전체 마운트 Zebrafish의 태아의 섬모 기관에서 면역 염색 포스 - 항원 결정기
Summary
기술은 전체 제브라 피쉬 배아에서 포스 항원을 발현 사이 다음 주 섬모와 같은 작은 세포 구조에서 두 색 형광 공 초점 현지화를 수행 할 수 있도록 서술되어있다. 고정 및 이미징 기술은 위치 특정 단백질의 모양이나 활성화 동력학을 정의 할 수있다.
Abstract
세포의 급속한 확산, 유전자의 조직 – 특이 적 발현 및 시그널링 네트워크의 출현은 모든 척추 동물의 초기 배아 발달의 특성. 심지어 하나의 셀 내에서 – – 반응 속도 및 신호의 위치 배아의 중요한 발달 유전자의 식별을 보완합니다. 면역 염색 기술이 차 섬모 작게 구조의 세포 내 및 전체 동물 신호의 동역학을 정의하기 위해 도시되었다는 것을 설명한다. 레이저 스캐닝 공 초점 복합 현미경을 사용하여 정착, 영상 및 이미지 처리 기술은 적은 36 시간 내에 완료 될 수있다.
제브라 피쉬 (다니오 레 리오)는 저렴하고 인간의 질병에 관련된 척추 동물 종에서 연구를 수행하고자 연구자를위한 바람직한 유기체이다. 유전자 녹아웃 또는 knockdowns는 실제 단백질 제품의 손실에 의해 확인되어야한다. 단백질 손실 이러한 확인여기에 설명 된 기술을 사용하여 달성 될 수있다. 신호 전달 경로에 대한 단서는 번역 후 인산화에 의해 변형 된 단백질과 반응하는 항체를 사용하여 해독 할 수 있습니다. 보존 및 에피토프의 인산화 상태를 최적화하는 것은이 결정하는 것이 중요하며,이 프로토콜에 의해 달성된다.
이 연구는 개발의 첫 72 시간 동안 배아를 해결하기 위해 기술을 설명 쿠퍼의 소포 (KV)의 섬모와 관련 항원의 다양한 신장과 내이 공동 현지화. 이러한 기술은 절개하지 않아도, 간단하고 비교적 짧은 시간에 완료 될 수있다. 하나의 이미지로 공 초점 이미지 스택을 투사하는 것은 이러한 데이터를 제시하는 유용한 수단이다.
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
PFA / 메탄올 방법은 제브라 피쉬 개발하는 동안 포스-T 287 -CaMK-II 에피토프의의 면역을 최적화의 주요 목적이 실험실에서 개발되었다. 이 방법은 성공적으로 제브라 피쉬의 KV, 14, 특히 KV 단계에서 신장. (13),이 기술이 필요했다 (12) 내이 등 여러 기관 섬모의 형성 동안 P-CaMK-II를 현지화. 이러한 방법의 성공으로 인해자가 형광의) 최소화, 포스 CaMK-II 에피토프 b) 보존…
Disclosures
The authors have nothing to disclose.
Acknowledgements
이 작품은 국립 과학 재단 (National Science Foundation) 부여 IOS-0817658에 의해 지원되었다.
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).
