Inmunotinción fosfo-epítopos en ciliadas Órganos de todo el montaje embriones de pez cebra
Summary
Se describen las técnicas de inmunotinción fosfo-epítopos en embriones de pez cebra enteros y luego llevar a cabo la localización de dos colores confocal de fluorescencia en las estructuras celulares tan pequeños como los cilios primarios. Las técnicas para la fijación y formación de imágenes pueden definir la ubicación y la cinética de la aparición o la activación de proteínas específicas.
Abstract
La rápida proliferación de las células, la expresión específica de tejido de genes y la aparición de las redes de señalización caracterizan el desarrollo embrionario temprano de todos los vertebrados. La cinética y la ubicación de las señales – incluso dentro de las células individuales – en el embrión en desarrollo complementa la identificación de genes de desarrollo importantes. técnicas de inmunotinción se describen que se han demostrado para definir la cinética de señales intracelulares y animales enteros en estructuras tan pequeñas como cilios primarios. Las técnicas de fijación, de imágenes y procesamiento de imágenes utilizando un microscopio compuesto confocal de escaneo láser se puede completar en tan sólo 36 horas.
El pez cebra (Danio rerio) es un organismo deseable para los investigadores que buscan llevar a cabo estudios en una especie de vertebrados que es asequible y relevante para las enfermedades humanas. knockouts o caídas genéticos deben ser confirmadas por la pérdida de la proteína producto real. Dicha confirmación de la pérdida de proteínasse puede lograr usando las técnicas descritas aquí. Pistas sobre las vías de señalización también se pueden descifrar mediante el uso de anticuerpos que son reactivos con proteínas que han sido modificados después de la traducción mediante fosforilación. Preservar y optimizar el estado fosforilado de un epítopo tanto, es crítico para esta determinación y se logra mediante este protocolo.
Este estudio describe las técnicas para solucionar los embriones durante las primeras 72 h de desarrollo y co-localizar una variedad de epítopos relevantes con los cilios en la vesícula Kupffer (KV), el riñón y el oído interno. Estas técnicas son sencillas, no requieren de disección y se puede completar en un período relativamente corto de tiempo. Proyección de pilas de imágenes confocal en una sola imagen es un medio útil para la presentación de estos datos.
Introduction
The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals.
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred.
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer’s Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer’s Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.
Protocol
Representative Results
Discussion
El método / metanol PFA fue desarrollado en este laboratorio con el objetivo principal de la optimización de la inmunolocalización de la fosfo-T 287 -CaMK-II epítopo durante el desarrollo de pez cebra. Este método localiza con éxito P-CaMK-II durante la formación de varios órganos incluyendo el ciliadas KV pez cebra, 12 oído interno 14 y el riñón. 13 En particular, en la etapa de KV, esta técnica era necesario. El éxito de este método es probablemente debido a un…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Este trabajo fue apoyado por la beca de la Fundación Nacional de Ciencia IOS-0817658.
Materials
| 1-phenyl-2-thiourea (PTU) | Sigma | P-7629 | 0.12% Stock solution. Dilute 1:40 in system water |
| Alexa488 anti-mouse IgG | Life Technologies | A11001 | Goat polyclonal, use at 1:500 |
| Alexa488 anti-rabbit IgG | Life Technologies | A11008 | Goat polyclonal, use at 1:500 |
| Alexa488 phalloidin | Life Technologies | A12379 | Preferentially binds to F-actin |
| Alexa568 anti-mouse IgG | Life Technologies | A11004 | Goat polyclonal, use at 1:500 |
| Alexa568 anti-rabbit IgG | Life Technologies | A11011 | Goat polyclonal, use at 1:500 |
| anti-acetylated a-tubulin | Sigma | T7451 | Mouse monoclonal, use at 1:500 |
| anti-phospho-T287 CaMK-II | EMD Millipore | 06-881 | Rabbit polyclonal, use at 1:20 |
| anti-total CaMK-II | BD Biosciences | 611292 | Mouse monoclonal, use at 1:20 |
| Ethanol | Fisher | S96857 | Lab grade, 95% denatured |
| Forceps | Fine Science Tools | 11252-20 | Dumont #5 |
| Glass coverslips | VWR | 16004-330 | #1 thickness |
| Glass microscope slides | Fisher | 12-550-15 | Standard glass slides |
| Methanol | Fisher | A411 | Store in freezer |
| Microcentrifuge tubes | VWR | 20170-038 | capped tubes, not sterile |
| Normal goat serum | Life Technologies | 16210-064 | Aliquot 1ml tubes, store in freezer |
| Paraformaldehyde | Sigma | P-6148 | Reagent grade, crystalline |
| Phosphate buffered saline (PBS) | Quality Biological | 119-069-131 | 10X stock solution or made in lab |
| Triton X-100 | Sigma | BP-151 | 10% solution in water, store at room temp |
| Tween-20 | Life Technologies | 85113 | 10% solution in water, store at room temp |
| Compound microscope | Nikon | E-600 | Mount on vibration-free table |
| C1 Plus two-laser scanning confocal | Nikon | C1 Plus | Run by EZ-C1 program, but upgrades use "Elements" |
References
- Webb, S. E., Miller, A. L. Calcium signalling during embryonic development. Nat Rev Mol Cell Biol. 4, 539-551 (2003).
- Webb, S. E., Miller, A. L. Ca2+ signalling and early embryonic patterning during zebrafish development. Clin Exp Pharmacol Physiol. 34, 897-904 (2007).
- Whitaker, M. Calcium at fertilization and in early development. Physiol. Rev. 86, 25-88 (2006).
- Yuen, M. Y., et al. Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development. Biochim Biophys Acta. 1833, 1641-1656 (2013).
- Tombes, R. M., Borisy, G. G. Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient. J. Cell Biol. 109, 627-636 (1989).
- Hudmon, A., Schulman, H. Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function. Annu. Rev. Biochem. 71, 473-510 (2002).
- Tombes, R. M., Faison, M. O., Turbeville, C. Organization and Evolution of Multifunctional Ca2+/CaM-dependent Protein Kinase (CaMK-II). Gene. 322, 17-31 (2003).
- Braun, A. P., Schulman, H. The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase: From Form to Function. Annu. Rev. Physiol. 57, 417-445 (1995).
- Rich, R. C., Schulman, H. Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 273, 28424-28429 (1998).
- Rothschild, S. C., et al. Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis. Dev Biol. 330, 175-184 (2009).
- Rothschild, S. C., Lister, J. A., Tombes, R. M. Differential expression of CaMK-II genes during early zebrafish embryogenesis. Dev Dyn. 236, 295-305 (2007).
- Francescatto, L., Rothschild, S. C., Myers, A. L., Tombes, R. M. The activation of membrane targeted CaMK-II in the zebrafish Kupffer’s vesicle is required for left-right asymmetry. Development. 137, 2753-2762 (2010).
- Rothschild, S. C., Francescatto, L., Drummond, I. A., Tombes, R. M. CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia. Development. 138, 3387-3397 (2011).
- Rothschild, S. C., et al. CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling. Dev Biol. 381, 179-188 (2013).
- Yuan, S., Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J Vis Exp. , e1113 (2009).
- Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. J Vis Exp. , e1115 (2009).
- Westerfield, M. . The Zebrafish Book: A guide for the laboratory use of zebrafish (Brachydanio rerio). , (1993).
- Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310 (1995).
- Obara, T., et al. Polycystin-2 immunolocalization and function in zebrafish. J Am Soc Nephrol. 17, 2706-2718 (2006).
- Chitramuthu, B. P., Bennett, H. P. High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function. J Vis Exp. , e50644 (2013).
- Thisse, C., Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature. 3, 59-69 (2008).
- Harris, P., Osborn, M., Weber, K. Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage. J Cell Biol. 84, 668-679 (1980).
