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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 李士傑 | |
| dc.contributor.author | I-Chen Hung | en |
| dc.contributor.author | 洪苡蓁 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:09:25Z | - |
| dc.date.available | 2022-02-08 | |
| dc.date.copyright | 2017-02-08 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-10-24 | |
| dc.identifier.citation | Baker, L.P., Daggett, D.F., Peng, H.B., 1994. Concentration of pp125 focal adhesion
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48792 | - |
| dc.description.abstract | 黏著斑激酶(focal adhesion kinase，FAK)在許多重要的細胞生理過程上扮演著重要的角色，其中包括了胚胎發育及器官發育等過程。儘管我們已知FAK在胚胎發育過程中的重要性，然究竟FAK如何調控早期胚胎發育，又如何在此一時期與其他訊息分子溝通的研究還是很有限。在本論文中，藉由比較fak1a與1b與人類FAK的氨基酸序列, 我發現兩者有高度的相似性。利用mopholino oligonucleotides (MO)抑制蛋白質生成，我觀察到魚胚(morphants)的外包作用(epiboly), 胚體趨中與延展作用(convergent extension)以及下胚層(hypoblast)細胞的移動都受到明顯的影響。藉由細胞的移植實驗, 我發現fak1a所調控的細胞移動受外界環境所影響，肌凝蛋白(filamentous actin，F-actin)在細胞外包作用時所產生的環狀構造(actin ring)在fak1a缺乏的胚胎是不完整的,進而推測其可能為導致胚外包作用缺失的原因。我同時也利用了CRISPR/Cas9的基因修改技術製作出了fak1a的突變魚(mutant)，但僅有少部分胚有腔腸化缺陷的表型。然而在mutants與mophants我發現相同的基因有上升之趨勢，此顯示相似的細胞傳導路徑受到了影響，更重要的是我發現Wnt5b可能位於fak1a的上游調控下游路徑以調節細胞移動的過程。藉由mRNA拯救的實驗我發現其下游因子是small GTPase，Rac1與CDC42。總結,我首次在斑馬魚胚腔腸化過程中發現了FAK與WNT5b共同藉由調控Rac1與CDC42影響了肌凝蛋白之動態平衡以控制細胞移動現象。 | zh_TW |
| dc.description.abstract | Focal adhesion kinase is known to mediate multiple vital cellular processes and be involved in embryogenesis and organ development. Despite its necessity, how FAK regulates and integrates with other cellular signals during early embryogenesis still remains poorly understood. Here, I first demonstrated the high sequence similarity of zebrafish fak1a and fak1b to human FAK. Using antisense morpholino (MO), I observed that the loss of Fak1a impaired epiboly, convergence and extension and hypoblast cell migration in a non cell-autonomous manner. Furthermore, I showed clear disturbance of the filamentous actin (F-actin) linkage bundles between actin-ring and yolk syncytial nuclei that appeared to affect epiboly in fak1a morphants. Genetic deletion of fak1a using CRISPR/Cas9 mediated gene editing reveals minor gastrulation defects than that of morphants, but some genes were induced both in morphants and mutants. It suggests that similar molecular pathways were affected. More importantly, I found that overexpression of fak1a or wnt5b mRNA could cross rescue convergence defects induced by wnt5b or fak1a MO, respectively. Both Wnt5b and Fak1a appeared to mediate gastrulation via Rac1 and Cdc42, since both small GTPases could synergistically rescue wnt5b and fak1a morphant phenotypes. Taken together, I demonstrate for the first time the missing functional interaction between Wnt and FAK signaling to mediate gastrulation cell movements via precise regulation of Rac1 and Cdc42 activities and subsequent actin dynamics. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:09:25Z (GMT). No. of bitstreams: 1 ntu-105-D99b41005-1.pdf: 4059343 bytes, checksum: c1783e38783a71039c79891ff36ff9ed (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 ................................................................................................................................... 4
ABSTRACT .................................................................................................................................. 5 Introduction .............................................................................................................................. 7 Materials and methods .......................................................................................................... 11 Zebrafish fak cloning and expression vector construction ...................................... 11 Immunofluorescence embryos staining .................................................................... 12 Cell transplantation ................................................................................................. 12 Immunoblotting ........................................................................................................ 13 Zebrafish maintenance and embryo culture ............................................................. 13 Microinjection .......................................................................................................... 14 Whole-mount in situ hybridization ........................................................................... 14 Rhodamine-phalloidin staining and confocal imaging analysis .............................. 15 Time-lapse DIC imaging and analysis ..................................................................... 15 Rac1 and Cdc42 activation assay ............................................................................ 16 Hae III mutagenesis assay and sequencing ............................................................. 17 Statistical analysis ................................................................................................... 18 Results ..................................................................................................................................... 18 Fak1a and Fak1b are maternally and ubiquitously expressed in zebrafish embryos .................................................................................................................................. 19 Loss Fak1a and Fak1b causes gastrulation defects ................................................ 19 Loss of Fak1a and Fak1b causes convergence and extension defect ...................... 21 Loss of Fak1a causes reduction of cortical actin fibers and uneven distribution of YSL nuclei ................................................................................................................ 22 Wnt5b act both upstream and parallel to Fak1a to regulate gastrulation .............. 26 CRISPR/Cas9 mediated fak1a deletion causes mild gastrulation defect ................. 28 Fak1a and Wnt5b cooperatively mediate gastrulation via modulating Rac1 and Cdc42 ....................................................................................................................... 32 Discussion ................................................................................................................................ 34 Tables ...................................................................................................................................... 39 Table 1. Quantification of convergence and extension defects caused by fak1a tMO and wnt5b tMO co-injection .................................................................................... 39 Table 2. Quantification of convergence and extension defects caused by wnt5b tMO with or without fak1a mRNA co-injection ...................................................... 40 Table 3. Quantification of convergence and extension defects caused by fak1a tMO with or without wnt5b mRNA co-injection .............................................................. 41 Table 4. Quantification of convergence and extension defects caused by fak1a tMO in wild type or fak1a mutant background ................................................................ 42 Table 5. Primers and MOs used in this study .......................................................... 43 Table 6. Guiding RNA in this study ........................................................................ 44 Table 7. Sequences of fak1a mutants ...................................................................... 45 References ............................................................................................................................... 46 Figures ..................................................................................................................................... 55 Figure 1. fak1a expression pattern in zebrafish .................................................. 55 Figure 2. fak1a tMO2 causes dose-dependent and specific inhibition on epiboly .................................................................................................................................. 56 Figure 3. Loss of fak1b causes dose-dependent and specific inhibition on epiboly ..................................................................................................................... 58 Figure 4. Loss of Fak1a perturbs the synchronized migration of enveloping and deep cell layers and F-actin network ............................................................ 60 Figure 5. Fak1a functions non cell-autonomously to regulate cell migration during gastrulation ................................................................................................ 62 Figure 6. Fak1a overexpression rescues defects in wnt5b mutants and morphants ............................................................................................................... 64 Figure 7. Knockdown of p53 could not rescued epiboly defect of wnt5b morphants ............................................................................................................... 65 Figure 8. Fak1a and wnt5b showed no synergistic effect during gastrulation . 66 Figure 9. Fak1a is down-regulated in wnt5b morphants ................................... 68 Figure 10. Fak1a and wnt5b reciprocally rescue convergence defect caused by antisense MO against each other .......................................................................... 69 Figure 11. Wnt5b partially rescued epiboly defect of fak1a morphants ........... 70 Figure 12. CRISPR/Cas9-mediated deletion of Fak1a resulted in minor defect in gastrulation......................................................................................................... 72 Figure 13. CRISPR/cas9 mediated fak1a ablation causes minor effect on gastrulation ............................................................................................................. 73 Figure 14. Fak1a deletion resulted in compensatory changes in fak1b and wnt5b ....................................................................................................................... 75 Figure 15. CRISPR/cas9 mediated fak1a interference causes gastrulation defect ....................................................................................................................... 76 Figure 16. CRISPR/cas9 mediated fak1b interference causes gastrulation defect in F0 embryos .............................................................................................. 77 Figure 17. Low concentration of Rac1 and Cdc42 mRNA rescues wnt5b and fak1a morphants during gastrulation .................................................................. 79 Figure 18. Rac1 and Cdc42 are downstream of wnt5b and fak1a during gastrulation in zebrafish embryos ........................................................................ 81 Appendix Figures .................................................................................................................... 82 | |
| dc.language.iso | en | |
| dc.title | Fak1a與Wnt5b經由Rac1與Cdc42聯合調控斑馬魚胚早期腔腸化時之細胞遷徙過程 | zh_TW |
| dc.title | Focal adhesion kinase 1a and Wnt5b cooperatively mediate gastrulation cell movements via Rac1 and Cdc42 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 沈湯龍,鍾邦柱,鄭邑荃,黃聲蘋 | |
| dc.subject.keyword | FAK,Wnt5b,Rac1,Cdc42,胚體趨中與延展作用,外包現象,腔腸化,斑馬魚, | zh_TW |
| dc.subject.keyword | FAK,Wnt5b,Rac1,Cdc42,convergent extension,epiboly,gastrulation,zebrafish, | en |
| dc.relation.page | 92 | |
| dc.identifier.doi | 10.6342/NTU201603706 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-10-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| dc.date.embargo-terms | 2300-01-01 | |
| dc.date.embargo-lift | 2300-01-01 | - |
| Appears in Collections: | 生命科學系 | |
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