Article
Reference
Combining BrdU-Labeling to Detection of Neuronal Markers to Monitor Adult Neurogenesis in Hydra
BUZGARIU, Wanda Christa, et al.
Abstract
The nervous system is produced and maintained in adult Hydra through the continuous production of nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogenesis. Upon continuous [...]
BUZGARIU, Wanda Christa, et al . Combining BrdU-Labeling to Detection of Neuronal Markers to Monitor Adult Neurogenesis in Hydra. Methods in Molecular Biology , 2020, vol. 2047, p.
3-24
DOI : 10.1007/978-1-4939-9732-9_1 PMID : 31552646
Available at:
http://archive-ouverte.unige.ch/unige:124006
Disclaimer: layout of this document may differ from the published version.
Combining BrdU-labeling to detection of neuronal markers to monitor adult neurogenesis in Hydra
Wanda Buzgariu°, Marie-Laure Curchod, Chrystelle Perruchoud and Brigitte Galliot°
Department of Genetics and Evolution, iGE3, Faculty of Sciences, University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland. ° Corresponding authors: Brigitte.Galliot@unige.ch;
Wanda.Buzgariu@unige.ch
Key words: Hydra nervous system, interstitial stem cells, neurogenesis, nematogenesis, in situ hybridization, immunofluorescence, Hydroxyurea, BrdU, prdl-a, Hym-355, RFamide
ABSTRACT
The nervous system is maintained in adult Hydra through the continuous production of nerve cells and mechano-sensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as RFamide or Hym-355. This method can be performed on whole- mount animals as well as on macerated tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling can be combined to the analysis of gene expression by whole mount in situ hybridization. This co-immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogenesis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic or developmental conditions.
1. INTRODUCTION
1.1. Anatomy of the nervous system, neurogenesis and nematogenesis in Hydra
The freshwater hydrozoan Hydra belongs to Cnidaria, an early-branched eumetazoan phylum, sister group to bilaterians. Hydra is an attractive model for biologists not only for its outstanding regenerative properties but also for its highly dynamic neurogenesis. Indeed the nervous system is continuously renewed with nerve precursors produced in the body column, then displaced towards the extremities where they terminally differentiate to replace the nerve cells that die [1] (Fig.1A). In addition, animals bisected at any level along the body column replace the missing part in few days, e.g. a new head equipped with a complete nervous system (Fig.1B). The Hydra nervous system is made of nerve cells and mechano- sensory cells (named nematocytes or cnidocytes) that form a nerve net much denser at the apical and basal extremities of the animal. There it controls a series of more or less complex behaviors such as peristaltic movements, walking, prey capture, food ingestion, reaction to light.
Figure 1. Adult neurogenesis in Hydra and cell types that form its nervous system
(A, B) Neurogenesis in adult intact (A) and in head-regenerating (B) Hydra polyp (modified from ref.[42]). Only nerve cells are shown, denser in the apical (top) and basal (bottom) regions than in the body column where neurogenesis takes place. The interstitial stem cells (ISCs) provide precursors for all nerve cells and nematocytes, which migrate towards the extremities [48]. Terminal differentiation of nerve cells predominantly takes place at the extremities. In head-regenerating Hydra (B) interstitial cells and derivatives are first eliminated upon injury- induced cell death after mid-gastric bisection [33] and de novo neurogenesis takes place at the tip where the apical nervous system get built within two days, with precursors detected after 24 hours [14,20]. (C) ISCs divide every 24-30 hours and frequently appear as pairs (top-left panel). Nematogenesis starts with a series of synchronous syncytial divisions that lead to the formation of nematoblast clusters that contain 4x, 8x, 16x or even 32x cells. At any stage, a cluster can stop proliferating to enter differentiation, i.e. each nematoblast forms a venom capsule named nematocyst (arrows) and a cnidocil (not visible), both fully mature in nematocytes [22,23]. Note the moon-shape nucleus in nematocytes, compressed by the nematocyst. Hydra differentiate different classes of neurons: sensory, sensory-motor bipolar, and multipolar ganglia neurons (right panels). Cells obtained after tissue maceration [16], were immunodetected with an anti a-tubulin antibody followed by an anti-mouse secondary antibody coupled with Alexa-488 (green). The nuclei were counterstained with DAPI (light blue). Pictures were taken on a SP8 Leica confocal microscope and optimized on Photoshop with channelmixer to lighten the dark blue color and M-curves to increase the contrast. Scale bar: 10 µm.
Cnidarian neurons are equipped with neurites but no axons, therefore not polarized and often named “nerve cells”. Every nerve cell is able to function as sensory, sensory-motor or interneuron. Nevertheless, nerve cells show diverse anatomies, sensory, bipolar, or multipolar as ganglia neurons (Fig.1C), and the neuronal phenotype of a given cell can change according to its position along the body axis [2]. At the base of the apex, ganglia neurons can form a nerve ring that allows coordinated behaviors [3]. Although cnidarian nervous systems are highly peptidergic with peptide-gated ion channels as receptors [4,5], they share with bilaterians all the basic properties of synaptic conduction and chemical neurotransmission [6,7], even though the neuromuscular junction might have evolved independently in cnidarians and bilaterians [8]. Still, the transcriptional factors that control neurogenesis are largely evolutionarily-conserved [9–16] and cnidarian nervous systems might represent the first evolutionary attempt of centralized nervous system in eumetazoans [3].
Figure 2. Molecular markers of neurogenesis and nematogenesis in Hydra
A partial list of genes expressed during neurogenesis (upper) and/or nematogenesis (lower). Gene products detected with antibodies are written plain. ISC: interstitial stem cells; pc: precursor cell; nb: nematoblasts. The spatial and cell-type expression pattern of genes presumably involved in neurogenesis and neurotransmission (103 and 156 respectively) was identified by RNAseq transcriptomics [44], and for any Hydra gene of interest the spatial and cell-type expression patterns are now available on the HydrAtlas server (https://hydratlas.unige.ch/blast/blast_link.cgi) [32]. Specific signatures were obtained by analyzing the expression patterns of (i) neuropeptides such as RGamide, RFamide, KVamide or LWamide [45–47], the neuropeptide Hym-355 enhancing neuronal differentiation [48], (ii) 86 genes identified in a microarray screened with cRNAs from nerve-less animals [49], (iii) neurogenic genes that regulate the proliferation and/or the differentiation of progenitors such as CnASH [9,50], prdl-a [29], COUP-TF, prdl-b [52], hyZic [51], cnox-2 [44], CREB [52], Myc1 [53,54], FoxN1, PaxA, PaxB, Pou4F2 [44], (iv) the two Piwi proteins (HyWi, HyLi) strongly expressed in ISCs and nematoblasts [55], (v) some gap junction proteins, innexin-2 being involved in neurotransmission [56]. No pan-neuronal marker was identified in Hydra yet. As early markers of nematocyst differentiation, the minicollagens N-COL1 and NOWA [57,58] are components of the wall, spinalin of the spines [59], N-COL15 of the tubule [60], nematogalectins A and B of the tubule from distinct types of nematocysts [61].
Finally, a proteomic analysis of the venom identified 410 secreted proteins in nematocysts [62] (not shown here).
The two myoepithelial layers of the body wall, the epidermis and the gastrodermis, are made of cells that derive from three not interchangeable stem cell populations: the two unipotent
ectodermal and endodermal epithelial stem cells (ESCs) and the multipotent interstitial stem cells (ISCs). ISCs, predominantly located in the central body column, give rise to a variety of cell types, including the two cell lineages that build up the nervous system (Fig.1A, Fig2): the rare nerve cells and the abundant nematocytes, which represent 3% and 35% of the total cell number respectively [17,18]. The nerve cells directly differentiate from migrating precursors and then get displaced towards the extremities where they form a dense diffuse nerve net [18–21]. In intact animals (homeostasis), neurogenesis is a slow process, leading to the replacement of the nerve cells that get sloughed off at the extremities, while after bisection a faster de novo neurogenesis process takes place in the regenerating structure (Fig.3A).
In contrast to neurogenesis, nematogenesis is a multiple step process where interstitial progenitors synchronously divide up to five times, providing clusters that contain 4, 8, 16 or 32 nematoblasts (Fig.1B, Fig.2) [22]. Cells from a given cluster can exit the cell cycle at any time from the 4-cell stage to differentiate as nematocytes equipped with a sensory cnidocil and a vacuole named cnidocyst that contains a paralyzing venom, the latter structure being the hallmark of cnidarians [23]. The turnover of nematocytes is highly dynamic as once the venom capsule is discharged, the nematocyte is eliminated and replaced. Interestingly, ISCs cycle three to four times faster than ESCs and can thus be easily eliminated upon pulse treatments of antimitotic drugs [24–26]. Elimination of the cycling interstitial cells leads to the loss of the nervous system, after several weeks in intact animals, within few days in regenerating ones, as such animals regenerate a missing part that lacks a nervous system (Fig.3B, Fig.4). Nerve-free animals actually maintain their developmental properties if they are force-fed, pointing to an important morphogenetic role played by the epithelial cells [25].
The Hydra Peptide Project identified about 500 peptides, half neuropeptides, half epithelio- peptides, with specific roles in neurotransmission, morphogenesis and differentiation [27,28].
However not much is known concerning the role and the regulation of the neuronal-epithelial cross-talk on the slow and fast modes of neurogenesis in intact and regenerating animals respectively. These questions require the monitoring of the expression of neuronal markers in proliferating and differentiating progenitors as well as in fully mature nerve cells.
1.2. Principles of immunodetection on whole mount and macerated tissues in Hydra
The Hydra nervous system is well visualized with the immunostaining procedures applied either on whole animals or on cells that keep their morphology after tissue maceration [17].
In both contexts, the procedure is based on the specific antigen-antibody recognition followed by different detection techniques to visualize the cells that express or over-express a known antigen. The immunodetection procedure follows the classical steps: fixation and permeabilization of the tissue, saturation of unspecific sites, antigen recognition by a specific antibody followed by detection and visualization. The expression patterns obtained in intact or regenerating Hydra as well as in specific cell types can also be verified at the transcriptomic level on the HydrAtlas server (https://hydratlas.unige.ch) [29].
Immunodetection on whole mounts: The whole-mount immunodetection procedure is a robust method to detect with specific antibodies the spatial or temporal expression pattern of neurogenic proteins or neuropeptides. In complement the same procedure can be used to detect with antibodies raised against tagged material incorporated into macromolecules such as Bromodeoxyuridine (BrdU) incorporated into newly synthesized DNA [30] or digoxygenin (DIG)-labelled riboprobes that bind transcripts [31]. As a consequence, immunodetection allows the visualization of proteins, DNA, transcripts in tissues that show a preserved architecture.
Figure 3. The homeoprotein prdl-a as a marker of apical neurogenesis
(A) Double immunostaining of BrdU (green) and prdl-a (red) expressed in apical neuronal progenitors or nerve cells of homeostatic animals (upper panel) or animals having regenerated their head (lower panel). All animals were incubated with BrdU for four hours and then washed out to remain intact or to undergo mid-gastric bisection (red arrow). White arrow heads indicate the prdl-a+/BrdU+ cells, which are more numerous in the regenerated than in the homeostatic head. (B) De novo neurogenesis evidenced by prdl-a (red) immunostaining in the presence (top panel) or the absence (lower panel) of interstitial progenitors after hydroxyurea (HU) treatment. The animals were exposed or not to 10 mM HU in three rounds (2x 24 hours and 1x 32 hours). After the third HU treatment, animals were transferred to HM, bisected (red arrow) and let to regenerate for seven days. Note the absence of prdl-a+ cells in HU-treated animals. Nuclei were counterstained with DAPI (blue).
Image acquisition was done on a Leica LSM700 confocal microscope. Scale bar: 100 µm.
Immunodetection on cells obtained after tissue maceration: To get a more accurate analysis at the cellular level, the immunodetection procedure can be performed on histological sections [10,32] or on cells directly fixed on slides after tissue maceration [17]: In the presence of acetic acid and glycerol, the tissues are dissociated into single cells or small clusters, which
are subsequently fixed with paraformaldehyde (pFA) that preserves their typical cellular architecture. Hydra-specific or exogenous epitopes as BrdU can next be immunodetected. The maceration technique can be adapted for small amount of tissue such as head- or foot- regenerating tips [33]. The strengths of this method are multiple: it identifies cell types and specific subcellular localization, it allows the precise quantification of each expressing cell type, including neurons, interstitial cells or clusters of nematoblasts (Fig.1B).
Antibodies: As mentioned above, the Hydra nervous system produces numerous neuropeptides. Since 1982, their immunodetection on intact animals proved to be extremely useful to identify the anatomical organization and localization of specific subsets of neurons in Hydra [34,35]. The vasopressin-like or RFamide sera were critical to monitor the phenotypic conversion of neurons as they get displaced along the body axis [2]. With the development of bio-informatic tools and genomics, evolutionarily-conserved regions can be easily mapped and commercially available antibodies raised against such mammalian epitopes can be used to cross-react against Hydra proteins. Alternatively monoclonal [36] or polyclonal antibodies can be raised against Hydra proteins, either lab-made such as the sera against the RFamide neuropeptide [34], the transcription factor CREB [37], the homeoprotein prdl-a [10], or commercially produced. The most efficient way to validate the specificity of a given antibody is to perform gene silencing, usually through RNA interference in Hydra, to assess the decrease in protein abundancy by Western analysis or immunofluorescence [38].
Figure 4. Loss of terminal neuronal differentiation in Hydra undergoing apical or basal regeneration in the absence of ISCs or interstitial progenitors.
HU treatment and regeneration were performed as in Figure 3. The apical (A) and basal (B) nerve nets were identified after RFamide immunostaining (green) and nuclear DAPI counterstaining (purple). Note the sparse RFamide+ nerve cells in apical (A) and basal (B) regions regenerated after HU treatment. Image acquisition was done on a Leica LSM700 confocal microscope. Scale bars: 100 µm.
Tissue fixation and tissue permeabilization are crucial steps as they allow the preservation of cells and tissue architecture. Paraformaldehyde (pFA) or formaldehyde (FA) are two cross- linking agents commonly used as fixatives; they ensure a good penetrability and preserve the antigens by forming chemical bonds between the proteins. In contrast, alcohols such as ethanol and methanol, or acetic acid are precipitating fixatives that disrupt the hydrophobic bonds and thus denature the tertiary structure of proteins. In fact, ethanol or methanol allow a good permeabilization by precipitating membrane proteins, leading to the formation of pores in the membrane, thus contributing to the leakage of RNA or cytoplasmic proteins and to a poor cellular preservation. Therefore, the commonly used method to fix Hydra polyps for
whole-mount immunodetection or in situ hybridization (ISH) combines pFA and alcohol fixatives. As an alternative the Lavdowsky fixative that combines ethanol, FA, acetic acid and water (50/3.7/4/42), also takes advantage of the properties of cross-linking and precipitating agents [36]. A variant of the Lavdowsky fixative that does not contain acetic acid is often used to detect nuclear antigens. Finally, the mercury-containing “Helly” fixative or the Zinc–FA fixative used on insect brains to preserve morphology and improves immunodetection at synapses [39], can be used efficiently when the other fixatives fail. For each new epitope/antibody several fixatives need to be tested together with the fixing conditions such as temperature (4°C, 18°C, 37°C) or duration of fixation. Tissue permeabilization is usually completed by adding a detergent such as Triton X-100 or Tween 20, again applied for a period of time that needs to be adapted to each antigen of interest.
Antigen detection can be made directly with the primary antibody or indirectly through a secondary antibody that binds to the primary one. The indirect method is preferred as Hydra specific antibodies conjugated with fluorochrome are rare and immunodetection is more sensitive when indirect as the signal is amplified. A large choice of secondary antibodies coupled to fluorochromes or enzymes are available. Amplification is optimal when a Tyramide detection system is used with conditions adapted for each antibody (see supplemental Figure 1 in [13]). Unspecific binding of antibodies to reactive sites of proteins is blocked by BSA (bovine serum albumin) or normal serum from the species used to raise the secondary antibody. A common counterstaining is made by nuclear staining, which allows a better cell- type and tissue-layer identification through the shape and size of nuclei.
Figure 5. BrdU immunostaining combined with Hym-355 detection in homeostatic animals.
The neuropeptide Hym-355 is expressed in a subset of apical and basal neurons. The animals were exposed to BrdU for 4 hours, washed and subsequently maintained in HM for two days and then fixed. After the in situ hybridization procedure to detect Hym-355 expressing nerve cells (A, purple), the animals were immunolabeled for BrdU (B-C, green). In homeostatic condition, only few apical neurons do express Hym-355 and are positive for BrdU (arrows). The bright field (A) and fluorescence (B) images were acquired with a Leica DM5500 fluorescence microscope. Box in B corresponds to panel C. Scale bars: 100 µm (A, B) and 20 µm (C).
2. Materials
All stock solutions are prepared with MilliQ water in sterilized bottles with screw-cup, then autoclaved or filtered through a 0.22 µm Steritop filter, and finally stored at 4°C or at room temperature (RT) depending on the buffers.
After opening, the stock solution should be checked regularly to be discarded before they become viscous or turbid, with visible signs of contamination. Experiments are always performed with fresh solutions, i.e. 10x stock solutions freshly diluted in sterile dishware with MilliQ water to prepare the requested amount of 1x solution for a single experiment.
2.1. Solutions for Hydra maceration
1. Hydra medium (HM): 1 mM NaCl, 1 mM CaCl2, 0.1 mM KCl, 0.1 mM MgSO4, 1 mM Tris pH 7.6 [40]. HM is prepared from three stock solutions that can be stored several weeks at RT once autoclaved:
For the stock solution A (0.5 M Tris, 500x) dissolve 60.57 g Tris-base in 900 ml H2O, adjust the pH to 7.7 and complete the volume to 1000 ml. For the stock solution B (1 M MgSO4, 10’000x) dissolve 61.6 g MgSO4×7H2O in 250 ml H2O.
For the stock solution C (0.5 M CaCl2, 0.5 M NaCl, 0.05 M KCl, 500x) dissolve 54.77 g CaCl2×6H2O, 14.6 g NaCl, 1.85 g KCl in 500 ml H2O. Autoclave the solutions A, B and C. To prepare 1 liter HM solution, dilute 2 ml solution A, 100 µl solution B and 2 ml solution C in 1000 ml H2O.
2. Macerating solution (MS): 7% acetic acid, 7% glycerol in H2O. Add 0.7 ml glycerol and 0.7 ml glacial acetic acid to 8.6 ml H2O. Use the fume hood for preparation. Do not autoclave. Store at RT no longer than two weeks.
3. 10% Tween 80: Add 1 ml Tween 80 to 9 ml H2O (see Note 1). Do not autoclave.
Keep the solution for one month at RT.
2.2. Solutions for fixation
1. Urethane 4%: Dissolve 1 g urethane in 25 ml HM. Store at 4°C. Always wear gloves as urethane is carcinogenic.
2. Lavdowsky fixative: 50% ethanol, 3.7% FA, 4% acetic acid. Add 1 ml 37% FA to 5 ml ethanol, 0.4 ml acetic acid and 4 ml H2O. Prepare it fresh, do not store.
1. Lavdowsky fixative without acetic acid: 50% ethanol, 3.7% FA. Add 1 ml 37%
FA to 5 ml ethanol and 4 ml H2O. Prepare it fresh and do not store.
2. Paraformaldehyde (PFA) 8%: Dissolve 8 g pFA in 80 ml HM pre-heated at 65- 70°C in a glass bottle and stir the mixture on a waterbath maintained at 70°C under the hood. Add 100 µl 1N NAOH and stir the solution until the solution becomes clear. After cooling to RT, adjust the volume to 100 ml with HM and adjust the pH to 7.5 with 0.1N NaOH. Filter the solution using a steriflip filter.
Store at 4°C for two weeks maximum (see Note 2).
3. PFA 4%: Dilute 50 ml 8% pFA with 50 ml H2O and readjust the pH to 7.5 with 0.1N NaOH.
4. Formaldehyde (FA) 3.7%: Add 1 ml 37% FA to 9 ml H2O.
2.3. Solutions for BrdU immunodetection
BrdU (5-Bromo-2’-deoxyuridine) is an analog of thymidine that is rapidly incorporated into DNA during the replication phase of the cell cycle.
1. BrdU solution (10 mM): Add 154 mg BrdU to 50 ml HM in a 50 ml centrifugation tube, protect the tube from light and stir the solution that can be stored at 4°C for a few days (see Note 3).
2. 10x PBS buffer: 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4, 11 mM KH2PO4. Dissolve 80 g NaCl, 2 g KCl, 29 g Na2HPO4 12H2O and 2 g KH2PO4 in 1000 ml H2O. Verify that pH is 6.8. Autoclave and keep at RT (see Note 4).
3. PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.1 mM KH2PO4. Dilute 50 ml 10x PBS to 500 ml (see Note 4).
4. PBST: 0.5% (v/v) Triton X-100 in PBS. Add 0.5 ml Triton X-100 to 100 ml PBS (see Note 1).
5. Blocking solution: 2% BSA. Dissolve 1 g BSA in 50 ml PBS and stir until the solution becomes clear. Filter through a Steriflip and store at 4°C (see Note 5).
6. 2N HCl: Add 2 ml 37% HCl to 10 ml of H2O (see Note 6).
7. DAPI (4,6-diamindino-2-phenylindoline) stock solution (1 mg/ml): Dissolve 10 mg DAPI in 10 ml H2O. Aliquot and store at -20°C.
8. DAPI working solution (1 µg/ml): Dilute 1µl DAPI 1mg/ml into 1ml PBS.
2.4. Antibodies
1. The polyclonal anti-prdla antibody was produced in rabbit after immunization with the N-terminal fragment of prdl-a fused with 6HIS [29].
The polyclonal anti-RFamide antibody was produced in rabbits and kindly provided by Cok Grimmelikhuijzen [34]. For BrdU immunodetection the BrdU labeling and detection kit from Roche (ref 11444611001) provides the most reliable results. Dilute the antibody 1:20 in the labeling solution found in the kit (see Note 7).
2. The optimal titer for each primary antibody should be established after testing a series of dilutions; too high concentrations increase the background staining as the antibody accumulate at the cell or tissue surface, too low concentrations provide low-intensity signals due to a reduced antigen- antibody interaction.
3. In the indirect immunostaining procedure, the secondary antibody needs to be chosen according to the available filters and lasers that equip the fluorescent/confocal microscopes. If a double antigen detection is required, select the secondary antibodies considering the possible crosstalk between the excitation and emission wavelengths of each fluorochrome.
2.5. Mounting medium:
The samples should be mounted in a medium adapted for fluorescence, i.e.
characterized by a high refractory index, no autofluorescence and protecting against photobleaching. The MOWIOL mounting medium (polyvinyl alcohol) fulfills these criteria. It is a widely-used lab-made preparation, less expensive than the commercially available ones.
1. Tris 0.2 M pH 8.5: Dissolve 2.42 g Tris base in 100 ml of H2O. Adjust in a fume hood the pH to 8.5 with approximately 710 µl of 37% HCl solution.
2. Add 24 g MOWIOL 4-88 to 60 g glycerol and stir well. Subsequently add 60 g H2O and mix well for another two hours at RT.
3. Add 100 ml 0.2 M Tris pH 8.5 and continuously stir at 52°C on a heating plate until the powder is completely dissolved. This might take 4-5 hours.
4. Dispatch the mix in 50 ml centrifugation tubes and centrifuge at 5000 g for 15 min.
5. Carefully collect the supernatant and aliquot into 15 ml conical tubes.
6. Store the Mowiol mounting medium at -20°C up to one year. For current use, keep aliquots at 4°C.
2.6. Equipment:
1. Stereomicroscope.
2. Pasteur glass pipette.
3. 360° vertical rotator.
4. Shaker.
5. Superfrost Plus microscope slides (Thermo Scientific, Gerhard Menzel).
6. Coverslips: 22 x 40 mm, 0.13-0.17 thickness.
7. Surgical scalpel N°3.
8. Surgical blades N°10 (Ruttgers Solingen).
9. Plastic Petri dishes (6 and 9 cm diameter).
10. Silicon bulbs, 5 mm diameter for Pasteur pipette.
11. Steriflip and steritope for filtration (Millipore).
12. Hydrophobic pen (DAKO Pen).
13. Slide staining tray, Simport Scientific.
3. Methods
3.1. Hydra tissue maceration protocol and fixation
1. Collect five intact animals in a 1.5 ml Eppendorf tube with the help of a Pasteur pipette. Remove the medium and replacing it with 400 µl of fresh HM, repeat the washing 2-3x times (see Note 8).
2. After the last wash, eliminate carefully all the liquid and add 100 µl MS. Let the tube on a rack and mix gently, from time to time, until the tissue dissociates into a homogenous cell suspension (see Note 9).
3. After 30-60 minutes, fix the cell suspension by adding 100 µl 8% pFA, mix gently and let incubate for one hour at RT.
4. Add 10 µl Tween 80 10% and mix gently.
5. With the DAKO PEN, label a square of about 25 x 20 mm on a Superfrost Plus slide. Let it dry for about 10 minutes.
6. Add the cell suspension drop by drop on the surface delimitated by the hydrophobic marker lines.
7. Let the slides dry for about two days at RT.
8. The slides are ready to be processed or can be stored into a box at -20°C.
3.2. Hydra fixation for ISH and whole-mount immunostaining
1. Collect 15-20 intact animals with a glass Pasteur pipette in a graded 2 ml Eppendorf tube and adjust the final volume to 1 ml HM.
2. Add 1 ml 10 mM BrdU solution and transfer the animals in a plastic Petri wish (6 cm diameter) that contains 10 ml 5 mM BrdU solution. Protect from light and incubate for the desired period of time (see Note 3).
3. To wash out BrdU, collect the animals in 2 ml tube and wash them several times in HM by gently aspirating the liquid and replacing it with fresh medium.
4. To initiate regeneration, transfer the animals in a plastic Petri dish (9 cm diameter) in 50 ml HM and place the dish under the stereomicroscope. Let the animals relax for a few minutes.
5. Bisect the animals with a scalpel at half of the body column length and transfer the head regenerating halves to a new Petri dish filled with 50 ml HM.
Let the animals to regenerate (see Note 10).
6. Collect 15-20 intact or regenerated animals in a graded 2ml Eppendorf tube and wash them several times with HM as described at step 3.
7. Adjust the final volume to 1 ml. Let the animals to relax a few minutes.
8. Immediately before fixation, add 1 ml 4% urethane to reach a 2% final concentration. Mix gently and wait for 60 seconds until the animals are completely relaxed (see Note 11).
9. Remove 1 ml urethane solution and immediately add 1 ml 8% pFA to reach a 4% final concentration. Mix well and let the animals fall to the bottom of the tube and aspirate the maximum amount of the liquid.
10. Wash 3-4 times with 4% PFA by eliminating each time the liquid and replacing it with fresh fixative.
11. Place the tubes on a shaker/rotator and fix the animals for four hours at RT (see Note 12).
12. Aspirate the PFA and replace it with ethanol 100%; replace several times the ethanol until the brown pigment is solubilized.
13. Store the fixed samples at -20°C in ethanol until the ISH procedure.
3.3. Hydra fixation for BrdU immunostaining on whole-mount
1. For BrdU incubation and animal fixation, proceed as in 3.2. from steps 1 to 8.
2. Remove the maximum amount of the urethane solution and replace it with Lavdowsky fixative without acetic acid. Wash several times with the fixative.
3. Place the tubes on a shaker or rotator and let the animals fix either for one hour at 37°C or overnight at 4°C (see Note 12). Proceed immediately after fixation with the immunodetection.
3.4. Immunodetection of Hydra cells after tissue maceration
The immunodetection procedure is performed in a dark humidity chamber (see for example the StainTray slide staining system proposed by Sigma).
During antibody incubation, the atmosphere is kept humid by adding PBS in the bottom of the chamber.
1. Rehydrate the cells by washing 3x 10 min with PBS (see Note 13).
2. Incubate the cells with 0.1% Triton X-100 in PBS for 30 minutes (see Note 14).
3. Add the blocking solution 2% BSA in PBS for one hour at RT (see Note 15).
4. Dilute the primary antibody as desired in 100-150 µl blocking solution 5. Gently remove the blocking solution and add the primary antibody solution.
Incubate overnight at 4°C or alternatively 3-4 hours at RT (see Note 16).
6. Wash the slides 4x 10 minutes with PBS.
7. Dilute the secondary antibody in PBS as recommended by the supplier 8. Remove PBS, add the secondary antibody solution and incubate for 2-4 hours
protected from light.
9. Wash the slides 4x 10 minutes with PBS.
10. Incubate with 1 µg/ml DAPI for 10 minutes (see Note 17).
11. Wash the slides 2x 5 minutes with PBS.
12. Wash fast the slides with H2O and let them dry at RT from 15-20 minutes protected from light.
13. Once dried, add one drop of mounting medium and apply the coverslip by holding it at 45°C. Gently descend the coverslip allowing the mounting medium to cover the surface delimitated by the square. Avoid producing air bubbles (see Note 18).
3.5. Immunostaining on whole mount Hydra
The procedure presented below is a general protocol, which has to be adapted for each antibody. Unless specified, the incubation steps are performed at RT in 1.5ml Eppendorf tubes. The protocol should be adjusted according to the fixative. If samples are freshly fixed with Lavdowsky fixative,
with or without acetic acid, start with step 1. If samples are stored in ethanol after pFA fixation, directly proceed to step 2.
1. Remove the Lavdowsky fixative and replace it with PBS. Repeat quickly the washing step twice. Proceed to step 3.
2. Rehydrate the pFA-fixed animals stored in ethanol by washing successively in 75%, 50% and 25% ethanol, each step for 5-10 min (see Note 8).
3. Wash 4x 10 min with PBS.
4. Remove PBS, add PBST to permeabilize for 30 minutes (see Note 14).
5. Optional for nuclear antigen detection. Otherwise proceed to step 7. Remove PBST and incubate in 2N HCl for 30 min (see Note 19).
6. Remove HCl and wash quickly 6x in PBS to eliminate all HCl traces. Complete with 2x 5 min washes in PBS (see Note 20).
7. Add the blocking solution 2% BSA in PBS for 60 minutes at RT (see Note 15).
8. Dilute the primary antibody as desired in 100 µl blocking solution
9. Remove the blocking solution, add at least 100 µl primary antibody solution.
Incubate overnight at 4°C or alternatively 3-4 hours at RT (see Note 21).
10. Remove the primary antibody, wash quickly twice in PBS, then 4x 10 min.
11. Dilute the secondary antibody in PBS as recommended by the supplier 12. Remove PBS, add the secondary antibody solution and incubate for 2-4 hours
protected from light (see Note 22).
13. Remove the secondary antibody, wash quickly twice in PBS, then 4x 10 min.
14. Remove PBS and add 300 µl 1µg/ml DAPI for 10 minutes (see Note 23).
15. Wash quickly twice with PBS, then 2x 5 min.
16. Wash quickly with H2O.
17. Mount the animals on glass slide with the mounting medium. Gently descend the coverslip allowing the mounting medium to cover the surface. Avoid producing air bubbles (see Note 24).
3.6. Immunostaining on whole mount Hydra after in situ hybridization
The immunodetection protocol described above can be applied to samples previously processed for in situ hybridization (ISH) to combine the detection of both protein and gene expression. Since the 1990s, gene expression patterns can be investigated in whole-mount Hydra. The classical procedure comprises fixation of the samples, hybridization of Digoxygenin (DIG) or Fluorescein labeled riboprobes to the targeted transcript, and immunodetection of these hybridized riboprobes with an anti-DIG or an anti- Fluorescein antibody coupled to alkaline phosphatase, followed by a colorimetric detection of the coupled enzyme with NBT/BCIP substrate (for a detailed protocol, see [31]). The presentation of the complete ISH procedure is omitted here and only the steps that link the two methods are detailed.
1. At the end of the ISH procedure, stain the samples with NBT/BCIP as in [31]
and follow carefully the development of the staining. Once the pattern is obtained, wash out as indicated the NBT/BCIP substrate to block the staining.
2. Post-fix the samples in 3.7% FA for 30 minutes at RT.
3. Wash out FA by rinsing two to three times with methanol.
4. Incubate the samples for 10 minutes in methanol.
5. Rinse three-four times with PBST and proceed with step 4 from section 3.5.
Figure 6. Pitfalls of the BrdU labeling procedure on whole-mount Hydra
Detection of BrdU-labeled cells (green) on whole mount animals requires a strong DNA denaturation obtained with HCl treatment (A). It can fail after a too partial DNA renaturation followed by an extensive washing step (B), or after an insufficient permeabilization of the tissues, which leads to the lack of penetration of the anti- BrdU antibody (C). Images were acquired with a Leica DM5500 fluorescence microscope. Scale bars: 50 µm.
4. Notes
1. Tween 80 and Triton X-100 are viscous detergents and therefore the pipetting should be done gently in order to take up the requested volume. The solution should be stirred at low speed to ensure a proper solubilization and to circumvent foam and bubbles formation.
2. Wear gloves and mask when preparing the PFA solution as pFA is a hazardous chemical. All manipulations should be performed in a fume hood. PFA wastes should be discarded in accordance with the safety procedure established at your institution. Given the low osmolarity of Hydra cells the pFA is diluted in HM and not in PBS as for mammalian tissues [41]. As HM has a lower buffering capacity than PBS, a critical step is pH adjustment, which should be done precisely.
3. BrdU (5-bromo-2’-deoxyurdine) is light sensitive and mutagenic; chemical and solutions should be manipulated with gloves. During incubation of live animals in the BrdU solution, protect the dishes from light by covering them with aluminum foil.
4. The pH of the 10x PBS stock solution is approximately 6.8; it does not need to be adjusted if the components were correctly weighted. After dilution to 1x, the pH will increase to 7.4.
5. When preparing the BSA solution, add first the powder into a 50 ml centrifugation tube, then carefully pour the buffer. Stir at low speed to avoid foam and formation of bubbles. After filtration, the BSA solution can be stored at 4°C for several months.
6. 2N HCl should always be diluted freshly from a 12N HCl stock solution that is stable at RT. Do not use old 2N HCl solution as it is unstable, thus less efficient to denature DNA in nuclei.
7. The most effective anti-BrdU antibody for Hydra contains nucleases that cleave DNA and generate single-stranded DNA, thus enhancing the exposure of the sites that have incorporated BrdU. Always use the buffer supplied by the manufacturer when diluting the anti-BrdU antibody. If multiple antigens are detected, dilute the second primary antibody in the buffer supplied for BrdU. To avoid unspecific binding caused by the second primary antibody, pre-incubate it with fixed Hydra polyps for few hours at 4°C.
8. To avoid animal loss during manipulations: (1) always wait few seconds after each washing step to let the animals settle at the bottom of the tube, then aspirate carefully the washing solution and replace it with the next solution; (2) cover part of the bench with a black sticky back plastic
9. During the maceration procedure, dissociate the tissues by gently pipetting up and down every 10 minutes and by flicking the tube. A too strong pipetting might break the large epithelial cells as well as the dendrites that are fragile, or disrupt the large nematoblast clusters. In contrast, interstitial cells, nematoblasts and nematocytes are resistant to mechanical pressure.
10. To correctly estimate the amputation level when bisecting Hydra for regeneration, the animals should be relaxed to reach their maximal length. This is easily obtained by placing them in a Petri dish under the stereomicroscope and letting them stretching out for a few minutes under light. Orientate the scalpel perpendicular to the body axis and cut fast. If the animals are floating in the dish, transfer them with a low amount of liquid in the cover lid of the Petri dish and cut them there under the light. After bisection, transfer back the halves into dishes prefilled with HM, usually 1 ml/animal as when animal density is too high, regeneration is delayed [32].
11. Urethane added for one minute is used to relax the myoepithelial layers [42]. When animals are not sufficiently relaxed at the time the fixative is added, permeabilization is reduced, antibodies accumulate at the animal surface and no specific signal is obtained. However, a too long exposure to urethane damages the cell membranes, therefore this step should be limited to 2 minutes. Always wear gloves, urethane is highly carcinogenic.
12. Alternatively, the samples can be fixed overnight at 4°C. The fixation time should not exceed 16 hours for PFA, 24 hours for modified Lavdowsky (no acetic acid).
13. To wash the cells attached to the slide, add the liquid by gently pipetting within the surface delimitated by the hydrophobic square. Reverse the slide to get the liquid and replace it with fresh PBS. Wipe the excess of liquid found outside of the square limits with a tissue paper and avoid touching the hydrophobic rim.
14. Triton X-100 concentration is indicative, as the optimal concentration depends on the antigen localization in the cell. In case of a poor permeabilization or reduced antigen accessibility, a concentration range varying between 0.1% - 2% might be tested. Alternatively, the incubation time with the detergent can be increased. Tissue permeabilization also depends on the thickness of the mucus and cuticle layer that protect the animals. Finally, Triton concentration should be adapted to the Hydra strain, as the cuticle layer varies between different strains/species.
15. BSA is a widely used blocking agent to prevent the potential unspecific binding of antibodies. As an alternative, the serum from the species where the secondary antibody was raised, would be preferred.
However normal serum from any other sources can be used as it contains albumin and other proteins that bind non-specifically to the reactive sites of proteins.
16. When the signal to noise ratio is inappropriate meaning a high background is noticed, the detection might be improved by pre-adsorbing the primary antibody on Hydra tissue (see note 7) or by varying the environmental conditions such as lowering the temperature or shortening the antibody incubation time. The primary antibody dilution can be reused several times over several weeks, if stored at 4°C.
17. DAPI is a widely used fluorescent DNA dye that is excited by UV and has a broad emission spectrum.
Alternatively, other DNA stains such as Hoechst 33258 (bis-benzimidazole), TOPRO 3 can be selected.
The option has to be taken according to the availability of the filters/lasers, and to the selected fluorophores used for the antigen detection.
18. Once dried, the slides can be stored at -20°C for long, although the fluorescence preservation depends on the nature of the selected fluorochromes. Alexa dyes are very stable and samples can be stored for years at -20°C with fluorescence preservation.
19. When antigens are nuclear, i.e. transcription factors, DNA-tagged molecules, a DNA denaturation step is required to expose the reactive sites to the antibody. The most common DNA denaturation agent is HCl (2N), usually applied 30 minutes at RT. However, depending on the environmental conditions (season, temperature), the denaturation process can be faster (20 min) or slower (40 min). The duration of the denaturation step is critical, as a too long treatment destroys and fragments the DNA, while a too short exposure is not sufficient to reveal the antibody binding sites. Examples of insufficient denaturation and permeabilization are given in Fig.6.
20. HCl should be removed fast and the washing step should not take more than 15 minutes, as longer washing time might allow renaturation of the DNA and thus reduce the accessibility of antibody to the incorporated BrdU (Fig.6).
21. When double immunostaining is done, the primary antibodies can be incubated simultaneously if the species in which the antibodies were raised are not closely related and do not cross-react. For instance, a combo of rat and mouse primary antibodies should be avoided, as both the anti-mouse and the anti- rat secondary antibodies recognize the antibodies from the other species.
22. When selecting the secondary antibodies, consider those that recognize specifically the species in which the primary antibody was raised. Moreover, as a large choice of antibodies coupled with different fluorochromes are commercially available, select the antibodies labeled with fluorochromes that ensure a bright and stable fluorescence with a reduced background, according to the available equipment for image acquisition. Fluorochromes are sensitive to light, to avoid photobleaching, protect from light the samples at all steps following incubation with the secondary antibody by covering the tubes/slides with aluminium foil, or hiding them in a drawer.
23. When BrdU immunodetection is performed, DAPI is a convenient nuclear stain. In contrast, Hoechst- 33258 or Hoechst-33342 should be avoided as the intensity of the fluorescence staining is quenched by the thymidine analogs such as BrdU incorporated into DNA [43].
24. When mounting, collect the animals in the minimum amount of liquid and place them onto the slide.
Remove all excess of the liquid by pipetting up or by draining with a tissue paper. Add one or two drops of mounting medium and place the animals by softly pushing them with a pincer. When applying the coverslip, avoid the formation of air bubbles. Let the slides dry for a few hours at RT protected from light and transfer them at -20°C.
Acknowledgements
This work was supported by the Swiss National Science Foundation (SNF grants 31003A_149630, 31003_169930), the Claraz donation and the Canton of Geneva.
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