Name: translating ribosome affinity purification (trap) to investigate arabidopsis thaliana root development at a cell type-specific scale
Uploaded: 2020-05-14T12:30:00
Description: Watch this Scientific Journal Video about Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale at JoVE.com

We would like to thank Jean-Claude Walser of the Genetic Diversity Center Zurich for crucial expert advice in the early phase of this project. Work in the Vermeer lab was supported by an SNF Professorship grant (PP00P3_157524) and a R&#39;EQUIP equipment grant (316030_164086) from the Swiss National Science Foundation (SNSF) awarded to JEMV.

1. Cloning of transgene, transgenic line production and selection
<ol>
	<li>Clone the promoter of choice in the appropriate entry vector. Use a recombination-based cloning method (Table of Materials) and recombine the promoters in pDONRP4-P1r. Clone RPL18 (with affinity tag or fluorescent protein of choice) using recombination-based cloning in pDONRP1-P249.</li>
	<li>Combine the entry vector containing RPL18 with the promoter-containing entry vector in a two fragment re...

Verification of RPL18 localization pattern 
Crucial to avoid misinterpretation of data from any TRAP experiment is the proper expression pattern of the tagged ribosomal subunit. Therefore, the incorporation of GFP as an epitope tag to RPL18 very elegantly allows verification of the desired expression pattern and consecutively, pulldown of the polysome fraction from the same tissue. More invasive approaches to assure proper promoter patterns are followed by Jiao and Mayerowitz 2010, which requires GU...

Driven by the increasing application of next-generation sequencing techniques, spatial resolution in developmental biology could be augmented. Contemporary studies aim at dissecting tissues down to specialized cell types, if not single-cell level1,2,3,4. To this end, a plethora of different methods has been devised over the last fifty years (see Figure 1A)5,6,7,8,9,10,11,12,13,14,15.
Many tools in plant science have been adaptations of techniques that were pioneered in animal research. This is not the case for the method we are introducing in detail here. In 2005, equipped with a strong background in protein translation, the Bailey-Serres Lab set out to engineer ribosomal proteins for subsequent affinity purification16. Thus, they could avoid time-consuming and labor-intensive polysome profiling, which is based on ultracentrifugation with a sucrose gradient and was used to assess translating ribosomes since the 1960s17,18. The method has since been referred to as translational ribosome affinity purification (TRAP)16. After successful translatome studies in plants, Heiman et al. adapted TRAP for animals19 and others extended its application to yeast20, Drosophila21, Xenopus22 and zebrafish23,24.
Although genetic modification of the model system is a prerequisite for TRAP, which limits its application to species amenable to genetic transformation, one can simultaneously harness this objection to target subsets of cells that are of special interest and otherwise extremely difficult to isolate from the intact tissue/organ25 (e.g., highly branched dendritic cells in a mouse brain or fungal hyphae in infected plant tissue). In plants, all cells are held in place via cell walls that form the basis of the hydrostatic skeleton26. To free a plant cell from this matrix, scientists have either physically cut the cell out of its surrounding tissue through laser capture microdissection (LCM)27 or performed enzymatic digestion of the cell walls28. Among the latter cells, so-called protoplasts, the population of interest is fluorescently labeled and can be separated via fluorescence-activated cell sorting (FACS)7. LCM usually requires a sample to be fixed and embedded in wax, which ultimately deteriorates the quality of its RNA29. FACS-based methods yield high-quality RNA, but the process of protoplasting itself introduces differences in gene expression30 and tissues with modified and thick secondary cell walls are notoriously difficult to treat. Moreover, many developmental processes in plants are assumed to rely on mechanically transmitted signals and therefore the integrity of the cell wall is of paramount importance31. Two methods, which use a shortcut to circumvent cell isolation by operating on the level of nucleii, are fluorescence-activated nuclear sorting (FANS) and isolation of nuclei tagged in specific cell types (INTACT). As in TRAP, they use cell type-specific promoters to mark nuclei, that subsequently get enriched via sorting or pull down, respectively8,15. A major challenge for all these approaches is to get sufficient RNA material from subsets of cells in a tissue. As TRAP captures only a fraction of the cellular RNAs, sample collection is a considerable bottleneck. Therefore, especially sensitive library preparation protocols are needed to produce high-quality data from low input amounts.
Since its establishment, TRAP has been either used in combination with DNA microarrays or, as sequencing costs dropped significantly in recent years, RNA-seq10,32,33. A multitude of research questions has already been elucidated as reviewed in Sablok et al.34. We are convinced that more reports will follow in coming years as the technique is very versatile when combining different promoters to target specific cell types. Eventually, this will be done even in an inducible way, and may be combined with probing the plant&#39;s reaction to many biotic and abiotic stress factors. Additionally, where stable transgenic lines are not available, hairy root expression systems have also been successfully used to perform TRAP in tomato and medicago35,36.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60919/60919fig1b.jpg" /> 
Figure 1: Translating ribosome affinity purification (TRAP) complements the &#34;omics&#34; analysis portfolio. A. Increasing levels of analytical precision, down to single-cell or even subcellular resolution can be achieved by a plethora of methods or combinations thereof. The scheme gives an overview of currently available tools in the plant and animal field. Tissue collection at cellular resolution can be achieved by protocols like LCM or FACS, which are then coupled to standard transcriptome or polysome profiling/translatome analysis. TRAP and INTACT integrate both tissue capture and RNA isolation as they are based on epitope-tagging. However, INTACT samples only cell nuclei and constitutes, therefore, a special case of transcriptome analysis. A small rabbit icon marks newly developed methods in the animal field: While SLAM-ITseq and Flura-seq rely on metabolic targetting of nascent RNAs with modified uracil bases in cells expressing the permissive enzyme, Slide-seq makes use of a coated glass slide with DNA barcodes that provide positional information in the cellular range. A proximity-labeling approach is followed in APEX-seq to sample RNAs in specific subcellular compartments. Notably, increased resolution often requires the generation of transgenic material (asterisks) and these methods are thus predominantly used for model species. TRAP is especially suited for plant science studies involving cell wall (CW) or mechanic signaling as well as cell species that are difficult to release from their CW matrix. B. Detailed wet-lab steps of the TRAP procedure: Seedlings expressing GFP-tagged ribosomal protein in distinct cell types (e.g. root endodermis) are grown on Petri dishes for seven days and root material harvested by snap freezing. A total RNA control sample is collected from the homogenized crude extract before pelleting the debris via centrifugation. Magnetic anti-GFP beads are added to the cleared extract to perform immunoprecipitation. After incubation and three wash steps, the polysome-associated RNA (TRAP/polysome RNA) is directly obtained via phenol-chloroform extraction. LCM: laser capture microdissection, FACS/FANS: fluorescence-activated cell/nuclear sorting, APEX-seq: method based on engineered ascorbate peroxidase, INTACT: isolation of nuclei tagged in specific cell types, SLAM-ITseq: thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue, Flura-seq: fluorouracil-labeled RNA sequencing (Created with Biorender.com) <a href="https://www.jove.com/files/ftp_upload/60919/60919fig1blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The goal of this article is to supply a detailed description of the TRAP method, to highlight critical steps and to provide guidance for a possible library preparation method.
A generic TRAP experiment will essentially consist of the following steps (see also Figure 1B): (1) Preparation of plant material including cloning of ribosome-tagging construct, transgenic line production and selection, growing and bulking up of seeds, sterilization and plating, and stress application/treatment (optional) and tissue harvesting; (2) immunopurification including tissue homogenization and clearing of the crude extract, bead wash and immunopurification, and wash steps; (3) RNA extraction and quality assessment; and (4) library preparation.
The Arabidopsis root has been a model system to study plant development ever since its introduction as a model plant37,38. Here, the application of TRAP is showcased in the context of plant lateral root development. In plants, the buildup of the entire root system relies on the execution of this program and is therefore very important for the survival of the organism39. In Arabidopsis, lateral roots originate from pericycle tissue that resides next to xylem vessels and therefore is termed xylem pole pericycle (XPP; see Figure 2C)40. Some XPP cells, which are located deep inside the root, acquire a founder cell identity and, upon a local hormonal trigger, start to proliferate by swelling and dividing anticlinally41. However, due to the presence of a rigid cell wall matrix, this process exerts mechanical stress on the surrounding tissues. In particular, the overlying endodermis is affected, as it is in the way of the lateral root growth axis42,43,44. Indeed, the newly forming primordium will have to grow through the overlying endodermis cell (Figure 2C2) whereas cortex and epidermis cells are just pushed aside for the primordium to finally emerge45,46. Recent work in our lab has shown that the endodermis is actively contributing to accommodate the proliferation in the pericycle. Targeted blocking of endodermal hormonal signaling is sufficient to inhibit even the very first division in the XPP cells47. Hence, pericycle-endodermis communication constitutes a very early checkpoint for lateral root development in Arabidopsis. It is, however, not known how this crosstalk is performed. To unravel this mystery, we chose the TRAP-seq approach to target XPP and endodermal cells. To enrich for cells in the lateral root program, we mimicked the hormonal trigger by exogenously applying an auxin analog (1-naphthaleneacetic acid, NAA)48, which at the same time allowed to temporally resolve the initial phase of lateral root formation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sterilization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bleach, 13%</td>
			<td>Sigma</td>
			<td>71696</td>
			<td></td>
		</tr>
		<tr>
			<td>beaker</td>
			<td>VWR</td>
			<td>214-1172/74/75</td>
			<td></td>
		</tr>
		<tr>
			<td>desiccator with porcelaine plate (DURAN)</td>
			<td>Sigma/Merck</td>
			<td>Z317454-1EA/Z317594-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH, p.a.</td>
			<td>Honeywell</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl, 37%</td>
			<td>Roth</td>
			<td>4625.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate growth + harvesting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS salts, basal salt mixture, incl. MES buffer</td>
			<td>Duchefa</td>
			<td>M0254</td>
			<td></td>
		</tr>
		<tr>
			<td>agar plant for cell culture</td>
			<td>Applichem/Panreac</td>
			<td>A2111.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>forcepts</td>
			<td>Rubis Switzerland</td>
			<td>5-SA model</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Fluka</td>
			<td>60370</td>
			<td></td>
		</tr>
		<tr>
			<td>micropore/surgical tape</td>
			<td>3M</td>
			<td>1530-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NAA</td>
			<td>Duchefa</td>
			<td>N0903</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dishes 120x120 mm</td>
			<td>Greiner bio-one</td>
			<td>688102</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>VWR/Swann-Morton</td>
			<td>233-5454</td>
			<td></td>
		</tr>
		<tr>
			<td>tissues, neutral, two-layered</td>
			<td></td>
			<td></td>
			<td>any supplier of your choice</td>
		</tr>
		<tr>
			<td>Immunoprecipitation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-beads: gtma-100 GFP-Trap_MA</td>
			<td>Chromotek</td>
			<td>e.g. gtma-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Brij-35</td>
			<td>Sigma</td>
			<td>P1254-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tubes (in accordance with centrifuge)</td>
			<td>Beckman Coulter</td>
			<td>357001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Applichem</td>
			<td>C0378-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton gloves</td>
			<td>VWR</td>
			<td>113-7355</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide, HPLC grade</td>
			<td>Sigma</td>
			<td>01810-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>VWR</td>
			<td>E174</td>
			<td>might have long delivery times</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fluka</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3054.3</td>
			<td></td>
		</tr>
		<tr>
			<td>homogenizers DUALL 23</td>
			<td>KONTES GLASS CO (via VWR)</td>
			<td>SCERSP885450-0023 (set)</td>
			<td>SCERSP885451-0023 pestle only - SCERSP885452-0023 cylinder only; long delivery times</td>
		</tr>
		<tr>
			<td>Igepal CA-360</td>
			<td>Sigma</td>
			<td>I3021-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 hexahydrat</td>
			<td>Roth</td>
			<td>2189.2</td>
			<td></td>
		</tr>
		<tr>
			<td>mortar and pestle</td>
			<td>VWR</td>
			<td>470148-960 &#38; 470019-978</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Roche</td>
			<td>10 837 091 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-(10)-tridecylether/PTE</td>
			<td>Sigma</td>
			<td>P2393-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Roth</td>
			<td>T143.3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAZap</td>
			<td>Thermo Fisher</td>
			<td>AM9780/AM9782</td>
			<td>for cleaning surfaces</td>
		</tr>
		<tr>
			<td>Tris, &#62;99.3%</td>
			<td>Roth</td>
			<td>AE15.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka</td>
			<td>T8787-250ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol, p.a.</td>
			<td>Sigma</td>
			<td>33539-1L-GL-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform, HPLC grade</td>
			<td>Scharlau</td>
			<td>CL02181000</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH, p.a.</td>
			<td>Honeywell</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>low-retention microcentrifuge tubes, 1.5 ml</td>
			<td>Eppendorf/Sigma</td>
			<td>Z666548-250EA</td>
			<td>LoBind</td>
		</tr>
		<tr>
			<td>RNase-free DNase set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy MiniElute Cleanup Kit</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>ThermoFisher/Ambion</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Library preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15/50 mL Tube Magnetic Separator</td>
			<td>Abraxis</td>
			<td>PN 472250</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Index Kit A</td>
			<td>Illumina</td>
			<td>FC-131-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Index Kit D</td>
			<td>Illumina</td>
			<td>FC-131-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>neodymium magnets</td>
			<td>Amazon/other</td>
			<td>6 x 1.5 mm range: N42 (NdFeB)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT kit</td>
			<td>Illumina</td>
			<td>FC-131-1024/1096</td>
			<td><a href="https://emea.support.illumina.com/sequencing/sequencing_kits/nextera_xt_dna_kit/documentation.html" target="_blank">https://emea.support.illumina.com/</a></td>
		</tr>
		<tr>
			<td>PCR strips</td>
			<td>ThermoScientific</td>
			<td>AB-0266</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer v4 kit</td>
			<td>Takara Bioscience</td>
			<td>634892</td>
			<td><a href="https://www.takarabio.com/products/next-generation-sequencing/rna-seq/smart-seq-v4-for-mrna-seq" target="_blank">https://www.takarabio.com/</a></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>2100 Bioanalyzer Instrument</td>
			<td>specialized equipment for RNA/DNA quality control</td>
		</tr>
		<tr>
			<td>Tapestation</td>
			<td>Agilent</td>
			<td>4200 Tapestation Instrument</td>
			<td>specialized equipment for RNA/DNA quality control</td>
		</tr>
		<tr>
			<td>Fragment Analyzer</td>
			<td>Agilent</td>
			<td>5400 Fragment Analyzer System</td>
			<td>specialized equipment for RNA/DNA quality control (high throughput)</td>
		</tr>
		<tr>
			<td>LabChip</td>
			<td>PerkinElmer</td>
			<td>LabChip GX Touch Nucleic Acid Analyzer</td>
			<td>specialized equipment for RNA/DNA quality control (high throughput)</td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>ThermoFisher</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/Q33239" target="_blank">Q33239</a></td>
			<td>specialized equipment for RNA/DNA concentration determination</td>
		</tr>
		<tr>
			<td>qRT-PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GATA23</td>
			<td>Microsynth</td>
			<td></td>
			<td>fwd: AGTGAGAATGAA 
			AGAAGAGAAGGG; 
			rev: GTGGCTGCGAAT 
			AATATGAATACC</td>
		</tr>
		<tr>
			<td>GH3.3</td>
			<td>Microsynth</td>
			<td></td>
			<td>fwd: CAAACCAATCCT 
			CCAAATGAC; 
			rev: ACTTATCCGCAA 
			CCCGACT</td>
		</tr>
		<tr>
			<td>LBD29</td>
			<td>Microsynth</td>
			<td></td>
			<td>fwd: TCTCCAACAACA 
			GGTTGTGAAT; 
			rev: AAGGAGCCTTAG 
			TAGTGTCTCCA</td>
		</tr>
		<tr>
			<td>UBC21</td>
			<td>Microsynth</td>
			<td></td>
			<td>fwd: TGCGACTCAGGG 
			AATCTTCT; 
			rev: TCATCCTTTCTT 
			AGGCATAGCG</td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green</td>
			<td>Bio-Rad</td>
			<td>#172-5270</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript Adv cDNA Kit</td>
			<td>Bio-Rad</td>
			<td>#172-5038</td>
			<td></td>
		</tr>
		<tr>
			<td>miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 ml, Cellstar</td>
			<td>Greiner bio-one</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 ml, Cellstar</td>
			<td>Greiner bio-one</td>
			<td>210261</td>
			<td></td>
		</tr>
		<tr>
			<td>filter tips 1 ml</td>
			<td>Axygen</td>
			<td>TF-1000-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>filter tips 10 &#181;l</td>
			<td>Axygen</td>
			<td>TF-10-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>filter tips 100 &#181;l</td>
			<td>Axygen</td>
			<td>TF-100-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>filter tips 20 &#181;l</td>
			<td>Axygen</td>
			<td>TF-20-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>filter tips 200 &#181;l</td>
			<td>Axygen</td>
			<td>TF-200-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge tubes 1.5 ml</td>
			<td>SARSTEDT</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing company</td>
			<td>Novogene</td>
			<td></td>
			<td><a href="http://en.novogene.com" target="_blank">en.novogene.com</a></td>
		</tr>
	</tbody>
</table>

translating ribosome affinity purification (trap) to investigate arabidopsis thaliana root development at a cell type-specific scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

For quality assessment, the above-mentioned procedure should be probed at several intermediate steps: expression pattern validation in planta, quality control of the isolated polysomal RNA as well as of the final libraries. qRT-PCR using known marker genes can, in addition, be performed to confirm the response to the treatment condition or to fine-tune the experimental conditions.
Confocal analysis of GFP signal distribu...

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Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

With TRAP, we can investigate developmental processing at cellular resolution. We use cell-type-specific promoters to drive a tagged ribosomal sub-unit, and from there we can isolate polysome RNA. This protocol yields high quality RNA, but in very limited quantities, therefore, methods that are specialized for ultra-low input are needed to reliably produce high quality RNA libraries.TRAP is an ideal tool for plant research, because many developmental processes rely on cell wall-related and mechanical signaling pathways. We use TRAP to study cell-cell communication in lateral root formation. As obtaining good quality RNA proved not as straight-forward as we anticipated, we hope that this step-by-step video instruction will increase TRAP applicability and help promote this powerful technique.To sterilize the propagated homozygous lines, spread less than 300 microliters of seeds evenly on 12 by 12 centimeter squared Petri dishes, and stack the dishes on a 60 liter desiccator. After overnight gas sterilization, let the gas evaporate before collecting the seeds into a 50 milliliter storage tube. Before plating, dilute the sterilized seeds with 0.1%agar to obtain one milliliter of imbibed seed mix per plate.To plate the seed mix, use square Petri dish lids to create a laminated template holder for planting three rows of seeds per plate, and place empty agar plates into the template holder. Distribute one milliliter of the imbibed seeds evenly onto three rows of plates. And place the processed plates in stacks in a laminar flow, until the seeds are dry.When seeds have stuck to the agar's surface, close the lids and seal each plate with micropore tape. For exogenous treatment of Arabidopsis roots with an agent of interest, prepare 1.5 to two centimeter-wide, 10 centimeter-long, strips of tissue paper. Soak the tissue paper in 10 micromolar NAA, and use tweezers to apply a strip of tissue paper to each row of roots.Then, gently press out any air bubbles, and empty the excess liquid from the plate, before labeling the plate with the time. At the appropriate experimental end point, use forceps to carefully remove the tissue paper strips from each plate without detaching the roots, and use a surgical blade to cut once per row along the shoot-root junction, in a single determined stroke. Use tweezers to swipe along the roots of each row, to collect the roots in three bundles.And empty the the roots into a 50 milliliter tube filled with liquid nitrogen, for snap freezing. When all of the root samples per treatment have been collected, decant the excess liquid nitrogen using the tube lid to prevent the roots from spilling. And place the tube into a doer vessel, for a 80 degrees celsius storage.For tissue grinding and homogenization, wear cotton gloves under standard lab gloves, and add PMSF to the polysome extraction buffer. Empty the tissue sample into a mortar containing liquid nitrogen, and use a cooled pestle to carefully grind the tissues until all the material appears as a white powder. When all of the tissue has been ground, add five milliliters of polysome extraction buffer to the sample, and quickly mix the frozen tissue fragments with the powder before the buffer freezes.As soon as the mixture can be transferred, empty the slurry into a glass homogenizer on ice, and use an additional two milliliters of polysome extraction buffer to rinse the mortar and pestle. Manually grind the slurry until the extract is homogenous. Then, pour the crude root extract into a 50 milliliter centrifuge tube on ice, and process the next sample as demonstrated.For total RNA collection, transfer 200 microliter aliquots of each crude sample into individual clean, pre-cooled, labeled microcentrifuge tubes. To remove cell walls, debris, and large organelles, centrifuge the samples and decant the supernatant into fresh, pre-cooled conical tubes for a second centrifugation. While the tubes are spinning, aliquot 60 microliters of magnetic GFP beads per sample, into a 1.5 milliliter tube.And place the tube on a magnetic stand, to facilitate supernatant removal. Then, wash the beads two times in one milliliter of cold wash buffer per wash, and re-suspend the beads in 60 microliters of wash buffer per sample. Immediately after the centrifugation, decant the cleared supernatant into labeled 15 milliliter tubes, and add 60 microliters of washed beads to each sample.Then, place all of the samples horizontally into an ice bucket, for a two hour incubation with rocking. At the end of the incubation, collect the beads on a magnetic stand on ice, and add PMSF to the remaining polysome extraction buffer. After discarding the supernatants, add approximately five milliliters of polysome extraction buffer to the samples, and re-suspend the beads within the samples with gentle tilting.Rock the samples for 15 minutes on the shaker on ice, followed by two washes with fresh wash buffer on the magnet, as demonstrated. After the last wash, transfer the samples in one milliliter of wash buffer, into a 1.5 milliliter tube. And collect the beads one more time on the magnetic stand, to remove all of the supernatant.Then, place the tubes on ice until all of the samples have been processed. For reconditioning of the lab supplies, first, rinse all of the equipment into the chemical waste. Hand wash the mortars, pestles, and homogenizers with soap, before rinsing each instrument thoroughly.Next, place the equipment in a heat-safe container, and bake the materials overnight at greater than 220 degrees celsius. Brush all of the centrifuge tubes clean with detergent, before placing the tubes onto an autoclaveable tray with a rim and a fume hood. Pour a one milliliter of liquid diethyl pyrocarbonate and one liter of deionized water solution onto the tubes for three to 18 hours.Then, collect the solution before sterilizing the tray of tubes in the autoclave. Here, representative plants with GFP signals are shown. Counter-staining with propidium iodide marks the cell wall outlines.The lines correspond to the localization pattern of the endodermis, and the xylem-pole pericycle. In these representative measurements obtained from polysome RNA, most samples showed very little degradation, with RNA integrity numbers ranging from nine to 10. In these graphs, traces of successfully prepared libraries can be observed, highlighting the robustness of the procedure, despite scaled-down reaction volumes.Before a genome-spanning dataset is produced, TRAP RNA from a pilot experiment can be probed by quantitative RTPCR to validate the treatment success and/or the experimental conditions, as well as the tissue specificity. In this analysis, the testing of three auxin-responsive genes confirmed a successful hormone induction after two hours of treatment. Tissue-specific marker gene profiles retrieved from the sequencing data confirmed the cell-type specificity.Follow good practice advice when you handle RNA. Work at a sterile bench, clean your equipment with RNA-removing solution, wear gloves and immediately change them when they get contaminated. Be sure to appropriately handle and discard all toxic chemicals and gases that have been used during the procedure, especially phenol-DEPC chlorine gas and liquid nitrogen.In the portfolio of omic studies, TRAP occupies an important niche, and has already been used to answer many biological questions, especially in developmental biology, TRAP has a huge potential.

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Research

JoVE Journal

Developmental Biology

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...