Studying Annelida Regeneration in a Novel Model Organism: The Freshwater Aeolosoma viride

Chen, Chiao-Ping; Fok, Sheridan Ke-Wing; Chen, Cheng-Yi; Hsu, Fei-Man; Hsieh, Yu-Wen; Chen, Jiun-Hong

doi:10.1007/978-1-0716-2172-1_9

Chiao-Ping Chen⁴^na1,
Sheridan Ke-Wing Fok⁴^na1,
Cheng-Yi Chen⁴^na1,
Fei-Man Hsu⁴^na1,
Yu-Wen Hsieh⁴^na1 &
…
Jiun-Hong Chen⁴^na1

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

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Abstract

Aeolosoma viride, a globally distributed freshwater annelid, has a semitransparent appearance with 10 to 12 segments, about 2 to 3 mm in length. It is easy to raise and handle in laboratory conditions. Due to its robust regenerative capacity and applicability of various molecular tools including EdU labeling, whole-mount in situ hybridization (WISH), and RNA interference (RNAi), it rises as a promising model for studying whole-body regeneration.

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Key words

1 Introduction

Earthworms are known form their regenerative capabilities [1, 2]. However, after testing several earthworm species in Taiwan, we could not find a robust model for studying annelid regeneration. The possibility of interrogating annelid regeneration came true with the presence of Aeolosoma viride . The discovery of this worm was an unexpected event when we collected Daphnia sp. from water ponds at National Taiwan University. Since these annelids exhibit asexual fission, and regeneration is recognized as one type of asexual reproduction [3], we inferred that this annelid may have regenerative abilities, which, to our knowledge, was not systematically documented previously. We tested its regenerative capability by amputating at the foregut-midgut or midgut-hindgut junctions (Fig. 1). Surprisingly, we found that this annelid can regrow anterior and posterior segments within 1 week.

A. viride is a semitransparent, freshwater annelid of length 2–3 mm, comprising 10–12 segments [4]. Phylogenetically, A. viride is defined as a “clitellate-like polychaete ” [5, 6]. Consistent with previous reports, A. viride reproduces exclusively by paratomic fission under our laboratory conditions (Fig. 1) [3, 7]. Paratomy is a form of agametic reproduction that produces multiple zooids simultaneously by fission in posterior segment. Species reproducing by paratomy have different regenerative capacities to regenerate anterior and posterior segments [8, 9]. Given their small size and transparency, as well as their strong regenerative ability together with applicability of various molecular tools including EdU labeling, whole-mount in situ hybridization (WISH), and RNA interference (RNAi) [4, 10], we anticipate that A. viride will be informative in comparative studies focused on whole-body regeneration. In this chapter, we will provide detailed steps on how to manipulate and conduct this novel model in regenerative research.

2 Materials

All solutions are prepared using analytical grade reagents and dissolved in deionized ultrapure water at room temperature (RT).

2.1 Regeneration in Aeolosoma viride

1.
Artificial spring water (ASW): 48 mg/L NaHCO₃, 24 mg/L CaSO₄•2H₂O, 30 mg/L MgSO₄ • 7H₂O, 2 mg/L KCl. Adjust to pH 7.4 using 1 M HCl, sterilize before use.
2.
1× phosphate buffered saline (PBS): 40 g/L NaCl, 1 g/L KCl, 7.2 g/L Na₂HPO₄, 1.2 g/L KH₂PO₄. Sterilize before use.
3.
Ground oatmeal.
4.
Saturated menthol in ASW (see Note 1).
5.
4% (w/v) paraformaldehyde (PFA) in saturated menthol (see Note 2).
6.
4% (w/v) PFA in 1× PBS.
7.
Mounting solution (e.g., 100% glycerol).
8.
Sterile needles 27 G × 1/2″.
9.
Microscope glass slides (e.g., 8 well-slide).
10.
Culture plate (e.g., 6, 12 or 24 wells).
11.
25 °C incubator.
12.
Dissection microscope (e.g., WILD M8, Leica).
13.
DIC microscope.

2.2 Cell Proliferation Assay

1.
0.5% (v/v) Triton X-100 in 1× PBS.
2.
PBS-T: 0.1% (v/v) Triton X-100 in 1× PBS.
3.
0.1 mM 5-ethynyl-2′-deoxyuridine (EdU) in 10% (v/v) DMSO.
4.
3% (w/v) bovine serum albumin (BSA) in 1× PBS.
5.
EdU Imaging kit (e.g., Click-iT^® EdU Imaging kit, Invitrogen).
6.
18 mg/mL Hoechst 33342.
7.
Mounting solution (e.g., Fluoromount-G™, eBioscience).
8.
1× EdU buffer additive: 2 μL 10× ascorbic acid stock solution, 18 μL ddH₂O. Prepare fresh every time.
9.
EdU reaction cocktail: 172 μL 1× EdU reaction buffer, 8 μL 100 mM CuSO₄, 0.48 μL Alexa Fluor^® azide, 20 μL 1× EdU buffer additive. Prepare fresh every time.
10.
Fluorescent microscope.

2.3 Whole-Mount In Situ Hybridization (WISH)

1.
Primers for target gene.
2.
T7 primer.
3.
Trizol (e.g., TRIzol reagent).
4.
Chloroform.
5.
Isopropanol.
6.
0.0001% (v/v) diethyl pyrocarbonate (DEPC)-H₂O.
7.
75% ethanol in DEPC-H₂O.
8.
Reverse transcriptase (e.g., SuperScript III).
9.
DNA polymerase (e.g., SuperTherm Taq).
10.
Thymine and adenine (TA) cloning vector (e.g., Yeastern yT&A).
11.
T7 RNA polymerase (e.g., Ambion).
12.
RNA digoxigenin (DIG) labeling mix (e.g., Roche).
13.
DNase I (e.g., Promega).
14.
0.5 M EDTA in DEPC-H₂O, pH 8.4.
15.
6 M lithium chloride (LiCl) in DEPC-H₂O.
16.
Anhydrous alcohol.
17.
1× PBS in DEPC-H₂O.
18.
10 μg/mL proteinase K in PBS-T: dilute from 10 mg/mL proteinase K stock solution. Prepare fresh before use.
19.
20× saline sodium citrate (SSC): 3 M NaCl, 0.3 M sodium citrate in DEPC-H₂O.
20.
HYB⁺ buffer: 5 mL 100% formamide, 2.5 mL 20× SSC, 0.1 mL 5 mg/mL heparin, 0.1 mL 50 mg/mL torula RNA type VI, 92 μL 1 M citric acid, 0.1 mL 10% tween-20 in DEPC-H₂O. Add 2.108 mL DEPC-H₂O to a final 10 mL solution, store at 4 °C.
21.
HYB⁻ buffer: HYB⁺ buffer without heparin and torula RNA.
22.
HYB⁺ W/O: HYB⁺ without DEPC-H₂O.
23.
0.1% tween-20 in DEPC- H₂O.
24.
2× SSCTW: 10% (v/v) 20× SSC in 0.1% tween-20.
25.
0.2× SSCTW: 1% (v/v) 20× SSC in 0.1% tween-20.
26.
5% (w/v) BSA in PBS-T.
27.
1:5000 anti-DIG-alkaline phosphatase (AP) Fab fragments in 5% BSA. Prepare fresh every time.
28.
Staining buffer: 100 mM Tris-HCl, pH 9.0, 150 mM NaCl, 1 mM MgCl₂ in DEPC-H₂O. Prepare fresh every time before use.
29.
Alkaline phosphatase substrate: 50 mg/mL nitro blue tetrazolium (NBT), 50 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). Prepare fresh every time.
30.
65 °C incubator.

2.4 RNA Interference (RNAi)

1.
PCR product of target gene.
2.
L4440 plasmid.
3.
HT115 (DE3) competent cells.
4.
Sterilized LB broth.
5.
20 mg/mL ampicillin in ddH₂O.
6.
LB agar plate: 1.5% (w/v) agar with 20 μg/mL ampicillin in LB broth. Pour hot in 9 cm petri dishes, store at 4 °C after solidification.
7.
1 M IPTG in ddH₂O.
8.
Electroporation system (e.g., Bio-Rad Pulse Controller Plus).
9.
Glass capillary: borosilicate thin wall with filament, outside diameter 1.0 mm, inside diameter 0.78 mm, length 150 mm.
10.
Needle puller (e.g., P-97, Sutter Instrument).
11.
Microinjector (e.g., Nanoliter 2000 microinjector).
12.
1.5% agarose-based plates: 1.5% (w/v) agar in ASW. Pour hot in 6 cm petri dishes, store at 4 °C after solidification.

3 Methods

3.1 Animal Husbandry

1.
Raise worms (see Note 3) in ASW under a regime of 12:12 h day–night cycles at 22 ± 1 °C (see Note 4).
2.
Provide 20 mg ground oatmeal (dry weight) to 500 ± 200 worms three to five times per week.
3.
Replace one-half to one-fifth of old ASW with new ASW every week (see Note 5).
4.
Check weekly the cultures for healthy 2–3 mm long actively swimming worm populations.
5.
Discard unhealthy cultures.
6.
If necessary, pick healthy worms in fresh ASW to create new culture.

3.2 Regeneration in Aeolosoma viride

1.
Move worms to fresh tap water for 10 min to remove coexisting protozoa.
2.
Move the worms into ASW.
3.
Starve overnight to excrete their ingested food.
4.
To synchronize the growth phase of posterior segments and remove potential fission progeny, amputate worms at the segment anterior to the fission zone by sterile needles on microscope glass slide under stereo microscope (Fig. 1).
5.
Move the amputated worms into ASW for synchronization.
6.
Keep at 25 °C for 3 days.
7.
Amputate the worms again at the fourth segment for anterior regeneration experiment. or at the segment posterior to the midgut for posterior regeneration experiment.
8.
Transfer the regenerating animals to fresh ASW in culture plate.
9.
Keep at 25 °C for subsequent experiment and observation (see Notes 6 and 7).

3.3 Animal Fixation

1.
Place a maximum of 10 intact or amputated worms in 20 μL ASW per well on a 8 well-slide under a dissection microscope.
2.
Wash each well twice with 20 μL ASW.
3.
Add 20 μL cold (4 °C) saturated menthol for anaesthetization.
4.
Gently pipette for around 20 s to prevent the worms from curling up.
5.
Remove 20 μL of the menthol solution.
6.
Add 20 μL 4% PFA in saturated menthol.
7.
Pipette gently for around 30 s.
8.
Move the worms into 1.5 mL microcentrifuge tube with 200 μL 4% PFA in PBS.
9.
Keep at 4 °C until further treatment.
10.
Transfer to a microscope glass slide using a pipette.
11.
Mount the worms in mounting solution on the slide.
12.
Monitor its morphology using a DIC microscope (Fig. 2).

3.4 Cell Proliferation Assay

1.
Incubate worms with 0.1 mM EdU in culture plate at 25 °C for 12 h (see Notes 8 and 9).
2.
Follow steps 1 to 8 in Subheading 3.3 to fix the animals.
3.
Wash the worms with PBS-T five times, for 5 min each time.
4.
Remove the PBS-T.
5.
Incubate with 3% BSA for 5 min.
6.
Remove the 3% BSA.
7.
Incubate with 0.5% Triton X-100 for 20 min.
8.
Remove the 0.5% Triton X-100.
9.
Wash twice with 3% BSA, for 5 min each time.
10.
Remove the 3% BSA.
11.
Add EdU reaction cocktail, incubate in the dark for 30 min.
12.
Remove the EdU reaction cocktail.
13.
Wash five times with PBS-T, for 5 min each time.
14.
Remove the PBS-T.
15.
Add 18 ng/μL Hoechst 33342, incubate in the dark for 30 min.
16.
Remove the Hoechst solution.
17.
Wash the worms five times with PBS-T, for 5 min each time.
18.
Mount the worms in mounting solution on slide.
19.
Images are collected using a fluorescent microscope (Fig. 3).

3.5 Whole-Mount In Situ Hybridization (ISH)

1.
Design gene specific primers for target gene according to an unpublished transcriptome database from A. viride . Target length is around 200–800 bp. For example, Avi-caspase X [11].
2.
Place a maximum of 50 intact or amputated worms in 200 μL ASW per tube.
3.
Wash twice with ASW, for 5 min each time.
4.
Remove the ASW and add 200 μL TRIzol.
5.
Vortex for 20–40 s until the tissue is completely liquefied without visible particles.
6.
Add 40 μL chloroform.
7.
Gently invert 10 times.
8.
Keep at 25 °C for 15 min.
9.
Centrifuge tubes at 10,000 rcf at 4 °C for 15 min.
10.
Transfer 120 μL of the clear supernatant to a new tube.
11.
Add 120 μL isopropanol.
12.
Gently invert 10 times.
13.
Incubate for 30 min at −20 °C to precipitate total RNA.
14.
Centrifuge tubes at 10,000 rcf at 4 °C for 30 min.
15.
Remove the supernatant carefully.
16.
Wash twice with 200 μL cold (−20 °C) 75% ethanol, for 5 min each time.
17.
Remove the supernatant carefully and air dry.
18.
Dissolve RNA pellet with 10 μL DEPC-H₂O.
19.
Quantify the RNA concentration.
20.
Synthesize cDNA from 0.1–5 μg total RNA using a reverse transcriptase.
21.
Amplify DNA with gene specific primers and DNA polymerase by using PCR.
22.
Clone riboprobe fragment into TA vector.
23.
Select sense and antisense clone from TA vector.
24.
Amplify DNA with T7 primers and DNA polymerase by using PCR.
25.
Synthesize sense and antisense RNA probe using T7 RNA polymerase with RNA DIG labeling mix.
26.
Digest DNA template with DNase I at 37 °C, for 20 min.
27.
Mix with EDTA, LiCl and anhydrous alcohol sequentially to reach 50 mM, 100 mM, and 75% final concentration.
28.
Precipitate sense and antisense RNA probes at −20 °C for 30 min.
29.
Repeat steps 14–18 to produce RNA probe.
30.
Quantify the RNA concentration.
31.
Add HYB⁺ W/O to store at −20 °C.
32.
Wash the fixed worms in microcentrifuge tube (see Subheading 3.3) five times with PBS-T, for 5 min each time.
33.
Dilute proteinase K with PBS-T to 10 μg/mL.
34.
Treat the worms with diluted proteinase K at room temperature for 10 min.
35.
Replace proteinase K with 4% PFA.
36.
Incubate at room temperature for 20 min.
37.
Remove 4% PFA.
38.
Wash five times with PBS-T, for 5 min each time.
39.
Replace with HYB⁺ buffer.
40.
Incubate the microcentrifuge tube 65 °C for 1 to 3 h (see Note 10).
41.
Hybridize worms with sense or antisense riboprobes (1 to 3 ng/μL) in HYB⁺ at 65 °C for 16–24 h (see Note 11).
42.
Remove HYB⁺ buffer with riboprobes under dissection microscope carefully.
43.
Add HYB⁻ buffer at 65 °C for 5 min.
44.
Remove HYB⁻ buffer under dissection microscope carefully.
45.
Transfer serially (5 min each time) into 66%, and 33% HYB⁻ buffer in 2× SSCTW at 65 °C.
46.
Remove supernatant.
47.
Wash the worms with 2× SSCTW at 65 °C for 5 min.
48.
Remove 2× SSCTW.
49.
Wash twice in 0.2× SSCTW at 65 °C, for 15 min each time.
50.
Remove 0.2× SSCTW.
51.
Transfer serially (for 5 min each time) into 66%, and 33% 0.2× SSCTW in PBS-T at room temperature.
52.
Remove supernatant.
53.
Wash the worms with PBS-T at room temperature for 5 min.
54.
Block with 5% BSA at 4 °C overnight or at room temperature for 2 h.
55.
Dilute anti-DIG-AP Fab fragments 1:5000 with 5% BSA.
56.
Remove 5% BSA.
57.
Incubate overnight with 1:5000 anti-DIG-AP Fab fragments at 4 °C.
58.
Remove anti-DIG-AP Fab fragments.
59.
Wash ten times with PBS-T, for 5 min each time.
60.
Prepare fresh staining buffer.
61.
Remove PBS-T.
62.
Wash the worms twice with staining buffer, for 5 min each time.
63.
Prepare fresh staining solution, add NBT (final 220 μg/mL) and BCIP (final 170 μg/mL) to staining buffer in the dark.
64.
Remove the staining buffer.
65.
Stain the worms with staining solution at 4 °C or room temperature in the dark.
66.
Check the staining status under a dissection microscope.
67.
Mount the worms in mounting solution.
68.
Images are collected using a microscope (Fig. 4).

3.6 RNA Interference (RNAi) by Feeding Method

1.
Design 300 bp gene specific primers for yellow fluorescent protein (YFP, as control group) and target gene. For example, Avi-beta tubulin isoform 1 [4].
2.
Amplify DNA with gene specific primers and DNA polymerase by using PCR.
3.
Clone DNA fragment into L4440 plasmid (see Note 12).
4.
Transform the L4440 vector (1 μL containing 100 ng) into 50 μL HT115 (DE3) competent cell through electroporation system under the following conditions: 2.0 kV, 100 Ω, and 25 μF.
5.
Add 200 μL of LB broth to a cuvette.
6.
Mix gently by pipetting.
7.
Transfer to 1.5 mL microcentrifuge tube.
8.
Shake vigorously (200 rpm) on an orbital shaker at 37 °C for 1 h.
9.
Dilute the cells 20 times with LB broth.
10.
Spread cells on LB agar plates.
11.
Incubate at 37 °C for 16 h to overnight.
12.
Pick single colony from LB plate into an LB broth containing 20 μg/mL ampicillin.
13.
Incubate at 37 °C for 16 h.
14.
Subculture at a 1:100 dilution with shaking until OD₆₀₀ reaches 0.4–0.6.
15.
Add 1 M IPTG to reach 0.1 mM final concentration.
16.
Express target dsRNA at 37 °C for 4 h.
17.
Collect the bacteria expressing the dsRNA into 2 mL tubes.
18.
Spin at 3000 rcf for 1 min.
19.
Replace the supernatant with fresh LB broth.
20.
Repeat steps 18 and 19 to wash bacteria a second time.
21.
Dilute 1 μL of bacteria in 99 μL of LB broth.
22.
Plate the serially diluted bacteria on an LB agar plate.
23.
Incubate at 37 °C for 16 h.
24.
Count the number of colony forming units (CFU).
25.
This is the number of CFU per μL in the original solution.
26.
Place the undiluted bacteria in their 2 mL tubes on a hot plate at 100 °C for 10 min.
27.
Cool the tubes on ice for 5 min.
28.
Store the bacteria at −20 °C (see Note 13).
29.
Fed worms with 1 × 10⁸ CFU/mL bacteria containing dsRNA for three consecutive days, renewed every 24 h.
30.
To validate the knock-down efficiency of the target gene, real time quantitative polymerase chain reaction (RT-qPCR) or ISH (see Subheading 3.5) can be performed after the feeding procedure (see Note 14).
31.
Mount the fed worms in mounting solution.
32.
Images are collected using a microscope (Fig. 5).

3.7 RNAi by Microinjection

1.
Produce control and target dsRNA from L4440 vector (follow steps 1 and 2 in Subheading 3.6 and steps 23–26 in Subheading 3.5).
2.
Dissolve RNA pellet with DEPC-H₂O.
3.
Store at −20 °C.
4.
Pull the microinjection pipette with the following settings: pressure 500, heat 640, pull 125.
5.
Set the microinjector in slow mode with an injection volume of 27.6 nL. Each injection will contain roughly 100 ng dsRNA.
6.
Open the tip of the microinjecting pipette using a pair of tweezers.
7.
Load the dsRNA into the microinjecting pipette.
8.
Move the worms onto a 1.5% agarose-based plate.
9.
Remove ASW to limit movements of the worms on the agar surface.
10.
Inject the dsRNA at the fourth segment of the worms (see Note 15) for two consecutive days to optimize intake of the dsRNA.
11.
Allow the worms to recover in fresh ASW for 24 h after the final injection .
12.
Validate the knock-down efficiency of the target gene by RT-qPCR or ISH (see Subheading 3.5).
13.
Mount the injected worms in mounting solution.
14.
Images are collected using a microscope (Fig. 5).

4 Notes

1.
Preparation of saturated menthol in ASW by add 0.2 g menthol in 10 mL ASW, and placed at 4 °C for more than 24 h, make sure at least a piece of crystal menthol is still visible in the saturated solution before use.
2.
Prepare 4% (w/v) PFA by adding 2 g PFA in 40 mL ASW, mix well and adjust to pH 11 using 1 M NaOH to help dissolution. Incubate at 65 °C overnight, adjust to pH 7.4 and then add ASW to a final volume of 50 mL. Finally, filter this 4% (w/v) PFA by 0.22 μm membrane and store at 4 °C.
3.
Sample worms for initiating a laboratory culture of A. viride can be obtained from our lab, from Dr. Dian-Han Kuo at National Taiwan University or from Dr. Chia-Liang Cheng at National Dong Hwa University. Please contact the corresponding author if interested.
4.
In our laboratory, A. viride was cultured in a glass bottle with ASW, the suitable volume of ASW filling about 70% capacity of the glass bottle. For bulk culture, we initially mix 200 worms in 30 mL ASW with 320 mL ASW in a 500 mL glass bottle. After 3 weeks, we can harvest around 30 times more worms for the experiment. In the meantime, we will change one-half to one-fifth of fresh ASW according to the water quality every week.
5.
One habitual behavior of this worm is to adhere to the wall of the glass container. Therefore, most A. viride will remain on the wall when the old ASW is poured out.
6.
Two morphological and behavioral criteria were used to confirm complete regeneration: (1) bulged head formation with a circular mouth at the ventral side of the peristomium; and (2) free swimming.
7.
More than 80% of worms can complete anterior regeneration from the remaining 6 or 9 segments at 120 h postamputation (hpa) [1].
8.
Both bromodeoxyuridine (BrdU) and EdU are commonly used reagents for incorporation in most vertebrates and invertebrates [12, 13]. However, we found that BrdU labeling did not work well in A. viride .
9.
Commonly applied concentrations and duration of EdU labeling are 1–10 μM for 0.5–5 h in cell lines and annelids [14, 15]. However, these conditions did not work for A. viride . Instead, we used a high concentration (100 μM) and long incorporation time (12 h), which resulted in the regenerating blastema being observed at 48 hpa (Fig. 3).
10.
The hybridization temperature commonly used in our lab was 65 °C, however, it depends on the condition of each riboprobe.
11.
The probe concentration commonly used in our lab was 1 ng/μL, however, it depends on the concentration of each riboprobe. Typically, we use 3 ng/μL of newly synthesized riboprobe.
12.
There are various ways to produce double-stranded RNA. The most common way is to amplify the target gene fragment with PCR using specific primers that contain SP6, T3, or T7 promoters. Then, both strands of the gene fragment can be synthesized using specific in vitro transcription enzymes. Finally, the two products can be mixed to form dsRNA. This approach has the benefit that each strand can be proofread by gel electrophoresis and the concentration of each strand can be varied. However, producing both strands separately using different enzymes is time-consuming. Both strands can be produced simultaneously using only T7 RNA transcriptase and L4440 vector. Gel electrophoresis can be used to separate the two strands, enabling validation that both strands have been synthesized successfully.
13.
To confirm the expression of dsRNA of the target gene, collect 1 mL cultured bacteria and lyse with Trizol to extract total RNA. Finally, assess the quality and length of the dsRNA by gel electrophoresis using 1% (w/v) agarose gel.
14.
Evaluate the knock-down efficiency of dsRNA by feeding or microinjection method through RT-qPCR and WISH (see Subheading 3.5). In RT-qPCR, Avi-actin was selected as internal control with specific primers: 5′-AT GGAGAAGATCTGGCATCA-3′ and 5′-GGAGTACTTGCGCTCAGGTG-3′ designed from Avi-actin (NCBI # KY079092.1). The gene expression relative to Avi-actin was calculated by the ΔΔCT method.
15.
The injected material diffuses through the entire body cavity of annelids because they lack internal septa.

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Author information

Chiao-Ping Chen and Sheridan Ke-Wing Fok have contributed equally to this work.

Authors and Affiliations

Department of Life Science, National Taiwan University, Taipei, Taiwan
Chiao-Ping Chen, Sheridan Ke-Wing Fok, Cheng-Yi Chen, Fei-Man Hsu, Yu-Wen Hsieh & Jiun-Hong Chen

Authors

Chiao-Ping Chen
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Sheridan Ke-Wing Fok
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Cheng-Yi Chen
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Fei-Man Hsu
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Yu-Wen Hsieh
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Jiun-Hong Chen
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Corresponding author

Correspondence to Jiun-Hong Chen .

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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

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Chen, CP., Fok, S.KW., Chen, CY., Hsu, FM., Hsieh, YW., Chen, JH. (2022). Studying Annelida Regeneration in a Novel Model Organism: The Freshwater Aeolosoma viride . 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_9

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DOI: https://doi.org/10.1007/978-1-0716-2172-1_9
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

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