Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Podsumowanie

Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Streszczenie

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Wprowadzenie

Drosophila tissues such as the central nervous system contain a complex mixture of cell types. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it is first necessary to isolate a homogenous population of specific cells in sufficient quantities to enable downstream applications. Methods to isolate cells from intact tissues include laser microdissection, and fluorescence-activated cell sorting (FACS) of whole cells. While FACS has been used to isolate cells and nuclei from Drosophila embryos and from Caenorhabditis elegans for gene expression and chromatin profiling^1-3, FACS and laser microdissection can be difficult to perform successfully in tissues that contain highly intermixed cell types or that contain cells with irregular morphology, such as neurons. To overcome this difficulty, nuclei rather than cells can be isolated from specific cell types and used for subsequent gene expression profiling. Importantly, microarray-based mRNA expression analysis using nuclear RNA samples shows generally comparable results with that performed using total RNA^4,5. Moreover, gene expression analysis using nuclear RNA has been successfully used to study gene expression in multiple organisms including C. elegans, Arabidopsis thaliana, Drosophila, and humans^4,52,3.

Several approaches have recently been described for the isolation of specific populations of labeled nuclei from Drosophila tissues that are suitable for gene expression analysis and/or chromatin immunoprecipitation. The batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) method utilizes FACS to isolate fixed nuclei on the basis of cell-type specific expression of nuclear-localized GFP². This approach has been successfully used to analyze the distribution of histone modifications and transcription factors using chromatin immunoprecipitation of isolated nuclei from the mesoderm of Drosophila embryos². However, FACS-based approaches may be less suitable for isolating labeled nuclei that constitute only a small proportion of a mixed population due to the increased sort time needed to obtain suitable numbers for downstream applications. To overcome these limitations, several groups have utilized affinity-based isolation techniques to purify nuclei that are labeled with a specific epitope in a particular cell type. The isolation of nuclei tagged in specific cell types (INTACT) method developed for use in Arabidopsis thaliana^6,7 has recently been adapted for use in Drosophila⁸. In this method, a nuclear envelope fusion protein that is a substrate for in vivo biotinylation is coexpressed with the Escherichia coli biotin ligase BirA in specific cell types. Biotin-labeled nuclei can be subsequently purified from mixed populations using streptavidin-based affinity isolation. Using this approach, nuclei were successfully labeled and isolated from the mesoderm of Drosophila embryos in which a nuclear envelope fusion protein was expressed under control of a mesoderm-specific enhancer⁸. The authors also generated nuclear envelope fusion proteins that can be expressed in any cell type under control of the Gal4 regulatory sequence, UAS⁹. This approach is capable of rapidly isolating subsets of labeled nuclei from mixed populations, but requires three separate transgenic constructs and may therefore be unsuitable for particular genetic applications. Recently, an approach has been described in which SUN (Sad1 and UNC-84) domain-containing proteins that localize to the inner membrane of the nuclear envelope were tagged with fluorescent proteins and expressed under control of the Gal4/UAS system¹⁰. Nuclei were isolated in the presence of nonionic detergent to remove the outer membrane of the nuclear envelope, and affinity-purified using magnetic beads coupled to anti-GFP antibodies. This approach was successfully used to isolate small populations of labeled nuclei from specific neuronal subtypes within the adult brain of Drosophila¹⁰.

Here, a protocol for isolation of labeled nuclei from a mixed population of cells from Drosophila larval tissue is described. This method was developed independently, but is similar to the approach described by Henry et al.¹⁰ First, nuclei were labeled with a fluorescent tag that is expressed only in specific cell types in Drosophila melanogaster to facilitate the subsequent isolation of tagged nuclei from mixed populations using an affinity-based approach. To label nuclei, the KASH (Klarsicht/Anc-1/Syne homology) domain was utilized. The KASH domain is a transmembrane domain that localizes to the outer membrane of the nuclear envelope, in part through interactions with SUN domain-containing proteins within the perinuclear space¹¹. The C-terminal KASH domain of proteins such as Drosophila Msp-300 and Klarsicht anchors these proteins to the outer nuclear membrane, while their N-terminal domains interact with cytoskeleton proteins such as actin or microtubules in the cytoplasm^12-14. Constructs were generated in which the KASH domain of Drosophila Msp-300 was fused to the C-terminus of EGFP, under control of the Gal4 regulatory sequence, UAS in the pUAST-attB vector^15,16. Using phiC31 site-specific integration, transgenic flies were generated in which the UAS-EGFP::Msp-300^KASH transgene was inserted in the attP2 loci on chromosome 3L¹⁷. The UAS-EGFP::Msp-300^KASH flies can be crossed with flies that express the Gal4 driver in a particular cell type, resulting in the targeted expression of EGFP on the outer nuclear membrane in the cell type of interest. Labeled nuclei can then be purified from mixed cell populations using antibodies against GFP coupled to magnetic beads. In this approach, the use of nonionic detergent is not required because the EGFP tag is localized to the cytoplasmic side of the outer nuclear membrane, and is therefore accessible to antibodies.

The protocol described below can be used to isolate/enrich EGFP-labeled nuclei from specific cell types in Drosophila larval tissues, to quantify the purity and yield of isolated nuclei, and to extract nuclear RNA suitable for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) gene expression analysis. The isolation and affinity-purification of nuclei (excluding tissue dissection) can be performed in less than one hour. Results are presented showing that glial nuclei can be successfully isolated from the larval optic lobe and eye imaginal disc, and used for subsequent gene expression analysis. It is anticipated that this approach will be useful for the isolation/enrichment of labeled nuclei from embryonic and larval tissues in which the target cells constitute less than 5% of the overall population. All Drosophila stocks and plasmids generated in this study are available from the authors upon request.

Access restricted. Please log in or start a trial to view this content.

Protokół

1. Generation and Characterization of EGFP::KASH Drosophila

1. Cross Gal4 driver flies to UAS-EGFP::Msp-300^KASH flies. Alternatively, recombinant flies that carry both the Gal4 driver and the UAS-EGFP::Msp-300^KASH transgene can be generated using standard genetic techniques. This second approach is advantageous if large numbers of progeny are required.
2. Characterize expression of the EGFP::KASH marker using standard microscopy techniques. Note: EGFP expression can be observed by standard microscopy techniques in dissected, fixed Drosophila tissues, or in whole larvae, and does not require use of an antibody.
   1. Determine the expression pattern of the EGFP::KASH marker in the entire organism under a fluorescence dissecting microscope (see Materials PDF). Note: Many Gal4 drivers, including the repo-GAL4 driver shown in Figure 1, exhibit nonspecific patterns of expression in other larval tissues such as the salivary gland (see Results).
   2. Analyze the EGFP::KASH expression pattern in the dissected tissue of interest using confocal microscopy.
      1. Fix the tissue of interest using formaldehyde at a final concentration of 4%, and stain DNA using DAPI at a final concentration of 0.1 μg/ml to visualize the localization of the EGFP::KASH tag relative to DNA.
      2. Acquire images using either a 40X / 1.30 oil immersion objective or a 20X / 0.75 oil immersion objective, depending on the size of the tissue of interest. The following excitation/emission conditions can be used for image acquisition: 408 nm/425-475 nm (DAPI); 488 nm /525-550 nm (GFP).

2. Isolate Nuclei from Drosophila Larval Tissues

1. Prepare equipment for the larval tissue dissection and subsequent nuclei isolation. Note that whole dechorionated embryos could also be used as starting material if desired. It is recommended to use partially dissected larval tissues rather than entire larvae if the cell type of interest is present in a specific tissue such as the imaginal disc. This reduces nonspecific binding, and also eliminates problems that can be caused by expression of some Gal4 drivers in nontarget cells.
   1. Prepare two pairs of sharp forceps (see Materials PDF), one siliconized 9-well glass plate (see Materials PDF), and PBT (1x PBS, pH 7.4; 0.1% Tween-20).
   2. Prebind the GFP antibody to the magnetic beads. This step should be started before beginning the dissection. Add 10 μl of the magnetic beads (Invitrogen, see Materials PDF) and 2 μg of GFP antibody (Roche, see Materials PDF) to 400 μl of wash buffer (1x PBS, pH 7.4; 2.5 mM MgCl₂) in a 1.5 ml tube. The amount of beads and antibody used can be optimized if necessary based on the amount of target in the sample and the binding affinity of the antibody to the beads.
   3. Incubate the beads and antibody with rotation for at least 30 min at room temperature.
   4. Place the 1.5 ml tube containing the beads and antibody in the magnetic rack (see Materials PDF) and allow the beads to bind to the magnetic for approximately 1-2 min. Remove the supernatant containing the unbound antibody and wash buffer using a 1 ml pipette. The beads will remain bound to the wall of the 1.5 ml tube on the side adjacent to the magnet.
   5. Rinse the beads twice with 1 ml of wash buffer each time as described in section 2.1.4. Remove the 1.5 ml tube from the magnetic rack and invert briefly to mix the beads and wash buffer during each wash step.
   6. Store the antibody-bound beads in wash buffer until ready to continue with step 2.7. The beads should not be allowed to dry out at any stage of the protocol.
2. Dissect larval tissues in PBT and transfer dissected tissues to one well of the 9-well dish. Typically, dissection of the optic lobe and eye imaginal disc from 100 third instar larvae provides sufficient material for downstream gene expression analysis following nuclei isolation.
3. Rinse the isolated tissue in nuclear extraction buffer (10 mM HEPES-KOH, pH 7.5; 2.5 mM MgCl₂; 10 mM KCl) in a 1.5 ml tube. Transfer isolated larval tissues to a 1 ml Dounce Homogenizer (see Materials PDF) on ice that contains 1 ml of fresh nuclear extraction buffer. Incubate on ice for 5 min. It is not necessary to add protease inhibitors if RNA is to be extracted following nuclei isolation.
4. Disrupt the tissue by 20 strokes with the loose pestle and then incubate the sample on ice for 10 min to allow the cells to swell. Extract the nuclei from the cells using 15 strokes with the tight pestle. The number of strokes with the loose and tight pestles needs to be optimized for different tissues and cell types; excess homogenization can result in sheared nuclei, while insufficient homogenization will not extract all nuclei from the tissue.
5. Filter the homogenate, which contains the nuclei and cellular debris, through a 40 μm pore-size strainer (see Materials PDF) that has been prerinsed with nuclear homogenization buffer. Collect the filtered homogenate in a fresh tube.
6. Collect pre-isolation samples for subsequent analysis to determine nuclei yield, nuclei integrity, and to determine transcript levels of target genes prior to nuclei isolation.
   1. Transfer 50 μl of the filtered homogenate to a fresh 1.5 ml tube using a pipette. This is the pre-isolation sample. Add 500 μl of Trizol reagent (see Materials PDF, CAUTION), invert to mix, and store at -20 °C for subsequent RNA isolation (step 3.1).
   2. Transfer 20 μl of the filtered homogenate to a fresh 1.5 ml tube.
      1. Add DAPI to a final concentration of 0.1 μg/ml, and incubate sample on ice for 10 min.
      2. Transfer DAPI-stained nuclei to a poly-lysine coated coverslip, and place on a microscope slide. Seal the coverslip using clear nail polish.
      3. Check the nuclei integrity, and presence of GFP-tagged nuclei of interest using fluorescent microscope. Note: It is not necessary to fix the nuclei, or to stain for GFP if these slides are analyzed reasonably quickly following preparation (within 24 hr).
   3. Transfer 10 μl of the filtered homogenate to a fresh 1.5 ml tube and add 90 μl of wash buffer (1x PBS, pH 7.4; 2.5 mM MgCl₂). Add DAPI to a final concentration of 0.1 μg/ml, and incubate on ice for 10 min. Determine the nuclei yield in the pre-isolation sample using a hemocytometer to count the DAPI-positive nuclei using epifluorescence under a 10X air objective.
7. Place the 1.5 ml tube containing the antibody-bound beads (prepared in section 2.1.6) in a magnetic rack, and allow the magnetic beads to bind to the magnet for at least 2 min as described in section 2.1.4. Remove the wash buffer from the antibody-bound beads using a 1 ml pipette, and add the filtered homogenate from step 2.5 to the 1.5 ml tube using a 1 ml pipette.
8. Gently invert the tube several times to mix, and incubate at 4 °C for 30 min with end-over-end agitation. Avoid bubbles in the tube if possible since these can result in clumping of the nuclei.
9. Place the 1.5 ml tube containing the antibody-bound beads and homogenate in a magnetic rack, and allow the magnetic beads to bind to the magnet for at least 2 min as described in section 2.1.4. Remove the homogenate containing the unbound nuclei fraction using a 1 ml pipette.
10. Wash the sample 3x with 1 ml of wash buffer each time. Remove the 1.5 ml tube containing the beads, bound nuclei and wash buffer from the rack and invert to mix several times, then place the tube in the magnetic rack as described in step 2.9 and remove the supernatant using a 1 ml pipette.
11. Add 150 μl of wash buffer to the beads, which should be bound to the target nuclei. This is the post-isolation sample. Prepare samples for microscopy analysis and subsequent RNA extraction.
    1. Transfer 20 μl of the post-isolation sample to a fresh 1.5 ml tube and stain with DAPI and analyze as described in section 2.6.2. Count the GFP+/DAPI+ and GFP-/DAPI+ nuclei captured by magnetic beads to determine the purity of the enriched GFP+ nuclei in the post-isolation sample.
    2. Add 1 ml of Trizol reagent to the remainder of the post-isolation sample, invert to mix, and store at -20 °C for subsequent RNA isolation (step 3.1). Note: Samples in Trizol can be stored for several weeks if necessary at -20 °C. It is not necessary to elute the nuclei from the magnetic beads prior to RNA extraction using Trizol reagent, because the beads do not interfere with the subsequent RNA isolation.

3. Determine Transcript Levels in Nuclei Isolated from Larval Tissues

1. Isolate RNA from the pre-isolation and post-isolation samples prepared in sections 2.6.1 and 2.11.2 using the standard protocol described for Trizol-based RNA extraction (see Materials PDF) with the following modifications:
   1. Following precipitation of the RNA pellet, add 19.2 μl of RNase-free water and 0.8 μl of RNASecure reagent (see Materials PDF) to the pellet and incubate at 60 °C for 20 min to inactivate RNAses.
2. Determine the RNA concentration with a fluorescent-RNA binding dye such as the Qubit RNA assay kit (see Materials PDF) using the QubitR 2.0 Fluorometer following the manufacturer’s protocol.
3. Synthesize cDNA using an amplifying reverse transcriptase such as the EpiScript Reverse Transcriptase kit and oligodT primers following the manufacturer’s instructions. Note: Typically, 50 to 100 ng of RNA generates sufficient cDNA to perform multiple gene expression analyses. Equivalent amounts of pre-isolation and post-isolation RNA should be used to generate cDNA.
4. Add 180 μl of nuclease-free water to the 20 μl cDNA sample to dilute this 10-fold prior to real-time quantitative PCR (qPCR) analysis. This dilution is an important step, because undiluted cDNA samples can inhibit the qPCR.
5. Prepare a qPCR master mix with polymerase and dNTPs, 4 pmoles each of forward and reverse primer, made up with sterile water to the final reaction volume of 20 μl.
   1. Design qPCR primers to target genes that are expected to be expressed in the cell type of interest, and at the developmental stage at which the tissue is collected. Design primers to span an intron if possible, so that genomic and cDNA PCR products can be distinguished. Note: If the gene expression profile of the target cell type is not known, primers against eGFP, which should be expressed specifically in the tagged nuclei population, can be used to optimize the protocol for a particular cell type and tissue (Table 1).
6. Determine the relative transcript levels of each gene of interest in the pre-isolation and post-isolation samples by comparison to a dilution series of cDNA prepared from the whole tissue. Transcript levels of target genes should be normalized to a reference gene such as Rpl32 (or preferably several reference genes).

Access restricted. Please log in or start a trial to view this content.

Wyniki

The repo-GAL4 driver (Bloomington stock number 7415) is specifically expressed in glial cells in the Drosophila nervous system at multiple stages of development¹⁸. Flies were generated that stably express UAS-EGFP::Msp-300^KASH under control of the repo-GAL4 driver using standard genetic techniques. To characterize the expression pattern of the EGFP::KASH transgene in these flies, the pattern of GFP expression in the dissected optic lobe and eye imaginal disc from ...

Access restricted. Please log in or start a trial to view this content.

Dyskusje

This protocol can be used to isolate or highly enrich specifically tagged nuclei from mixed cell populations in Drosophila embryonic or larval tissues. Using this protocol, nuclei can be isolated from dissected tissues in approximately 1 hr. The purity and yield of the isolated nuclei must be experimentally determined for each cell type. It can be difficult to quantify the bead-bound nuclei at the post-isolation stage of the protocol using a hemocytometer because of the beads present in the sample. Thus, if exac...

Access restricted. Please log in or start a trial to view this content.

Materiały

Name	Company	Catalog Number	Comments
Anti-chaoptin antibody (mouse, monoclonal)	Developmental Studies Hybridoma Bank	mAB24B10
Anti-GFP antibody (mouse, monoclonal)	Roche	11814460001	This specific antibody is recommended for this protocol. However, alternative antibodies could also be used.
2x Bullseye EvaGreen qPCR Mastermix with ROX	MidSci	BEQPCR-R
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride salt)	Biotium	40011
Dounce Homogenizer (1 ml)	VWR	62400-595
Dumont #5 Tweezers, 11 cm	World Precision Instruments	14095
Dynabeads Protein G for Immunoprecipitation	Invitrogen	10003D	This specific brand of magnetic beads is recommended for this protocol.
EpiScript Reverse Transcriptase	Epicentre	ERT12910K	http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf
Falcon cell strainers, 40 μm pore size	VWR	21008-949	Alternative pore sizes could be used, depending on the size of the target cell nuclei.
Magnetic stand (8 x 1.5 ml tubes)	Millipore	LSKMAGS08
Mini tube rotator	Genemate	H-6700
Qubit starter kit	Life Technologies	Q32871	Similar fluorescence-based methods for quantifying RNA yield could be used. http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdfZ
RNAsecure (Ambion)	Life Technologies	AM7010	The use of this reagent to inactivate RNAses is optional.
RT-PCR machine Thermal Cycler	BioRad	CFX Connect Thermal Cycler
Siliconized 9-well glass plate	Hampton Research	HR3-134
Stereomicroscope Fluorescence Adapter (Royal Blue (440-460 nm) excitation + yellow barrier	Nightsea		This adaptor enables GFP fluorescence to be examined using a typical dissecting microscope (eg Nikon)
StereoZoom Microscope 83604 Set with 10X Wide Field Eyepieces and Universal Boom Stand	Nikon	SMZ 745
Thermal Cycler	BioRad	T100
Trizol reagent	Life Technologies	15596018	CAUTION; Alternative RNA extraction methods can be used. http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf
Tween 20	Amresco	0777-1L