pubmed

開設課程

  • 基因表現分析及實驗
    Gene Expression Analysis And Laboratory
  • 碩士班專題討論
    Seminar (M.S)
  • 發生學專題討論
    Seminar in Developmental Biology
  • 早期胚胎發生學
    Early Developmental Embryology
  • 書報討論(一)
    Seminar (B.S.)(1)
  • 魚類功能性基因體分析實驗
    Laboratory of Functional Genomics Analysis in Fish
  • 細胞訊息傳導
    Cellular Signaling

Courses

  • Gene Expression Analysis And Laboratory
  • Seminar (M.S)
  • Seminar in Developmental Biology
  • Early Developmental Embryology
  • Seminar (B.S.)(1)
  • Laboratory of Functional Genomics Analysis in Fish
  • Cellular Signaling