Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio)
Las enfermedades del neurodesarrollo causan alteraciones en el sistema nervioso de los individuos que las padecen. El pez cebra (Danio rerio) como modelo animal es útil para el estudio de la patología y sus tratamientos, dado su rápido desarrollo y su homología genética con los seres humanos (Vaz, H...
- Autores:
-
López Sanmiguel, Alejandra
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2024
- Institución:
- Universidad de los Andes
- Repositorio:
- Séneca: repositorio Uniandes
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.uniandes.edu.co:1992/73661
- Acceso en línea:
- https://hdl.handle.net/1992/73661
- Palabra clave:
- Neurodesarrollo
MAP2
Pez cebra
Inmunofluorescencia
Histología
Biología
- Rights
- openAccess
- License
- Attribution-NonCommercial-NoDerivatives 4.0 International
| id |
UNIANDES2_ec177b240c1f82850bc6f845abd1a52f |
|---|---|
| oai_identifier_str |
oai:repositorio.uniandes.edu.co:1992/73661 |
| network_acronym_str |
UNIANDES2 |
| network_name_str |
Séneca: repositorio Uniandes |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| title |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| spellingShingle |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) Neurodesarrollo MAP2 Pez cebra Inmunofluorescencia Histología Biología |
| title_short |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| title_full |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| title_fullStr |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| title_full_unstemmed |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| title_sort |
Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio) |
| dc.creator.fl_str_mv |
López Sanmiguel, Alejandra |
| dc.contributor.advisor.none.fl_str_mv |
Garavito Aguilar, Zayra Viviana |
| dc.contributor.author.none.fl_str_mv |
López Sanmiguel, Alejandra |
| dc.contributor.researchgroup.none.fl_str_mv |
Facultad de Ciencias::Laboratorio de Biología del desarrollo (Bioldes) |
| dc.subject.keyword.spa.fl_str_mv |
Neurodesarrollo MAP2 Pez cebra Inmunofluorescencia Histología |
| topic |
Neurodesarrollo MAP2 Pez cebra Inmunofluorescencia Histología Biología |
| dc.subject.themes.none.fl_str_mv |
Biología |
| description |
Las enfermedades del neurodesarrollo causan alteraciones en el sistema nervioso de los individuos que las padecen. El pez cebra (Danio rerio) como modelo animal es útil para el estudio de la patología y sus tratamientos, dado su rápido desarrollo y su homología genética con los seres humanos (Vaz, Hofmeister, & Lindstrand, 2019). Durante el desarrollo del sistema nervioso los microtúbulos cumplen roles importantes al actuar en la proliferación, diferenciación y migración de las neuronas, así como la orientación y formación de axones, ramificación de dendritas y sinapsis (Lasser, Tiber, & Lowery, 2018). Unas de las proteínas que conforman estos microtúbulos son las proteínas asociadas a microtúbulos (MAP). Estas son esenciales para el ensamblaje, dinamicidad, fragmentación, estabilización y el transporte intracelular de la tubulina (Lasser, Tiber, & Lowery, 2018); de estas la más caracterizada y prominente en las neuronas es la proteína MAP2. Por lo tanto, el objetivo de este trabajo fue estandarizar el uso del anticuerpo MAP2 (Cell Signaling Technology #8707), diseñado con reactividad contra ratón y rata, para inmunofluorescencia en embriones de pez cebra, y determinar su patrón de expresión normal en el desarrollo del sistema nervioso entre las 24 y 60 horas postfertilización (hpf). Con este fin, se realizó un alineamiento entre la proteína del pez y la proteína de la rata y ratón y se hicieron pruebas de inmunofluorescencia, en los que se encontró la aplicabilidad de este anticuerpo en el pez. Finalmente, se determinó un protocolo con resultados óptimos, el cual se aplicó en diferentes estadios del desarrollo del pez para caracterizar la expresión normal de la proteína en embrión completo y en secciones histológicas. Se observó que la expresión de la proteína coincide con los procesos de neurogénesis primaria en pez cebra para los estadios menores a 60 hpf y secundaria para 60 hpf. Sin embargo, el anticuerpo parece no tener reactividad contra las neuronas sensoriales primarias en comparación con el marcaje que se obtiene con el anticuerpo para tubulina acetilada, el cual se ha utilizado para el estudio de la neurogenesis (Zebrafish UCL, s.f). |
| publishDate |
2024 |
| dc.date.accessioned.none.fl_str_mv |
2024-01-31T13:28:32Z |
| dc.date.available.none.fl_str_mv |
2024-01-31T13:28:32Z |
| dc.date.issued.none.fl_str_mv |
2024-01-29 |
| dc.type.none.fl_str_mv |
Trabajo de grado - Pregrado |
| dc.type.driver.none.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
| dc.type.version.none.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.coar.none.fl_str_mv |
http://purl.org/coar/resource_type/c_7a1f |
| dc.type.content.none.fl_str_mv |
Text |
| dc.type.redcol.none.fl_str_mv |
http://purl.org/redcol/resource_type/TP |
| format |
http://purl.org/coar/resource_type/c_7a1f |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://hdl.handle.net/1992/73661 |
| dc.identifier.instname.none.fl_str_mv |
instname:Universidad de los Andes |
| dc.identifier.reponame.none.fl_str_mv |
reponame:Repositorio Institucional Séneca |
| dc.identifier.repourl.none.fl_str_mv |
repourl:https://repositorio.uniandes.edu.co/ |
| url |
https://hdl.handle.net/1992/73661 |
| identifier_str_mv |
instname:Universidad de los Andes reponame:Repositorio Institucional Séneca repourl:https://repositorio.uniandes.edu.co/ |
| dc.language.iso.none.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.none.fl_str_mv |
Abcam. (n.d.). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488). Revisado Enero 5, 2024, de https://www.abcam.com/en-co/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-488-ab150077 Alunni, A., Coolen, M., Foucher, I., & Bally-Cuif, L. (2020). Neurogenesis in zebrafish. Patterning and Cell Type Specification in the Developing CNS and PNS, 643–697. doi:10.1016/b978-0-12-814405-3.00026-6 Amidzadeh, Z., Behbahani, A. B., Erfani, N., Sharifzadeh, S., Ranjbaran, R., Moezi, L., Aboualizadeh, F., Okhovat, M. A., Alavi, P., & Azarpira, N. (2014). Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry. Avicenna journal of medical biotechnology, 6(1), 38–46. Bally-Cuif, L., & Vernier, P. (2010). Organization and physiology of the zebrafish nervous system. In Fish Physiology (Vol. 29, pp. 25–80). https://doi.org/10.1016/S1546- 5098(10)02902-X Boulanger-Weill, J., & Sumbre, G. (2019). Functional integration of newborn neurons in the zebrafish optic tectum. Frontiers in Cell and Developmental Biology. Frontiers Media S.A. https://doi.org/10.3389/fcell.2019.00057 Bradford, Y.M., Van Slyke, C.E., Ruzicka, L., Singer, A., Eagle, A., Fashena, D., Howe, D.G., Frazer, K., Martin, R., Paddock, H., Pich, C., Ramachandran, S., Westerfield, M. (2022) Zebrafish Information Network, the knowledgebase for Danio rerio research. Genetics. 220(4). Cell Signaling Technology. (s.f). MAP2 (D5G1) XP® Rabbit mAb (#8707) Datasheet. https://www.cellsignal.com/datasheet.jsp?productId=8707&images=1&size=A4 Chapouton, P., & Bally-Cuif, L. (2004). Neurogenesis. Methods in Cell Biology. Academic Press Inc. https://doi.org/10.1016/s0091-679x(04)76010-0 Chitnis, A. B., & Kuwada, J. Y. (1990). Axonogenesis in the brain of zebrafish embryos. Journal of Neuroscience, 10(6), 1892–1905. https://doi.org/10.1523/jneurosci.10-06- 01892.1990 Cucun, G., Köhler, M., Pfitsch, S., & Rastegar, S. (2023). Insights into the mechanisms of neuron generation and specification in the zebrafish ventral spinal cord. FEBS Journal. John Wiley and Sons Inc. https://doi.org/10.1111/febs.16913 Danesin, Cathy & Soula, Cathy. (2017). Moving the Shh Source over Time: What Impact on Neural Cell Diversification in the Developing Spinal Cord?. Journal of Developmental Biology. 5. 10.3390/jdb5020004. de Abreu, M. S., Genario, R., Giacomini, A. C. V. V., Demin, K. A., Lakstygal, A. M., Amstislavskaya, T. G., ... Kalueff, A. V. (2020). Zebrafish as a Model of Neurodevelopmental Disorders. Neuroscience. Elsevier Ltd. https://doi.org/10.1016/j.neuroscience.2019.08.034 Dehmelt, L., & Halpain, S. (2005). The MAP2/Tau family of microtubule-associated proteins. Genome Biology. https://doi.org/10.1186/gb-2004-6-1-204 Gladysheva, J., Evnukova, E., Kondakova, E., Kulakova, M., & Efremov, V. (2021). Neurulation in the posterior region of zebrafish, Danio rerio embryos. Journal of Morphology, 282(10), 1437–1454. https://doi.org/10.1002/jmor.21396 Hammond-Weinberger, D. R., & Zeruth, G. T. (2019). Whole mount immunohistochemistry in zebrafish embryos and larvae. Journal of Visualized Experiments, 2020(155). https://doi.org/10.3791/60575 Harada, A., Teng, J., Takei, Y., Oguchi, K., & Hirokawa, N. (2002). MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction. Journal of Cell Biology, 158(3), 541–549. https://doi.org/10.1083/jcb.200110134 Hawkins, Tom. (s.f.). Whole-mount antibody staining. Zebrafish UCL, ZebrafishBrain: a neuroanatomical atlas of the developing zebrafish brain. (Revisado en 2023) Hobro, A. J., & Smith, N. I. (2017). An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging. Vibrational Spectroscopy, 91, 31–45. https://doi.org/10.1016/j.vibspec.2016.10.012 Karlsson, J., Von Hofsten, J., & Olsson, P. E. (2001). Generating transparent zebrafish: A refined method to improve detection of gene expression during embryonic development. Marine Biotechnology, 3(6), 522–527. https://doi.org/10.1007/s1012601-0053-4 Kimmel, C. B., Hatta, K., & Eisen, J. S. (1991). Genetic control of primary neuronal development in zebrafish. Development, 113(2 SUPPL.), 47–57. https://doi.org/10.1242/dev.113.supplement_2.47 Lasser, M., Tiber, J., & Lowery, L. A. (2018). The role of the microtubule cytoskeleton in neurodevelopmental disorders. Frontiers in Cellular Neuroscience. Frontiers Media S.A. https://doi.org/10.3389/fncel.2018.00165 Liedtke, Daniel. (2008). Functional divergence of Midkine growth factors : Non-redundant roles during neural crest induction, brain patterning and somitogenesis. Macdonald, R. (1999). Zebrafish immunohistochemistry. Methods in Molecular Biology (Clifton, N.J.). https://doi.org/10.1385/1-59259-678-9:77 MAP2, NCBI Orthologs [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [2023 12 22]. Available from: https://www-ncbi-nlm-nih-gov.ezproxy.uniandes.edu.co/gene/4133/ortholog/?scope=7776 Morris-Rosendahl, D. J., & Crocq, M. A. (2020). Neurodevelopmental disorders-the history and future of a diagnostic concept. Dialogues in Clinical Neuroscience, 22(1), 65–72. https://doi.org/10.31887/DCNS.2020.22.1/macrocq National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [2023 12 22]. Available from: https://www.ncbi.nlm.nih.gov/ OLeary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, ONeill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD, Pruitt KD. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016 Jan 4;44(D1):D733-45 PubMed PubMedCentral Otálora Tarazona. (2018). Caracterización estructural del desarrollo pronefrico del Pez Cebra (Danio rerio). Universidad de los Andes. Papadopoulos, J. S., & Agarwala, R. (2007). COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics (Oxford, England), 23(9), 1073–1079. https://doi-org.ezproxy.uniandes.edu.co/10.1093/bioinformatics/btm076 Perdiz, D., Mackeh, R., Poüs, C., & Baillet, A. (2011). The ins and outs of tubulin acetylation: More than just a post-translational modification? Cellular Signalling. https://doi.org/10.1016/j.cellsig.2010.10.014 Preibisch, S., Saalfeld, S., & Tomancak, P. (2009). Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics, 25(11), 1463–1465. doi:10.1093/bioinformatics/btp184 Ross, L. S., Parrett, T., & Easter, S. S. (1992). Axonogenesis and morphogenesis in the embryonic zebrafish brain. Journal of Neuroscience, 12(2), 467–482. https://doi.org/10.1523/jneurosci.12-02-00467.1992 Sakai, C., Ijaz, S., & Hoffman, E. J. (2018). Zebrafish Models of Neurodevelopmental Disorders: Past, Present, and Future. Frontiers in Molecular Neuroscience. Frontiers Media S.A. https://doi.org/10.3389/fnmol.2018.00294 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. Nature Methods. https://doi.org/10.1038/nmeth.2019 Shihan, M. H., Novo, S. G., Le Marchand, S. J., Wang, Y., & Duncan, M. K. (2021). A simple method for quantitating confocal fluorescent images. Biochemistry and Biophysics Reports, 25. https://doi.org/10.1016/j.bbrep.2021.100916 Soltani, M. H., Pichardo, R., Song, Z., Sangha, N., Camacho, F., Satyamoorthy, K., ... Setaluri, V. (2005). Microtubule-associated protein 2, a marker of neuronal differentiation, induces mitotic defects, inhibits growth of melanoma cells, and predicts metastatic potential of cutaneous melanoma. American Journal of Pathology, 166(6), 1841–1850. https://doi.org/10.1016/S0002-9440(10)62493-5 Vallee, R. B. (1984). MAP2 (microtubule-associated protein 2). Cell and Muscle Motility. https://doi.org/10.1007/978-1-4684-4592-3_8 Vaz, R., Hofmeister, W., & Lindstrand, A. (2019). Zebrafish models of neurodevelopmental disorders: Limitations and benefits of current tools and techniques. International Journal of Molecular Sciences. MDPI AG. https://doi.org/10.3390/ijms20061296 Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)(4)., Univ. of Oregon Press, Eugene White, R. J., Collins, J. E., Sealy, I. M., Wali, N., Dooley, C. M., Digby, Z., ... Busch- Nentwich, E. M. (2017). A high-resolution mRNA expression time course of embryonic development in zebrafish. ELife, 6. https://doi.org/10.7554/eLife.30860 Wilson, S. W., Brand, M., & Eisen, J. S. (2002). Patterning the zebrafish central nervous system. Results and Problems in Cell Differentiation. https://doi.org/10.1007/978-3- 540-46041-1_10 Wilson, S. W., Ross, L. S., Parrett, T., & Easter, S. S. (1990). The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio. Development, 108(1), 121–145. https://doi.org/10.1242/dev.108.1.121 Wright Cell Imaging Facility. (2004). Autofluorescence: Causes and Cures. Internet, 1–8. Retrieved from https://hwpi.harvard.edu/files/iccb/files/autofluorescence.pdf?m=1465309329 Zebrafish UCL, ZebrafishBrain: a neuroanatomical atlas of the developing zebrafish brain. (Revisado en 2023). Anti-acetylated tubulin. http://zebrafishucl.org/antibodies- 1/tubulin |
| dc.rights.en.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International |
| dc.rights.uri.none.fl_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| dc.rights.accessrights.none.fl_str_mv |
info:eu-repo/semantics/openAccess |
| dc.rights.coar.none.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.none.fl_str_mv |
74 páginas |
| dc.format.mimetype.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Universidad de los Andes |
| dc.publisher.program.none.fl_str_mv |
Biología |
| dc.publisher.faculty.none.fl_str_mv |
Facultad de Ciencias |
| dc.publisher.department.none.fl_str_mv |
Departamento de Ciencias Biológicas |
| publisher.none.fl_str_mv |
Universidad de los Andes |
| institution |
Universidad de los Andes |
| bitstream.url.fl_str_mv |
https://repositorio.uniandes.edu.co/bitstreams/3c2114f9-c505-48b9-bcc3-f9c113332e04/download https://repositorio.uniandes.edu.co/bitstreams/cce42731-a152-4492-a303-833ff691600f/download https://repositorio.uniandes.edu.co/bitstreams/72fa0eff-b613-4441-b7de-3404538c5a22/download https://repositorio.uniandes.edu.co/bitstreams/e95c94c8-dff8-4e70-ad8a-2c10d6d96aa6/download https://repositorio.uniandes.edu.co/bitstreams/7b793577-8b76-42f2-b24a-0f8c9ff843c3/download https://repositorio.uniandes.edu.co/bitstreams/b1e53169-52b7-4273-b387-b69059dd04df/download https://repositorio.uniandes.edu.co/bitstreams/4942deec-3b62-46af-885b-d21df3b0fdb3/download https://repositorio.uniandes.edu.co/bitstreams/6a92a7bd-038a-46f1-bb2f-43ddcd4207ee/download |
| bitstream.checksum.fl_str_mv |
88d000df052d841806574fe1f33114bb 8c723950cd3a4d0aad268d4eed8809ca 4460e5956bc1d1639be9ae6146a50347 ae9e573a68e7f92501b6913cc846c39f 0faeeda9724ff5d6b1061647b1a75cbe 569744e5f4a7fdbf4c6dd1263390b2e8 4d3d2cff9578a73fd3a97f2de63327df 44d909ed7d9561b315ad3ac45aa437eb |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio institucional Séneca |
| repository.mail.fl_str_mv |
adminrepositorio@uniandes.edu.co |
| _version_ |
1812133818749943808 |
| spelling |
Garavito Aguilar, Zayra VivianaLópez Sanmiguel, AlejandraFacultad de Ciencias::Laboratorio de Biología del desarrollo (Bioldes)2024-01-31T13:28:32Z2024-01-31T13:28:32Z2024-01-29https://hdl.handle.net/1992/73661instname:Universidad de los Andesreponame:Repositorio Institucional Sénecarepourl:https://repositorio.uniandes.edu.co/Las enfermedades del neurodesarrollo causan alteraciones en el sistema nervioso de los individuos que las padecen. El pez cebra (Danio rerio) como modelo animal es útil para el estudio de la patología y sus tratamientos, dado su rápido desarrollo y su homología genética con los seres humanos (Vaz, Hofmeister, & Lindstrand, 2019). Durante el desarrollo del sistema nervioso los microtúbulos cumplen roles importantes al actuar en la proliferación, diferenciación y migración de las neuronas, así como la orientación y formación de axones, ramificación de dendritas y sinapsis (Lasser, Tiber, & Lowery, 2018). Unas de las proteínas que conforman estos microtúbulos son las proteínas asociadas a microtúbulos (MAP). Estas son esenciales para el ensamblaje, dinamicidad, fragmentación, estabilización y el transporte intracelular de la tubulina (Lasser, Tiber, & Lowery, 2018); de estas la más caracterizada y prominente en las neuronas es la proteína MAP2. Por lo tanto, el objetivo de este trabajo fue estandarizar el uso del anticuerpo MAP2 (Cell Signaling Technology #8707), diseñado con reactividad contra ratón y rata, para inmunofluorescencia en embriones de pez cebra, y determinar su patrón de expresión normal en el desarrollo del sistema nervioso entre las 24 y 60 horas postfertilización (hpf). Con este fin, se realizó un alineamiento entre la proteína del pez y la proteína de la rata y ratón y se hicieron pruebas de inmunofluorescencia, en los que se encontró la aplicabilidad de este anticuerpo en el pez. Finalmente, se determinó un protocolo con resultados óptimos, el cual se aplicó en diferentes estadios del desarrollo del pez para caracterizar la expresión normal de la proteína en embrión completo y en secciones histológicas. Se observó que la expresión de la proteína coincide con los procesos de neurogénesis primaria en pez cebra para los estadios menores a 60 hpf y secundaria para 60 hpf. Sin embargo, el anticuerpo parece no tener reactividad contra las neuronas sensoriales primarias en comparación con el marcaje que se obtiene con el anticuerpo para tubulina acetilada, el cual se ha utilizado para el estudio de la neurogenesis (Zebrafish UCL, s.f).Neurodevelopmental disorders lead to disruptions in the nervous system of affected individuals. The zebrafish (Danio rerio), as an animal model, proves valuable for studying pathology and its treatments due to its rapid development and genetic homology with humans (Vaz, Hofmeister, & Lindstrand, 2019). Microtubules play crucial roles during nervous system development, influencing neuron proliferation, differentiation, migration, as well as axon guidance, dendritic branching, and synapse formation (Lasser, Tiber, & Lowery, 2018). Microtubule associated proteins (MAP) constitute a key component of these structures, with MAP2 being particularly characterized and prominent in neurons. These proteins are essential for tubulin assembly, dynamics, fragmentation, stabilization, and intracellular transport (Lasser, Tiber, & Lowery, 2018). This study aimed to standardize the use of the MAP2 antibody (Cell Signaling Technology #8707), designed for reactivity against mouse and rat, for immunofluorescence in zebrafish embryos. The objective was to determine its normal expression pattern during nervous system development between 24 and 60 hours post-fertilization (hpf). Alignment between zebrafish and mouse/rat proteins was performed, followed by immunofluorescence tests, demonstrating the antibody's applicability in zebrafish. A protocol with optimal results was established and applied to different developmental stages to characterize the normal protein expression in whole embryos and histological sections. The findings revealed that protein expression aligns with primary neurogenesis in zebrafish up to 60 hpf and secondary neurogenesis at 60 hpf. However, the antibody appears non-reactive towards primary sensory neurons compared to the labeling obtained with the antibody for acetylated tubulin, commonly used in neurogenesis studies (Zebrafish UCL, n.d.).BiólogoPregrado74 páginasapplication/pdfspaUniversidad de los AndesBiologíaFacultad de CienciasDepartamento de Ciencias BiológicasAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Caracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio)Trabajo de grado - Pregradoinfo:eu-repo/semantics/bachelorThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_7a1fTexthttp://purl.org/redcol/resource_type/TPNeurodesarrolloMAP2Pez cebraInmunofluorescenciaHistologíaBiologíaAbcam. (n.d.). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488). Revisado Enero 5, 2024, de https://www.abcam.com/en-co/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-488-ab150077Alunni, A., Coolen, M., Foucher, I., & Bally-Cuif, L. (2020). Neurogenesis in zebrafish. Patterning and Cell Type Specification in the Developing CNS and PNS, 643–697. doi:10.1016/b978-0-12-814405-3.00026-6Amidzadeh, Z., Behbahani, A. B., Erfani, N., Sharifzadeh, S., Ranjbaran, R., Moezi, L., Aboualizadeh, F., Okhovat, M. A., Alavi, P., & Azarpira, N. (2014). Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry. Avicenna journal of medical biotechnology, 6(1), 38–46.Bally-Cuif, L., & Vernier, P. (2010). Organization and physiology of the zebrafish nervous system. In Fish Physiology (Vol. 29, pp. 25–80). https://doi.org/10.1016/S1546- 5098(10)02902-XBoulanger-Weill, J., & Sumbre, G. (2019). Functional integration of newborn neurons in the zebrafish optic tectum. Frontiers in Cell and Developmental Biology. Frontiers Media S.A. https://doi.org/10.3389/fcell.2019.00057Bradford, Y.M., Van Slyke, C.E., Ruzicka, L., Singer, A., Eagle, A., Fashena, D., Howe, D.G., Frazer, K., Martin, R., Paddock, H., Pich, C.,Ramachandran, S., Westerfield, M. (2022) Zebrafish Information Network, the knowledgebase for Danio rerio research. Genetics. 220(4).Cell Signaling Technology. (s.f). MAP2 (D5G1) XP® Rabbit mAb (#8707) Datasheet. https://www.cellsignal.com/datasheet.jsp?productId=8707&images=1&size=A4Chapouton, P., & Bally-Cuif, L. (2004). Neurogenesis. Methods in Cell Biology. Academic Press Inc. https://doi.org/10.1016/s0091-679x(04)76010-0Chitnis, A. B., & Kuwada, J. Y. (1990). Axonogenesis in the brain of zebrafish embryos. Journal of Neuroscience, 10(6), 1892–1905. https://doi.org/10.1523/jneurosci.10-06- 01892.1990Cucun, G., Köhler, M., Pfitsch, S., & Rastegar, S. (2023). Insights into the mechanisms of neuron generation and specification in the zebrafish ventral spinal cord. FEBS Journal. John Wiley and Sons Inc. https://doi.org/10.1111/febs.16913Danesin, Cathy & Soula, Cathy. (2017). Moving the Shh Source over Time: What Impact on Neural Cell Diversification in the Developing Spinal Cord?. Journal of Developmental Biology. 5. 10.3390/jdb5020004.de Abreu, M. S., Genario, R., Giacomini, A. C. V. V., Demin, K. A., Lakstygal, A. M., Amstislavskaya, T. G., ... Kalueff, A. V. (2020). Zebrafish as a Model of Neurodevelopmental Disorders. Neuroscience. Elsevier Ltd. https://doi.org/10.1016/j.neuroscience.2019.08.034Dehmelt, L., & Halpain, S. (2005). The MAP2/Tau family of microtubule-associated proteins. Genome Biology. https://doi.org/10.1186/gb-2004-6-1-204Gladysheva, J., Evnukova, E., Kondakova, E., Kulakova, M., & Efremov, V. (2021). Neurulation in the posterior region of zebrafish, Danio rerio embryos. Journal of Morphology, 282(10), 1437–1454. https://doi.org/10.1002/jmor.21396Hammond-Weinberger, D. R., & Zeruth, G. T. (2019). Whole mount immunohistochemistry in zebrafish embryos and larvae. Journal of Visualized Experiments, 2020(155). https://doi.org/10.3791/60575Harada, A., Teng, J., Takei, Y., Oguchi, K., & Hirokawa, N. (2002). MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction. Journal of Cell Biology, 158(3), 541–549. https://doi.org/10.1083/jcb.200110134Hawkins, Tom. (s.f.). Whole-mount antibody staining. Zebrafish UCL, ZebrafishBrain: a neuroanatomical atlas of the developing zebrafish brain. (Revisado en 2023)Hobro, A. J., & Smith, N. I. (2017). An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging. Vibrational Spectroscopy, 91, 31–45. https://doi.org/10.1016/j.vibspec.2016.10.012Karlsson, J., Von Hofsten, J., & Olsson, P. E. (2001). Generating transparent zebrafish: A refined method to improve detection of gene expression during embryonic development. Marine Biotechnology, 3(6), 522–527. https://doi.org/10.1007/s1012601-0053-4Kimmel, C. B., Hatta, K., & Eisen, J. S. (1991). Genetic control of primary neuronal development in zebrafish. Development, 113(2 SUPPL.), 47–57. https://doi.org/10.1242/dev.113.supplement_2.47Lasser, M., Tiber, J., & Lowery, L. A. (2018). The role of the microtubule cytoskeleton in neurodevelopmental disorders. Frontiers in Cellular Neuroscience. Frontiers Media S.A. https://doi.org/10.3389/fncel.2018.00165Liedtke, Daniel. (2008). Functional divergence of Midkine growth factors : Non-redundant roles during neural crest induction, brain patterning and somitogenesis.Macdonald, R. (1999). Zebrafish immunohistochemistry. Methods in Molecular Biology (Clifton, N.J.). https://doi.org/10.1385/1-59259-678-9:77MAP2, NCBI Orthologs [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [2023 12 22]. Available from: https://www-ncbi-nlm-nih-gov.ezproxy.uniandes.edu.co/gene/4133/ortholog/?scope=7776Morris-Rosendahl, D. J., & Crocq, M. A. (2020). Neurodevelopmental disorders-the history and future of a diagnostic concept. Dialogues in Clinical Neuroscience, 22(1), 65–72. https://doi.org/10.31887/DCNS.2020.22.1/macrocqNational Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [2023 12 22]. Available from: https://www.ncbi.nlm.nih.gov/OLeary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, ONeill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD, Pruitt KD. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016 Jan 4;44(D1):D733-45 PubMed PubMedCentralOtálora Tarazona. (2018). Caracterización estructural del desarrollo pronefrico del Pez Cebra (Danio rerio). Universidad de los Andes.
Papadopoulos, J. S., & Agarwala, R. (2007). COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics (Oxford, England), 23(9), 1073–1079. https://doi-org.ezproxy.uniandes.edu.co/10.1093/bioinformatics/btm076Perdiz, D., Mackeh, R., Poüs, C., & Baillet, A. (2011). The ins and outs of tubulin acetylation: More than just a post-translational modification? Cellular Signalling. https://doi.org/10.1016/j.cellsig.2010.10.014Preibisch, S., Saalfeld, S., & Tomancak, P. (2009). Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics, 25(11), 1463–1465. doi:10.1093/bioinformatics/btp184Ross, L. S., Parrett, T., & Easter, S. S. (1992). Axonogenesis and morphogenesis in the embryonic zebrafish brain. Journal of Neuroscience, 12(2), 467–482. https://doi.org/10.1523/jneurosci.12-02-00467.1992Sakai, C., Ijaz, S., & Hoffman, E. J. (2018). Zebrafish Models of Neurodevelopmental Disorders: Past, Present, and Future. Frontiers in Molecular Neuroscience. Frontiers Media S.A. https://doi.org/10.3389/fnmol.2018.00294Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. Nature Methods. https://doi.org/10.1038/nmeth.2019Shihan, M. H., Novo, S. G., Le Marchand, S. J., Wang, Y., & Duncan, M. K. (2021). A simple method for quantitating confocal fluorescent images. Biochemistry and Biophysics Reports, 25. https://doi.org/10.1016/j.bbrep.2021.100916Soltani, M. H., Pichardo, R., Song, Z., Sangha, N., Camacho, F., Satyamoorthy, K., ... Setaluri, V. (2005). Microtubule-associated protein 2, a marker of neuronal differentiation, induces mitotic defects, inhibits growth of melanoma cells, and predicts metastatic potential of cutaneous melanoma. American Journal of Pathology, 166(6), 1841–1850. https://doi.org/10.1016/S0002-9440(10)62493-5Vallee, R. B. (1984). MAP2 (microtubule-associated protein 2). Cell and Muscle Motility. https://doi.org/10.1007/978-1-4684-4592-3_8Vaz, R., Hofmeister, W., & Lindstrand, A. (2019). Zebrafish models of neurodevelopmental disorders: Limitations and benefits of current tools and techniques. International Journal of Molecular Sciences. MDPI AG. https://doi.org/10.3390/ijms20061296Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)(4)., Univ. of Oregon Press, EugeneWhite, R. J., Collins, J. E., Sealy, I. M., Wali, N., Dooley, C. M., Digby, Z., ... Busch- Nentwich, E. M. (2017). A high-resolution mRNA expression time course of embryonic development in zebrafish. ELife, 6. https://doi.org/10.7554/eLife.30860Wilson, S. W., Brand, M., & Eisen, J. S. (2002). Patterning the zebrafish central nervous system. Results and Problems in Cell Differentiation. https://doi.org/10.1007/978-3- 540-46041-1_10Wilson, S. W., Ross, L. S., Parrett, T., & Easter, S. S. (1990). The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio. Development, 108(1), 121–145. https://doi.org/10.1242/dev.108.1.121Wright Cell Imaging Facility. (2004). Autofluorescence: Causes and Cures. Internet, 1–8. Retrieved from https://hwpi.harvard.edu/files/iccb/files/autofluorescence.pdf?m=1465309329Zebrafish UCL, ZebrafishBrain: a neuroanatomical atlas of the developing zebrafish brain. (Revisado en 2023). Anti-acetylated tubulin. http://zebrafishucl.org/antibodies- 1/tubulin201911053PublicationORIGINALCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdfCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdfapplication/pdf16723260https://repositorio.uniandes.edu.co/bitstreams/3c2114f9-c505-48b9-bcc3-f9c113332e04/download88d000df052d841806574fe1f33114bbMD51autorizacion tesis (1).pdfautorizacion tesis (1).pdfHIDEapplication/pdf413815https://repositorio.uniandes.edu.co/bitstreams/cce42731-a152-4492-a303-833ff691600f/download8c723950cd3a4d0aad268d4eed8809caMD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8805https://repositorio.uniandes.edu.co/bitstreams/72fa0eff-b613-4441-b7de-3404538c5a22/download4460e5956bc1d1639be9ae6146a50347MD53LICENSElicense.txtlicense.txttext/plain; charset=utf-82535https://repositorio.uniandes.edu.co/bitstreams/e95c94c8-dff8-4e70-ad8a-2c10d6d96aa6/downloadae9e573a68e7f92501b6913cc846c39fMD54TEXTCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdf.txtCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdf.txtExtracted texttext/plain95288https://repositorio.uniandes.edu.co/bitstreams/7b793577-8b76-42f2-b24a-0f8c9ff843c3/download0faeeda9724ff5d6b1061647b1a75cbeMD55autorizacion tesis (1).pdf.txtautorizacion tesis (1).pdf.txtExtracted texttext/plain2045https://repositorio.uniandes.edu.co/bitstreams/b1e53169-52b7-4273-b387-b69059dd04df/download569744e5f4a7fdbf4c6dd1263390b2e8MD57THUMBNAILCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdf.jpgCaracterización de la expresión de la proteína asociada a los microtúbulos 2 (map2) en el desarrollo neural del pez cebra (Danio rerio).pdf.jpgGenerated Thumbnailimage/jpeg6509https://repositorio.uniandes.edu.co/bitstreams/4942deec-3b62-46af-885b-d21df3b0fdb3/download4d3d2cff9578a73fd3a97f2de63327dfMD56autorizacion tesis (1).pdf.jpgautorizacion tesis (1).pdf.jpgGenerated Thumbnailimage/jpeg10900https://repositorio.uniandes.edu.co/bitstreams/6a92a7bd-038a-46f1-bb2f-43ddcd4207ee/download44d909ed7d9561b315ad3ac45aa437ebMD581992/73661oai:repositorio.uniandes.edu.co:1992/736612024-02-16 14:40:47.056http://creativecommons.org/licenses/by-nc-nd/4.0/Attribution-NonCommercial-NoDerivatives 4.0 Internationalopen.accesshttps://repositorio.uniandes.edu.coRepositorio institucional Sénecaadminrepositorio@uniandes.edu.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 |