Zic4 is required for tentacle development and tentacle maintenance
Zic4 is required for tentacle development and tentacle maintenance
To explore Zic4 function, we knocked down Zic4 by RNAi. We measured the efficiency of Zic4 RNAi at different time points during the procedure and detected a significant down-regulation of Zic4 starting 1 day after the first electroporation (EP1) and lasting until at least 11 days after the procedure has been initiated (Fig. 3A). Thereafter, we consistently performed experiments within this time window. Three days after EP3, intact Zic4(RNAi) animals exhibit tentacles with half the length of those in control animals, while the overall tentacle number is not affected (100%; n = 100) (Fig. 3B and fig. S5A). In contrast, apical-regenerating Zic4(RNAi) animals regenerate not only shorter but also 25% fewer tentacles (100%; n = 99) (Fig. 3C and fig. S5B). Overactivation of Wnt signaling through ALP treatment leads normally to ectopic tentacle formation (35). We found that when ALP treatment is combined with Zic4 down-regulation through RNAi, the development of ectopic tentacles along the body column is strongly impaired, pointing out to a strong genetic interaction between Wnt/β-catenin signaling and Zic4 (Fig. 3D and fig. S5C). We also noticed that the expression of two components of the head organizer, Wnt3 and HyBra1 (34), is not affected in intact or regenerating Zic4(RNAi) animals, suggesting that Zic4 is required neither for the maintenance nor for the formation of a functional head organizer (Fig. 3E and fig. S6). When we transplanted apical tissue from control and Zic4(RNAi) animals into actin:GFP transgenic animals, we observed a similar induction of a secondary body axis, a hallmark of a functional head organizer (Fig. 3F). We concluded that Zic4 operates downstream of the head organizer and is required for tentacle maintenance and tentacle formation.
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Zic4 actively prevents the basal disc differentiation program in tentacles
In addition to the active role of Zic4 in tentacle formation and maintenance, we also identified a suppressive role for Zic4, evidenced by the transdifferentiation of TBCs in BDCs when Zic4 is down-regulated . This process is rapid as within a week, a cluster of adjacent transdifferentiated cells in tentacles can form and behave as a fully functional basal disc, able to attach on solid substrates. We could monitor this change of cell identity in epidermal epithelial cells of tentacles that display all the features of terminally differentiated TBCs. In original tentacles of Zic4(RNAi) and Zic4/Sp5(RNAi) Hydra as well as in transplanted tentacles containing GFP+-labeled TBCs submitted to Zic4/Sp5(RNAi), we identified different types of mixed cell identities. As markers of cell fate change, we found that fully differentiated TBCs first lose expression of ks1 and HyAlx while entering the cell cycle without dividing, subsequently express Crim-1, form droplets with peroxidase activity at the apical pole, and finally rapidly organize into a compact mass of adjacent transformed cells. In parallel, nematocytes embedded in the TBCs disappear as observed histologically and confirmed by the loss of Nem-A expression, while the typical tentacle architecture becomes disorganized with the disappearance of the large extracellular space between TBCs.
These results enable us to propose a five-step scenario to switch a TBC to a BDC where the coexpression of basal markers in cells that still exhibit a typical TBC organization supports the criteria of transdifferentiation We inferred from this scenario that Zic4 actively prevents the basal disc differentiation program in the proximal region of tentacles. However, we cannot exclude that a low level of Zic4 activity in the tentacle zone also leads epidermal epithelial cells that are still cycling there and committed to becoming TBCs but not yet differentiated to transform into BDCs. In addition, it should be noted that we never observed cell division among tentacle BrdU-positive cells, and we never detected mitotic activity in tentacles of Zic4(RNAi) or Zic4/Sp5(RNAi) Hydra. Therefore, we concluded that cells that reenter S phase undergo endoreplication, a process observed in developmental as well as injury and stress contexts associated with regeneration Endoreplication allows cells to speed-up protein production without spending time dividing, therefore accelerating regeneration. Previous studies have identified transdifferentiation in Hydra, but never in epithelial cells, rather in cells that belong to the interstitial lineages, e.g., between neuronal subtypes or ganglion cells converting into epidermal sensory cells or zymogen gland cells transforming into granular mucous cells. As Zic4 is also expressed in nerve cells, it remains to be investigated whether Zic4 activity in neurons acts as a safeguard to protect their fate.
The basal disc epithelial cell fate appears as a default state in Hydra
The main finding of this study is that Zic4 acts as a master regulator that controls the choice between two epidermal cell fates: tentacle battery cells when Zic4 level is high on the one hand, and basal mucous cells when Zic4 level is low on the other. When Zic4 expression is reduced, the gene expression profile of these cells indicates not only a lower expression of TBC markers but also a concomitant up-regulation of BDC markers. Therefore, the BDC status appears as a default state of epidermal epithelial terminal differentiation in Hydra possibly reflecting an ancestral differentiation fate of multifunctional epithelial cells in early metazoans. The fact that ectopic basal disc tissue can also develop at any place along the body column, mostly when Sp5 or Zic4 and Sp5 are knocked down, indicates that different constraints apply on the epithelial differentiation programs, with BDC differentiating in many places along the body axis and TBC differentiating only in tentacles. Several studies reported tentacle inhibition in Hydra however, ectopic basal disc formation was never noticed in these contexts, suggesting that the transient genetic perturbations leading to tentacle inhibition were downstream of Zic4 and/or Sp5 activity, thus keeping active the Zic4-dependent repression of BDC differentiation.
If we assume that the default state of BDC is similarly repressed in the tentacles and in the body column, then by analogy with the blocking of this default state by TBCs in the tentacles, we can assume that the epidermal ESCs along the body column may play the same role, preventing BDC differentiation in this region, possibly via Sp5 that is expressed at high levels there. At the molecular level, we can consider two possibilities, either Zic4 directly represses the set of genes involved in basal disc differentiation (active model) or the Zic4-induced tentacle differentiation program suffices to prevent basal disc differentiation (passive model). Once a specific anti-HyZic4 antibody is made available, further ChIP-seq experiments combined with RNA-seq analysis will tell us when and where Zic4 acts as an activator or a repressor. The identification of direct Zic4 and Sp5 target genes in the apical and body column regions would help dissect this cell fate regulation. The transdifferentiation phenotype obtained in animals submitted to the double Zic4/Sp5 knockdown is enhanced and extended to the body column when compared to the single Zic4 knockdown. This result is coherent with an epistatic relationship between Sp5 and Zic4, as well as between Zic4 and HyAlx. The relationship with Notch signaling involved in tentacle formation requires further investigation Furthermore, histone modifications such as H3K27 methylation appear critical when Wnt signaling is activated , and it would be interesting to investigate whether Zic4 activity interferes with such H epigenetic mechanisms.
The Wnt/β-catenin/Sp5/Zic4 cascade might act as an evolutionarily conserved switch of epithelial cell fate
Hydra genome contains four members of the Zic family, HyZic (also named Zic1) involved in nematocyte differentiation and Zic2/ZNF143 and Zic3 predominantly expressed in the gastrodermal epithelial cells, with Zic3 expressed at higher levels at the base of tentacles pointing to a possible role of Zic3 in tentacle development. In the sea anemone Nematostella, several Zic genes are expressed during tentacle formation in distinct tentacle cell types suggesting that the Zic4 function in tentacle maintenance and tentacle formation is shared among cnidarians. In bilaterians, zinc finger transcription factors play a crucial role in cell fate stability, acting as not only versatile multifunctional proteins, classical DNA binding proteins that regulate the expression of the ascidian Brachyury or the mammalian Oct4 and Nanog but also transcriptional coactivators via their protein-protein interaction with transcription factors such as Gli, TCF, Smad, Pax, Cdx, and SRF as well as chromatin remodeling factors contributing to enhancer functions In vertebrates, the human Zfp521 promotes the conversion of fibroblasts to neural stem cells ex vivo and Zic proteins can inhibit the Wnt/β-catenin signaling pathway compete with Sp transcription factors on promoter sequences or associate with Geminin in cell cycle regulation. The question now is whether, as in Hydra, the genetic circuit involving the Wnt/β-catenin/Sp5/Zic4 cascade acts in other cnidarian or bilaterian organisms as a switch regulating epithelial cell fate.
MATERIALS AND METHODS
Animal culture and drug treatment
All experiments were performed with strains of Hydra vulgaris (Hv) either Basel1, AEP2 (kindly provided by R. Steele), or Hm-105 (53), and the cultures were maintained in Hydra medium (HM) [1 mM NaCl, 1 mM CaCl2, 0.1 mM KCl, 0.1 mM MgSO4, and 1 mM tris (pH 7.6)]. The animals were fed two to three times per week with freshly hatched Artemia nauplii and starved for 4 days before starting the experiments. For ALP treatments, Hv_Basel were treated for 2 days with 5 μM ALP (Sigma-Aldrich) diluted in HM. All experiments were performed in accordance with ethical standards and the Swiss national regulations for the use of animals in research.
Reaggregation assays
Hm-105 animals were electroporated twice with siRNAs targeting β-catenin. One day after EP2, 100 animals were dissociated in 10 ml of dissociation medium (DM) [3.6 mM KCl, 6 mM CaCl2, 1.2 mM MgSO4, 6 mM Na-citrate, 6 mM pyruvate, 4 mM glucose, and 12.5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 6.9)] and the cell suspension was centrifuged at 4°C, 1400 rpm for 45 min. The pellet was resuspended in 2 ml of DM, and the suspension was equally distributed into 1.5-ml tubes. After a second round of centrifugation at 4°C, 1400 rpm for 45 min, the tubes were laid down on the bench for 4 hours to detach the reaggregates. The reaggregates were then kept overnight at 18°C in six-well plates filled with 75% DM/HM, then for 4 hours in 50% DM/HM, and afterward transferred to HM.
Lateral transplantation experiments
A transgenic reporter line that expresses a GFP in the epidermis under the control of the actin promoter (actin::GFP kindly provided by T. Bosch) was used as a host to monitor the induction of a secondary body axis. To prepare the donor tissue, Hv_AEP2 animals were electroporated three times with scramble or Zic4 siRNAs. Three days later, host animals were carefully wounded in the central body column with a scalpel. Immediately after wounding, hypostomal tissue of scramble and Zic4(RNAi) animals was excised and inserted with tweezers into these small wounds. The donor and the host tissue were allowed to heal together, and 24 hours after grafting, the animals were carefully transferred into fresh HM.
Whole-mount in situ hybridization
Whole-mount ISH with the bromochloroindolyl phosphate–nitro blue tetrazolium (BCIP-NBT) substrate was performed as recently described in detail (6). All polymerase chain reaction (PCR) products were cloned into pGEM-T-Easy (Promega) to generate riboprobes. Primer sequences are listed in table S2. For the double detection of Nematocilin A/Crim-1 and Wnt3-Crim-1, the Nematocilin A and Wnt3 riboprobes labeled with fluorescein were first developed with BCIP-NBT, while Crim-1 labeled with digoxigenin was developed in a second step with Fast Red. The BCIP-NBT reaction was stopped by washing the animals in NTMT [0.1 M NaCl, 0.1 M tris-HCl (pH 9.5), and Tween 0.1%] six times for 1 min. The samples were then incubated in 100 mM glycine (pH 2.2) supplemented with 0.1% Tween for 10 min, followed by washes in MAB-Buffer1 (1× MAB and 0.1% Tween) four times for 30 s and two times for 10 min. Next, the samples were incubated in MAB-Buffer2 (1× MAB, 10% sheep serum, and 0.1% Tween) for 1 hour, and the anti–DIG-AP antibody (1:4000; Roche) was added for overnight incubation at 4°C. On the next day, the samples were washed in MAB-Buffer2 two times for 1 min, MAB-Buffer1 two times for 1 min, and 0.1 M tris-HCl (pH 8.2) three times for 10 min and developed with Fast Red. To prepare the Fast Red solution, 1 tablet of buffer from the Sigmafast Fast Red TR/Naphthol AS-MX kit (Sigma-Aldrich) was dissolved in 1 ml of Milli-Q water and the Fast Red tablet was added. NaCl with a final concentration of 0.3 M was added to improve the strain. The reaction was stopped by washing the samples in 0.1 M tris-HCl (pH 8.2) three times for 1 min. The samples were postfixed in 3.7% formaldehyde diluted in Milli-Q water for 10 min, washed in Milli-Q water two times for 1 min, and mounted in Mowiol. All double ISHs were performed using Hv_AEP2.
ISH on macerates
Macerates of head and foot tissue were prepared with the maceration solution (glycerol:acetic acid:water = 1:1:13) as described in the section on immunostaining on macerates. ISH was performed as described in with a few modifications. In short, samples were rehydrated through a series of EtOH, PBS-Tw [0.1% Tween 20 in phosphate-buffered saline (PBS)] washes (75, 50, and 25%) for 5 min each, followed by washes in PBS-Tw three times for 5 min. The samples were then incubated in heparin (10 μg/ml) for 5 min and kept in hybridization buffer [100 mM dithiothreitol, heparin (100 ng/ml), and 3× SSC] containing 2 μg/ml of each riboprobe for 4 hours at 65°C. The Nematocilin A riboprobe was labeled with digoxygenin, and the Crim-1 riboprobe was labeled with fluorescein. Next, the samples were washed with 2× SSC two times for 20 min at 65°C and kept in 2× SSC overnight at 4°C. On the next day, the samples were washed with MAB-Buffer1 two times for 10 min, MAB-Buffer2 (1× MAB, 10% sheep serum, and 0.1% Tween) for 1 hour and incubated in MAB-Buffer2 with an anti–Fluo-AP antibody (1:4000; Roche) for 4 hours. The samples were then washed with MAB-Buffer1 six times for 15 min, NTMT [0.1 M NaCl, 0.1 M tris-HCl (pH 9.5), and Tween 0.1%] for 5 min, NTMT with 1 mM levamisole two times for 5 min, and the staining solution [0.1 mM tris-HCl (pH 9.5), 0.1 mM NaCl, 7.8% polyvinyl alcohol (PVA), and 1 mM levamisole] containing BCIP-NBT was added. The colorimetric reaction was stopped by washing the samples with NTMT six times for 1 min. The Fast Red staining was performed as described above (see the previous section). The samples were postfixed in 3.7% formaldehyde diluted in Milli-Q water for 10 min, washed in Milli-Q water two times for 1 min, and mounted in Mowiol. All steps were performed at room temperature (RT) except stated otherwise.
Hybridization chain reaction RNA-FISH
The HCR RNA-FISH protocol has been adapted for Hydra based on the manufacturer’s instructions (Molecular Instruments) and provided solutions. All incubations are performed at RT unless otherwise specified. Hydra were relaxed in 2% urethane/HM for 1 min before fixation for 10 min in 4% paraformaldehyde (PFA)/PBS (v/v). The fixative was washed away with PBS/0.1% Tween 20 solution (PBST) twice, for 10 min each time. Tissues were then incubated for 1 hour in a 70% ethanol/PBST (v/v) solution, followed by an incubation of 5 min in a 35% ethanol/PBST (v/v) solution at 4°C. Ethanol solutions have been cooled down at 4°C before use. Samples were washed twice for 10 min with PBST before incubation in 250 μl of HCR probe hybridization buffer, without probe, for 30 min at 37°C. Samples were then incubated with 250 μl of HCR probe hybridization buffer containing 2 pmol of ks1 hybridization probe overnight (12 to 16 hours) at 37°C. The probe was synthesized by Molecular Instruments (lot number PR0588). Hybridization probe has been washed out by five washes of 30 min in 500 μl of HCR probe wash buffer at 37°C, followed by two washes of 5 min with a 5× sodium chloride sodium citrate/0.1% Tween 20 (SSCT) solution. Signal was preamplified with 250 μl of amplification buffer for 30 min. Amplifying hairpin RNAs, provided by Molecular Instruments, were heated at 95°C for 1.5 min and allowed to cool down in the dark for 30 min. The preamplification solution was replaced by 250 μl of amplification buffer containing the two hairpins, and samples were incubated overnight (12 to 16 hours) in the dark. Excess was washed out with several washes in 5× SSCT solution in the dark: twice for 5 min, three times for 30 min, once for 30 min with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml), and once for 5 min. Samples were mounted on a slide with ProLong Gold Antifade.
Quantitative PCR
The extraction of total RNA was done with E.Z.N.A. Total RNA Kit I (Omega), and complementary DNA (cDNA) was synthesized using the qScript cDNA SuperMix (Quanta Biosciences). The cDNA samples were diluted to 1.6 ng/μl, and gene-specific primers were designed with Primer3. The primer efficiencies were determined on the basis of standard dilution series. All quantitative PCRs (qPCRs) were performed with the CFX96 Real-Time system (Bio-Rad), and the amplifications were done with the SYBR Select Master Mix (Applied Biosystems). The thermal cycling conditions were composed of 50°C for 2 min, 95°C for 2 min, 39 cycles at 95°C for 15 s, 60°C for 30 s, and 65°C for 5 s. Relative gene expression levels were calculated with a model based on PCR efficiency and crossing point deviation (58). TBP was used as an internal reference gene to normalize all data.
RNA interference
RNAi was performed as previously described with minor modifications. In short, Hydra were briefly washed and incubated for 1 hour in Milli-Q water. Twenty animals per condition were then transferred into a 0.4-cm gap electroporation cuvette (Bio-Rad), the remaining Milli-Q water was removed, and 200 μl of siRNA solution was added, i.e., a mix of three distinct gene-specific siRNAs, 1.33 μM each in sterilized Hepes solution (pH 7). Control RNAi animals were electroporated with scramble siRNA (4 μM). For Zic4/Sp5 double knockdown experiments, Zic4 siRNAs were mixed with Sp5 siRNAs (0.7 μM of Zic4 siRNA-1, siRNA-2, and siRNA-3 + 0.7 μM of Sp5 siRNA-1, siRNA-2, and siRNA-3). The electroporation was performed with a Bio-Rad GenePulser Xcell electroporation system, and the following conditions were applied: voltage: 150 V, pulse length: 50 ms, number of pulses: 2, and pulse intervals: 0.1 s. Animals that were directly imaged after RNAi were first relaxed in 2% urethane/HM for 1 min, fixed for 2 hours at RT, and mounted in Mowiol. RNAi animals that were used for drug treatments were kept for 18 hours in HM with 5 μM ALP, followed by fixation for 2 hours at RT. For hoTG electroporations, RNAi animals were electroporated with 40 μg of hoTG plasmid under the same conditions as described above. Hv_Basel were used for all experiments, except stated otherwise.
Reporter constructs
To generate the HyActin:mCherry::HyZic4-3505:EGFP (enhanced green fluorescent protein) construct (subsequently named Zic4-3505:GFP), the HyWnt3 promoter of the plasmid HyActin-RFP::HyWnt3FL-EGFP (a kind gift from T. Holstein) (61) was replaced with a 3505-bp-long HyZic4 promoter fragment that was amplified from Hm-105 genomic DNA. RFP was replaced with mCherry that was codon-optimized for Hydra expression (GenScript). To generate the HyZic4-3505:Luciferase construct (Zic4-3505:Luc), 3505 bp of the Hydra Zic4 promoter were PCR-amplified from Hm-105 genomic DNA and subcloned into a pGL3 reporter construct (a kind gift from Z. Kozmik) (62). pcDNA-Wnt3 (huWnt3) was obtained from Addgene (plasmid no. 35909) (63), pcDNA6-huLRP6-v5 (huLRP6) was kindly provided by B. Williams (64), and pFLAG-CMV-hu-β-CateninΔ45 (huΔβcat) by A. Ruiz i Altaba (65). To generate the construct encoding the TCCS as designed by Sakaue-Sawano et al. (66), we amplified from Hm-105 genomic DNA 180 bp of the N-terminal part of Hydra Geminin-like (A0A8B7E3E1_HYDVU) that contains the destruction box required for the M-G1 phase–specific APC/C ubiquitin ligase and fused it with GFP. As second sensor, we amplified from Hm-105 genomic DNA 579 bp of the N-terminal part of Hydra DNA replication factor 1 (Cdt1, XP_012559344.1) that contains the conserved PIP box (PCNA interacting domain) and fused it to mCherry. Each of the two sensors was subsequently placed under the control of the Hydra actin promoter (1437 bp) and assembled into a unique construct. To prove the functionality of this construct, we electroporated it into Hydra and found that transfected epidermal epithelial stem cells transiently coexpress cytoplasmic/nuclear GFP and nuclear mCherry.
Luciferase assays in human HEK293T cells
HEK293T cells were cultured in DMEM high glucose, 1 mM Na pyruvate, 6 mM l-glutamine, and 10% fetal bovine serum. Cells were seeded into 96-well plates (5000 cells per well) and 18 hours later transfected with an X-tremeGENE HP DNA transfection reagent (Roche). The following plasmid amounts were transfected: 1 ng of pGL4.74[hRluc/TK] (Promega), 40 ng of HyZic4-3505:Luc, 10 ng of huΔβcat, 40 ng of huLRP6, 40 ng of huWnt3, 20 ng of HySp5-420, and 20 ng of HySp5-ΔDBD. The total plasmid amount was adjusted with pTZ18R to 100 ng per well. Extracts were prepared 24 hours after transfection with the Dual-Luciferase Reporter Assay System (Promega) and transferred to a white OptiPlate-96 (PerkinElmer). Firefly and Renilla luciferase activities were immediately measured on a VICTOR X5 multilabel plate reader (PerkinElmer).
Generation of Hydra transgenic lines
Male and female polyps of the strain Hv_AEP2 were cultured together and fed three times a week, which resulted in the regular production of fertilized eggs. The Zic4-3505:GFP construct was injected into one-cell–stage embryos; out of 424 injected eggs, 71 embryos were hatched, and 1 of 71 embryos showed epidermal GFP expression in the tentacle region. Concerning the transgenic TCCS line, the plasmid encoding the TCCS was injected into one-cell–stage embryos; out of 78 injected eggs, five embryos hatched and one embryo showed epidermal epithelial Geminin-GFP/Cdt1-mCherry expression, nuclear and cytoplasmic GFP, and nuclear mCherry as expected. A stable transgenic TCCS line was derived from this embryo.
Whole-mount immunofluorescence
Intact transgenic TCCS Hydra were relaxed in 2% urethane/HM for 2 min, fixed in 4% PFA for 3 hours, dehydrated in MeOH, and kept at −20°C until processed for immunostaining. To immunodetect GFP, the animals were rehydrated with 75, 50, and 25% MeOH in Milli-Q water for 10 min, washed with PBS five times for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 30 min, and blocked with 2% bovine serum albumin (BSA), 0.5% Triton X-100 in PBS for 60 min. Next, samples were incubated in rabbit anti-GFP primary antibody (1:500; Novus Biological) diluted in 2% BSA, 0.5% Triton X-100 in PBS for 60 min at 37°C and then overnight at 4°C. The next day, the samples were washed in PBS four times for 10 min and incubated in anti-rabbit immunoglobulin G (IgG)–Alexa 488 (1:500; Thermo Fisher Scientific) diluted in PBS for 4 hours. The samples were then washed in PBS three times for 10 min, stained with DAPI (1 μg/ml; Roche) for 10 min, washed three times for 2 min in PBS, and mounted in Mowiol. All steps were performed at RT, except stated otherwise. For the phospho-histone H3 detection, Hv-AEP2 animals were processed as above, except that fixation in 4% PFA was done overnight at 4°C, and the permeabilization and blocking steps were done in 1% Triton X-100 in PBS. The mouse anti-histone H3 phospho-S10 antibody (Abcam, 14955) was diluted 1:1000 in 2% BSA, 1% Triton X-100 and subsequently detected with the anti-mouse Alexa 555 (Invitrogen, A-31570) diluted 1:500 in PBS. For actin staining, samples were incubated with 200 pM Phalloidin–Atto 565 (Sigma-Aldrich) in PBS for 10 min in the dark and then washed 3× in PBST (PBS + 0.1% Tween 20). When needed, tentacles and basal regions were dissected and mounted between coverslips using a ProLong Gold Antifade mounting medium (Thermo Fisher Scientific).
Immunostaining on macerates
Intact transgenic TCCS Hydra were decapitated, and eight heads per replicate were pooled and incubated in 30 μl of maceration solution (glycerol:acetic acid:water = 1:1:13) for 1 to 3 hours and gently pipetted up and down from time to time to dissociate the tissue. Cells were fixed in 4% PFA for 1 hour, then Tween 80 (final 0.5%) was added, and the cell suspension was spread on freshly prepared gelatin-coated slides as described in (67) with a 1.0 × 1.0 Gene Frame (Thermo Fisher Scientific). Samples on the slides were dried for 1 to 2 days, then washed in PBS three times for 10 min, and incubated in 0.5% Triton X-100 in 0.2% citrate buffer (pH 6) for 2 min at RT and then for 7 min at 70°C. After washing in PBS six times for 5 min, the cells were permeabilized with 0.5% Triton X-100 in PBS for 30 min, and the unspecific binding was blocked with 2% BSA and 0.5% Triton X-100 in PBS for 60 min. To detect GFP and tubulin, a primary antibody mixture of rabbit anti-GFP (1:500; Novus Biological) and mouse anti-tubulin (1:1000; Sigma-Aldrich) was added, and the cells were first incubated for 1 hour at 37°C and then overnight at 4°C. Primary antibodies were diluted in 2% BSA and 0.5% Triton X-100 in PBS. The cells were washed in PBS four times for 10 min and incubated for 4 hours in a mixture of secondary antibodies, anti-rabbit IgG–Alexa 488 (1:500; Thermo Fisher Scientific), and anti-mouse IgG–Alexa 555 (1:500; Thermo Fisher Scientific) diluted in PBS. After several fast washes in PBS, cells were counterstained with DAPI (1 μg/ml; Roche) for 10 min, washed in PBS three times for 30 s, dried, and mounted in Mowiol. All steps were performed at RT, except stated otherwise.
Detection of BrdU labeling
For all conditions of BrdU detection, we used BrdU Labeling and Detection Kit I (reference: 11296736001) from Roche with the anti-BrdU antibody diluted 1:20 in BrdU buffer. All steps were performed at RT, except stated otherwise.
Detection of BrdU labeling in whole-mount transgenic TCCS animals
Transgenic TCCS Hydra were incubated in 5 mM BrdU for 2 or 5 hours, washed five times in HM, relaxed for 2 min in 2% urethane/HM, fixed overnight at 4°C in 4% PFA, washed five times with MeOH, and stored in MeOH at −20°C for several days. For BrdU and GFP immunostaining, the samples were successively rehydrated in 75, 50, and 25% MeOH, each step for 10 min, and then washed in PBS five times for 10 min. Next, the samples were permeabilized in 1% Triton X-100 in PBS for 60 min and washed fast with PBS, and DNA was denatured with 2.5 N HCl for 30 min. After extensive washing in PBS for 20 min, the samples were incubated with a mixture of the mouse anti-BrdU and rabbit anti-GFP (1:500; Novus Biological) antibodies diluted in BrdU buffer for 1 hour at 37°C and overnight at 4°C. The next day, the samples were washed four times for 10 min in PBS, incubated in secondary anti-mouse Alexa Fluor 647 (1:500; Thermo Fisher Scientific) and anti-rabbit Alexa Fluor 488 (1:500; Thermo Fisher Scientific) antibodies for 4 hours, then washed in PBS four times for 10 min, counterstained with DAPI (1 μg/ml; Roche) for 10 min, washed again two times for 3 min in PBS and once with Milli-Q water, and mounted in Mowiol.
Detection of nuclear BrdU labeling in macerated tissues
Live Hv_Basel animals were incubated in 5 mM BrdU/HM (Sigma-Aldrich) for 16 hours and washed in HM two times for 5 min, and 20 body columns were macerated in 100 μl of maceration solution (as described above) for 45 min. The cell suspension was fixed in 4% PFA for 15 min, Tween 80 was added (final 1%) and spread on Epredia SuperFrost Plus Adhesion slides (Thermo Fisher Scientific) with a 1.7 × 2.8 Gene Frame (Thermo Fisher Scientific). The cells were dried for 2 days, washed in PBS three times for 10 min, treated with 2 N HCl for 30 min to denature DNA, then washed in PBS three times for 5 min, and incubated in 2% BSA/PBS for 1 hour and then with the anti-BrdU antibody overnight at 4°C in a wet chamber. On the next day, cells were washed in PBS three times for 10 min, incubated in anti–Alexa Fluor 488 antibody (1:600; Molecular Probes) for 2 hours, washed again in PBS three times for 10 min, DAPI-stained (0.2 μg/ml; Roche) for 10 min, washed in Milli-Q water two times for 5 min, and mounted in Mowiol. Similarly, transgenic TCCS Hydra first exposed to 5 mM BrdU for 5 hours were then decapitated, and six to seven heads per replicate were pooled, dissociated in 75 μl of maceration solution for 4 hours, fixed, and spread on slides as described above. After drying for 3 days, the samples were washed three times for 10 min in PBS, permeabilized for 10 min with MeOH, washed three times for 10 min in PBS, and incubated with 0.5% Triton X-100 in PBS for 15 min. After a short rinse in PBS, samples were treated with 2.5 N HCl for 30 min, washed extensively in PBS for 15 min, then incubated with a mixture of mouse anti-BrdU and rabbit anti-GFP antibodies, and detected as for whole mounts.
Detection of cell death
Intact transgenic TCCS Hydra were decapitated, and four to six heads per replicate were pooled and macerated for 3 hours in 40 μl of maceration solution as described above. The cells were fixed in 4% PFA for 1 hour, Tween 80 was added (final 0.5%), and the cell suspension was spread on freshly prepared gelatin-coated slides with a 1.5 × 1.6 cm Gene Frame (Thermo Fisher Scientific). The slides were dried for 1 to 2 days, then washed three times for 10 min in PBS, and incubated in 1% Triton X-100 in 0.2% citrate buffer (pH 6) for 2 min and at 70°C for 10 min. After washing six times for 5 min in PBS, the cells were incubated for 90 min at 37°C in a wet chamber in the dark in the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) mix prepared according to the supplier’s instructions (Sigma-Aldrich, reference: 11684795910). After washing three times for 10 min in PBS, cells were blocked with 2% BSA, 0.5% Triton X-100 in PBS for 30 min, and then incubated for 60 min at 37°C in a mixture of rabbit anti-GFP (1:500; Novus Biological) and mouse anti-tubulin (1:1000; Sigma-Aldrich) antibodies diluted in 2% BSA and 0.5% Triton X-100 in PBS. The cells were washed with PBS four times for 10 min and incubated for 3 hours in a mixture containing the anti-rabbit IgG coupled with Alexa 555 (1:400; Thermo Fisher Scientific) and anti-mouse IgG coupled with Alexa 647 (1:400; Thermo Fisher Scientific) secondary antibodies diluted in PBS. After several washes in PBS for 30 min, the cells were stained with DAPI (1 μg/ml; Roche) for 10 min, washed in PBS three times for 15 s, and mounted in Mowiol. All steps were performed at RT, except stated otherwise.
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