体外模拟旁非规范Wnt信号传导
Özet
本研究概述了一种高度可重复和易于处理的体外研究旁分泌非典型Wnt信号事件的方法 。 该协议用于评估旁分泌Wnt5a信号传导对小鼠神经嵴细胞和成肌细胞的影响。
Abstract
非规范Wnt信号调节胚胎发生过程中细胞内肌动蛋白丝组织和祖细胞的极化迁移。这个过程需要信号发送和信号接收细胞之间复杂而协调的旁分泌相互作用。鉴于这些相互作用可能发生在不同谱系的各种类型的细胞之间，细胞特异性缺陷的 体内 评估可能具有挑战性。本研究描述了一种高度可重复的体外评估旁分泌非典型Wnt信号传导 的方法。 该协议的设计能够（1）对任何两种感兴趣的细胞类型之间的非规范Wnt信号传导进行功能和分子评估;（2）剖析信号发送分子与信号接收分子在非规范Wnt信号通路中的作用;（3）使用标准分子或药理学方法进行表型挽救实验。
该协议用于评估成肌细胞中神经嵴细胞（NCC）介导的非规范Wnt信号传导。NCC 的存在与成肌细胞中鬼笔环肽阳性细胞丝状伪足和板状伪足的数量增加以及伤口愈合测定中肌母细胞迁移的改善有关。 Wnt5a-ROR2 轴被确定为NCC和第二心野（SHF）心肌母细胞祖细胞之间关键的非规范Wnt信号通路。总之，这是一个高度易于处理的方案，用于研究 体外旁分泌非规范Wnt信号传导机制。
Introduction
非规范Wnt信号传导是一种进化保守的途径，可调节细胞丝组织和定向迁移。该途径与多种生物学过程有关，包括胚胎组织形态发生1，2，3，淋巴和血管生成4，5，6，7以及癌症生长和转移8，9，10.在细胞水平上，非规范的Wnt信号是通过信号发送和信号接收细胞之间的协调旁分泌相互作用进行的。这些相互作用经常发生在不同谱系或类型的细胞之间，并涉及多种分子网络，包括多达19个配体和多个受体，共受体和下游信号转导效应子11。使这种信号传导过程进一步复杂化的是，先前的研究表明，配体-受体组合可以以上下文和组织依赖性的方式变化12，13，并且驱动信号接收细胞中非规范Wnt信号传导的相同源配体可以由多种信号发送细胞类型产生14，15.鉴于与非规范Wnt信号传导相关的细胞和分子复杂性，研究体内个体和临床相关机制的能力受到限制。
已经尝试使用体外细胞培养技术研究非规范的Wnt信号传导。例如，在细胞单层中进行的伤口愈合测定已被用于功能评估细胞定向迁移4，16，17，18，19。免疫染色技术已被用于对表面蛋白表达进行空间分析，以评估非规范Wnt诱导的细胞形态变化7，10，结构和不对称极化18，19，20。 尽管这些方法为表征信号接收细胞中的Wnt相关表型提供了重要工具，但这些协议中缺乏信号发送组件限制了它们准确模拟体内观察到的旁分泌信号机制的能力。因此，仍然迫切需要开发体外系统，该系统允许对非规范Wnt途径的信号发送和接收细胞之间的旁分泌信号传导相互作用进行稳健和可重复的评估，特别是不同细胞类型的细胞。
为此，本研究的主要目的是建立一种方案来模拟体外旁分泌非规范Wnt信号相互作用。我们开发了一种非接触式共培养系统，该系统概括了这些相互作用的信号发送和信号接收成分，并允许使用标准的分子、遗传或药理学方法来独立研究非规范 Wnt 途径中的特定配体受体机制。使用已建立的鼠细胞系在成肌细胞中检查NCC介导的Wnt信号传导的机制。作为原理证明，该模型用于证实先前小鼠体内研究的结果，这些研究涉及Wnt5a-ROR2轴是NCC 21和SHF心肌母细胞祖细胞3，22，23之间的相关非规范Wnt信号通路。
Protocol
1. 细胞的实验前扩增和传代 C2C12细胞培养：将 Dulbecco 的改良鹰培养基 （DMEM） 与 10% 胎牛血清 （FBS） 和 1% 青霉素/链霉素混合，制备 500 mL C2C12 培养基。 在37°C水浴中解冻一小瓶C2C12细胞。当 C2C12 细胞解冻时，将 5 mL C2C12 培养基加入 15 mL 锥形管中。立即使用 P1000 移液器将解冻的细胞转移到 15 mL 管中。注意：C2C12细胞是鼠肌母细胞，以前已被使用并验证为模拟…
Representative Results
NCC对鼠成肌细胞迁移能力的影响该测定首先用于评估NCC对成肌细胞迁移能力的影响。 图1 概述了该测定的示意图模型。为了测试这种影响，使用单独生长的成肌细胞（没有NCC插入物）与在插入物存在下生长的成肌细胞进行了划痕测定。作为阳性对照，将500 ng/mL重组Wnt5a（rWnt5a）加入到带有NCC插入物的腔室孔中。rWnt5a的浓度是通过在C2C12细胞中进行的剂量反应分…
Discussion
非规范Wnt/平面细胞极性（PCP）信号通路是一种至关重要的细胞信号通路，与多种发育过程24，25和疾病过程24，26有关。在胚胎发育过程中，非规范的Wnt信号涉及来自信号发送细胞的分子信号的广泛网络，最终诱导信号接收细胞的形态，不对称组织和定向迁移的变化11。先前的研究表明?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH授予O.T.的F30HL154324以及S.R.K.的K08HL121191和R03HL154301的部分支持。作者要承认，本手稿中图 1 中的原理 图 是用 biorender.com 创建的。
Materials
| 2-Mercaptoethanol | Sigma Aldrich | M-7522 | |
| Antifade mounting medium with DAPI | Vector Laboratories | H-1200-10 | Stored at 4 °C |
| Bovine serum albumin | Santa Cruz Biotechnology | sc-2323 | Stored at 4 °C |
| C2C12 murine myoblast cell line | ATCC | CRL-1772 | |
| Cell culture flasks, 75 cm2 | ThermoFisher Scientific | 156499 | |
| Chamber Slide System, 4-well | ThermoFisher Scientific | 154526 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (4.5 g/L), L-glutamine (2 mM) | Corning | 10-017-CV | Stored at 4 °C |
| Falcon conical centrifuge tubes, 15 mL | Fisher Scientific | 14-959-53A | |
| Falcon permeable support for 24-well plate with 0.4 µM transparent PET membrane | Corning | 353095 | |
| Fetal bovine serum | Fisher Scientific | W3381E | Stored in 50 mL aliquots at -20 °C |
| Gelatin solution, 0.1% | ATCC | PCS-999-027 | Stored at 4 °C |
| Graduated and sterile pipette tips, 10 µL | USA Scientific | 1111-3810 | |
| Leukemia inhibitory factor (LIF), 106 unit/mL | Millipore Sigma | ESG1106 | |
| L-glutamine 200 mM (100x) | Gibco | 25030-081 | |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | 13778-075 | |
| MEM non-essential amino acids (MEM NEAA) 100x | Gibco | 11140-050 | |
| Minimum essential medium (MEM) | Corning | 10-022-CV | |
| Mitomycin C | Roche | 10107409001 | |
| Non-stick auto-glass coverslips, 24 x 55 mm | Springside Scientific | HRTCG2455 | |
| O9-1 neural crest cell line | Millipore Sigma | SCC049 | |
| Opti-MEM I, 1x | Gibco | 31985-070 | |
| Paraformaldehyde solution in PBS, 4% | Santa Cruz Biotechnology | sc-281692 | Stored at 4 °C |
| Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) | Fisher Scientific | W3470H | Stored in 10 mL aliquots at -20 °C |
| Phalloidin-iFluor 488 | Abcam | ab176753 | Stored at -20 °C, Keep out of light |
| Phosphate-buffer saline (PBS), 1x, without calcium and magnesium, pH 7.4 | Corning | 21-040-CV | Stored at 4 °C |
| Recombinant human fibroblast growth factor-basic (rhFGF-basic) | R&D Systems | 233-FB-025 | |
| Recombinant human/mouse Wnt5a protein | R&D Systems | 645-WN-010 | |
| Sodium pyruvate, 100 mM | Gibco | 11360-070 | |
| Square Petri dish with grid | Thomas Scientific | 1219C98 | |
| STO murine fibroblast feeder cells | ATCC | CRL-1503 | |
| Triton X-100 solution | Sigma Aldrich | X100-100ML | |
| Trypsin-EDTA, 0.25% | Fisher Scientific | W3513C | Stored at 4 °C |
| Zeiss Apotome.2 fluoresence microscope | Carl Zeiss AG | ||
| Zeiss inverted Axio Vert.A1 light microscope | Carl Zeiss AG | ||
| Zen lite 2012 microscopy software | Carl Zeiss AG | imaging software |
Referanslar
- Ho, H. Y. H., et al. Wnt5a-Ror-Dishevelled signaling constitutes a core developmental pathway that controls tissue morphogenesis. Proceedings of the National Academy of Sciences of the United States of America. 109 (11), 4044-4051 (2012).
- Čapek, D., et al. Light-activated Frizzled7 reveals a permissive role of noncanonical wnt signaling in mesendoderm cell migration. Elife. (8), 42093 (2019).
- Li, D., et al. Planar cell polarity signaling regulates polarized second heart field morphogenesis to promote both arterial and venous pole septation. Development. 146 (20), 181719 (2019).
- Lutze, G., et al. Noncanonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling. Scientific Reports. 9 (1), 4739 (2019).
- Buttler, K., et al. Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice. Gelişim Biyolojisi. 381 (2), 365-376 (2013).
- Betterman, K. L., et al. Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow. Journal of Clinical Investigation. 130 (6), 3315-3328 (2020).
- Descamps, B., et al. Frizzled 4 regulates arterial network organization through noncanonical Wnt/planar cell polarity signaling. Circulation Research. 110 (1), 47-58 (2012).
- Weeraratna, A. T., et al. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell. 1 (3), 279-288 (2002).
- Henry, C., et al. Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both β-catenin dependent and independent Wnt signalling. Journal of Cancer Research and Clinical Oncology. 141 (2), 243-254 (2014).
- Anastas, J. N., et al. A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression. Oncogene. 31 (32), 3696-3708 (2012).
- Niehrs, C. The complex world of WNT receptor signalling. Nature Reviews Molecular Cell Biology. 13 (12), 767-779 (2012).
- Dong, B., et al. Functional redundancy of frizzled 3 and frizzled 6 in planar cell polarity control of mouse hair follicles. Development. 145 (19), (2018).
- Bernascone, I., et al. Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels. Nature Communications. 10 (1), 2481 (2019).
- Hendrickx, G., et al. WNT16 requires Gα subunits as intracellular partners for both its canonical and noncanonical WNT signalling activity in osteoblasts. Calcified Tissue International. 106 (3), 294-302 (2020).
- Avgustinova, A., et al. Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness. Nature Communications. (7), 10305 (2016).
- Tseng, J. C., et al. CAPE suppresses migration and invasion of prostate cancer cells via activation of noncanonical Wnt signaling. Oncotarget. 7 (25), 38010-38024 (2016).
- Wang, Q., et al. A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer. PLoS One. 5 (5), 10456 (2010).
- Gibbs, B. C., et al. Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects. Biology Open. 5 (3), 323-335 (2016).
- Cui, C., et al. a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton. PLoS Biology. 11 (11), 1001720 (2013).
- Gombos, R., et al. The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila. The Journal of Neuroscience. 35 (28), 10154-10167 (2015).
- Toubat, O., et al. Neural Crest Cell-derived Wnt5a Regulates Planar Cell Polarity in Cranial Second Heart Field Progenitor Cells. Circulation. 142, 12540 (2020).
- Li, D., et al. Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment. Gelişim Biyolojisi. 412 (1), 18-31 (2016).
- Sinha, T., et al. Loss of Wnt5a disrupts second heart field cell deployment and may contribute to OFT malformations in DiGeorge syndrome. Human Molecular Genetics. 24 (6), 1704-1716 (2015).
- Humphries, A. C., et al. From instruction to output: Wnt/PCP signaling in development and cancer. Current Opinion in Cell Biology. (51), 110-116 (2018).
- Shi, D. L. Decoding Dishevelled-Mediated Wnt Signaling in Vertebrate Early Development. Frontiers in Cell and Developmental Biology. (8), 588370 (2020).
- Butler, M. T., et al. Planar cell polarity in development and disease. Nature Reviews Molecular Cell Biology. 18 (6), 375-388 (2017).
- Bradshaw, L., et al. Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H. Journal of Anatomy. 214 (2), 245-257 (2009).
- Kodo, K., et al. Regulation of Sema3c and the interaction between cardiac neural crest and second heart field during outflow tract development. Scientific Reports. 7 (1), 6771 (2017).
- Waldo, K. L., et al. Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field. Gelişim Biyolojisi. 281 (1), 66-77 (2005).
- Schleiffarth, J. R., et al. Wnt5a is required for cardiac outflow tract septation in mice. Pediatric Research. 61 (4), 386-391 (2007).
- Nguyen, B. H., et al. Culturing and Manipulation of O9-1 Neural Crest Cells. Journal of Visualized Experiments. (140), e58346 (2018).
- Suarez-Arnedo, A., et al. An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. PLoS One. 15 (7), 0232565 (2020).
- Martinotti, S., et al. Scratch wound healing assay. Methods in Molecular Biology. (2109), 225-229 (2020).
Bu Makaleden Alıntı Yapın
Toubat, O., Choi, J., Kumar, S. R. Modeling Paracrine Noncanonical Wnt Signaling In Vitro. J. Vis. Exp. (178), e63247, doi:10.3791/63247 (2021).