Chapter 12 Hox Transcriptomics in Drosophila Embryos Maria Polychronidou and Ingrid Lohmann Abstract Hox proteins are evolutionarily conserved homeodomain containing transcription factors that specify segment identities along the anteroposterior axis of almost all bilaterian animals. They exert their morphogenetic role by transcriptionally regulating a large battery of downstream target genes. Therefore the dissection of transcriptional networks regulated by Hox proteins is an essential step towards a mechanistic understanding of how these transcription factors coordinate multiple developmental and morphogenetic processes. High-throughput techniques allowing whole-transcriptome mRNA expression profiling are powerful tools for the genome-wide identification of Hox downstream target genes in a variety of experimental settings. Here, we describe how to quantitatively identify Hox downstream genes in Drosophila embryos by performing a Hox transcriptome analysis using microarrays. Key words Hox, Drosophila, Transcription, Microarray, Transcriptome, Expression profiling
1
Introduction Hox genes are expressed along the anteroposterior axis of animal embryos where they coordinate morphogenetic processes in a segment-specific manner [1, 2]. The essential role of Hox genes in specifying segment identities is evidenced by the “homeotic transformations” that are frequently observed upon disruption of Hox function in Drosophila [1]. Hox genes encode homeodomain transcription factors that exert their regulatory functions in diverse cell and tissue types by activating or repressing downstream target genes [3]. Identification of Hox target genes is crucial for elucidating how Hox proteins carry out their regulatory role in vivo and how distinct morphogenetic programs are executed under the control of Hox proteins in the different embryonic segments. The complexity of Hox regulatory networks in combination with the frequently encountered and degenerated Hox-binding sites and the involvement of co-regulatory factors in the transcriptional regulation of Hox target genes present significant limitations for the identification of Hox target genes using in silico or in vitro
Yacine Graba and René Rezsohazy (eds.), Hox Genes: Methods and Protocols, Methods in Molecular Biology, vol. 1196, DOI 10.1007/978-1-4939-1242-1_12, © Springer Science+Business Media New York 2014
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approaches [4, 5]. Consequently, it is required to employ in vivo strategies in order to identify Hox-responsive gene regulatory networks. Studies in Drosophila have provided a substantial amount of knowledge on the molecular mechanisms underlying Hox protein functions. In recent years, high-throughput analyses of Hoxregulated transcriptional networks using genome-wide methods have shed light on the mechanistic basis of how Hox proteins acquire their high specificity and selectivity (reviewed in refs. 5, 6). A number of studies in which a Hox transcriptome analysis was performed in Drosophila allowed the genome-wide identification of Hox downstream genes [7–11]. Importantly, the quantitative identification of target genes for six out of eight Drosophila Hox proteins, by performing transcriptome analysis using microarrays and whole-Drosophila embryos ubiquitously overexpressing each of the selected Hox proteins [10], demonstrated that Hox proteins control the expression of hundreds of genes and that different Hox proteins, despite their similar DNA-binding properties in vitro, show highly specific effects on the transcriptome (reviewed in refs. 5, 12). For a variety of reasons, in experiments aiming at target gene identification Hox overexpression is advantageous in comparison to Hox loss of function. First of all, the extensive cross-regulation of Hox genes introduces complications in the interpretation of the results from experiments using mutants [13]. An additional limitation arises from the fact that Hox proteins are only expressed in small subsets of cells [2] and therefore the changes in gene expression taking place in the Hox-deficient cells will be diluted in the heterogeneous RNA mixture isolated from whole embryos. Performing the transcriptome analysis using isolated cells in which the Hox protein of interest is active could solve this problem. Considering that cellular and nuclear mRNA pools are very comparable [14], isolated nuclei are a reliable source of cell-typespecific transcripts. Thus, the recently developed methods allowing isolation of pure populations of nuclei from selected cell types [15, 16] could be employed. Importantly, given the context-dependent activity of Hox proteins, these methods offer the possibility to identify targets of a given Hox protein in different tissue types. In this chapter we provide the protocol for identification of Hox target genes in Drosophila embryos by performing a transcriptome analysis using microarrays. The procedure does not only apply to whole embryos but can also be used with different experimental settings, i.e., when using larval tissues or purified cell populations. Principally, each gene of a given genome is represented on a microarray chip, in the case of Affymetrix Chips by a series of different oligonucleotide probes that serve as unique, sequencespecific detectors. The chip is hybridized with biotinylated cRNA
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that was generated from oligo-dT-primed cDNA of a given tissue. Each sample (for example, “wild-type” versus “mutant” tissue) is hybridized to a separate array. After hybridization, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. The staining protocol includes a signal amplification step that employs anti-streptavidin antibody (goat) and biotinylated goat IgG antibody. After washing, transcript levels are calculated by reference to cRNA spikes of known concentration added to the hybridization mixture. Differences in mRNA levels between samples are determined by comparison of any two hybridization patterns produced on separate arrays of the different samples. Here, we describe the procedures for collecting the Drosophila embryos, isolating the total RNA, synthesizing the complementary DNA (cDNA) and the complementary RNA (cRNA), controlling the quality of the cRNA probes, performing the hybridization to the microarrays, and scanning the probes.
2
Materials
2.1 Drosophila Embryo Collection
2.2 Isolation of RNA from Drosophila Embryos 2.3 Double-Stranded cDNA (ds-cDNA) and cRNA Synthesis and Cleanup, cRNA Fragmentation 2.4 Agarose Gel Analysis
Apple juice agar plates: Add 24 g agar to 740 ml ultrapure water and autoclave. Keep warm so that it does not become solid. Boil 250 ml apple juice with 25 g sugar and add to the autoclaved agar. Stir on a magnetic plate until the mixture had cooled down to approximately 50 °C and pour in petri dishes. The plates can be stored at 4 °C. Fresh yeast paste is spread on the plates directly before use. 100 % bleach is used for dechorionating the embryos. 1. RNeasy Mini Kit (QIAGEN). 2. Polypropylene micropestle (Eppendorf). 3. QIAshredder spin columns (QIAGEN). MessageAmp™ II-Biotin Enhanced, Single Round aRNA Amplification Kit (Ambion). All reagents required for the synthesis and cleanup of ds-cDNA and biotinylated cRNA are provided by the kit.
1. Denaturing gel loading buffer: 0.5 μl ethidium bromide (1 mg/ml), 1 μl 10× MOPS buffer, 5 μl formamide, 5 μl formaldehyde (37 %), 3 μl RNA loading dye (50 % glycerol containing 2.5 mg/ml bromophenol blue and xylene cyanol, each). 2. RNA gel: It is not necessary to run a denaturing RNA gel. Prepare a 1 % agarose gel for the unfragmented samples and 2 % agarose gel for the fragmented samples.
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2.5 Hybridization, Washing, Staining, and Scanning
Additional arrays and staining solutions can be stored at 4 °C until needed. 1. Arrays: GeneChip Drosophila Genome 2.0 Array (Affymetrix). 2. Streptavidin-phycoerythrin (SAPE) solution (final volume 1,200 μl): 600 μl 2× 2-(N-morpholino)ethanesulfonic acid (MES) stain buffer, 120 μl acetylated bovine serum albumin (BSA) (20 mg/ml), 12 μl SAPE, 468 μl water. Split in two aliquots of 600 μl that will both be used for one array (see Note 1). 3. Antibody solution (final volume 600 μl): 300 μl 2× MES stain buffer, 60 μl acetylated BSA (20 mg/ml), 6 μl goat IgG (10 mg/ml), 3.6 μl biotinylated antibody (0.5 mg/ml), 230.4 μl water. 4. 2× Hybridization buffer: 200 mM MES, 0.5 M NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.005 % Tween-20. 5. 20× SSPE buffer (1 l): 175.3 g sodium chloride, 27.6 g sodium phosphate monobasic, 9.4 g ethylenediaminetetraacetic acid (EDTA). 6. Wash buffer A: Non-stringent wash buffer consisting of 6× SSPE buffer (use the 20× SSPE stock described in item 5) and 0.01 % Tween-20.
3
Methods
3.1 Drosophila Embryo Collection
1. Two days before starting the embryo collection transfer newly hatched adult flies into a collection cage and cover it with an apple juice agar plate with yeast paste (see Note 2). Collection and aging times depend on the desired embryonic stage(s). When collecting embryos keep in mind that it is recommended to perform microarray experiments using biological triplicates. 2. Add water to the apple juice agar plate and brush the embryos to release them from the agar surface. Transfer the embryos to a sieve, rinse thoroughly with water, and dechorionate in 100 % bleach. Check under the microscope that the chorion has been completely removed. Wash the embryos well with water and remove as much liquid as possible by blotting the sieve with a paper towel. Using a brush, transfer the embryos in a preweighed microcentrifuge tube, measure the weight, and snapfreeze in liquid nitrogen. At this point the embryos can be stored at −80 °C.
3.2 Isolation of RNA from Drosophila Embryos
Use of RNeasy Mini Kit (QIAGEN) is recommended for the isolation of total RNA. Make sure that sterile and nuclease-free glassware or plastic are used throughout the procedure (see Note 3) and follow the instructions for “isolation of total RNA from animal
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tissues.” The following procedure applies to isolation of RNA from whole embryos. The required modifications in cases when tissues (see Note 4) or sorted cells (see Note 5) are used are described separately. 1. Remove the frozen embryos from storage. Do not use more than 30 mg of embryos per sample. Do not allow the frozen embryos to thaw and proceed to step 2 as fast as possible. 2. Before use, ensure that β-mercaptoethanol is added to Buffer RLT (RNA easy mini kit, QIAGEN). Add 400 μl Buffer RLT to the embryos and lyse them by homogenizing in the microcentrifuge tube using a polypropylene pestle. Alternatively the embryos can be lysed in a Dounce homogenizer using a tight pestle. 3. Transfer the lysate to a new labeled tube. 4. Rinse both pestle and grinder with 200 μl RLT and combine the solution with the lysate of the previous step. 5. Incubate at RT for 5 min. 6. Centrifuge the lysate at maximum speed in a microcentrifuge for 3 min. 7. Carefully transfer the supernatant (cleared lysate) to a new microcentrifuge tube. 8. Add 600 μl of 70 % ethanol (prepared with diethylpyrocarbonate (DEPC)-treated water) to the cleared lysate and mix immediately by pipetting. 9. Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube. Centrifuge for 15 s at 8,000 × g and discard the flow-through. (If the sample volume exceeds 700 μl, centrifuge successive aliquots in the spin column.) 10. Add 700 μl Buffer RW1 (QIAshredder spin columns, QIAGEN) to the RNeasy spin column. Centrifuge for 15 s at 8,000 × g and discard the flow-through. 11. Before use, make sure that ethanol is added to Buffer RPE (RNA easy mini kit, QIAGEN). Add 500 μl Buffer RPE to the RNeasy spin column. Centrifuge for 15 s at 8,000 × g and discard the flow-through. 12. Repeat step 7 and centrifuge for 2 min at 8,000 × g. 13. Transfer the RNeasy spin column in a new 2 ml collection tube and centrifuge at full speed for 1 min to ensure no carryover of ethanol. 14. Place the RNeasy spin column in a new 1.5 ml microcentrifuge tube and add 30 μl RNase-free water directly to the membrane. Centrifuge for 1 min at 8,000 × g.
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15. Optional: Repeat step 10 using another 30–50 μl RNase-free water. Do not mix the two eluates before measuring the RNA concentration. 16. Measure RNA concentration and 260 nm/280 nm absorbance ratio (as a control of RNA purity) using Nanodrop. At this step the two eluates can be pooled together depending on their concentration. The RNA can be stored at −80 °C. 3.3 ds-cDNA Synthesis 3.3.1 First-Strand cDNA Synthesis
Use the “MessageAmpTm II-Biotin Enhanced, Single Round aRNA Amplification Kit” (Ambion). 1. Before you start prepare spike controls (see Note 6) according to the following dilution table. The first dilution can be stored at −20 °C and reused up to three times. Spike control dilution Total RNA (μg)
First dilution
Second dilution
Third dilution
Final dilution
1
1:20
1:50
1:25
1:25,000
5
1:20
1:50
1:5
1:5,000
10
1:20
1:50
1:2.5
1:2,500
2. Prepare the following mix and keep the amount of RNA constant for all samples: 10 μl total RNA (1 μg in 10 μl water is recommended when using the Ambion kit), 1 μl T7 Oligo(dT) primer, 1 μl spike controls (according to the dilution table). 3. Incubate RNA at 70 °C for 10 min. 4. Chill rapidly on ice. 5. For each sample, prepare a master mix containing the following reagents: 2 μl 10× first-strand buffer, 4 μl dNTP Mix, 1 μl RNase inhibitor, and 1 μl ArrayScript Reverse Transcriptase. 6. Add 8 μl master mix to each RNA sample (final volume 20 μl) and mix gently. Spin down briefly if necessary. 7. Incubate at 42 °C for 2 h. 3.3.2 Second-Strand cDNA Synthesis
1. Place the first-strand cDNA synthesized in Subheading 3.3.1 on ice. 2. For each sample, prepare a master mix containing the following reagents: 63 μl RNase-free water, 10 μl 10× second-strand buffer, 4 μl dNTP mix, 2 μl DNA polymerase, 1 μl RNase H. 3. Mix gently and spin down briefly if necessary.
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4. Add 80 μl master mix to each sample. Mix thoroughly by pipetting up and down two to three times. Spin down briefly if necessary. 5. Incubate at 16 °C for 2 h. Take care not to let the temperature of the samples rise above 16 °C. 3.3.3 ds-cDNA Cleanup
1. Before you begin preheat the nuclease-free water at 50–55 °C and make sure that ethanol has been added to the bottle of wash buffer. 2. Add 250 μl cDNA-binding buffer to each sample and mix thoroughly by pipetting up and down two to three times. Spin down briefly if necessary. 3. Apply the sample mix to a cDNA Cleanup Spin Column placed in a 2 ml collection tube. 4. Spin at 8,000 × g for 1 min and discard flow-through. 5. Apply 500 μl cDNA wash buffer to each spin column. 6. Spin at 8,000 × g for 1 min and discard flow-through. 7. Spin once again at 8,000 × g for 1 min in order to remove trace amounts of wash buffer. 8. Transfer the spin column into a new 1.5 ml collection tube and pipet 10 μl preheated nuclease-free water directly onto the center of the membrane. 9. Incubate for 2 min. 10. Spin at 8,000 × g for 1 min. 11. Pipet 12 μl preheated nuclease-free water directly onto the center of the membrane and incubate for 2 min. 12. Spin at 8,000 × g for 1 min. 13. The total elution volume will be 20 μl. At this point the dscDNA can be stored at −80 °C.
3.4
cRNA Synthesis
3.4.1 In Vitro Transcription
Use the “MessageAmpTm II-Biotin Enhanced, Single Round aRNA Amplification Kit” (Ambion). 1. Thaw ds-cDNA on ice and prepare the following in vitro transcription master mix for each sample: 12 μl biotin-NTP mix, 4 μl T7 10× reaction buffer, 4 μl T7 enzyme mix. 2. Add 20 μl in vitro transcription master mix to 20 μl ds-cDNA (final volume 40 μl) and mix gently. Spin down briefly if necessary. 3. Incubate at 37 °C overnight (approximately 16 h). 4. Add 60 μl nuclease-free water to each cRNA sample (final volume 100 μl). At this point the cRNA can be stored at −80 °C.
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3.4.2 cRNA Cleanup
1. Before you begin preheat nuclease-free water to 50–60 °C and assemble cRNA filter cartridges and tubes. 2. Add 350 μl cRNA-binding buffer to the cRNA and vortex. 3. Add 250 μl ethanol (>99 %) to each sample and mix thoroughly by pipetting the mixture up and down two to three times. Do not vortex and do not centrifuge the samples. 4. Apply sample mix (final volume 700 μl) to a cRNA Cleanup Spin Column placed in a 2 ml collection tube. 5. Spin at 8,000 × g for 1 min and discard flow-through. 6. Add 650 μl wash buffer (supplemented with ethanol) to each sample. 7. Spin at 8,000 × g for 1 min and discard flow-through. 8. Spin at 8,000 × g for 1 min to remove trace amounts of wash buffer. 9. Transfer spin column into new 1.5 ml collection tube and pipet 40 μl preheated nuclease-free water directly onto the membrane. 10. Incubate for 2 min. 11. Spin at 8,000 × g for 1 min to elute cRNA. 12. Transfer spin column into new 1.5 ml collection tube and repeat elution with an additional 60 μl preheated nuclease-free water. 13. Use 1 μl of the first eluate to measure cRNA concentration and 260/280 ratio. Optional steps, in case the cRNA concentration is lower than 1.0 μg/μl: 14. Combine the two cRNA eluates (final volume 100 μl). 15. Add 10 μl 5 M ammonium acetate and 275 μl 100 % ethanol. 16. Mix well and incubate at −20 °C for at least 30 min. 17. Centrifuge at full speed for 15 min at 4 °C and discard supernatant. 18. Wash pellet with 500 μl cold 70 % ethanol. 19. Centrifuge at full speed for 15 min at 4 °C or room temperature. 20. Remove 70 % ethanol. 21. Spin briefly, remove any residual fluid, and air-dry the pellet. 22. Resuspend the cRNA in an appropriate volume of nucleasefree water. 23. Use 1 μl to measure cRNA concentration and 260 nm/280 nm absorbance ratio.
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1. Set up the fragmentation reaction (final volume: 20 μl) by mixing the following: 15 μg cRNA in a total volume of 16 μl nuclease-free water and 4 μl 5× fragmentation buffer. 2. Incubate at 94 °C for 35 min. 3. Chill rapidly on ice.
3.6 Agarose Gel Analysis
It is recommended to check the quality of the synthesized cRNA and the fragmentation on an agarose gel in order to confirm the quality of the probe before starting the hybridization procedure. 1. Add 5 μl gel loading buffer to the following samples: (a) 2 μl unfragmented cRNA. (b) 2 μl fragmented cRNA. 2. Incubate at 65 °C for 10 min and chill on ice. 3. Run the unfragmented samples on a 1 % agarose gel and the fragmented samples on a 2 % agarose gel.
3.7 Preparation of Hybridization Cocktail and Hybridization
1. For each sample prepare a master mix containing the following reagents: 4.25 μl control oligo B2, 12.5 μl 20× eukaryotic hybridization controls, 2.5 μl salmon sperm DNA, 6.25 μl BSA (20 mg/ml), 125.0 μl 2× hybridization buffer, 25.0 μl dimethyl sulfoxide (DMSO), 56.5 μl water. 2. Add 232 μl master mix to the remaining 18 μl fragmented cRNA from each sample (final volume 250 μl). At this point the samples can be stored at −80 °C. 3. Before hybridization, denature the probe for 5 min at 95 °C, followed by 5 min at 45 °C. 4. Prehybridize the probe array with 250 μl 1× hybridization buffer at 45 °C for at least 10 min. 5. Hybridize the probe array for 16 h at 45 °C while shaking at 60 rpm.
3.8 Washing, Staining, and Scanning of the Arrays
Before you start enter experiment description into Affymetrix workstation (GCOS). Unless the experiments have been defined first, you will not be able to wash, stain, and scan your arrays. Wash and stain the probe array using the Affymentrix Fluidics Station 450, which is operated using GCOS/Microarray Suite. Throughout the procedure, follow the instructions in the LCD window on the fluidics station. 1. Prime fluidics station with wash buffers in order to make sure that the lines of the fluidics station are filled with the appropriate buffers. 2. Remove probe from arrays and fill arrays with wash buffer A. Take care not to introduce any bubbles. If necessary, the probes can be frozen at −80 °C at this point (see Note 7).
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3. Select the correct experiment name and the appropriate antibody amplification protocol to control the washing and staining of the probe array, from the drop-down Experiment list. 4. Load the three experiment microcentrifuge vials into the sample holders on the fluidics station as follows: Place one vial containing 600 μl of SAPE solution in sample holder 1, one vial containing 600 μl of antibody solution in sample holder 2, and one vial containing 600 μl of SAPE solution in sample holder 3. 5. Start the run. The Fluidics Station dialog box displays the status of the washing and staining during the procedure. After the run has finished, if you do not plan to scan the arrays immediately, keep them at 4 °C in the dark until ready for scanning. 6. Clean excess fluid from around septa on the back of the probe array cartridge. 7. Carefully apply one Tough-Spots to each of the two septa (see Note 8). 8. Insert the cartridge into the scanner and ensure that the spots lie flat. 9. Select the correct experiment name, insert the probe array into the holder, and start the scan. 10. After completion of the scan, the data is ready for analysis.
4
Notes 1. SAPE should be stored in the dark at 4 °C and should not be frozen. Before preparing staining solution take SAPE out of the refrigerator and mix well. The SAPE solution should always be prepared fresh. 2. The apple juice agar plates and yeast paste must be fresh and pre-warmed before use in order to maximize the yield of the collections. 3. To provide RNase-free conditions, bake all the glassware for 2 h at 300 °C, use DEPC-H2O for preparing the solutions, and wear gloves and a lab coat when working with RNA samples. 4. A few parameters need to be considered when isolating RNA from dissected tissues (i.e., imaginal discs or other larval tissues). During dissection, the tissues of interest should be kept on ice at all times so that the RNA does not degrade. Snapfreeze the tissue in liquid nitrogen as soon a possible. If the amount of dissected tissue is smaller than 20 mg, use 300 μl of RLT buffer. 5. When using isolated cells (i.e., isolated by FACS sorting), follow the protocol “Purification of Total RNA from Animal Cells”
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provided in the RNeasy Mini Kit (QIAGEN) and adjust the volume of RLT according to the number of cells as described in the manual. 6. The use of external RNA controls (“spikes”) allows assessing the performance of the microarray experiment. The probes that correspond to the spikes are informative of the fidelity of the microarray technology in determining their presence. It is recommended to spike external controls in an early step of sample processing in order to monitor several steps. 7. If the probes were frozen, equilibrate them to room temperature. 8. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly and you observe bumps, bubbles, tears, or curled edges, remove the spot and apply a new one. Do not attempt to smoothen the spot.
Acknowledgements We would like to thank Jan U. Lohmann and Markus Schmid for sharing protocols with us. References 1. Lewis EB (1978) A gene complex controlling segmentation in Drosophila. Nature 276: 565–570 2. McGinnis W, Krumlauf R (1992) Homeobox genes and axial patterning. Cell 68:283–302 3. Hombría JC-G, Lovegrove B (2003) Beyond homeosis—HOX function in morphogenesis and organogenesis. Differentiation 71:461–476. doi:10.1046/j.1432-0436.2003.7108004.x 4. Pearson JC, Lemons D, McGinnis W (2005) Modulating Hox gene functions during animal body patterning. Nat Rev Genet 6:893–904. doi:10.1038/nrg1726 5. Hueber SD, Lohmann I (2008) Shaping segments: Hox gene function in the genomic age. Bioessays 30:965–979. doi:10.1002/bies.20823 6. Polychronidou M, Lohmann I (2013) Celltype specific cis-regulatory networks: insights from Hox transcription factors. Fly (Austin) 7:13–17. doi:10.4161/fly.22939 7. Leemans R, Loop T, Egger B et al (2001) Identification of candidate downstream genes for the homeodomain transcription factor Labial in Drosophila through oligonucleotide-array transcript imaging. Genome Biol 2:RESEARCH0015 8. Mohit P, Makhijani K, Madhavi MB et al (2006) Modulation of AP and DV signaling
9.
10.
11.
12.
13.
14.
pathways by the homeotic gene Ultrabithorax during haltere development in Drosophila. Dev Biol 291:356–367. doi:10.1016/j.ydbio. 2005.12.022 Hersh BM, Nelson CE, Stoll SJ et al (2007) The UBX-regulated network in the haltere imaginal disc of D. melanogaster. Dev Biol 302:717–727. doi:10.1016/j.ydbio.2006.11.011 Hueber SD, Bezdan D, Henz SR et al (2007) Comparative analysis of Hox downstream genes in Drosophila. Development 134:381– 392. doi:10.1242/dev.02746 Pavlopoulos A, Akam M (2011) Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis. Proc Natl Acad Sci U S A 108:2855–2860. doi:10.1073/pnas.1015077108 Pavlopoulos A, Akam M (2007) Hox go omics: insights from Drosophila into Hox gene targets. Genome Biol 8:208. doi:10.1186/ gb-2007-8-3-208 Miller DF, Rogers BT, Kalkbrenner A et al (2001) Cross-regulation of Hox genes in the Drosophila melanogaster embryo. Mech Dev 102:3–16 Barthelson RA, Lambert GM, Vanier C et al (2007) Comparison of the contributions of
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the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8:340. doi:10.1186/1471-21648-340 15. Bonn S, Zinzen RP, Perez-Gonzalez A et al (2012) Cell type-specific chromatin immunoprecipitation from multicellular complex sam-
ples using BiTS-ChIP. Nat Protoc 7:978–994. doi:10.1038/nprot.2012.049 16. Steiner FA, Talbert PB, Kasinathan S et al (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res 22:766–777. doi:10.1101/gr.131748.111
1
Introduction Hox genes are expressed along the anteroposterior axis of animal embryos where they coordinate morphogenetic processes in a segment-specific manner [1, 2]. The essential role of Hox genes in specifying segment identities is evidenced by the “homeotic transformations” that are frequently observed upon disruption of Hox function in Drosophila [1]. Hox genes encode homeodomain transcription factors that exert their regulatory functions in diverse cell and tissue types by activating or repressing downstream target genes [3]. Identification of Hox target genes is crucial for elucidating how Hox proteins carry out their regulatory role in vivo and how distinct morphogenetic programs are executed under the control of Hox proteins in the different embryonic segments. The complexity of Hox regulatory networks in combination with the frequently encountered and degenerated Hox-binding sites and the involvement of co-regulatory factors in the transcriptional regulation of Hox target genes present significant limitations for the identification of Hox target genes using in silico or in vitro
Yacine Graba and René Rezsohazy (eds.), Hox Genes: Methods and Protocols, Methods in Molecular Biology, vol. 1196, DOI 10.1007/978-1-4939-1242-1_12, © Springer Science+Business Media New York 2014
197
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approaches [4, 5]. Consequently, it is required to employ in vivo strategies in order to identify Hox-responsive gene regulatory networks. Studies in Drosophila have provided a substantial amount of knowledge on the molecular mechanisms underlying Hox protein functions. In recent years, high-throughput analyses of Hoxregulated transcriptional networks using genome-wide methods have shed light on the mechanistic basis of how Hox proteins acquire their high specificity and selectivity (reviewed in refs. 5, 6). A number of studies in which a Hox transcriptome analysis was performed in Drosophila allowed the genome-wide identification of Hox downstream genes [7–11]. Importantly, the quantitative identification of target genes for six out of eight Drosophila Hox proteins, by performing transcriptome analysis using microarrays and whole-Drosophila embryos ubiquitously overexpressing each of the selected Hox proteins [10], demonstrated that Hox proteins control the expression of hundreds of genes and that different Hox proteins, despite their similar DNA-binding properties in vitro, show highly specific effects on the transcriptome (reviewed in refs. 5, 12). For a variety of reasons, in experiments aiming at target gene identification Hox overexpression is advantageous in comparison to Hox loss of function. First of all, the extensive cross-regulation of Hox genes introduces complications in the interpretation of the results from experiments using mutants [13]. An additional limitation arises from the fact that Hox proteins are only expressed in small subsets of cells [2] and therefore the changes in gene expression taking place in the Hox-deficient cells will be diluted in the heterogeneous RNA mixture isolated from whole embryos. Performing the transcriptome analysis using isolated cells in which the Hox protein of interest is active could solve this problem. Considering that cellular and nuclear mRNA pools are very comparable [14], isolated nuclei are a reliable source of cell-typespecific transcripts. Thus, the recently developed methods allowing isolation of pure populations of nuclei from selected cell types [15, 16] could be employed. Importantly, given the context-dependent activity of Hox proteins, these methods offer the possibility to identify targets of a given Hox protein in different tissue types. In this chapter we provide the protocol for identification of Hox target genes in Drosophila embryos by performing a transcriptome analysis using microarrays. The procedure does not only apply to whole embryos but can also be used with different experimental settings, i.e., when using larval tissues or purified cell populations. Principally, each gene of a given genome is represented on a microarray chip, in the case of Affymetrix Chips by a series of different oligonucleotide probes that serve as unique, sequencespecific detectors. The chip is hybridized with biotinylated cRNA
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that was generated from oligo-dT-primed cDNA of a given tissue. Each sample (for example, “wild-type” versus “mutant” tissue) is hybridized to a separate array. After hybridization, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. The staining protocol includes a signal amplification step that employs anti-streptavidin antibody (goat) and biotinylated goat IgG antibody. After washing, transcript levels are calculated by reference to cRNA spikes of known concentration added to the hybridization mixture. Differences in mRNA levels between samples are determined by comparison of any two hybridization patterns produced on separate arrays of the different samples. Here, we describe the procedures for collecting the Drosophila embryos, isolating the total RNA, synthesizing the complementary DNA (cDNA) and the complementary RNA (cRNA), controlling the quality of the cRNA probes, performing the hybridization to the microarrays, and scanning the probes.
2
Materials
2.1 Drosophila Embryo Collection
2.2 Isolation of RNA from Drosophila Embryos 2.3 Double-Stranded cDNA (ds-cDNA) and cRNA Synthesis and Cleanup, cRNA Fragmentation 2.4 Agarose Gel Analysis
Apple juice agar plates: Add 24 g agar to 740 ml ultrapure water and autoclave. Keep warm so that it does not become solid. Boil 250 ml apple juice with 25 g sugar and add to the autoclaved agar. Stir on a magnetic plate until the mixture had cooled down to approximately 50 °C and pour in petri dishes. The plates can be stored at 4 °C. Fresh yeast paste is spread on the plates directly before use. 100 % bleach is used for dechorionating the embryos. 1. RNeasy Mini Kit (QIAGEN). 2. Polypropylene micropestle (Eppendorf). 3. QIAshredder spin columns (QIAGEN). MessageAmp™ II-Biotin Enhanced, Single Round aRNA Amplification Kit (Ambion). All reagents required for the synthesis and cleanup of ds-cDNA and biotinylated cRNA are provided by the kit.
1. Denaturing gel loading buffer: 0.5 μl ethidium bromide (1 mg/ml), 1 μl 10× MOPS buffer, 5 μl formamide, 5 μl formaldehyde (37 %), 3 μl RNA loading dye (50 % glycerol containing 2.5 mg/ml bromophenol blue and xylene cyanol, each). 2. RNA gel: It is not necessary to run a denaturing RNA gel. Prepare a 1 % agarose gel for the unfragmented samples and 2 % agarose gel for the fragmented samples.
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2.5 Hybridization, Washing, Staining, and Scanning
Additional arrays and staining solutions can be stored at 4 °C until needed. 1. Arrays: GeneChip Drosophila Genome 2.0 Array (Affymetrix). 2. Streptavidin-phycoerythrin (SAPE) solution (final volume 1,200 μl): 600 μl 2× 2-(N-morpholino)ethanesulfonic acid (MES) stain buffer, 120 μl acetylated bovine serum albumin (BSA) (20 mg/ml), 12 μl SAPE, 468 μl water. Split in two aliquots of 600 μl that will both be used for one array (see Note 1). 3. Antibody solution (final volume 600 μl): 300 μl 2× MES stain buffer, 60 μl acetylated BSA (20 mg/ml), 6 μl goat IgG (10 mg/ml), 3.6 μl biotinylated antibody (0.5 mg/ml), 230.4 μl water. 4. 2× Hybridization buffer: 200 mM MES, 0.5 M NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.005 % Tween-20. 5. 20× SSPE buffer (1 l): 175.3 g sodium chloride, 27.6 g sodium phosphate monobasic, 9.4 g ethylenediaminetetraacetic acid (EDTA). 6. Wash buffer A: Non-stringent wash buffer consisting of 6× SSPE buffer (use the 20× SSPE stock described in item 5) and 0.01 % Tween-20.
3
Methods
3.1 Drosophila Embryo Collection
1. Two days before starting the embryo collection transfer newly hatched adult flies into a collection cage and cover it with an apple juice agar plate with yeast paste (see Note 2). Collection and aging times depend on the desired embryonic stage(s). When collecting embryos keep in mind that it is recommended to perform microarray experiments using biological triplicates. 2. Add water to the apple juice agar plate and brush the embryos to release them from the agar surface. Transfer the embryos to a sieve, rinse thoroughly with water, and dechorionate in 100 % bleach. Check under the microscope that the chorion has been completely removed. Wash the embryos well with water and remove as much liquid as possible by blotting the sieve with a paper towel. Using a brush, transfer the embryos in a preweighed microcentrifuge tube, measure the weight, and snapfreeze in liquid nitrogen. At this point the embryos can be stored at −80 °C.
3.2 Isolation of RNA from Drosophila Embryos
Use of RNeasy Mini Kit (QIAGEN) is recommended for the isolation of total RNA. Make sure that sterile and nuclease-free glassware or plastic are used throughout the procedure (see Note 3) and follow the instructions for “isolation of total RNA from animal
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tissues.” The following procedure applies to isolation of RNA from whole embryos. The required modifications in cases when tissues (see Note 4) or sorted cells (see Note 5) are used are described separately. 1. Remove the frozen embryos from storage. Do not use more than 30 mg of embryos per sample. Do not allow the frozen embryos to thaw and proceed to step 2 as fast as possible. 2. Before use, ensure that β-mercaptoethanol is added to Buffer RLT (RNA easy mini kit, QIAGEN). Add 400 μl Buffer RLT to the embryos and lyse them by homogenizing in the microcentrifuge tube using a polypropylene pestle. Alternatively the embryos can be lysed in a Dounce homogenizer using a tight pestle. 3. Transfer the lysate to a new labeled tube. 4. Rinse both pestle and grinder with 200 μl RLT and combine the solution with the lysate of the previous step. 5. Incubate at RT for 5 min. 6. Centrifuge the lysate at maximum speed in a microcentrifuge for 3 min. 7. Carefully transfer the supernatant (cleared lysate) to a new microcentrifuge tube. 8. Add 600 μl of 70 % ethanol (prepared with diethylpyrocarbonate (DEPC)-treated water) to the cleared lysate and mix immediately by pipetting. 9. Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube. Centrifuge for 15 s at 8,000 × g and discard the flow-through. (If the sample volume exceeds 700 μl, centrifuge successive aliquots in the spin column.) 10. Add 700 μl Buffer RW1 (QIAshredder spin columns, QIAGEN) to the RNeasy spin column. Centrifuge for 15 s at 8,000 × g and discard the flow-through. 11. Before use, make sure that ethanol is added to Buffer RPE (RNA easy mini kit, QIAGEN). Add 500 μl Buffer RPE to the RNeasy spin column. Centrifuge for 15 s at 8,000 × g and discard the flow-through. 12. Repeat step 7 and centrifuge for 2 min at 8,000 × g. 13. Transfer the RNeasy spin column in a new 2 ml collection tube and centrifuge at full speed for 1 min to ensure no carryover of ethanol. 14. Place the RNeasy spin column in a new 1.5 ml microcentrifuge tube and add 30 μl RNase-free water directly to the membrane. Centrifuge for 1 min at 8,000 × g.
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15. Optional: Repeat step 10 using another 30–50 μl RNase-free water. Do not mix the two eluates before measuring the RNA concentration. 16. Measure RNA concentration and 260 nm/280 nm absorbance ratio (as a control of RNA purity) using Nanodrop. At this step the two eluates can be pooled together depending on their concentration. The RNA can be stored at −80 °C. 3.3 ds-cDNA Synthesis 3.3.1 First-Strand cDNA Synthesis
Use the “MessageAmpTm II-Biotin Enhanced, Single Round aRNA Amplification Kit” (Ambion). 1. Before you start prepare spike controls (see Note 6) according to the following dilution table. The first dilution can be stored at −20 °C and reused up to three times. Spike control dilution Total RNA (μg)
First dilution
Second dilution
Third dilution
Final dilution
1
1:20
1:50
1:25
1:25,000
5
1:20
1:50
1:5
1:5,000
10
1:20
1:50
1:2.5
1:2,500
2. Prepare the following mix and keep the amount of RNA constant for all samples: 10 μl total RNA (1 μg in 10 μl water is recommended when using the Ambion kit), 1 μl T7 Oligo(dT) primer, 1 μl spike controls (according to the dilution table). 3. Incubate RNA at 70 °C for 10 min. 4. Chill rapidly on ice. 5. For each sample, prepare a master mix containing the following reagents: 2 μl 10× first-strand buffer, 4 μl dNTP Mix, 1 μl RNase inhibitor, and 1 μl ArrayScript Reverse Transcriptase. 6. Add 8 μl master mix to each RNA sample (final volume 20 μl) and mix gently. Spin down briefly if necessary. 7. Incubate at 42 °C for 2 h. 3.3.2 Second-Strand cDNA Synthesis
1. Place the first-strand cDNA synthesized in Subheading 3.3.1 on ice. 2. For each sample, prepare a master mix containing the following reagents: 63 μl RNase-free water, 10 μl 10× second-strand buffer, 4 μl dNTP mix, 2 μl DNA polymerase, 1 μl RNase H. 3. Mix gently and spin down briefly if necessary.
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4. Add 80 μl master mix to each sample. Mix thoroughly by pipetting up and down two to three times. Spin down briefly if necessary. 5. Incubate at 16 °C for 2 h. Take care not to let the temperature of the samples rise above 16 °C. 3.3.3 ds-cDNA Cleanup
1. Before you begin preheat the nuclease-free water at 50–55 °C and make sure that ethanol has been added to the bottle of wash buffer. 2. Add 250 μl cDNA-binding buffer to each sample and mix thoroughly by pipetting up and down two to three times. Spin down briefly if necessary. 3. Apply the sample mix to a cDNA Cleanup Spin Column placed in a 2 ml collection tube. 4. Spin at 8,000 × g for 1 min and discard flow-through. 5. Apply 500 μl cDNA wash buffer to each spin column. 6. Spin at 8,000 × g for 1 min and discard flow-through. 7. Spin once again at 8,000 × g for 1 min in order to remove trace amounts of wash buffer. 8. Transfer the spin column into a new 1.5 ml collection tube and pipet 10 μl preheated nuclease-free water directly onto the center of the membrane. 9. Incubate for 2 min. 10. Spin at 8,000 × g for 1 min. 11. Pipet 12 μl preheated nuclease-free water directly onto the center of the membrane and incubate for 2 min. 12. Spin at 8,000 × g for 1 min. 13. The total elution volume will be 20 μl. At this point the dscDNA can be stored at −80 °C.
3.4
cRNA Synthesis
3.4.1 In Vitro Transcription
Use the “MessageAmpTm II-Biotin Enhanced, Single Round aRNA Amplification Kit” (Ambion). 1. Thaw ds-cDNA on ice and prepare the following in vitro transcription master mix for each sample: 12 μl biotin-NTP mix, 4 μl T7 10× reaction buffer, 4 μl T7 enzyme mix. 2. Add 20 μl in vitro transcription master mix to 20 μl ds-cDNA (final volume 40 μl) and mix gently. Spin down briefly if necessary. 3. Incubate at 37 °C overnight (approximately 16 h). 4. Add 60 μl nuclease-free water to each cRNA sample (final volume 100 μl). At this point the cRNA can be stored at −80 °C.
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3.4.2 cRNA Cleanup
1. Before you begin preheat nuclease-free water to 50–60 °C and assemble cRNA filter cartridges and tubes. 2. Add 350 μl cRNA-binding buffer to the cRNA and vortex. 3. Add 250 μl ethanol (>99 %) to each sample and mix thoroughly by pipetting the mixture up and down two to three times. Do not vortex and do not centrifuge the samples. 4. Apply sample mix (final volume 700 μl) to a cRNA Cleanup Spin Column placed in a 2 ml collection tube. 5. Spin at 8,000 × g for 1 min and discard flow-through. 6. Add 650 μl wash buffer (supplemented with ethanol) to each sample. 7. Spin at 8,000 × g for 1 min and discard flow-through. 8. Spin at 8,000 × g for 1 min to remove trace amounts of wash buffer. 9. Transfer spin column into new 1.5 ml collection tube and pipet 40 μl preheated nuclease-free water directly onto the membrane. 10. Incubate for 2 min. 11. Spin at 8,000 × g for 1 min to elute cRNA. 12. Transfer spin column into new 1.5 ml collection tube and repeat elution with an additional 60 μl preheated nuclease-free water. 13. Use 1 μl of the first eluate to measure cRNA concentration and 260/280 ratio. Optional steps, in case the cRNA concentration is lower than 1.0 μg/μl: 14. Combine the two cRNA eluates (final volume 100 μl). 15. Add 10 μl 5 M ammonium acetate and 275 μl 100 % ethanol. 16. Mix well and incubate at −20 °C for at least 30 min. 17. Centrifuge at full speed for 15 min at 4 °C and discard supernatant. 18. Wash pellet with 500 μl cold 70 % ethanol. 19. Centrifuge at full speed for 15 min at 4 °C or room temperature. 20. Remove 70 % ethanol. 21. Spin briefly, remove any residual fluid, and air-dry the pellet. 22. Resuspend the cRNA in an appropriate volume of nucleasefree water. 23. Use 1 μl to measure cRNA concentration and 260 nm/280 nm absorbance ratio.
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1. Set up the fragmentation reaction (final volume: 20 μl) by mixing the following: 15 μg cRNA in a total volume of 16 μl nuclease-free water and 4 μl 5× fragmentation buffer. 2. Incubate at 94 °C for 35 min. 3. Chill rapidly on ice.
3.6 Agarose Gel Analysis
It is recommended to check the quality of the synthesized cRNA and the fragmentation on an agarose gel in order to confirm the quality of the probe before starting the hybridization procedure. 1. Add 5 μl gel loading buffer to the following samples: (a) 2 μl unfragmented cRNA. (b) 2 μl fragmented cRNA. 2. Incubate at 65 °C for 10 min and chill on ice. 3. Run the unfragmented samples on a 1 % agarose gel and the fragmented samples on a 2 % agarose gel.
3.7 Preparation of Hybridization Cocktail and Hybridization
1. For each sample prepare a master mix containing the following reagents: 4.25 μl control oligo B2, 12.5 μl 20× eukaryotic hybridization controls, 2.5 μl salmon sperm DNA, 6.25 μl BSA (20 mg/ml), 125.0 μl 2× hybridization buffer, 25.0 μl dimethyl sulfoxide (DMSO), 56.5 μl water. 2. Add 232 μl master mix to the remaining 18 μl fragmented cRNA from each sample (final volume 250 μl). At this point the samples can be stored at −80 °C. 3. Before hybridization, denature the probe for 5 min at 95 °C, followed by 5 min at 45 °C. 4. Prehybridize the probe array with 250 μl 1× hybridization buffer at 45 °C for at least 10 min. 5. Hybridize the probe array for 16 h at 45 °C while shaking at 60 rpm.
3.8 Washing, Staining, and Scanning of the Arrays
Before you start enter experiment description into Affymetrix workstation (GCOS). Unless the experiments have been defined first, you will not be able to wash, stain, and scan your arrays. Wash and stain the probe array using the Affymentrix Fluidics Station 450, which is operated using GCOS/Microarray Suite. Throughout the procedure, follow the instructions in the LCD window on the fluidics station. 1. Prime fluidics station with wash buffers in order to make sure that the lines of the fluidics station are filled with the appropriate buffers. 2. Remove probe from arrays and fill arrays with wash buffer A. Take care not to introduce any bubbles. If necessary, the probes can be frozen at −80 °C at this point (see Note 7).
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3. Select the correct experiment name and the appropriate antibody amplification protocol to control the washing and staining of the probe array, from the drop-down Experiment list. 4. Load the three experiment microcentrifuge vials into the sample holders on the fluidics station as follows: Place one vial containing 600 μl of SAPE solution in sample holder 1, one vial containing 600 μl of antibody solution in sample holder 2, and one vial containing 600 μl of SAPE solution in sample holder 3. 5. Start the run. The Fluidics Station dialog box displays the status of the washing and staining during the procedure. After the run has finished, if you do not plan to scan the arrays immediately, keep them at 4 °C in the dark until ready for scanning. 6. Clean excess fluid from around septa on the back of the probe array cartridge. 7. Carefully apply one Tough-Spots to each of the two septa (see Note 8). 8. Insert the cartridge into the scanner and ensure that the spots lie flat. 9. Select the correct experiment name, insert the probe array into the holder, and start the scan. 10. After completion of the scan, the data is ready for analysis.
4
Notes 1. SAPE should be stored in the dark at 4 °C and should not be frozen. Before preparing staining solution take SAPE out of the refrigerator and mix well. The SAPE solution should always be prepared fresh. 2. The apple juice agar plates and yeast paste must be fresh and pre-warmed before use in order to maximize the yield of the collections. 3. To provide RNase-free conditions, bake all the glassware for 2 h at 300 °C, use DEPC-H2O for preparing the solutions, and wear gloves and a lab coat when working with RNA samples. 4. A few parameters need to be considered when isolating RNA from dissected tissues (i.e., imaginal discs or other larval tissues). During dissection, the tissues of interest should be kept on ice at all times so that the RNA does not degrade. Snapfreeze the tissue in liquid nitrogen as soon a possible. If the amount of dissected tissue is smaller than 20 mg, use 300 μl of RLT buffer. 5. When using isolated cells (i.e., isolated by FACS sorting), follow the protocol “Purification of Total RNA from Animal Cells”
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provided in the RNeasy Mini Kit (QIAGEN) and adjust the volume of RLT according to the number of cells as described in the manual. 6. The use of external RNA controls (“spikes”) allows assessing the performance of the microarray experiment. The probes that correspond to the spikes are informative of the fidelity of the microarray technology in determining their presence. It is recommended to spike external controls in an early step of sample processing in order to monitor several steps. 7. If the probes were frozen, equilibrate them to room temperature. 8. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly and you observe bumps, bubbles, tears, or curled edges, remove the spot and apply a new one. Do not attempt to smoothen the spot.
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the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8:340. doi:10.1186/1471-21648-340 15. Bonn S, Zinzen RP, Perez-Gonzalez A et al (2012) Cell type-specific chromatin immunoprecipitation from multicellular complex sam-
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