激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具
Summary
Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.
Abstract
The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.
Introduction
在组织水平进行转录的研究，高度专业化但罕见的细胞类型的转录由更丰富周围细胞通常掩蔽。对于这样的高度特化的细胞类型的例子是在植物中女性生殖谱系（种系）的细胞。女性生殖正在开发胚珠中指定的，种子的前身花1,2的雌蕊内。的大孢子母细胞（MMC）是女性生殖系的第一个单元。它经过减数分裂形成大孢子减少了四分。通常情况下，只有这些大孢子的一个生存和有丝分裂不分裂细胞分裂， 即在一个合胞体。这些有丝分裂之后是细胞化以形成成熟配子体，其典型地包括四个细胞类型：三级antipodals，二助细胞，卵，和中央细胞。鸡蛋和中央细胞是获得由两个精子细胞中斗受精雌配子BLE受精以产生显影种子1,2的胚和胚乳。在性模型系统拟南芥中，只有约50种子每花发展而大约50 – 80种子每花发展密切相关的属Boechera。因此，女性生殖系仅由少数高度特化的细胞类型的，使得它的极好的模型来研究发育过程，诸如细胞说明书和分化。
此外，见解执政植物繁殖的基因调控过程中可以应用的价值。在植物中，可能会出现通过种子（无融合生殖）两种性与无性繁殖。而有性生殖群体中产生遗传多样性，无融合生殖导致形成克隆的后代是遗传上相同母株。因此，无融合生殖对农业和种子生产应用中的巨大潜力，因为即使是复杂的孕产妇的基因型几代3,4,5维持不变。因为无融合生殖不天然存在于任何主要作物物种，无融合生殖的作物的工程是极大的兴趣3,4,5。然而，这个长期目标是难以实现的，因为无融合生殖的潜在的遗传和分子基础是不足够详细6理解。
要深入了解转录的基础上使用激光辅助显微切割（LAM）和下一代测序（NGS）治无融合生殖繁衍，细胞类型特异性转录谱是一个非常有效的方法7,8。 LAM已先行建立了动物和生物医学研究。在过去的几年中一直LAM也被应用到植物生物学6,9,10。在与其它的方法使单个细胞和组织类型的分析，林不需要的标记线6,9,10的产生。因此，它可以是应用谎称，恕不另行分子知识的任何细胞或组织类型。林的另一个优点是，它可以使用，只要适用于任何类型的细胞作为细胞可以在干燥部分基于位置和/或结构特征进行识别。林具有附加的优点，即使用固定的组织，以防止在加工过程中的转录轮廓的变化。
的兴趣， 例如，花组织的组织，在石蜡包埋之前固定在非交联固定剂。在石蜡包埋可以手动完成，以下建立的协议9,11。然而，对于脱水和渗透的使用自动化组织处理器的与蜡通常导致更高的再现性在RNA的质量和组织形态的保护方面。包埋在树脂组织的替代策略也已成功地由LAM 8用于细胞类型特异性的分析。然而，使用一个奥波的在蜡包埋组织ated处理器是非常有效的时间，因为许多样品可在一次需要动手的时间最少的处理。而RNA的质量通常不显著损失固定和嵌入过程中发生，用切片机薄片的制备，尤其，安装在用于林的frameslides仍然是RNA的质量的保存的关键步骤。此以前已注意到与使用的磁带传送系统的被描述为导致更好的RNA的质量在此步骤12。然而，这增加了制备该幻灯片的期间额外耗时的步骤，也需要特殊的设备。下面所描述的优化的协议可重复产生的RNA是足够的质量与基因芯片和新一代测序（NGS）接近7,11,13,14转录谱。此外，与所使用的激光显微切割显微镜，分离的细胞类型的高纯度是击溃inely生产7,11,13,14。
属Boechera是研究无融合生殖的关键步骤一个很好的模型系统。在Boechera，各种不同的性和无融合生殖种质的已经确定，并可以用于比较分析15,16,17。在细胞的细胞类型特异性转录的从阴种系免受性拟南芥和无融合生殖Boechera的比较，我们鉴定的基因和途径被差异表达，从而鉴定调节过程的新的方面理事无融合生殖7。此外，本研究证实林的细胞类型特异性的转录小和稀有细胞类型分析的适用性。我们已经使用该协议为不同的细胞类型中的各种植物物种的分析，但物种和组织特异性的修改的协议可能在某些情况下是需要的。
Protocol
Representative Results
Discussion
该协议适用于不同的细胞和组织类型
林与转录结合由微阵列或分析RNA测序是洞察基因活性的调节发育或生理过程7-11,13,14特定模式的有价值的工具。然而，该方法对于任何给定细胞类型的适宜性是极其依赖于结构问题。细胞必须清晰可见，并用于LAM干节明确识别。在Boechera divaricarpa，配子体的形态允许易于识别的卵细胞（ 图2，图3）。?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Timothy F. Sharbel (IPK Gatersleben) for providing Boechera divaricarpa seeds and Sharon Kessler (University of Oklahoma) for critical reading and proofreading. Work on cell type-specific transcriptome analyses to study gametophyte development and apomixis in UG´s laboratory is supported by the University of Zürich, by a fellowship of the “Deutsche Forschungsgemeinschaft” and the Marie Curie project IDEAGENA to AS, by grants from the “Staatssekretariat für Bildung und Forschung” in the framework of COST action FA0903 (to UG and AS) and the Swiss National Foundation (to UG).
Materials
| Ethanol | VWR | 1,009,861,000 | absolute EMPROVE Ph Eur,BP,USP |
| 15 ml falcon centrifuge tubes | VWR | 62406-200 | |
| 2100 Bioanalyzer | Agilent | G2939AA | |
| Acetic Acid | Applichem | A3686,2500 | 100% Molecular biology grade |
| Ambion Nuclease free water | life technologies | AM9932 | |
| ASP200 S | Leica | 14048043624 | tissue processor |
| black cardboard | can be purchased in special paper shops | ||
| DNA- and RNAse-free Frame Slides | Micro Dissect GmbH | 1,4 µm PET-membrane; can also be purchased from Leica | |
| Dumond Forceps | Actimed | 0208-5SPSF-PS | |
| ethanol lamp | |||
| exsiccator | Sigma-Aldrich | Z354074-1EA | Nalgene Vaccuum Dessicator or similar equipment |
| filter tips 10 µl | Axon Lab AG | AL60010 | can be replaced by similar tips |
| filter tips 1000 µl | Axon Lab AG | AL60010 | can be replaced by similar tips |
| filter tips 20 µl | Axon Lab AG | AL60020 | can be replaced by similar tips |
| filter tips 200 µl | Axon Lab AG | AL60200 | can be replaced by similar tips |
| forceps precision | VWE | 232-1221 | |
| glass slide holder | Huber & Co.AG | 10.0540.01 | Färbekästen nach Hellendahl |
| glass staining trough | Huber & Co.AG | 10.0570.01 | Färbekasten |
| Heated Paraffin Embedding Module Leica | Leica | Leica EG 1150 H | blocking station, similar devises are suitable |
| Heating and Drying Table | Medax | 15501 | other models and/or suppliers are suitable |
| ice bucket | VWR ice bucket with lid | 10146-184 | similar buckets equally suitable |
| light table | UVP An Analytical Jena Company | TW-26 | white light transluminator |
| microscope slide | Thermo Scientific | 10143562CE | cut edges |
| microtome blade | Thermo Scientific | FEAHS35 | S35 microtome blade disposable |
| MMI Cell Cut Plus Instrument | MMI (Molecular Machines and Industries) | ||
| Non-stick, RNAse free Microfuge tubes, 2ml | life technologies | AM12475 | |
| Paraplast X-TRA | Roth | X882.2 | for histology |
| PicoPure RNA Isolation Kit | life technologies | KIT0204 | Arcturus PicoPure RNA Isolation Kit |
| plastic balancing trays | Semadeni AG | 2513 | |
| plastic box | Semadeni AG | 2971 | Plastikdose PS |
| plastic lid for heating plate | homemade | ||
| preparation needle | VWR | 631-7159 | |
| RNA 6000 Pico Kit | Agilent | 5067-1513 | |
| RNAse free microfuge tubes | life technologies | AM12400 | |
| RNAse ZAP Decontamination Solution | life technologies | AM9780 | |
| Semi-automated Rotary Microtome | Leica | RM2245 | similar devises are equally suitable |
| Tissue Loc Histo Screen Cassettes | Thermo Scientific | C-1000_AQ | similar cassettes of other suppliers are suitable |
| Tubes with adhesive lid, without diffusor 500 µl | MMI (Molecular Machines and Industries) | 50204 | |
| Xylol (Isomere) ROTIPURAN | VWR | 4436.2 | min. 99 %, p.a.,ACS, ISO SP |
| process embedding cassettes | Leica | 14039440000 | Leica Jet Cassette I without lid |
| Universal Oven | Memmert | UF55 | other models and/or suppliers are suitable |
References
- Maheshwari, P. . An Introduction to the Embryology of Angiosperms. , (1950).
- Willemse, M. T. M., Van Went, J. L., Johri, B. M. The female gametophyte. Embryology of Angiosperms. , 159-196 (1984).
- Koltunow, A. M., Bicknell, R. A., Chaudhury, A. M. Apomixis: Molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 108, 1345-1352 (1995).
- Vielle-Calzada, J. P., Crane, C., Stelly, D. M. Apomixis – the asexual revolution. Science. 274, 1322-1323 (1996).
- Grossniklaus, U., Koltunow, A., van Lookeren Campagne, M. A bright future for apomixis. Trends Plant Sci. 3, 415-416 (1998).
- Schmidt, A., Schmid, M. W., Grossniklaus, U. Plant germline formation: molecular insights define common concepts and illustrate developmental flexibility in apomictic and sexual reproduction. Development. 142, 229-241 (2015).
- Schmidt, A., et al. Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation. PLOS GENET. 10, e1004476 (2014).
- Okada, T., et al. Enlarging cells initiating apomixis in Hieractium praealtum transition to an embryo sac program prior to entering mitosis. Plant Physiol. 163, 216-231 (2013).
- Wuest, S. E., Grossniklaus, U. Laser-assisted microdissection applied to floral tissues. Methods Mol Biol. 1110, 329-344 (2014).
- Wuest, S. E., Schmid, M. W., Grossniklaus, U. Cell-specific expression profiling of rare cell types as exemplified by its impact on our understanding of female gametophyte development. Curr Opin Plant Biol. 16 (1), 41-49 (2013).
- Wuest, S. E., et al. Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr Biol. 20, 1-7 (2010).
- Cai, S., Lashbrook, C. C. Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling. Plant J. 48 (4), 628-637 (2006).
- Schmidt, A., Wuest, S. E., Vijverberg, K., Baroux, C., Kleen, D., Grossniklaus, U. Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germ line development. PLOS BIOL. 9, e1001155 (2011).
- Schmid, M. W., Schmidt, A., Klostermeier, U. C., Barann, M., Rosenstiel, P., Grossniklaus, U. A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing. PLOS ONE. 7, e29685 (2012).
- Mau, M., et al. Hybrid apomicts trapped in the ecological niches of their sexual ancestors. Proc Natl Acad Sci.U S A. 112 (18), 2357-2365 (2015).
- Aliyu, O. M., Seifert, M., Corral, J. M., Fuchs, J., Sharbel, T. F. Copy number variation in transcriptionally active regions of sexual and apomictic Boechera. demonstrates independently derived apomictic lineages. Plant Cell. 25 (10), 3808-3823 (2013).
- Corral, J. M., et al. A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species. Plant Physiol. 163 (4), 1660-1672 (2013).
- Deeken, R., Ache, P., Kajahn, I., Klinkenberg, J., Bringmann, G., Hedrich, R. Identification of Arabidopsis thaliana. phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments. Plant J. 55, 746-775 (2008).
- Schmid, M. W. . Genome Scale Quantitative Biology in Arabidopsis thaliana. , 40-104 (2015).
