An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Podsumowanie

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Streszczenie

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.

Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

Wprowadzenie

In flowering plants, the establishment of reproductive lineages begins with the differentiation of SMCs, female MMC and male microspore mother cell. The MMC develops from a sub-epidermal nucellar cell at the distal tip of the ovule primordium, and the microspore mother cell develops from sporogenous tissue in the anther locule, which are located deep inside the floral organs¹. SMCs undergo meiosis to produce haploid spores, which then give rise to the gametophytes upon mitosis. The female gametophyte, or embryo sac, consists of one egg cell, one central cell, two synergids and three antipodals. The male gametophyte, or pollen, is composed of one vegetative cell and two sperm cells. While the male gametophyte remains a relatively accessible object-of-study, the female gametophyte is embedded inside the ovule, itself enclosed in the flower carpel, and thus poses specific challenges to molecular and cytological analyses. Recently, however, laser-assisted microdissection offered an elegant solution allowing transcriptomic analyses in the MMC and female gametophytic cells^2-4. In addition to candidate gene expression analyses, using e.g. RNA in situ hybridization or reporter gene assays, cytological analyses allows investigating the dynamics of endogenous cellular components using specific direct cellular staining or indirect immunostaining. Particularly, cytogenetic staining using FISH and DNA staining, together with immunostaining of chromatin modifications or chromatin components are central approaches to elucidate chromatin dynamics and nuclear organization in Arabidopsis⁵. Typically, meiosis entails specific chromosome dynamics which has been well investigated in plant male meiocytes^6,7; further large-scale, cell-specific chromatin reorganization, likely reflecting dynamic epigenetic reprogramming has been described during pollen development^8-10. By contrast, due to the relative inaccessibility of the female meiocyte and gametophyte, these investigations remain technically difficult to apply, and often require sectioning or manual dissection and enzymatic digestion (see below). In addition, the prevalent lack of optical clarity in whole-mount is an obstacle to high-resolution imaging of reproductive cells in intact ovules.

A classical method for cytological analysis of chromosome organization in whole-mount ovules uses Feulgen’s staining^11-13. It involves acid hydrolysis (using hypochlorous acid) of the DNA which results in protein denaturation and thus causes destruction of the chromatin structure. Alternatively, chromosome organization in female meiocytes and gametophytic cells can be observed using DAPI staining and immunostaining on semi-thin sections or dissected embryo sacs and MMC (for instance see^14-18). Clearly, however, manual dissection and sectioning can be labor intensive and impedes on the qualitative and quantitative analysis of a large number of chromatin epitopes.

Here we provide an efficient protocol to prepare a large number of Arabidopsis ovules suitable for a variety of downstream cytological staining in whole-mount. In brief, flower buds are incubated in a fixative solution, rows of ovules are dissected from the carpel and embedded in acrylamide on slide as done for pollen meiocytes^19,20. The embedded ovules are further cleared and fixed in methanol, ethanol, and xylene before cell wall digestion and permeabilization. Possible variations of these steps are discussed. The samples can then be used for DNA staining, immunostaining, and FISH. The preparation mode is efficient and allows for parallel experimental set-up (up to 16 slides can be prepared in a day for different downstream analysis). The treatments described enable homogeneous signals in whole-mount and well preserved histological, cellular, and nuclear organization in reproductive cells and surrounding nucellar cells which benefit qualitative and quantitative comparisons between cell types. Calibrated, CLSM-based high-resolution imaging followed by 3-dimensional reconstruction enables meaningful quantitative measurements of fluorescent signals. We successfully used this procedure to analyze chromatin dynamics in the differentiating MMC²¹ and developing female gametophyte²²; we present here representative results of heterochromatin analysis, chromatin immunostaining, GFP immunostaining and FISH in whole-mount ovules. We further believe that our protocol will be suitable for other plant tissues and species.

Protokół

The procedure is described in the workflow in Figure 1, and the setup for dissection and embedding of tissues are presented in Figure 2.

1. Tissue Fixation

1. Collect 20-30 carpels in a microfuge tube containing freshly made BVO fixative buffer on ice.
2. Fix the tissue 30 min with gentle shaking at room temperature.
3. Spin the tubes containing the carpels in fixative in a benchtop microcentrifuge 1 min at 400 x g.
4. Remove carefully the fixative buffer and add 1 ml of PBT, place the tubes on ice.

2. Dissection and Embedding

1. Prepare five Eppendorf tubes with each 200 μl of a freshly made, 5% acrylamide mix.
2. Prepare five Superfrost slides pre-cleaned with 70% ethanol and labeled with a pencil.
3. Thaw one aliquot of 20% APS and 20% NaPS each, on ice.
4. Take 4-5 carpels with a cut-end tip, place them on a clean slide, remove the excess of liquid.
5. Make longitudinal cuts with a fine needle and detach the carpel walls to release rows of ovules as shown Figure 2, avoid drying by covering with PBS (not more than 10 μl).
6. Quickly add and mix 12 μl NaPS, 12 μl APS with an aliquot of 200 μl acrylamide mix.
7. Add 30 μl of the activated acrylamide onto the dissected ovules.
8. Cover with a 20 x 20 mm coverslip, let polymerize at room temperature, 45-60 min.
9. Remove the coverslip using a razor blade. At this stage, the samples can be kept overnight at 4 °C in a Coplin jar containing PBS.

3. Tissue Processing

NOTE: All steps except 3.2.1, 3.2.3, 3.3.2 and 3.4.3 are carried out in Coplin jars with 80 ml solution under the chemical hood at room temperature. Slides are transferred with a flat-tip forceps.

1. Tissue clarification and fixation:
   1. Incubate 5 min in methanol.
   2. Incubate 5 min in ethanol.
   3. Incubate 30 min in ethanol:xylene (1:1).
   4. Incubate 5 min in ethanol.
   5. Incubate 5 min in methanol.
   6. Incubate 15 min in methanol and PBT (1:1), complemented with 2.5% formaldehyde.
   7. Rinse 2 x 10 min in PBT. At this stage, slides can be kept overnight at 4 °C.
2. Cell wall digestion:
   1. Thaw an aliquot of the cell wall digestion mix on ice.
   2. Take a slide from the Coplin jar, drain the excess of liquid by placing it vertically on a paper towel.
   3. Add 100 μl of cell wall digestion mix over the acrylamide pad and cover with a 23 x 46 mm coverslip. Repeat for the other slides. Incubate for 2 hr at 37 °C in a moist chamber (described in Materials).
   4. Wash the slides 2 x 5 min in PBT.
3. RNase A treatment:
   1. Take a slide from the Coplin jar, drain the excess of liquid as before.
   2. Incubate each slide with 100 μl of RNAseA at 100 μg/ml in PBS with 1% Tween-20 for 1 hr at 37 °C in a moist chamber.
   3. Wash the slides for 2 x 5 min in PBT.
4. Post-fixation and permeabilization:
   1. Post-fix for 20 min in freshly made PBT-F.
   2. Rinse the slides for 10 min in PBT.
   3. Permeabilize for 2 hr in PBS with 2% Tween-20 at 4 °C.
   4. Rinse the slides for 2 x 5 min in PBT.

4. Immunostaining

NOTE: For this step, the optimal concentration of the primary antibody has to be tested by using different dilutions (1:200, 1:500, 1:1000) of the antibodies.

1. Incubate each slide with 100 μl of primary antibody diluted in PBS with 0.2% Tween-20 for 12-24 hr at 4 °C.
2. Wash the slides in PBT for 2-4 hr at room temperature under gentle shaking.
3. Apply the secondary antibody 1:200 in PBS + 0.2% Tween-20 for 24 hr at 4 °C.
4. Wash slides in PBT for 1 hr at room temperature under gentle shaking.
5. Counterstain with 10 μg/ml propidium iodide in PBS for 15 min, then rinse 15 min in PBS under gentle shaking, at room temperature.
6. Mount in anti-fading liquid mountant supplemented with 10 μg/ml propidium iodide. Let the mounting medium harden for 1 hr before acquiring images by CLSM.

5. Quantitative Imaging

1. Image acquisition:
   1. Acquire high resolution images using CLSM, ideally using a resonance scanning mode, which allows better preservation of fluorescent signals over prolonged imaging²³, and a 63X glycerol immersion lens.
   2. Test the acquisition parameters such as laser intensity, gain, pinhole, voxel size and zoom factor at the beginning of the experiment to define a standard acquisition procedure to strictly follow throughout all slides for consistent quantitative measurements.
   3. Verify the absence of cross-talk between fluorochromes. If present, set up a sequential scan. Acquire transmission images separately and not simultaneously.
   4. Perform serial, three-dimensional image acquisition with highest possible resolution in the x and y dimensions and with 2x oversampling in the z dimension (Nyquist’s rule).
2. Image processing:
   1. Reconstruct serial images in three-dimensions using commercial or open source software.
   2. Define contour surfaces around each nucleus (or cell) of interest in 3D.
   3. Quantify fluorescence in each channel as the sum of pixel intensities in each object.
   4. Export the data to Excel for statistical analyses. Normalize antibody signals against e.g. DNA staining signals.

Wyniki

We provide a robust protocol for large-scale preparation and processing of Arabidopsis ovules suitable for cytological staining in whole-mount. Thanks to the embedding, the ovules retain a 3-dimensional structure (Figure 3). Furthermore, the tissue processing including optical clarification enables imaging subcellular structures at high-resolution. Figure 4 shows DNA staining in whole-mount ovule primordia where heterochromatin appears as bright, well defined conspicuous foci (n...

Dyskusje

In flowering plants, the female reproductive lineage is surrounded by several cell layers including the nucellus and the ovule teguments, thus rendering cytological staining in whole-mount technically challenging. Here we present an efficient protocol enabling the preparation and processing of a large number of ovules suitable for cytological staining such as immunostaining, DNA staining and fluorescence in situ hybridization in whole-mount. We successfully used it for the analysis of the female reproductive ger...

Materiały

Name	Company	Catalog Number	Comments
Solutions
BVO Fixation Buffer (based on32)			2 mM EGTA, pH 7.5, 1% (v/v) formaldehyde, 10% DMSO, 1x PBS, 0.1% Tween-20
PBT			1x PBS, 0.1% Tween-20
PBT-F			1x PBT, 2.5% (v/v) formaldehyde
30% acrylamide:bisacrylamide			3 g acrylamide, 0.33 g bisacrylamide, 1x PBS (prepare 10 ml, store at 4 °C)
200 ml 5% acrylamide mix in PBS			34 ml 30% acrylamide:bisacrylamide, 166 ml 1x PBS (make fresh from 30% stock)
20% ammoniumpersulfte			0.2 g ammoniumpersulfte, 1 ml sterile water (prepare aliquots with 1 ml and store at -20 °C)
20% sodium sulfite			0.2 g sodium sulfite, 1 ml sterile water (prepare aliquots with 1 ml and store at -20 °C)
Cell wall enzyme mix			0.5% (w/v) cellulase, 1% (w/v) driselase, 0.5% (w/v) pectolyase
Reagents and Materials
Formaldehyde	Sigma-Aldrich	F1635
DMSO	Sigma	D5879
Tris	Amaresco	0497
Ethanol	Schaurlau	ET00102500
Methanol	Schaurlau	ME03062500
Xylene	ROTH	4436.1
Cellulase	Sigma	1794
Driselase	Sigma	D8037
pectolyase	Sigma	P5936
Tween-20	Merck	8.22184.0500
EGTA	Sigma	E-4378
acrylamide	Sigma	A-3553
bisacrylamide	Sigma	M2022	toxic
ammoniumpersulfate	Sigma	A9164
Sodium sulfite	Fluka	71988
Anti-trimethyl-Histone H3 (Lys4)	Upstate	07-473
Anti- monomethyl-Histone H3 (Lys27)	Upstate	07-448
Alexa Fluor 488~goat ~anti ~rabbit (H+L)	Molecular Probe	A11008
ProlongGold	Invitrogen	P36934
Propidium iodide	Sigma	P4170	toxic
DAPI	Sigma	D9542	toxic
RNAse A	Roche	10109169001
Coplin jar	Huber & CO	10.055
Forceps	DUMONT BIOLOGY
Shaker	Heidolph	543-12310-00-0
Moist chamber			A plastic box with damp paper towel inside, a plastic support is put into the box for supporting the slides and keep slides from the water.
Superfrost Plus slide	Thermo Fisher	J1800AMNZ	Menzel-Gläser
FISH Tag DNA KIt	Invitrogen	F32947
GFP booster	Chromotek