In Situ Hybridization to Identify Stem Cells in the Freshwater Sponge Ephydatia fluviatilis

Kojima, Chiaki; Funayama, Noriko

doi:10.1007/978-1-0716-2172-1_17

Chiaki Kojima⁴ &
Noriko Funayama⁴

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2450))

5378 Accesses
1 Altmetric

Abstract

Sponges (Porifera) are a large phylum that includes an enormous number of species. They are classified into four classes. Among these four classes, class Demospongiae is the largest and contains more than 90% of sponge species. In the last decade, methodologies for molecular studies and sequencing resources in sponge biology have dramatically advanced and made it possible to clearly define particular types of cells based on the genes they are expressing. Here we describe in detail the method of high-resolution WISH (whole mount in situ hybridization) and dual color fluorescent detection of in situ hybridization (dual color FISH) that we have established to detect particular types of cells, especially their stem cells known as archeocytes, in juveniles of freshwater demosponge, E. fluviatilis.

You have full access to this open access chapter, Download protocol PDF

In Situ Hybridization Techniques in the Homoscleromorph Sponge Oscarella lobularis

Fluorescence In Situ Hybridization (FISH) and Immunolabeling on 3D Preserved Nuclei

An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis

Article Open access 28 March 2015

Key words

1 Introduction

Molecular studies of sponges are important for both evolutionary developmental biology (since sponges are one of the earliest branching metazoan phyla) and ecological developmental biology (since sponges are sessile organisms). Furthermore, many sponges have high regenerative ability and thus potentially have totipotent/pluripotent stem cells. Uncovering the cellular and molecular bases of sponge stem cells will not only be crucial for understanding the ancestral gene repertoire of animal stem cells, but will also give us clues for understanding the evolution of molecular mechanisms for maintaining multipotency (pluripotency) and for elucidating the regulatory mechanisms of their differentiation.

Molecular and cellular studies in juveniles of the freshwater Ephydatia fluviatilis suggested that demosponges, which contain more than 90% of all sponge species, have two types of stem cells: mesenchymal Archaeocytes/Archeocytes and food-entrapping Choanocytes [1,2,3,4]. Recent studies of sponges in other classes, suggest that this model could be generalized at least in three classes of sponges, demosponges, calcareous sponges, and homoscleromorpha [4]. However, the type of cells (archaeocytes or choanocytes) that acts as stem cells seems variable, depending on the cellular organization of each class of sponges. Traditionally, both “archeocytes” and “archaeocytes” are used as terms meaning amoeboid cells that contain a large nucleus with a large nucleolus, and are capable of phagocytosis [5, 6]. These cells are suggested to be totipotent somatic stem cells based on microscopic analysis. They are suggested to produce both somatic differentiated cells and gametes [5,6,7], just like the multipotent stem cells “interstitial stem cells” in hydra, and “neoblasts” in planarians.

In situ hybridization enables the detection of mRNA and thus it is a powerful tool to characterize cells expressing a particular gene, or to identify specific types of cells that express a particular gene. Actually, by the establishment of the methods of WISH and FISH with high resolution ([8, 9] respectively), together with the identification of cell-type specific genes [8,9,10,11,12,13], cells with morphological features of Archaeocytes/Archeocytes were defined as at least multipotent stem cells that can undergo self-renewal and directly differentiate into multiple types of cells [1]. Thus, EflPiwiA-, EflPiwiB-, EflMusashiA-expressing cells have been defined as at least multipotent stem cells in demosponges [1,2,3,4, 11, 12]. It has also been suggested on the basis of their gene expression and microscopic analysis that these cells are in fact totipotent stem cells.

Here we describe in detail the method of WISH and dual color fluorescent detection of in situ hybridization (dual color FISH) that we have established to detect particular types of cells, especially stem cells, in juveniles of freshwater demosponge, E. fluviatilis .

2 Materials

Prepare all solutions using ultrapure water (deionized water) or RNase-free deionized water (when necessary) and analytical grade reagents.

1.
M-Medium: 1.47 g CaCl₂•2H₂O, 1.23 g MgSO₄•7H₂O, 0.71 g Na₂SiO₃•9H₂O, 0.42 g NaHCO₃, 0.037 g KCl in 10 L. Adjust to pH 7.3–7.8 using 2 M HCl.
2.
Stage 2 to 4 juvenile sponges (see Note 1).
3.
1/4 Holtfreter’s solution (HS): 875 mg NaCl, 12.5 mg KCl, 25 mg CaCl₂, 50 mg NaHCO₃ in 1 L.
4.
Fixative solution: 4% (w/v) paraformaldehyde in 1/4 HS.
5.
50% MetOH: 50% (v/v) MetOH in 1/4 HS.
6.
100% MetOH.
7.
0.5 μg template DNA for RNA probe synthesis (see Note 2).
8.
100 mM DTT Molecular Grade (e.g., Promega).
9.
Resuspension mix: 3 μL 100 mM DTT, 50 μL RNase-free water.
10.
40 U/μL RNase inhibitor (e.g., Toyobo).
11.
T3 and/or T7 polymerase with 5× transcription buffer (e.g., Takara Bio).
12.
Dig RNA labeling mix (e.g., Roche).
13.
RNA synthesis reaction mix: 0.5 μg template linear DNA, 4 μL 5× transcription buffer, 2 μL Dig RNA labeling mix, 2 μL 100 mM DTT, 0.5 μL 40 U/μL RNase inhibitor, 1.5 μL polymerase, 9 μL RNase-free Water. Prepare fresh.
14.
0.5 M EDTA: 46.5 g ethylenediaminetetraacetic acid (EDTA)•2Na•2H₂O in 250 mL ultrapure water. Adjust to pH 8.0 using 5 M NaOH.
15.
1 mg/mL yeast RNA: 1 mL commercial 10 mg/mL yeast RNA (e.g., Thermo Fisher scientific), 9 mL RNase-free water.
16.
10 M NH₄OAc: 77 g NH₄OAc in 100 mL ultrapure water.
17.
100% EtOH.
18.
70% EtOH: 70% (v/v) EtOH in ultrapure water.
19.
50× TAE: 242 g Tris (hydroxymethyl)aminomethane (TAE), 57.1 mL acetic acid, 100 mL 0.5 M EDTA in 1 L ultrapure water.
20.
1× TAE: Mix 20 mL 50× TAE and 980 mL ultrapure water.
21.
0.7% agarose gel for electrophoresis: 0.7% (w/v) agarose (e.g., Takara Bio) in 1× TAE.
22.
DNA loading buffer for electrophoresis (e.g., ThermoFisher).
23.
Nucleic acid stain reagent (e.g., Midori green Xtra, Nippon genetics).
24.
50% (v/v) xylene in EtOH.
25.
35 mm glass petri dishes (see Note 3).
26.
10% (v/v) Tween 20 in ultrapure water.
27.
10 mg/mL heparin in ultrapure water.
28.
Phosphate buffered saline (PBS): 0.2 g KCl, 8 g NaCl, 2.9 g NaHPO₄•12H₂O, 0.24 g KH₂PO₄. Adjust to pH 7.4 using 1 M HCl.
29.
TPBS: 0.1% (v/v) Tween 20 in 1 L PBS.
30.
20× SSC: 175.3 g NaCl, 88.2 g trisodium citrate dihydrate in 1 L. Adjust to pH 7.0 using 1 M HCl.
31.
1 M DTT: 15.4 g in 100 mL ultrapure water.
32.
Hybridization solution: 25 mL formamide, 500 μL 1 mg/mL yeast RNA, 500 μL 10 mg/mL heparin, 500 μL 10% Tween 20, 500 μL 1 M DTT, 12.5 mL 20× SSC, 10.5 mL H₂O.
33.
WASH buffer: 250 mL formamide, 125 mL 20× SSC, 5 mL 10% Tween 20, 120 mL freshly obtained ultrapure water.
34.
Buffer I: 11.6 g maleic acid, 8.8 g NaCl, 10 mL 10% (v/v) Triton X-100 in 1 L. Adjust to pH 7.5 using 1 N NaOH.
35.
Buffer II: 1% blocking reagent (e.g., Sigma Aldrich) in Buffer I. Sterilize by autoclaving 20 min at 121 °C and store at 4 °C.
36.
TMN: 0.1 M Tris-HCl pH 9.5, 0.05 M MgCl₂, 0.1 M NaCl in 10 mL.
37.
Alkaline phosphatase conjugated Anti-Digoxigenin antibody, Fab fragments (Roche).
38.
DMFA: N, N-Dimethylformamide (e.g., Roche).
39.
50 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) in DMFA.
40.
100 mg/mL 4-Nitro blue tetrazolium chloride (NBT): 100 mg/mL NBT, 70% (v/v) DMFA in ultrapure water.
41.
BCIP-NBT solution: 35 μL 50 mg/mL BCIP, 18 μL 100 mg/mL NBT in 10 mL TMN.
42.
TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 in 1 L.
43.
Peroxidase-conjugated Anti-Digoxigenin antibody, Fab fragments (e.g., Roche).
44.
Peroxidase-conjugated streptavidin (e.g., PerkinElmer).
45.
1% (v/v) peroxidase-conjugated streptavidin in Buffer II.
46.
Fluorescent Tyramide Signal Amplification (TSA) kit (e.g., Molecular Probes).
47.
1% (w/v) H₂O₂: 1% (w/v) H₂O₂ in TPBS.
48.
Nuclear staining dye (e.g., Hoechst 33342, Invitrogen) in TPBS.
49.
Anti-fade reagent (e.g., Fluoro KEEPER, Nacalai Tesque).
50.
Shaker/Rotator.
51.
Hybridization incubator with rocking platform.

3 Methods

3.1 Preparation of Sponge Samples

1.
Transfer juvenile sponges to a 24 well plate filled with fixative solution.
2.
Fix the animal overnight at 4 °C.
3.
Replace the fixative with 1/4 HS.
4.
Gently shake plate at 4 °C for 30 min.
5.
Replace the solution with ice-cold 50% MetOH.
6.
Gently shake plate at 4 °C for 30 min.
7.
Replace the solution with ice-cold 100% MetOH.
8.
Store the sample at −30 °C.

3.2 RNA Probe Synthesis

1.
Prepare the RNA synthesis reaction mix in sterile 1.5 mL or 250 μL tube (see Notes 4 and 5). High quality of the template DNA is important to obtain a high-quality RNA probe.
2.
Incubate the reaction mixture at 37 °C for 3 h for RNA synthesis.
3.
Add 2 μL of 0.5 M EDTA to stop the RNA synthesis.
4.
Add 2 μL of 1 mg/mL yeast RNA, 5.5 μL of 10 M NH₄OAc and 70 μL 100% EtOH for ethanol precipitation.
5.
Keep the reaction mixture at −80 °C for 10 min or − 30 °C for 30 min.
6.
Centrifuge for 10 min at 17,500 rcf, 4 °C.
7.
Remove the supernatant.
8.
Add 100 μL of ice-cold 70% EtOH.
9.
Centrifuge for 10 min, at 17,500 rcf, 4 °C.
10.
Remove the supernatant.
11.
Dry the pellet briefly on ice.
12.
Dissolve the pellet in RNase-free water.
13.
Load 1 μL of the reaction mixture (synthesized RNA probe) with DNA loading buffer according to manufacturer’s instruction in 0.7% agarose gel.
14.
Store the remaining reaction mixture at −30 °C.
15.
Perform electrophoresis using 1× TAE buffer.
16.
Stain the agarose gel using the nucleic acid stain reagent according to the manufacture’s instruction.
17.
Confirm the RNA probe is efficiently synthesized. You should see thick band of RNA probe.

3.3 Whole Mount In Situ Hybridization (BCIP-NBT Detection)

Specificity of RNA probes and the RNA expression pattern of the gene of interest had better to be first determined by NCBI-NBT detection. Since the fluorescent detection reaction using TSA system is very quick (the time window of the reaction is 5–10 min), nonspecific signals can easily be obtained. Thus, the conditions of fluorescent detection, such as RNA probe concentration and duration of detection reaction, should be optimized for each gene, by comparison of the signals of RNA expression detected by NCBI-NBT.

Day 1

1.
Prepare 100% EtOH in 35 mm glass petri dishes at room temperature (RT).
2.
Transfer specimens from −30 °C 100% MetOH to glass dishes containing 100% EtOH.
3.
Replace the solution to 50% xylene in EtOH in a chemical safety hood (see Note 6).
4.
Keep the petri dishes in a chemical safety hood at RT for 30 min.
5.
Prepare ice-cold 100% EtOH in each well of a 12-well plate.
6.
Transfer a specimen to each well.
7.
Gently shake the plate for 15 min at 4 °C.
8.
Replace the solution to ice-cold 75% EtOH in 1/4 HS.
9.
Gently shake the plate for 15 min at 4 °C.
10.
Replace the solution to ice-cold 50% EtOH in 1/4 HS.
11.
Gently shake the plate for 15 min at 4 °C.
12.
Replace the solution to ice-cold 25% EtOH in 1/4 HS.
13.
Gently shake the plate for 15 min at 4 °C.
14.
Replace the solution to ice-cold 1/4 HS.
15.
Gently shake the plate for 5 min at 4 °C.
16.
Fix specimen with ice-cold fixative solution for 30 min at 4 °C.
17.
Replace the solution to ice-cold TPBS.
18.
Gently shake the plate for 15 min at 4 °C.
19.
Replace the solution to hybridization solution.
20.
Incubate the plate at 50 °C (42–55 °C) with gentle rocking with a seesaw-like motion for 1 h using Hybridization incubator (e.g., TAITEC).
21.
Replace the solution in each well with 1 mL of hybridization solution containing RNA probe (see Note 7).
22.
Incubate the plate at 50 °C (42–55 °C) with gentle rocking with a seesaw-like motion for overnight.

Day 2

23.
Warm wash buffer to 50 °C (42–55 °C).
24.
Replace the solution with the prewarmed wash buffer.
25.
Gently rock a plate with a seesaw-like motion at 50 °C (42–55 °C) for 5 min using Hybridization incubator.
26.
Repeat steps 24 and 25 for 2 times (wash using wash buffer 3 times at 50 °C (42–55 °C), in total).
27.
Warm wash buffer to 65 °C during step 4.
28.
Replace the solution with 65 °C wash buffer.
29.
Gently rock plate with a seesaw-like motion at 65 °C (55–65 °C) for 10 min.
30.
Repeat steps 28 and 29 for 2 times (10 min × 3 times, in total).
31.
Replace solution with 50 °C (42–55 °C) wash buffer.
32.
Gently rock the plate with a seesaw-like motion at 50 °C (42–55 °C) for 30 min.
33.
Repeat steps 31 and 32 at least 11 times (at least 30 min × 12 times, in total).
34.
Replace solution with 1 mL of Buffer I.
35.
Gently rock the plate at RT for 5 min.
36.
Repeat steps 34 and 35 for 2 times (5 min × 3 times, in total).
37.
Replace solution to Buffer II.
38.
Incubate specimens for 1 h (pre-blocking).
39.
Replace solution to 1/2000 (v/v) anti-DIG-Alkaline phosphatase (AP) in Buffer II.
40.
Incubate at 4 °C overnight (about 16 h).

Day 3

41.
Replace solution to Buffer I.
42.
Gently shake the plate at RT for 5 min.
43.
Repeat steps 41 and 42 for 2 times (5 min × 3 times, in total).
44.
Change Buffer I.
45.
Gently shake the plate at RT for 60 min.
46.
Repeat steps 44 and 45 for at least 5 times (30 min × 6 times, in total).
47.
Replace the solution to TMN.
48.
Gently shake the plate at RT for 5 min.
49.
Repeat steps 47 and 48 for 2 times (5 min × 3 times, in total).
50.
Replace the solution to BCIP-NBT solution.
51.
Develop color at RT while preventing light exposure (shading) by wrapping a plate with aluminum foil until signals in each cell can be easily detected. Do not shake.
52.
Stop the coloring reaction by replacing the solution to TE at RT.
53.
Gently shake plate for 5 min.
54.
Repeat steps 52 and 53 for × 2 times (5 min × 3 times, in total).
55.
Examine signals using a high-resolution stereomicroscope.
56.
Take photos at least within several days.

3.4 Dual-Color Fluorescent Whole Mount In Situ Hybridization

1.
Prepare 100% EtOH in 35 mm glass petri dishes at room temperature (RT).
2.
Remove a gemmule coat from each sponge body using a sharpened tungsten needle (see Note 8).
3.
Follow the steps from steps 2 to 37 in Subheading 3.3, replacing the hybridization solution containing RNA probes in step 21 with a hybridization solution containing Dig-labeled RNA probe and biotin-labeled RNA probe.
4.
Incubate specimens with Buffer II by gently shaking at 4 °C overnight for pre-blocking.
5.
Transfer specimens on parafilm in the plastic dish.
6.
Cover the specimen with 100 μL the mixture of 1/100 (v/v) Anti-DIG-HRP in Buffer II.
7.
Keep the specimens at RT for 30–60 min.
8.
Add 2 mL TPBS in each well of 12 well plate.
9.
Transfer specimens to 12 well plate.
10.
Gently shake the plate at RT for 10 min.
11.
Change solution to TPBS.
12.
Gently shake the plate at RT for 10 min.
13.
Repeat steps 45 and 46 for 2 times (10 min × 3 times, in total).
14.
Transfer specimens on Parafilm in the plastic dish.
15.
Cover specimens with 100 μL of TSA mixture (e.g., Alexa 488) according to the manufacturer’s instructions.
16.
Keep the specimens at RT for 5–10 min while shading with aluminum foil, for first color detection. Hereafter, all the steps should be performed with shading.
17.
Add 2 mL TPBS in each well of 12-well plate.
18.
Transfer specimens to 12-well plate.
19.
Gently shake the plate at RT for 10 min.
20.
Change to TPBS.
21.
Gently shake the plate at RT for 10 min.
22.
Repeat steps 54 and 55 for 2 times (10 min × 3 times, in total).
23.
Replace the solution to 1 mL 1% H₂O₂ in TPBS.
24.
Gently shake at RT for 30 min.
25.
Replace the solution to 1 mL Buffer II for secondary pre-blocking.
26.
Gently shake at RT for 30 min.
27.
Transfer specimens on parafilm in the plastic dish.
28.
Cover the specimens with 100 mL 1% (v/v) peroxidase-conjugated streptavidin (see Note 9).
29.
Keep the specimens at RT for 30–60 min.
30.
Add 2 mL TPBS in each well of 12 well plate.
31.
Transfer specimens to 12 well plate.
32.
Gently shake the plate at RT for 10 min.
33.
Change solutions to TPBS.
34.
Gently shake the plate at RT for 10 min.
35.
Repeat steps 67 and 68 for 2 times (10 min × 3 times, in total).
36.
Transfer specimens on parafilm in the plastic dish.
37.
Cover specimens with 100 μL of TSA mixture (e.g., Alexa 594) according to the manufacturer’s instructions.
38.
Keep the specimens at RT for 5–10 min for second color detection.
39.
Repeat steps 65–70.
40.
Add 100 mL of nuclear staining dye to each specimen.
41.
Keep at RT for 30 min.
42.
Add 2 mL TPBS in each well of 12 well plate.
43.
Transfer specimens to 12 well plate.
44.
Gently shake the plate at RT for 10 min.
45.
Mount specimens on glass slides with antifade reagent.
46.
Examine the fluorescent signals using fluorescence microscopy.

4 Notes

1.
Gemmules were isolated as described previously [1]. For the samples for WISH and FISH, gemmules were cultivated on round coverslips (e.g., Fisherbrand), and allowed to develop in M-medium. Developmental stages of juvenile sponges were determined as described previously [1].
2.
High quality of template DNA is necessary for synthesis of high-quality RNA probes. Digested linearized plasmid DNA should be purified by phenol/chloroform extraction followed by EtOH precipitation to eliminate enzyme(s).

PCR DNA fragments should be purified by agarose gel electrophoresis followed by extraction from the gel to eliminate the template circular DNA that was used for PCR. Alternatively, plasmid DNA (G^mATC methylated DNA) in the reaction mixture of PCR can be specifically digested by DpnI (e.g., Takara Bio), and then PCR fragments can be column purified using a Gene gel/PCR Extraction kit (e.g., Nippon Genetics). Do not use the PCR reaction solution without such purification, because contamination by the RNA that was synthesized using the PCR template could cause nonspecific signals in WISH or FISH.
3.
To prepare RNase-free glass dishes, cover the glass dishes with aluminum foil and heat them at 200 °C for 2 h to eliminate RNase.
4.
For negative control probe, we usually prepare sense RNA probe of gene of interest, or sense RNA probe of GFP gene.
5.
For dual color FISH, use Biotin RNA Labeling Mix (Sigma-Aldrich) instead of Dig NTP mix for the second RNA probe.
6.
Since xylene dissolves plastics, steps 2 and 3 of Day 1 should be performed in RNase-free glass dishes.
7.
We generally use 1 μL of the reaction mixture of RNA probe synthesis in 1 mL of hybridization solution. When mRNA expression of the gene of interest seems to be high (too quick development of color during detection), we optimize the concentration of the reaction mixture of RNA probe synthesis from 0.1 to 1 μL/mL of hybridization solution. If the RNA expression of the gene of interest is low, the reaction mixture of RNA probe synthesis might be increased up to 10 μL/mL hybridization solution.
8.
Removing the gemmule coat from the sponge body is useful to avoid autofluorescence for FISH. Removing gemmules is not necessary for WISH .
9.
For dual color Fish detection, we previously used a biotin-labeled RNA probe and anti-biotin antibody [2, 4, 12], but the commercial production of anti-biotin antibody was terminated. Thus, we re-examined possible combinations of a nucleotide analogue and its detection (antibody or chemical binding between biotin and streptavidin, Fig. 1). Although the biotin and streptavidin-HRP combination can give high background signals in several freshwater organisms such as planarians (personal communication), that was not the case in juveniles of E. fluviatilis , and EflMusashiA-expressing archeocytes and EflSilicateinM1-expressing sclerocytes could be specifically detected (Fig. 2). Thus, recently we are using the combination of biotin-labeled RNA probe and streptavidin-HRP [3].

References

Funayama N, Nakatsukasa M, Mohri K, Masuda Y, Agata K (2010) Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges. Evol Dev 12:275–287
Article CAS Google Scholar
Funayama N (2010) The stem cell system in demosponges: insights into the origin of somatic stem cells. Dev Growth Differ 52:1–14
Article CAS Google Scholar
Funayama N (2013) The stem cell system in demosponges: suggested involvement of two types of cells: Archeocytes (active stem cells) and choanocytes (food-entrapping flagellated cells). Dev Genes Evol 223:23–38
Article Google Scholar
Funayama N (2018) The cellular and molecular bases of the sponge stem cell systems underlying reproduction, homeostasis and regeneration. Int J Dev Biol 62:513–525
Article CAS Google Scholar
Simpson TL (1984) The cell biology of sponges. Springer-Verlag, New York
Book Google Scholar
Ereskovsky A (2010) The comparative embryology of sponges. Springer, Berlin
Book Google Scholar
Leys SP, Ereskovsky AV (2006) Embryogenesis and larval differentiation in sponges. Can J Zool 84:262–287
Article Google Scholar
Funayama N, Nakatsukasa M, Hayashi T, Agata K (2005a) Isolation of the choanocyte in the fresh water sponge, Ephydatia fluviatilis and its lineage marker, Ef annexin. Dev Growth Differ 47:243–253
Article CAS Google Scholar
Mohri K, Nakatsukasa M, Masuda Y, Agata K, Funayama N (2008) Toward understanding the morphogenesis of siliceous spicules in freshwater sponge: differential mRNA expression of spicule-type-specific silicatein genes in Ephydatia fluviatilis. Dev Dyn 237:3024–3039
Article CAS Google Scholar
Nakayama S, Arima K, Kawai K, Mohri K, Inui C, Sugano W, Koba H, Tamada K, Nakata YJ, Kishimoto K, Arai-Shindo M, Kojima C, Matsumoto T, Fujimori T, Agata K, Funayama N (2015) Dynamic transport and cementation of skeletal elements building up pole-and-beam structured skeleton of sponges. Curr Biol 25:2549–2554
Article CAS Google Scholar
Funayama N, Nakatsukasa M, Kuraku S, Takechi K, Dohi M, Iwabe N, Miyata T, Agata K (2005b) Isolation of Ef silicatein and Ef lectin as molecular markers for sclerocytes and cells involved in innate immunity in the fresh water sponge, Ephydatia fluviatilis. Zoolog Sci 22:1113–1122
Article CAS Google Scholar
Okamoto K, Nakatsukasa M, Alié A, Masuda Y, Agata K, Funayama N (2012) The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state. Mech Dev 129:24–37
Article CAS Google Scholar
Alié A, Hayashi T, Sugimura I, Manuel M, Sugano W, Mano A, Satoh N, Agata K, Funayama N (2015) The ancestral gene repertoire of animal stem cells. Proc Natl Acad Sci 112(51):E7093–E7100
Article Google Scholar

Download references

Acknowledgments

I thank Drs. Simon Blanchoud and Brigitte Galliot for giving me the opportunity to contribute to this book. I also thank Dr. Elizabeth Nakajima for her proofreading of the manuscript. This work was supported by MEXT/JSPS KAKENHI grants 20H05942, 19H00994, and 17KT0019 to N. F.

Author information

Authors and Affiliations

Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, Japan
Chiaki Kojima & Noriko Funayama

Authors

Chiaki Kojima
View author publications
You can also search for this author in PubMed Google Scholar
Noriko Funayama
View author publications
You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Noriko Funayama .

Editor information

Editors and Affiliations

Department of Biology, University of Fribourg, Fribourg, Fribourg, Switzerland
Simon Blanchoud
Dept Génétique et Evolution, Université de Genève, Genève, Geneva, Switzerland
Brigitte Galliot

Rights and permissions

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Reprints and permissions

Copyright information

About this protocol

Cite this protocol

Kojima, C., Funayama, N. (2022). In Situ Hybridization to Identify Stem Cells in the Freshwater Sponge Ephydatia fluviatilis . In: Blanchoud, S., Galliot, B. (eds) Whole-Body Regeneration. Methods in Molecular Biology, vol 2450. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2172-1_17

Download citation

DOI: https://doi.org/10.1007/978-1-0716-2172-1_17
Published: 01 April 2022
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2171-4
Online ISBN: 978-1-0716-2172-1
eBook Packages: Springer Protocols

Publish with us

Policies and ethics