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分子與細胞生物學研究所
98 學年度論文壁報競賽評審結果
一、博士班
獎項
姓 名
傑出獎 黃倖妍
優等獎 黃春怡
佳作
徐綜遠
題目
Arl6ip1 Functions in Differentiation, Proliferation, and cell
survival during Retinogenesis of Zebrafish Embryos
A novel extrinsic apoptosis pathway in C.elegans
研究線蟲吞噬細胞辨識死細胞且經由 Integrin α-SRC 與 RTK
X-CED-2 傳遞訊號調控細胞吞噬作用
二、碩士班
獎項
姓 名
題目
傑出獎
鄭令欣 Functional study of Arabidopsis AtNRT1.13
傑出獎
Novel animal model to study the ER and ER-associated stresses:
劉育瑋 using zebrafish transgenic line carrying the human uORFchop
cassette
優等獎
吳如蕙
Specific and inducible inhibition of target gene through short
hairpin RNA synthesized by RNA polymerase II
優等獎
蕭崇景
microRNA miR-In300 抑制其標的基因 homer-1 而影響斑馬魚胚
胎肌肉發育
佳作
蔡孟庭
Drosophila Processing Body Regulates Expression pattern of
microtubule in Follicle cells
佳作
張俊賢 Stimulation of Chloroplast Protein Import by Monovalent Cations
1
Arl6ip1 Functions in Differentiation, Proliferation, and cell survival
during Retinogenesis of Zebrafish Embryos
Hsing-Yen Huang and Huai-Jen Tsai
Abstract
ADP-ribosylation factor-like 6 (Arl6) mutation is linked to human disease and Arl6 interacts
with Arl6 interacting protein 1 (Arl6ip1). However, the cellular and molecular mechanisms of
arl6ip1 gene during embryogenesis are unknown. In this study, we found that arl6ip1 was a
maternal expression gene which distributes around the whole embryo before 24-hpf, but after
48-hpf, arl6ip1 was exclusively expressed in brain, retina, heart and kidney. When Arl6ip1
translation was blocked by injecting arl6ip1-morphlino, many organs and tissues developed
abnormally, and defective embryos could not survive beyond 5-dpf. Next, we focused on the
retinal development to discuss the functional role(s) of Arl6ip1 that because small eyes were
easily observed in all phenotypic defects. We observed the failure of cell differentiation, the
reduction in cell number, and the imperfect progression of cell cycle in the defective retina.
Mosaic analysis demonstrated that Arl6ip1 was required for retinal differentiation in a
non-cell autonomous manner by cell transplantation. ER and Golgi were swollen,
mictochondria was pale and engorged, and bigger nucleus in the defective cells, suggesting
that Arl6ip1 is involved in the organization of organelles. The ER-stress response marker,
chop, was highly expressed in the defective retina, indicating that cells were undergoing ER
stress. Apoptosis signals were found in the retina of arl6ip1-morphants at lateral development.
This evidence suggested that Arl6ip1 is involved in the ER-stress mediated apoptosis during
retinogenesis. Interestingly, we performed the immunoprecipitation assay (IP), in which the
total proteins extracted from zebrafish embryos were mixed with anti-Arl6ip1
antibody-conjugated protein G beads, following LC-MS/MS analysis of IP product. We
identified Ras-related nuclear protein (Ran) was interacted with Arl6ip1 directly. Arl6ip1
interacted with Ran at centrosome during mitosis. Taken together, these evidences suggested
that Arl6ip1 secures cell-cycle progress and cooperates with Ran to regulate mitosis, resulting
in playing an important role in differentiation and mitosis during retinogenesis in zebrafish
embryos.
2
A novel extrinsic apoptosis pathway in C.elegans
Chun-Yi Huang, Yi-Chun Wu
Institute of Molecular and Cellular Biology, National Taiwan University
摘要內容保留至論文發表後再行公佈。
3
學生: 徐綜遠
學號 :d92b43002
指導教授: 吳益群 老師
中文題目: 研究線蟲吞噬細胞辨識死細胞且經由Integrin α-SRC與RTK X-CED-2傳遞訊
號調控細胞吞噬作用
英文題目: Engulfment of apoptotic cells in C. elegans is mediated by Integrin α-SRC
and RTK X-CED-2 signaling
Abstract
The engulfment of cell corpses is a prominent feature of programmed cell death
(PCD). Recent studies have shown that the molecular control of cell-corpse engulfment is
evolutionally conserved in metazoans. The Integrin receptor is a large family of αβ
heterodimeric cell surface receptors and that determine their ligands specificity and their
cytoplasmic interactions. Moreover, Integrin receptor (αvβ3 and αvβ5) has been
implicated in the phagocytosis of programmed cell death in vitro, but its in vivo evidence
is still not well understood. However, the loss of function in phosphatidylserine receptor-1
(PSR-1), a engulfment receptor, was showed that the number of cell corpses is less than
the engulfment defect of downstream genes. Therefore, we suspected that at least another
receptor is participated in this engulfment pathway.
We took used of allelic mutants, RNAi and 4-Dimensional method to identify
candidate receptors, function as cell-engulfment mechanism. To better understand the
potential roles of Integrin receptor and Receptor Tyrosine Kinase/RTK-X in cell
engulfment, we proofed in vivo evidence that integrin/ina-1 alpha subunit and rtk-x play a
vital role in the engulfment of cell corpses in C.elegans.
In our genetic and biochemical data specify that INA-1 and RTK-X likely function
parallel and act as an engulfment receptor upstream of the signaling complex
CED-2/CED-5 and CED-12 through a non-canonical FAK-independent manner mediated
by SRC-1 tyrosine kinase to activate CED-10 for cytoskeleton re-arrangement.
Furthermore, we also distinguished that INA-1 and RTK-X are participated at phase III of
distal tip cell (DTC ) migration in gonad. In addition, the receptor tyrosine kinase/ RTK-X
is one of the novel spatiotemporal regulatory which expressed middle stage of DTC to
mediate cell migration in C.elegans.
4
Functional study of Arabidopsis AtNRT1.13
STUDENT: Ling-Hsin Cheng
ADVISOR: Yi- Fang Tsay
摘要內容保留至論文發表後再行公佈。
5
Novel animal model to study the ER and ER-associated stresses: using
zebrafish transgenic line carrying the human uORFchop cassette
Yu-Wei Liu (劉育瑋), Hung-Chieh Lee (李鴻杰) and Huai-Jen Tsai (蔡懷楨)
Endoplasmic reticulum (ER) stress is suggested to be involved in many human
neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and fetal
alcohol syndrome. Although ER stress is well studied in vitro, few animal models have been
generated to monitor ER stress in vivo. As a result, the precise contribution of ER stress in
various disease processes is not clear. Therefore, we first tested the feasibility of using
zebrafish embryos as a platform for the study of ER stress in vivo. We first analyzed the
spatiotemporal expressions of chop (C/EBP homologous protein), which plays important
roles under ER stress during embryogenesis. We found that the chop gene was specifically
detected in the midbrain and hindbrain during 72 hpf. To understand whether endogenous
chop is induced by ER and ER-associated stresses, we treated embryos with the ER
stress-inducing drug thapsigargin or heat-shock treatment at 40℃ for 30 min during 72 hpf,
followed by an analysis of chop expression at 96 hpf. Since the chop transcript was greatly
induced, the zebrafish was determined to be an excellent model for the study of ER and
ER-associated stresses in vivo. Therefore, we continued to assess the effect of the upstream
open reading frame of chop (uORFchop), which is located at the 5’ UTR of both human and
zebrafish, on the translation of mRNA. To accomplish an animal model, we generated a
transgenic line, Tg(KY43-3), in which the human uORFchop was fused with GFP and driven by
cytomegarovirus (CMV) promoter. The GFP was not apparent under normal conditions, but
the gfp mRNA was transcribed throughout the embryos, indicating that the translation of gfp
mRNA was completely inhibited by the uORFchop cassette. Interestingly, when Tg(KY43-3)
embryos were treated with thapsigargin, the GFP was detected in the brain, indicating that ER
stress may direct the uORFchop cassette to start its translation. Moreover, the GFP signals in
the brain of the Tg(KY43-3) embryos could be induced by various types of stress, including
heat shock, alcohol treatment, and hypoxia. To understand which cell types in the brain of
Tg(KY43-3) embryos are GFP-positive, and thus induced by heat shock stress, HuC and
GFAP immunostaining were performed. Results showed that the GFP-positive cells were not
HuC-positive neuron cells, but rather, partial GFAP-positive glia cells, suggesting that some
glia cells and immature neural stem cells in brain may be the most sensitive in their response
to heat stress. Therefore, we concluded that transgenic line Tg(KY43-3) carrying human
uORFchop might be an excellent alternative animal model for studying the response of
embryos against ER and ER-associated stresses from environment. Moreover, it can be used
to study the uORFchop regulating mechanism in vivo under different stresses, and, as such,
screen drugs alleviating stress responses.
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Specific and inducible inhibition of target gene through short hairpin RNA
synthesized by RNA polymerase II
Ju-Hui Wu(吳如蕙), Ren-Jun Hsu(許仁駿) and Huai-Jen Tsai(蔡懷楨)
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
RNA interference (RNAi) is a powerful technique to silence target genes in
vitro. However, the short hairpin RNA (shRNA) for silencing gene is hardly
tissue-specific and inducible design. Even RNAi construct is too large.
Therefore, to overcome these limitations, we took advantage of the splicing
feature of SMT1 and SMT2 motifs to develop a tissue-specific and inducible
Tet-on-based RNAi system. To accomplish this, we inserted an
SMT1-cTnTshRNA-SMT2 cassette, in which shRNA was designed to silence
the zebrafish cardiac troponin T2 (cTnT), into a plasmid containing
heart-specific EGFP under a Tet-on controlling system. After the transgenic
zebrafish lines were generated, the transgenic F1 embryos were induced by
doxycycline, which resulted in a dramatic decrease of the target cTnT protein.
Next, we constructed plasmid pCS2+-GFP-SMT-VEGFshRNA, in which GFP
and SMT1-VEGFshRNA- SMT2 were driven by cytomegalovirus promoter, and
transfected into human embryonic kidney cell line HEK-293T, African green
monkey kidney fibroblast-like cell line COS-1 and human colorectal
adenocarcinoma cell line SW620, we found that the number of GFP-positive
cells were reduced 76± 0.10 %, 75± 0.12 % and 73± 0.04 % (n=3), respectively,
compared to each control group. In addition, only the 0.7 kb GFP mRNA, not
the 0.9 kb GFP-SMT-VEGFshRNA mRNA, was detected in the total RNA
extracted from the SW620 cells infected by pCS2+-GFP-SMT-VEGFshRNA,
indicating that GFP mRNA was cut from GFP-SMT-VEGFshRNA. When the
DNA
encoding
VEGF121
was
co-transfected
with
+
pCS2 -GFP-SMT-VEGFshRNA into HEK-293T and COS-1 cells, the VEGF
protein level was decreased 56 % and 75 %, respectively. Finally, we
constructed an inducible plasmid, in which SMT1-VEGFshRNA-SMT2 was
constructed in Tet-on system, and generated stable SW620 cell lines. After
doxycycline induction, the protein level of VEGF was decreased, indicating that
SMT1-VEGFshRNA-SMT2 suppressed the endogenous VEGF protein. This
line of evidences indicated that SMT1 and SMT2 motif can be served as a
cassette for carrying shRNA both in zebrafish embryos and in mammalian cell
lines. Therefore, we demonstrated an inducible controlling RNAi system which
might be potentially applied on gene therapy.
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microRNA miR-In300 抑制其標的基因 homer-1 而影響斑馬魚胚胎肌肉發育
蕭崇景, 蔡懷楨
microRNA-In300 為一種 intronic microRNA （miRNA），其位於斑馬魚肌肉專一表
現基因 myf5 intron-1 序列中 ，會與其宿主基因 myf5 同時經由 polymerase II 進行轉錄作
用而產生，且在斑馬魚胚胎 myf5 mRNA 表現開始消退時期，miR-In300 仍可持續在腦部
以及肌肉組織中被偵測到其表現。因此，我們欲探討在 myf5 不表現時期，miR-In300 是
否仍具有經由調控標的基因表現，而影響斑馬魚胚胎肌肉發育之功能。首先，我們選擇
myf5 表現消退時期 32 hpf 之斑馬魚胚胎進行 Labeled microRNA pull-down assay system，
並配合 microarray 分析，進而獲得斑馬魚 miR-In300 可能標的基因之資料庫，並篩選出
會表現於斑馬魚胚胎軀幹部肌肉組織基因 homer-1、col1a-2、trmt-2a、six-1b 以及 dnajc-10
進行研究。接著，我們將上述五個 miR-In300 可能標的基因之 3’-untranslated translated
region （3’UTR）構築入報導基因 luciferase（luc）下游進行 Dual-luciferase reporter system
分析。綜合於 HEK-293T 細胞株以及斑馬魚胚胎之實驗結果顯示，在給予外源性
miR-In300 狀況下，miR-In300 僅可透過 home-r1 3’UTR 序列抑制報導基因 luc 的表現。
且在斑馬魚胚胎不外加 miR-In300，僅具有內生性 miR-In300 情況下，報導基因 luc 表現
即可受到抑制。若注射 miR-In300 mopholino （MO）到斑馬魚胚胎以抑制內生性 mature
miR-In300 之生成，報導基因 luc 表現量則會顯著增加。顯示，在斑馬魚胚胎中，隨著
miR-In300 表現量的改變，含有 homer-1 3’UTR 之報導基因 luc 表現程度也會隨之受到
調控。進一步，在 western blot 實驗中，我們可藉由抑制內生性 miR-In300 表現，發現
Homer-1 蛋白質表現量較野生型（wild-type）斑馬魚胚胎表現量增加。另一方面，我們
利用全胚胎原位雜合反應（whole mount in situ hybridization，WISH）發現 miR-In300 以
及 homer-1 均可表現於軀幹部肌肉之快肌中，顯示兩者的表現區域具有同位性。而在斑
馬魚胚胎進行 miR-In300 以及 homer-1-MO 注射實驗，在分別過量表現 miR-In300 以及
抑制內生性 Homer-1 蛋白質的轉譯時，會造成斑馬魚胚胎體軸彎曲以及尾巴變短之相似
缺失。並且，在斑馬魚胚胎共同注射 homer-1 mRNA 與 miR-In300 狀況下，則可降低
miR-In300 過量表現造成之缺失比例。由以上實驗結果顯示，miR-In300 可經由 homer-1
3’UTR 序列而抑制 Homer-1 蛋白質的產生，造成斑馬魚軀幹部肌肉之形成具有影響性。
8
Drosophila Processing Body Regulates Expression pattern of microtubule
in Follicle cells
Meng-Ting Tsai and Tze-Bin Chou*, Institute of Molecular and Cellular Biology
Abstract:
摘要內容保留至論文發表後再行公佈。
9
Stimulation of Chloroplast Protein Import by Monovalent Cations
Jun-Shian Chang
Advisor: Dr. Hsou-min Li
Institute of Molecular and Cellular biology, National Taiwan University
Abstract
摘要內容保留至論文發表後再行公佈。
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