Adapting INTACT to analyse cell-type-specific transcriptomes and nucleocytoplasmic mRNA dynamics in the Arabidopsis embryo
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In the early embryo of vascular plants, the different cell types and stem cells of the seedling are specified as the embryo develops from a zygote towards maturity. How the key steps in cell and tissue specification are instructed by genome-wide transcriptional activity is poorly understood. Progress in defining transcriptional regulation at the genome-wide level in plant embryos has been hampered by difficulties associated with capturing cell-type-specific transcriptomes in this small and inaccessible structure. We recently adapted a two-component genetic nucleus labelling system called INTACT to isolate nuclei from distinct cell types at different stages of Arabidopsis thaliana embryogenesis. We have used these to generate a transcriptomic atlas of embryo development following microarray-based expression profiling. Here, we present a general description of the adapted INTACT procedure, including the two-component labelling system, seed isolation, nuclei preparation and purification, as well as transcriptomic profiling. We also compare nuclear and cellular transcriptomes from the early Arabidopsis embryo to assess nucleocytoplasmic differences and discuss how these differences can be used to infer regulation of gene activity.
KeywordsNuclear isolation Cell-type-specific Embryo Transcriptome analysis
With the advent of transcriptomics, plant research has gained important insights into the genetic regulatory mechanisms that underlie cell fate determination, pattern formation and cell–cell communication during plant development. This framework of developmental processes, crucial for the continuous formation of plant structures from stem cells, is first established in the early plant embryo (reviewed in Palovaara et al. 2016). As such, much effort has been made in recent years to adapt transcriptomic approaches to this tissue at a cellular resolution (e.g. Belmonte et al. 2013; Casson et al. 2005; Slane et al. 2014). Recently, we adapted one such approach, INTACT (isolation of nuclei tagged in specific cell types), to isolate cell-type-specific nuclei from the early embryo of the flowering plant Arabidopsis thaliana for transcriptomic profiling (Palovaara et al. 2017).
Enrichment methods to isolate nuclei or cells based on the expression of specific promoters are very powerful, as the selection of cells for transcriptomics does not require manual selection and cells are by definition united by the expression of at least one marker gene. For the embryo, however, there is an important difficulty: genes expressed in the tissue precursors of the embryo are usually also expressed in the corresponding tissue of the seed that the embryo resides in (Belmonte et al. 2013; Palovaara et al. 2017). Thus, when using only expression of a marker gene as a selection step, transcriptomes of embryo cells would be overshadowed by the seed cells if entire seeds are used as starting material. Rather than using dissected embryos, we therefore resorted to a two-component genetic labelling system, INTACT (Deal and Henikoff 2010). INTACT is based on the selective biotinylation of a unique target peptide (biotin ligase recognition—BLRP) by the BirA biotin ligase enzyme (from Escherichia coli). The BLRP is integrated in a nuclear targeting fusion (NTF) protein, which in addition carries a green fluorescent protein (GFP) and a nuclear lamina localization domain. Only when BirA and NTF are co-expressed in the same cells, will the latter be biotinylated. Since streptavidin has a high affinity for biotin, biotin-tagged nuclei can next be isolated from crude nuclear preparations using streptavidin-coated beads. This circumvents the need for BirA and NTF promoter expression being exclusive to specific cells or tissues in the embryo. Consequently, specificity increases since more markers become available for use. We exploited this combinatorial logic by expressing BirA from an embryo-enriched promoter and driving NTF expression from a large range of cell-type-specific promoters. This allowed us to generate a transcriptome atlas of early Arabidopsis embryo development using nuclei isolated from cell types necessary for root stem cell niche establishment (Palovaara et al. 2017). We identified shifts in cell-type-specific gene expression associated with the developmental stage of the embryo, and enrichment of transcription factors and biological processes important for cell fate determination. Our work provides a resource for further exploration into how gene activity shapes the formation of the first plant tissues.
Here, we present the INTACT-based approach used in Palovaara et al. (2017). We discuss two-component labelling and describe the adapted INTACT procedure, including generating microarray-based transcriptomes. In addition, we compare previously published nuclear and cellular transcriptomes from the early Arabidopsis embryo to illustrate how INTACT-generated data can be used to investigate nucleocytoplasmic differences in a cell-type-specific manner.
INTACT on Arabidopsis embryos
INTACT was initially developed for use on roots from Arabidopsis (Deal and Henikoff 2010, 2011), a popular model plant to study cell specification processes due to its highly invariant cell division patterns and the ease to genetically manipulate. Compared to roots, Arabidopsis embryos are small, contain few cells and are surrounded by the endosperm and seed coat. This affects the final yield and purity of INTACT when performed according to the original protocol (Palovaara et al. 2017). Thus, it was necessary to adapt the INTACT protocol when isolating cell-type-specific nuclei from the early Arabidopsis embryo.
The adapted INTACT procedure and the approach used to generate transcriptomic profiles are described below. In theory, the procedure can be applied to any Arabidopsis embryo cell type, using our INTACT lines or other lines with a suitable cell-type-specific promoter, and can be combined with several downstream applications (Fig. 1). A detailed step-by-step protocol will be published separately.
Adapted INTACT procedure
The workflow can be divided into three separate sections: isolation of seeds with embryos of a known developmental stage, crude preparation of nuclei and purification of biotin-tagged nuclei. Recipes for buffers are presented in Supplementary Table 1.
For seed isolation, a set number of flowers from an appropriate NTF/BirA transgenic plant line are emasculated based on expected yield of bead-bound nuclei. Manual pollination of the exposed stigmas is performed the next day in less than 1 h to synchronize embryo development. We have consistently performed these steps with multiple people to minimize the time between the first and last pollination. Siliques are collected after a defined time interval at which they contain seeds with embryos at the development stage of interest. To avoid circadian rhythm effects, collection should be performed at the same time point of the day for each experiment. Siliques are adhered to slides with double-sided tape and seeds are exposed by opening the silique with a needle. Slides are then transferred to a 120 × 120 mm square Petri dish and placed under a stereo microscope. Next, seeds are collected from siliques onto a 20-μm nylon net filter using a suctioning apparatus consisting of a Pasteur pipette, a 25-mm filter holder and a vacuum pump. To avoid damage and dehydration of the seeds, a vacuum pressure at or under 40 mbar is used and 1 × PBS (pH 7.0) is continuously added to the Petri dish. Using this setup, it is possible to process siliques at a rate of 10 siliques per minute.
To prepare a crude nuclear extract, seeds are washed off the filter in 2-ml tubes containing 1 × PBS. The buffer is removed with a pipette, and a 4.8-mm stainless steel ball is added to each tube before flash-freezing them in liquid nitrogen. Seeds are homogenized in a mixer mill with pre-cooled adaptor racks (30 Hertz for 2 × 30 s) and resuspended with 10 ml ice-cold nuclear purification buffer (NPB) and 200 units of a RNase inhibitor in a 15-ml tube. The suspension is filtered through a 40-μm cell strainer and centrifuged at 1200g at 4 °C for 7 min to pellet nuclei. Nuclei are resuspended with 1 ml of NBP and transferred to a 1.5-ml tube. Ten microlitres of washed M-280 streptavidin-coated Dynabeads is added to the nuclei and incubated for 30 min at 4 °C with end-over-end rotation to bind beads to the nuclei.
Purification is performed by using a column system where the bead-bound nuclei are captured when they flow past a strong magnet. Columns are prepared by treating 1-ml pipette tips with NPB containing 1% (w/v) Casein for 20 min. Casein treatment prevents adhesion of non-bead-bound nuclei to the tip wall. A tip is inserted into an OctoMACS separator magnet placed vertically in a 4 °C cold room and then rinsed with ice-cold NPB containing 0.1% (v/v) Triton X-100 (NPBt). A two-way stopcock is attached to the narrow end of the pipette tip to control liquid flow rate. The bead and nuclei mixture is diluted with 9 ml NPBt and drawn into a plastic 10-ml serological pipette with a Parafilm-wrapped tip. The 10-ml pipette is fastened to the top of the 1-ml tip, and the mixture is flowed past the magnet at a rate of 0.75 ml min−1. This allows for efficient capture of bead-bound nuclei to the tip wall. The 1-ml tip is removed when empty, and the inner tip wall is washed by drawing NPBt in and out without disturbing the attached beads. The bead-bound nuclei are rinsed with 1 ml NPBt and diluted with 9 ml NPBt for a second round of purification, which is necessary for achieving maximum purity. After washing and rinsing the tip a second time, the bead-bound nuclei are collected by centrifugation (1000g at 4 °C for 5 min) and resuspended in 25 μl NPB. This solution contains the purified biotin-tagged nuclei, which can be directly used in downstream applications.
The purity of biotin-tagged nuclei is determined by the ratio of DAPI-stained (2 μg ml−1) bead-bound (biotinylated) and unbound (non-biotinylated) nuclei. Recovery efficiency is estimated by the ratio of bead-bound nuclei to the expected number of biotin-tagged nuclei in the starting material.
Generating transcriptomic profiles
One downstream application of the purified biotin-tagged nuclei is to generate and compare whole-genome expression data from various cell types. In Palovaara et al. (2017), this was performed using a microarray-based approach.
RNA is obtained from bead-bound nuclei isolated from different cell types and embryonic stages following TRIzol-based RNA extraction, DNase I treatment and RNA purification and concentration (Palovaara et al. 2017). To increase RNA amounts, reduce technical variances and avoid batch-to-batch effects (Clément-Ziza et al. 2009; Morse et al. 2010), RNA from biological replicates was pooled to 3–4 samples and then simultaneously amplified with an oligo(dT)/random primer mixture using a kit designed for small RNA quantities (Ovation Pico WTA System V2). This resulted in the amplification of both nascent transcripts and mature messenger RNA (mRNA). After labelling, amplified cDNA was hybridized to Arabidopsis Gene 1.1 ST 24-Array plates, which covers 84.2% (28,501) of all annotated genes (TAIR10). Analysis of the plates was performed using the MADMAX pipeline (Lin et al. 2011), with values normalized by RMA (Irizarry et al. 2003), to determine gene expression and differentially expressed genes. Further analyses were performed to determine dominant gene expression patterns by manual selection or hierarchical clustering (Orlando et al. 2009), biological significance of co-expressed genes (agriGO; Du et al. 2010; Tian et al. 2017) and enrichment of transcription factors (AtTFDB; Palaniswamy et al. 2006). Finally, a web-based tool was established at http://www.albertodb.org (ALBERTO) to display, compare and share the transcriptomic profiles.
In addition to our work, there are other publications concerning nuclear transcriptomics where RNA isolation, amplification methods and sequencing technologies differ (e.g. Deal and Henikoff 2010; Reynoso et al. 2018a, b; Slane et al. 2014; Zhang et al. 2008). For example, in Reynoso et al. (2018b) they demonstrate a method to remove ribosomal RNA (rRNA) from RNA isolated from INTACT-purified nuclei to produce a sample optimal for RNA-Seq. The preferred amplification method of nuclear RNA is oligo(dT)/random primer-based since it amplifies mRNA transcripts at different stages of processing. However, the highly abundant rRNAs are also amplified, which has a negative impact on sequencing coverage. In theory, this method could be combined with our approach to generate high-quality RNA-Seq data.
Comparing nuclear and cellular transcriptomes
INTACT relies on profiling RNA not yet exported to the cytosol, which raises the question if nuclear RNA is representative for the transcriptomic output of a cell. Several studies, including our own, have established that nuclear RNA is a reasonable proxy for steady-state transcript levels regardless of the experimental approach (Deal and Henikoff 2010; Lake et al. 2017; Palovaara et al. 2017; Slane et al. 2015; Zhang et al. 2008). However, there are differences between nuclear and total transcriptomes, which suggest selective compartmentalization of RNA in the nucleus and the cytosol. This may impact cell fate during development since post-transcriptional regulation, critical for protein translation and expression level, typically takes place in the cytoplasm. Despite this, relatively few studies have investigated the differences between nuclear, cytosolic and total transcriptomes in detail, especially in plants (see e.g. Barthelson et al. 2007; Chen and van Steensel 2017; Djebali et al. 2012; Bahar Halpern et al. 2015; Reynoso et al. 2018a; Solnestam et al. 2012). Until recently, the primary reason for this has been the lack of technologies that facilitate such a comparison, particularly at the tissue or cellular level.
RNA processing and maturation is a unidirectional process. Pre-mRNAs are spliced in the nucleus, capped and poly-adenylated, and exported to the cytosol for translation. Differences in nuclear/cytosolic abundance ratios between mRNAs may therefore derive from selective nuclear export, from stabilization or degradation, among others. A simple explanation for enrichment of a transcript in nuclear RNA pools would be that the mature mRNA is unstable and would quickly disappear from the cytosolic pool. Conversely, if an mRNA is exceptionally stable in the cytosol, its cytosolic levels will tend to be enriched. To determine if mRNA (in)stability could be a dominant cause of the observed enrichment, we investigated mRNA decay rates among cytosol- or nucleus-enriched transcripts using a mRNA decay data set from 5-day-old seedlings (Sorenson et al. 2018). We found that the cellular population had longer median mRNA half-life (142 min) compared to the nuclear population (92 min), with no apparent correlation to UTR lengths or coding region length (Supplementary Table 2c). This was especially evident when only transcription factors were analysed (101 vs. 41 min). This suggests that transcript enrichment in the cellular population may indeed reflect a relatively low decay rate. Earlier analyses have shown that high-flux RNAs associate with rapid signalling response pathways, including communication, hormone response and biotic/abiotic stress-related response (Narsai et al. 2007; Sorenson et al. 2018). Together, this demonstrates genome-wide differences between nuclear and cellular RNA that yield distinct functional characteristics.
The stage-specific clustering seen for the nuclear-enriched transcripts appears to be the result of rapidly changing expression levels between developmental stages: overall gene expression decreased from one stage (16-cell, early globular) to the next (late globular) as revealed by the expression heat map in Fig. 3b and a lower median expression value (21 vs. 14 signal intensity; Supplementary Table 2d). This, together with the low overall expression of the cellular-enriched genes in the atlas, supports the conclusion that RNAs with shorter half-lives and higher transcription rates are more common in the nuclear than the cellular RNA population. A list of “nuclear” or “cellular” genes that were enriched in the various cell types of the atlas is presented in Supplementary Table 2e.
From these results, we propose that selective nucleocytoplasmic enrichment of RNAs, through nuclear retention and post-transcriptional regulation, is a tissue- or even a cell-type-specific characteristic that directly impacts cell fates in the early plant embryo. Indeed, it is already known that microRNAs are involved in the establishment and maintenance of the shoot apical meristem in the Arabidopsis embryo (Palovaara et al. 2016; Takanashi et al. 2018) and that targeted RNA degradation influences embryonic stem cell fate in mammals (Li et al. 2015; Lou et al. 2014, 2016). However, to confirm our hypothesis, future work should compare the nuclear to the cytosolic RNA at a cellular resolution in wild-type embryos and embryos defective in post-transcriptional regulation. This could be achieved by using INTACT and another method, TRAP (translating ribosome affinity purification), to target the same tissue or cell type. TRAP allows for the isolation of actively translated transcripts after affinity purification of tagged ribosomal protein (Mustroph et al. 2009). Such a study would generate a comparative read-out of pre-processed (nuclear) and translated (cytosolic) mRNA, revealing the levels of regulation that direct gene expression in an individual cell. This read-out includes transcript sequence (size, codons, UTR regions) and isoforms, both of which can influence post-transcriptional regulation (e.g. Chen 2010; Hartmann et al. 2018; Theil et al. 2018), if RNA-Seq is used as the primary sequencing platform.
INTACT is a versatile tool that has been used for transcriptomic, epigenomic and proteomic studies of tissue- and cell-type-specific nuclei in plants and animals (Amin et al. 2014; Foley et al. 2017; Henry et al. 2012; Moreno-Romero et al. 2017; Park et al. 2016; Reynoso et al. 2018a; Ron et al. 2014; Steiner et al. 2012). In fact, a recent publication showed that several of these -omics studies can be performed on the same pool of INTACT-isolated nuclei (Mo et al. 2015). Here, we have presented how INTACT can be used to provide a snapshot of the nuclear transcriptome at a cellular resolution in the early Arabidopsis embryo. This has provided valuable information regarding mRNA synthesis. However, to fully explore the transcriptional networks that govern cell fate in the plant embryo we need to capture the full dynamics of the mRNA life cycle, especially at the cellular resolution. Therefore, the next logical step is to use INTACT in conjunction with a method such as TRAP and other -omics approaches to evaluate multiple levels of mRNA regulation. This would be a powerful addition to current research in plant embryos, where focus is on how cell fates are reprogrammed to establish the first tissues of the plant.
The authors thank Yanbo Mao and Tatyana Radoeva for providing the Arabidopsis plant and seed image, respectively, in Fig. 1. This work was supported by the Federation of European Biochemical Societies (FEBS) to J.P. and by the European Research Council (ERC; Starting Grant “CELLPATTERN”; Contract number 281573) and ERA-CAPS (EURO-PEC; 849.13.006) to D.W.
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