細胞骨架在生物體中擔任維持細胞形狀、協助胞器移動及幫助有絲分裂和減數分裂進行的重要功能，由微管、中間絲與微絲所組成，其中微絲為細胞骨架中分布廣泛且直徑最小，並可以與其他相關actin binding protein進行結合，調控微絲的組裝進而影響細胞的機械特性。Palladin是一種actin binding protein，為palladin/myotilin/myopalladin家族中的一員，並在細胞移動與細胞貼附中扮演重要角色，而斑馬魚早期胚胎發育的時期需通過細胞骨架組織架構，協助胚胎進行移動及分裂，因此可利用斑馬魚進行細胞骨架相關研究。
Palladin曾經被報導在小鼠中經由alternative splicing產生許多不同的isoform；以及透過不同的transcription start site產生 transcription variant。首先我們使用NCBI資料庫所提供的資訊，繪製在斑馬魚中palladin所有的isoform，但是因為此資料庫中與palladin相關的資訊來源均為expression sequencing tag預測所提供，因此並沒有辦法確認以片段序列組成的基因型態是否正確。我們將探討各isoform的表現形態，利用PCR放大目標片段並送至定序。結果顯示在斑馬魚早期胚發育palladin isoform X1、X2及X4均同時存在，且缺乏exon11，並以isoform X2為dominant form。而palladin isoform X1、X2及X3在斑馬魚早期胚胎於maternal時期及zygotic時期表現。isoform X4則是在早期胚胎授精後24小時表現。此外，因transcription start site不同所產生的transcription variant isoform X5僅於zygotic時期表現，isoform X6則在maternal時期表現量低，於zygotic時期表現量高。透過全胚胎原位雜交的技術也發現palladin在maternal時期表現於全胚胎，而在斑馬魚受精後24及48小時則表現在體節與心臟。
在小鼠胚胎進行palladin的基因剔除會使神經管的閉合出現異常，導致臟器外露的現象，使小鼠在胎內死亡而無法進後續實驗，因此我們利用斑馬魚早期胚胎透明及生長周期短的特性作為材料進行palladin基因剔除，使用CRISPR-Cas9的系統對斑馬魚胚胎進行注射，分別執行兩種形式的基因剔除，首先執行以exon16為目標的點突變，但是在zygotic homozygous魚隻並未出現表現型異常的情況，接著進行palladin長片段基因剔除，並持續觀察後續魚隻情況，加以分析。

The dynamic organization of the cytoskeleton in eukaryotic cells is important for maintaining cell morphology, organelle movement, cell division, and cell motility. Cytoskeleton consists of three main elements: microtubules, intermediate filaments, and actin filaments. Actin filaments are approximately 7nm in diameter, the smallest type among cytoskeletons. The actin binding protein can regulate actin assembly and further influence mechanical properties of a cell. Palladin is an actin-associated protein, which belongs to palladin/myotilin/myopalladin family, and plays a role in cell movement and cell adhesion. Dynamic cytoskeleton organization involved in cell movement and cell division during zebrafish early embryonic development, which is a good model for cytoskeleton research.
Based on previous studies, three major palladin isoforms exist due to alternative splicing: 200-kDa isoform, 140-kDa isoform, and 90-kDa isoform. To investigate palladin isoforms in zebrafish early embryonic development, we acquired palladin isoforms sequence from NCBI. However, different isoform or transcription variants were predicted by the comparison to the EST database and is not completely analyzed. The target fragments of different isoform were amplified by PCR.. The results showed that palladin isoform X1, X2 (dominant form), X3, and X6 in zebrafish existed at both maternal and zygotic stage, while palladin isoform X4, and X5 only expressed at zygotic stage. By using whole-mount in situ hybridization, we can find palladin exist at whole embryos at the maternal stage and expressed in heart and somite at 24 hpf.
Palladin null mutant mice exhibit some fatal defect: neural tube closure defect, inguinal and hepatic herniation, etc, while these defects were hard to be observed and analyzed. Thus, we generated palladin knockout zebrafish by CRISPR-Cas9 system with point mutation or large fragment mutations in conserved region on palladin gene. The results display that the zebrafish homozygous embryo did not exhibit any defect.

劉子毓，小鼠palladin於肌生成時之啟動子與基因表現分析，國立成功大學生命科學系碩士論文，2015。

高臆茹，人類PALLD基因於肌原母細胞之鑑定及表徵分析，國立成功大學生命科學系碩士論文，2017。

Artelt, N., T. A. Ludwig, H. Rogge, P. Kavvadas, F. Siegerist, A. Blumenthal, J. v. d. Brandt, C. A. Otey, M.-L. Bang, K. Amann, C. E. Chadjichristos, C. Chatziantoniou, K. Endlich and N. Endlich (2018). "The Role of Palladin in Podocytes." J Am Soc Nephrol, 29: 1662–1678.

Azatov, M., S. M. Goicoechea, C. A. Otey and A. Upadhyaya (2016). "The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts." scientific report, 6:28805.

Black, D. L. (2003). "Mechanisms of Alternative Pre-Messenger RNA Splicing." Microbiology, Immunology, and Molecular Genetics, 72 (c).

Boskovic, S., R. n. Marı´n-Juez, J. Jasnic, S. Reischauer, H. E. Sammak, A. Kojic, G. Faulkner, D. Radojkovic, D. Y. R. Stainier and S. Kojic (2018). "Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise." PLoS ONE, 13(9).

Brentnall, T. A., L. A. Lai, J. Coleman, M. P. Bronner, S. Pan and R. Chen (2012). "Arousal of Cancer-Associated Stroma: Overexpression of Palladin Activates Fibroblasts to Promote Tumor Invasion." PLoS ONE,7(1).

Cannon, A. R., M. K. Owen, M. S. Guerrero, M. L. Kerber, S. M. Goicoechea, K. C. Hemstreet, B. Klazynski, J. Hollyfield, E. H. Chang, R. F. Hwang, C. A. Otey and H. J. Kim (2015). "Palladin Expression is a Conserved Characteristic of the Desmoplastic Tumor Microenvironment and Contributes to Altered Gene Expression." cytoskeleton. 72:402–411

Carreira-Barbosa, F., M. Kajita, V. Morel, H. Wada, H. Okamoto, A. M. Arias, Y. Fujita, S. W. Wilson and M. Tada (2009). "Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation." Development, 136, 383-392.

Chang, E. H., A. H. Gasim, M. L. Kerber, J. B. Patel, S. A. Glaubiger, R. J. Falk, J. C. Jennette and C. A. Otey (2014). "Palladin is Upregulated in Kidney Disease and Contributes to Epithelial Cell Migration After Injury." scientific report, 5 : 7695.

Chen, X., X. Fan, J. Tan, P. Shi, X. Wang, J. Wang, Y. Kuang, J. Fei, J. Liu, S. Dang and ZhugangWang (2016). "Palladin is involved in platelet activation and arterial thrombosis." Thrombosis Research, TR 6513.

Chin, Y. R. and A. Toker (2010). "The Actin-Bundling Protein Palladin Is an Akt1-Specific Substrate that Regulates Breast Cancer Cell Migration." Molecular Cell, 38, 333–344.

Chin, Y. R. and A. Toker (2011). "Akt isoform-specific signaling in breast cancer Uncovering an anti-migratory role of palladin." Cell Adhesion & Migration, 5:3, 211-214.

Cui, Y., M. Cai and H. E. Stanley (2017). "Comparative Analysis and Classification of Cassette Exons and Constitutive Exons." Hindawi BioMed Research International, Volume 2017.

Dhanda, A. S., A. W. Vogl, S. E. Albraiki, C. A. Otey, M. R. Beck and J. A. Guttmana (2018). "Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections." amrican society for microbiology, Volume 9 Issue 2 e02259-17.

el-Brolosy, M. A., Z. Kontarakis, A. rossi, c. Kuenne, S. Günther, N. Fukuda, K. Kikhi, G. l. M. Boezio, c. M. takacs, S.-l. lai, r. Fukuda, c. Gerri, A. J. Giraldez and D. Y. r. Stainier (2019). "Genetic compensation triggered by mutant mRNA degradation." nature communication, Volume 568,193-197.

Gilam, A., J. o. Conde, D. Weissglas-Volkov, N. Oliva, E. Friedman, N. Artzi and N. Shomron1 (2016). "Local microRNA delivery targets Palladin and prevents metastatic breast cancer." nature communication, 7:12868.

Goicoechea, S., B. Bednarski, R. Garcı´a-Mata, H. Prentice-Dunn, H. Kim and C. Otey (2009). "Palladin contributes to invasive motility in human breast cancer cells." Oncogene, 28, 587–598.

Goicoechea, S. M., D. Arneman and C. A. Otey (2008). "The role of palladin in actin organization and cell motility." Eur J Cell Biol, 87(8-9):517-25.

Goicoechea, S. M., B. Bednarski, C. Stack, D. W. Cowan, K. Volmar, LeighThorne, E. Cukierman, A. K. Rustgi, T. Brentnall, R. F. Hwang, C. A. G., McCulloch, J. J. Yeh, D. J. Bentrem, S. N. Hochwald, S. R. Hingorani, H. J. Kim and C. A. Otey (2010). "Isoform-Specific Upregulation of Palladin in Human and Murine Pancreas Tumors." PLoS ONE, 5(4): e10347.

Goleya, E. D., A. Rammohanb, E. A. Znameroskia, E. N. Firat-Karalara, D. Septb and M. D. Welch (2009). "An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability." PNAS, vol. 107, 8159–8164.

Heisenberg, C.-P., M. Tada, G.-J. r. Rauch, L. S. de, M. L. Concha, R. Geisler, D. L. Stemple, J. C. Smith and S. W. Wilson (2000). "Silberblick/Wnt11mediates convergent extension movements during zebrafish gastrulation." letter to nature, VOL 405.

Henderson-jackson, e. b., j. helm, j. strosberg, n. a. nasir, t. j. yeatman, l. k. kvols, d. coppola and a. nasir (2011). "Palladin is a Marker of Liver Metastasis in Primary Pancreatic Endocrine Carcinomas." anticancer research, 31: 2957-2962.

Hong, S.-K., K. Tanegashima and I. B. Dawid (2011). "XIer2 is required for convergent extension movements during Xenopus development." Int J Dev Biol, 55(10-11-12): 917–921.

Huang, H., F.-I. Lu, S. Jia, S. Meng, Y. Cao, Y. Wang, W. Ma, K. Yin, Z. Wen, J. Peng, C. Thisse, B. Thisse and A. Meng (2007). "Amotl2 is essential for cell movements in zebrafish embryo and regulates c-Src translocation." Development , 134, 979-988.

Jin, L. (2011). "The actin associated protein palladin in smooth muscle and in the development of diseases of the cardiovasculature and in cancer." J Muscle Res Cell Motil, 32:7–17.

Jin, L., Q. Gan, B. J. Zieba, S. M. Goicoechea, G. K. Owens, C. A. Otey and A. V.Somlyo (2010). "The Actin Associated Protein Palladin Is Important for the Early Smooth Muscle Cell Differentiation." PLoS ONE, 5(9).

Koji, S. a. (2010). "MARP protein family a possible role in molecular mechanisms of tumorigenesis." J Med Biochem, 29: 157–164.

Lawton, A. K., A. Nandi, M. J. Stulberg, N. Dray, M. W. Sneddon, W. Pontius, T. Emonet and S. A. Holley (2013). "Regulated tissue fluidity steers zebrafish body elongation." Development, 140, 573-582.

Lee, S.-J. (2014). "Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly." Biochemical and Biophysical Research Communications, 452, 1–7

Link, V., A. Shevchenko and C.-P. Heisenberg (2006). "Proteomics of early zebrafish embryos." BMC Developmental Biology, 6:1.

Liu, X.-S., X.-H. Li, Y. Wang, R.-Z. Shu, L. Wang, S.-Y. Lu, H. Kong, Y.-E. Jin, L.-J. Zhang, J. Fei, Sai-Juan Chen, Z. Chen, M.-M. Gu, Z.-Y. Lu and Z.-G. Wang (2007). "Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver." Blood, 110:870-876.

Li-Villarreal, N., M. M. Forbes, A. J. Loza, J. Chen, T. Ma, K. Helde, C. B. Moens, J. Shin, A. Sawada, A. E. Hindes, J. Dubrulle, A. F. Schier, G. D. Longmore, F. L. Marlow and L. Solnica-Krezel (2016). "Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis." The Company of Biologists, 143, 1832.

Luo, H., X. Liu, F. Wang, Q. Huang, S. Shen, L. Wang, G. Xu, X. Sun, H. Kong, M. Gu, S. Chen, Z. Chen and Z. Wang (2005). "Disruption of palladin results in neural tube closure defects in mice." Mol. Cell. Neurosci, 507–515.

Meeker, S. A. Hutchinson, L. Ho and N. S. Trede (2007). "Method for isolation of PCR-ready genomic DNA from zebrafish tissues." BioTechniques, 43:610-614.

Najm, P. and M. El-Sibai (2014). "Palladin regulation of the actin structures needed for cancer invasion." cell Adhesion & Migration, 8:1, 29–35.

Nandelstadh, P. v., E. Gucciardo, J. Lohib, R. L. N. Sugiyama, O. Carpend and K. Lehti (2014). "Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton." Molecular Biology of the Cell, Volume 25 2556-2570.

Niedenberger, b. a., v. k. chappell, e. p. kaye, r. h. renegar and c. b. geyer (2013). "Nuclear Localization of the Actin Regulatory Protein Palladin in Sertoli Cells." Molecular Reproduction & Development, 80:403–413.

Otey, C. A., A. Rachlin, Monica Moza, D. Arneman and O. Carpen (2005). "The Palladin/Myotilin/Myopalladin Family of Actin‐Associated Scaffolds." International Review of Cytology, 0074-7696/05.

Otey, C. A., A. Rachlin, M. Moza, D. Arneman and O. Carpen (2005). "The Palladin/Myotilin/Myopalladin Family of Actin‐Associated Scaffolds." International Review of Cytology, 246.

Parast, M. M. and C. A. Otey (2000). "Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions." The Journal of Cell Biology, 150, 643-655.
Pogue-Geile, K. L., R. Chen, M. P. Bronner, T. Crnogorac-Jurcevic, K. W. Moyes, S. Dowen, C. A. Otey, D. A. Crispin, R. D. George, D. C. Whitcomb and T. A. Brentnall (2006). "Palladin Mutation Causes Familial Pancreatic Cancer and Suggests a New Cancer Mechanism."PLoS Med, 3(12),e516.

Qian, X., D. D. Mruk, Y. H. Cheng and C. Y. Cheng (2013). "Actin cross-linking protein palladin and spermatogenesis." Spermatogenesis, 3:1, e23473.

Qian, X., D. D. Mruk, E. W. P. Wong, P. P. Y. Lie and C. Y. Cheng (2013). "Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes." Endocrinology, 154(5):1907–1920.

Samuel M. Peterson, J. L. F. (2009). "RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis." Journal of Visualized Experiments, JoVE. 30.

Sato, D., T. Tsuchikawa, T. Mitsuhashi, Y. Hatanaka, K. Marukawa, A. Morooka, T. Nakamura, T. Shichinohe, Y. Matsuno and S. Hirano1 (2016). "Stromal Palladin Expression Is an Independent Prognostic Factor in Pancreatic Ductal Adenocarcinoma."PLoS ONE, 11(3): e0152523.

Shindo, A. (2018). "Models of convergent extension during morphogenesis." Dev Biol, 7:e293.

Shu, R.-Z., F. Zhang, X.-S. Liu, C.-L. Li, L. Wang, Y.-L. Tai, X.-L. Wu, X. Yang, X.-D. Liao, Y. Jin, M.-M. Gu, L. Huang, X.-F. Pang and Z.-G. Wang (2009). "Target Deletion of the Cytoskeleton-Associated Protein Palladin Does Not Impair Neurite Outgrowth in Mice." PLoS ONE, 4(9): e6916.

Spence, R., G. Gerlach, C. Lawrence and C. Smith (2008). "The behaviour and ecology of the zebrafish, Danio rerio." Biol. Rev, 83, pp. 13–34.

Tada, M. and C.-P. Heisenberg (2012). "Convergent extension: using collective cell migration and cell intercalation to shape embryos." Development, 139, 3897-3904.

Tan, J., X.-J. Chen, C.-L. Shen, H.-X. Zhang, L.-Y. Tang, S.-Y. Lu, W.-T. Wu, Y. Kuang, J. Fei and Z.-G. Wang (2017). "Lacking of palladin leads to multiple cellular events changes which contribute to NTD." nature development, 12:4.

Umegaki, Y., A. M. n. Brotons, Y. Nakanishi, Z. Luo, H. Zhang, X. Bonni and c. Ikeuchi (2018). "Palladin Is a Neuron-Specific Translational Target of mTOR Signaling That Regulates Axon Morphogenesis." Cellular/Molecular, 38(21):4985– 499.

Wallmeyer, B., S. Trinschek, S. Yigit, U. Thiele and T. Betz (2017). "Collective Cell Migration in Embryogenesis Follows the Laws of Wetting." Biophysical, 114, 213–222.

Wang, H.-V. and M. Moser (2008). "Comparative Expression Analysis of the
Murine Palladin Isoforms." Developmental dynamics, 237: 3342–3351.

Yanfang, J. A. Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung and J. D. Sander (2013). "High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells." Nat Biotechnol, 31(9): 822–826.

Zhang, X., X. Chen, J. Liu, X. Xu, Y. Zhang, Z. Ruan, Y. Xie, Q. Huang, T. Yin, Z. Chen and S. Chen (2017). "Palladin is a novel microtubuleassociated protein responsible for spindle orientation." scientific report, 7: 11806.