Studying Xenacoelomorpha WBR Using Isodiametra pulchra

Abstract

Xenacoelomorpha are a phylogenetically and biologically interesting, but severely understudied group of worm-like animals. Among them, the acoel Isodiametra pulchra has been shown to be amenable to experimental work, including the study of stem cells and regeneration. The animal is capable of regenerating the posterior part of the body, but not its head. Here, methods such as nucleic acid extractions, in situ hybridisation, RNA interference, antibody and cytochemical stainings, and the general handling of the animals are presented.

1 Introduction

Xenacoelomorpha are one of the few remaining phyla with an unresolved, contested position in the Tree of Life. The group is either recovered as sister group of all other bilaterian animals, or as a member of Deuterostomia [1, 2]. Three groups constitute the Xenacoelomorpha: Xenoturbellida with 6 described species in one genus, Nemertodermatida with 18 described species, and Acoela with more than 300 described species being by far the largest and best known of the three groups. Their simple body plan—lacking a coelom, a circulatory system, a skeleton or respiratory organs other than the epidermis—can either be seen as plesiomorphic, or as a series of reductions [3]. In either case, they are an interesting and still poorly studied group of almost exclusively marine animals.

The regeneration capacity of the few studied xenacoelomorphs varies, where only a few species were shown to be able to completely regenerate their head, including brain and statocyst (a gravity sensing organ), such as Hofstenia miamia [4]. Regeneration capacity is possibly linked to the mode of reproduction, where obligatorily sexually reproducing species are often less capable of regeneration than asexually reproducing species. In different acoels, all modes of asexual reproduction occur: architomy (fission happens before new organs have been built), paratomy (fission happens after new organs have been built), and budding [5].

Regeneration, growth and homeostasis in acoels is powered by neoblast stem cells, the only proliferating cells in the body, located exclusively in the mesenchymal space and thus lacking in the epidermis [4,5,6,7].

One of the better studied acoels is Isodiametra pulchra , an animal less than a millimeter in length, transparent, bearing a single statocyst near the anterior end (Fig. 1). It belongs to the species-rich family Isodiametridae (comprising about 100 species), and can be cultured in large numbers in the laboratory. It is sexually reproducing, and cannot regenerate its head, but posterior body parts [8]. The following protocols are tested with adult and juvenile I. pulchra , or its close (and even smaller) relative, Aphanostoma pisae, or both [6,7,8,9,10,11,11].

Fig. 1

(a) Squeeze preparation of a live adult specimen of Isodiametra pulchra . (b) Same specimen as in a, nuclei of the epidermis stained blue with DAPI. Anterior is up. dp digestive parenchyma, eg developing eggs, fg female genital opening, mg male genital apparatus, mo mouth, mp male genital opening, st statocyst. Scale bar is 100 μm

In particular, RNA and DNA extraction, anesthesia, amputation, fixation, in situ hybridization (Fig. 2), RNA interference, and antibody and cytochemical stainings (Fig. 3) are covered in this chapter. While all methods included here have been published elsewhere, this chapter serves to bring them together in a compact format and to provide tricks and tips and notes on critical steps.

Fig. 2

Wholemount in situ hybridisation of an adult specimen of Isodiametra pulchra against Ipiwi1, a stem-cell gene. (Picture is courtesy of Thomas Zauchner). Anterior is up. Scale bar is 100 μm

Fig. 3

Confocal laser scanning projection of the body wall and genital musculature of Aphanostoma pisae stained with rhodamine-conjugated phalloidin. (Picture is courtesy of Thomas Zauchner). Anterior is up. mg male genital apparatus, sb seminal bursa. Scale bar is 50 μm

2 Materials

2.1 Nucleic Acid Extractions

Use nuclease-free (but not DEPC (diethyl pyrocarbonate)-treated) water. Only use nuclease-free sterile tubes, pestles and pipet tips. Only use molecular biology graded reagents. Work under the fume hood if indicated on the reagent’s safety data sheet.

• 1. Isodiametra pulchra worms (see Note 1) and culture system (see Note 2).

• 2. DNA/RNA extraction buffer (e.g., TRIzol, Thermo Fisher Scientific; TRI Reagent, Sigma-Aldrich): store at 4 °C.

• 3. Glycogen, nuclease-free (e.g., Thermo Fisher Scientific, AM9510). Keep at −20 °C.

• 4. Isopropanol (2-propanol).

• 5. 80% (v/v) Ethanol.

• 6. Sodium dodecyl sulfate (SDS) buffer: 0.5% (w/v) SDS, 200 mM Tris, 25 mM ethylenediaminetetraacetic acid (EDTA), 250 mM NaCl. Store at RT.

• 7. Protease XIV stock solution: 20 mg/mL protease XIV. Aliquot in 10 μL and store at −20 °C.

• 8. Protease XIV working solution: 1% (v/v) protease XIV stock solution in PBS-Tx. Prepare fresh.

• 9. 25:24:1 (v/v/v) Phenol–chloroform–isoamyl alcohol: either prepare the mixture yourself, or purchase a premixed solution. Store at 4 °C.

• 10. 3 M sodium acetate: 40.83 g sodium acetate in 80 mL deionized water (dH₂O). Adjust to pH 5.2 with glacial acetic acid, fill up to 100 mL with deionized water, autoclave and store aliquots at −20 °C.

2.2 Antibody and Cytochemical Stainings

There is no requirement to use purified water other than dH₂O.

• 1. Artificial seawater (ASW): 3.2% (w/v) aquarium salt in dH₂O. Mix well and let oxygenize for at least 6 h.

• 2. MgCl₂: 7% (w/v) MgCl₂ • 6 H₂O in dH₂O.

• 3. 10× PBS: 2.4 g KH₂PO₄, 14.4 g Na₂HPO₄, 2 g KCl, 80 g NaCl in 800 mL dH₂O. Adjust pH to 7.4 with HCl, fill up to 1 L with dH₂O, autoclave, store at RT.

• 4. Formaldehyde (FA): 4% (w/v) paraformaldehyde in 1× PBS. Dissolve at 53 °C for 1 h, shake every 15 min, adjust to pH 7.4 with HCl and NaOH and store 1 mL aliquots at −20 °C.

• 5. PBS-Tw: 0.1% (v/v) Tween in 1× PBS.

• 6. PBS-Tx: 0.1% (v/v) Triton X-100 in 1× PBS.

• 7. BSA-Tx: 1% (w/v) bovine serum albumin (BSA) in PBS-Tx. Dissolve BSA powder by stirring, store at 4 °C and renew solution every 2 weeks.

• 8. 5-bromo-2′-deoxyuridine (BrdU) stock solution: 50 mM BrdU in dH₂O. Store at −20 °C.

• 9. 5 mM BrdU working solution: 10% (v/v) 50 mM BrdU stock solution in ASW. Prepare fresh.

• 10. 5-Ethynyl-2′-deoxyuridine (EdU): prepare and store all solutions of the Click-iT kit according to manufacturer’s protocol (Thermo Fisher Scientific, C10337).

• 11. 0.4 mM EdU working solution: 25% (v/v) 10 mM EdU stock in ASW. Prepare fresh.

• 12. 2 M HCl: 16.6 mL 37% (v/v) HCl in 83.4 mL water.

• 13. Primary antibodies: mouse-anti-BrdU and rabbit-anti-pH 3.

• 14. Primary antibodies solution: mouse anti-BrdU antibody 1:600, rabbit anti-pH3 antibody 1:150 in BSA-Tx (see Note 3). Prepare fresh.

• 15. Secondary antibodies: goat anti-mouse FITC-conjugated and swine anti-rabbit TRITC-conjugated.

• 16. Secondary antibodies solution: goat anti-mouse FITC-conjugated 1:250, swine anti-rabbit TRITC-conjugated 1:250 in BSA-Tx (see Note 4). Prepare fresh.

• 17. TRITC-conjugated phalloidin.

• 18. 4′,6-Diamidino-2-phenylindole (DAPI).

• 19. Triple staining solution: 1:500 phalloidin TRITC-conjugated, 1:10,000 DAPI in Click-iT solution according to the manufacturer’s instructions. Prepare fresh.

• 20. Mounting medium (e.g., Vectashield, VectorLabs, or 80% (v/v) glycerol in PBS).

2.3 In Situ Hybridization and RNA Interference

All solutions are to be prepared with either nuclease-free or DEPC-treated water (1 mL DEPC per liter solution; stir over night and autoclave).

• 1. 100%, 75%, 50%, 25% methanol.

• 2. Proteinase K stock solution: 10 mg/mL proteinase K in 1× PBS-Tw. Aliquot in 10 μL and store at −20 °C.

• 3. 20 μg/mL proteinase K working solution: 10 μL proteinase K stock solution in 5 mL PBS-Tw. Prepare fresh right before use.

• 4. 4% (w/v) glycine stock solution: 4 g glycine in 100 mL. Filter-sterilize and store at 4 °C.

• 5. 4 mg/mL glycine working solution: 50 μL glycine stock solution in 450 μL PBS-Tw. Prepare fresh right before use.

• 6. 1 M triethanolamine (TEA) stock solution: 18.57 g TEA in 100 mL. Adjust pH to 7.8, Filter-sterilize and store at RT.

• 7. 0.25% acetic anhydride: 0.25% (v/v) acetic anhydride in TEA. Prepare fresh.

• 8. 0.5% acetic anhydride: 0.5% (v/v) acetic anhydride in TEA. Prepare fresh.

• 9. 1% (w/v) heparin: 1 g heparin in 100 ml. Filter-sterilize and store at −20 °C.

• 10. 10× 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) stock solution: 1 g CHAPS in 100 mL. Filter-sterilize, aliquot in 50 mL and store at −20 °C.

• 11. 20× saline sodium citrate buffer (SSC): 175.3 g NaCl, 88.2 g sodium citrate in 1 L. Adjust to pH 7.0, treat with 1 mL DEPC, stir over night, autoclave and store at RT.

• 12. SSC–CHAPS: 10% (v/v) 20× SSC, 10% (v/v) 10× CHAPS. Prepare fresh.

• 13. 50× Denhardt’s solution: 1% (w/v) nuclease-free BSA, 1% (w/v) Ficoll 400, 1% (w/v) PVP-40, stir strongly until dissolved. Store at −20 °C.

• 14. 1% tRNA stocks: 1 g commercially available tRNA in 100 mL, shake over night at 60 °C. Store 1 mL aliquots at −20 °C.

• 15. Hybridisation mix (hybmix): 1000 mL 100% formamide, 500 mL 20× SSC, 40 mL 1% tRNA, 2 mL 100% Tween, 200 mL 10× CHAPS, 40 mL 50× Denhardt’s, 20 mL 1% heparin, 198 mL water. Store in 50 mL aliquots at −80 °C. Can be stored for 1 month at −20 °C.

• 16. Hybmix/PBS-Tw: 50% (v/v) hybmix in PBS-Tw. Prepare fresh.

• 17. 75% (v/v), 50% (v/v), 25% (v/v) hybmix in 20× SSC. Prepare fresh.

• 18. 100 mM maleic acid buffer (MAB): 11.62 g maleic acid, 8.76 g NaCl in 1 L. Adjust pH to 7.5, treat with 1 mL DEPC overnight, autoclave, and store at RT.

• 19. 10× blocking solution: 10 g blocking reagent (Roche, 11,096,176,001) in MAB. Heat at 60 °C until dissolved, autoclave and store at −20 °C.

• 20. Alkaline phosphatase stock I: 1 M NaCl. Autoclave and store at RT.

• 21. Alkaline phosphatase stock II: 0.5 M MgCl₂ • 6H₂O. Autoclave and store at RT.

• 22. Alkaline phosphatase stock III: 1 M Tris. Adjust pH to 9.5, autoclave and store at RT.

• 23. Alkaline phosphatase buffer (NTMT): 5 mL alkaline phosphatase stock I, 5 mL alkaline phosphatase stock II, 5 mL alkaline phosphatase stock III, 50 μL Tween. Prepare fresh.

• 24. Anti-digoxigenin-alkaline phosphatase antibody (anti-DIG-AP antibody).

• 25. Anti-DIG-AP staining solution: 1:2000 anti-DIG-AP antibody in blocking solution. Prepare fresh every time.

• 26. NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) stock solution: 18.75 mg/mL NBT, 9.4 mg/mL BCIP, 67% (v/v) dimethyl sulfoxide (DMSO).

• 27. NBT/BCIP working solution: 15 μL NBT/BCIP stock solution, 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl in 1 mL. Prepare fresh.

• 28. In vitro RNA production kit.

• 29. 3–50 μg/μL gene-specific double-stranded RNA (dsRNA) (see Note 5).

• 30. 10 mg/mL antibiotic stock solutions: kanamycin, streptomycin, and ampicillin in separate stocks.

3 Methods

Work at RT and use a pipette, if not stated otherwise.

3.1 Anesthesia (Relaxation), Amputation, and Fixation

The soft-bodied animals will contract to unsightly balls when exposed to a fixative without prior anesthesia. In the literature and in the following protocols, anesthesia is referred to as “relaxation.” Relaxing animals is not only necessary before fixation but also comes in handy for amputations to stop the animals from bending and turning around.

• 1. Isolate culture worms to be processed in an unfed culture vessel.

• 2. Starve the worms for 2 days.

• 3. Transfer animals to an embryo dish filled with ASW using a pipette.

• 4. Remove most ASW, so that the animals are barely covered in ASW.

• 5. Add 500 μL of MgCl₂ over the animals (see Note 6).

• 6. Repeat steps 4 and 5 two times.

• 7. Wait 10 min for the animals to relax (see Note 7).

• 8. Clean a razor blade with 70% ethanol to remove oil (see Note 8).

• 9. Wait 2 min for the ethanol on the razor blade to evaporate.

• 10. Transfer a single relaxed animal to an object slide in a small, flat droplet.

• 11. Amputate animal with a razor blade at the desired body level.

• 12. Quickly add a drop of ASW onto the amputated animal on the object slide.

• 13. Return the amputated animal to a petri dish or well plate filled with ASW.

• 14. Let the animals regenerate for a desired period of time.

• 15. Repeat steps 3 to 7.

• 16. Remove most MgCl₂ (see Note 9).

• 17. Immerse animals in cold FA (4 °C) (see Note 10).

• 18. Incubate for 1 h at RT.

• 19. Remove FA.

• 20. Rinse specimens in PBS-Tx for 5 min.

• 21. Repeat step 20 five times.

3.2 In Situ Hybridization

In situ hybridization is used to detect mRNA in the tissue where it is expressed, using labeled RNA probes. Probe design and synthesis can be done after standard protocols (e.g., [6]).

If not otherwise specified, procedures are done at RT. Pipet liquids, not the animals, that is, the animals stay in the same container (microcentrifuge tube, petri, or embryo dish if not otherwise specified. Liquids are to be removed before adding new liquids.

• 1. Wash fixed animals with distilled water for 1 min.

• 2. Dehydrate animals in 50% methanol for 10 min.

• 3. Repeat step 2 in a graded methanol series (70%, 90%, 100% methanol).

• 4. Store animals in 100% methanol in microcentrifuge tubes at −20 °C overnight or until further use (see Note 11).

• 5. Wash in 100% methanol for 5 min (see Note 12).

• 6. Wash in a graded methanol series (75%, 50%, 25%) for 5 min each.

• 7. Wash animals with PBS-Tw for 5 min.

• 8. Repeat step 7 five times.

• 9. Incubate animals in proteinase K working solution for 3–4 min at 25 °C (see Note 13).

• 10. Quickly stop proteinase treatment with glycine working solution for 20 min.

• 11. Wash with PBS-Tw for 5 min.

• 12. Repeat step 11 five times.

• 13. Wash in TEA for 5 min.

• 14. Repeat step 13.

• 15. Incubate in 0.25% acetic anhydride for 5 min.

• 16. Incubate in 0.5% acetic anhydride for 5 min.

• 17. Washes in PBS-Tw for 5 min.

• 18. Repeat step 17.

• 19. Postfix in FA for 20 min.

• 20. Wash with PBS-Tw for 5 min.

• 21. Repeat step 20 five times.

• 22. Incubate in PBS-Tw at 80 °C for 20 min (heat fixation).

• 23. Incubate in hybmix/PBS-Tw for 10 min.

• 24. Incubate in hybmix at 55 °C for 10 min.

• 25. Incubate in new hybmix for 2 h at 55 °C.

• 26. Denature mRNA probe for 7 min at 96 °C.

• 27. Snap chill probe on ice.

• 28. Add probe to the specimens in hybmix.

• 29. Hybridize at 55 °C for 1–2 days, shaking on an orbital shaker at 300 rpm (see Note 14).

• 30. Wash out probe with new hybmix at 62 °C for 5 min.

• 31. Wash with 75% hybmix at 62 °C for 5 min (see Note 15).

• 32. Wash with 50% hybmix at 62 °C for 5 min.

• 33. Wash with 25% hybmix at 62 °C for 5 min.

• 34. Wash with SSC–CHAPS at 62 °C for 30 min.

• 35. Repeat step 34.

• 36. Wash with MAB for 10 min.

• 37. Repeat step 36.

• 38. Incubate in blocking solution at 4 °C for 2 h.

• 39. Incubate in anti-DIG-AP staining solution at 4 °C overnight.

• 40. Wash with MAB for 10 min.

• 41. Repeat step 40 five times.

• 42. Develop colour with NBT/BCIP working solution until pattern emerges.

• 43. Stop colour development with 100% ethanol for 5 min.

• 44. Wash in PBS-Tw for 15 min.

• 45. Repeat step 44.

• 46. Mount animals on object slides with mounting medium.

3.3 RNA Interference (RNAi)

This method is used to knock down expression of targeted genes in vivo with double-stranded RNA (dsRNA). In Isodiametra, RNAi can be simply performed by soaking the animals in a seawater solution with dsRNA. Use 25–40 animals per well of a 24-well plate (see Note 16).

• 1. Add 400 μL ASW per well of a 24-well plate.

• 2. Transfer live animals from culture dishes to the prepared wells.

• 3. Add the dsRNA to a well at a final concentration of 3–50 ng/μL.

• 4. Add 2 μL of one of the antibiotic stocks to each well.

• 5. Put lid on well plate and place it in the culture room at 20 °C.

• 6. Wait for 24 h.

• 7. Observe behavioral or morphological changes.

• 8. Replace ASW containing dsRNA and antibiotics with 400 μL of fresh ASW.

• 9. Repeat steps 3 to 8, alternating the type of antibiotics, until the dsRNA treatment is over.

• 10. Transfer animals to embryo dishes filled with ASW.

• 11. Process the animals as required for downstream analysis.

3.4 Antibody Stainings

Different to in situ hybridization reagents, there is no requirement for using nuclease-free or DEPC-treated water. While many combinations of antibody stainings are possible, here a double fluorescent wholemount staining using antibodies against BrdU and phosphorylated histone H3 (pH3) is presented.

• 1. Incubate live animals in 5 mM BrdU working solution for 1 h at RT in darkness (see Note 17).

• 2. Wash animals with ASW to remove excessive BrdU.

• 3. Repeat step 2.

• 4. Follow steps 4 to 21 in Subheading 3.1 to fix the animals,

• 5. Incubate animals in protease XIV working solution for 20 min at 37 °C under visual control (see Note 18).

• 6. Add 1 mL 2 M HCl to stop the protease treatment when epidermis becomes slightly ragged, that is, the smooth line of the epidermis on the side of the animals turns into a slightly rippled line.

• 7. Replace 2 M HCl once.

• 8. Incubate in 2 M HCl for 1 h at 37 °C (see Note 19).

• 9. Wash with PBS-Tx for 5 min (see Note 20).

• 10. Repeat step 9 five times.

• 11. Block in BSA-Tx for 30 min.

• 12. Incubate in primary antibody solution at 4 °C overnight.

• 13. Transfer antibody dilution to a microcentrifuge tube for recycling (see Note 21).

• 14. Wash animals with PBS-Tx for 5 min.

• 15. Repeat step 14 five times.

• 16. Block in BSA-Tx for 30 min.

• 17. Incubate in secondary antibody solution for 1 h at RT in darkness.

• 18. Wash with PBS-Tx for 5 min.

• 19. Repeat step 18 five times.

• 20. Coverslip with mounting medium (see Note 22).

3.5 Cytochemical Stainings

Again, a great variety of cytochemical stainings can be performed. Here, a triple fluorescent wholemount staining using EdU, DAPI and phalloidin is presented.

• 1. Incubate live animals in EdU working solution for 1 h at RT in darkness.

• 2. Follow steps 2 to 4 in Subheading 3.4.

• 3. Follow steps 9 to 11 in Subheading 3.4.

• 4. Incubate in triple staining solution for 1 h at RT in darkness.

• 5. Follow steps 18 to 20 in Subheading 3.4.

3.6 RNA Extraction

When preparing animals for an RNAseq experiment, antibiotics can be used to remove (or reduce) bacterial RNA. Animals can be starved for several days to avoid contaminating algal RNA. Take care not to breathe in opened tubes to prevent RNases from breaking up RNA.

• 1. Pipette live animals (typically 10–100) into a microcentrifuge tube (see Note 23).

• 2. Carefully remove as much ASW as possible with a pipette.

• 3. Add 1 mL RNA extraction buffer.

• 4. Use a clean (RNase-free) pestle to mash tissue.

• 5. Pipette up and down to separate tissue clumps.

• 6. Centrifuge at max speed at 4 °C for 10 min.

• 7. Let tube rest at RT for 6 min.

• 8. Add 200 μL chloroform.

• 9. Close tube tightly.

• 10. Vigorously shake tube for 1 min (see Note 24).

• 11. Let tube rest at RT for 10 min.

• 12. Centrifuge at max speed at 4 °C for 15 min.

• 13. Transfer upper (transparent) phase (ca. 500 μL) into new tube (see Note 25).

• 14. Add 10 μg glycogen and mix.

• 15. Add 1 mL isopropanol.

• 16. Incubate at RT for 8 min.

• 17. Centrifuge at max speed at 4 °C for at least 1 h.

• 18. Take off supernatant (be careful not to pipette away pellet).

• 19. Add 1 mL 80% ethanol.

• 20. Centrifuge at max speed at 4 °C for 20 min.

• 21. Repeat steps 18 to 20.

• 22. Take off all supernatant.

• 23. Let air dry for 10–30 min (see Note 26).

• 24. Add 40–100 μL nuclease-free water.

• 25. Let rest on ice for 5 min.

• 26. Store at −80 °C.

3.7 DNA Extraction

For genome sequencing, antibiotics can be used on live animals to remove or reduce bacterial contamination.

• 1. Pipette live animals (typically 10–100) in microcentrifuge tube (see Note 27).

• 2. Carefully remove as much ASW or ethanol as possible.

• 3. Add 500 μL SDS buffer.

• 4. Add 5 μL 20 mg/mL protease XIV stock solution.

• 5. Incubate at 50 °C for at least 1 h to dissolve tissue.

• 6. Add 240 μL phenol–chloroform–isoamyl alcohol.

• 7. Tightly close tube.

• 8. Invert tube several times.

• 9. Centrifuge tube at max speed at RT for 20 min.

• 10. Transfer upper transparent phase into new tube (see Note 28).

• 11. Add 50 μL sodium acetate.

• 12. Mix by inverting tube.

• 13. Add 830 μL chilled 100% ethanol.

• 14. Leave at −20 °C overnight.

• 15. Centrifuge at max speed for 1 h.

• 16. Take off ethanol.

• 17. Add 1 mL 80% ethanol.

• 18. Centrifuge at max speed for 20 min.

• 19. Repeat steps 16 to 18.

• 20. Take off all ethanol.

• 21. Let air dry for at least 30 min.

• 22. Elute in 40–100 μL nuclease-free water.

• 23. Let rest on RT for 5 min.

• 24. Store at 4 °C if you plan to use the DNA within the next month, otherwise store at, −20 °C or − 80 °C.

4 Notes

• 1. Isodiametra pulchra was originally described as Convoluta pulchra and can be found in marine muddy sand beaches on the North American east coast [12]. Currently, lab cultures can be obtained from Andreas Hejnol (University of Bergen, Norway), Pedro Martinez (University of Barcelona, Spain), Simon Sprecher (University of Fribourg, Switzerland), and Peter Ladurner and Bernhard Egger (University of Innsbruck, Austria).

• 2. Isodiametra pulchra can be maintained in glass or plastic Petri dishes with 3.2% ASW, feeding on the diatom Nitzschia curvilineata in a light/dark cycle of 10/14 h at 20 °C. Isodiametra is an obligatorily sexually reproducing hermaphrodite. For detailed culture conditions, especially for the algae, see [13].

• 3. Avoid freeze–thaw cycles whenever possible; either store thawed tubes at 4 °C until further use or make small aliquots that will be used up for a single experiment.

• 4. Pretty much any kind of conjugation can be used with secondary antibodies; the presented protocol presents the combination most often used in Isodiametra. It is important to avoid light as much as possible to ensure the longevity of the fluorophores.

• 5. Gene-specific double-stranded RNA (dsRNA) first needs to be synthesized for RNAi interference experiments. We had good results following [6], using an in vitro RNA transcription system.

• 6. If the experimenter is skilled, it is preferable to amputate without relaxing animals in MgCl₂. Take care to add the relaxant gradually to avoid “freezing” the animals in awkward positions. Toward this end, also make sure to add the fixative at RT, to prevent animals contracting ring muscles in the middle of the body (“wasp waist”).

• 7. Incubating animals in relaxans for an extended period of time (longer than 20 or 30 min) in MgCl₂ will lead to loss of epidermal cells and eventually to the partial dissociation of the whole animal; younger animals are more susceptible to MgCl₂ than older animals.

• 8. It is important to clean razor blades from oil (that is covering the blades to prevent rust) with 70% ethanol, but care has to be taken not to blunt the blade during cleaning, and to let the ethanol evaporate before the blade is being used for amputations; after cleaning the blades, they will start rusting. For better handling, double-sided razor blades can be broken into four smaller pieces by bending the ends toward each other by touching them on the unsharpened sides; take special care not to cut yourself! The blades can be used in a guillotine-like movement to separate body parts of the specimens, or attached to the end of a small chopstick for a halberd-like amputation movement. Immediately after amputation, ASW needs to be returned on the object slide to prevent surface tension to destroy the animals.

• 9. Never remove the complete liquid from the dish/tube before fixation, as surface tension will flatten the animals and may even rip them apart. It is better to exchange the fixative shortly after adding it to dilute the remaining relaxant.

• 10. Reagents prepared for in situ hybridisation, such as FA, can also be used for antibody stainings, but not the other way round. It is better to use FA made in the lab from PFA instead of buying FA as a solution. For best results, prepare fresh FA for each use.

• 11. Even if you can continue immediately, always store animals at −20 °C in methanol at least over night before continuing with the in situ protocol; for rehydration of stored specimens, ethanol can be used instead of methanol.

• 12. All steps can be done in 1.5 mL tubes or in 24-well plates with or without meshes, but also embryo dishes can be used for better visibility of the specimens. Specimens are denser than the surrounding liquid and sink to the bottom of the container (e.g., tube). After pipetting or steps where specimens were disturbed, it is advisable to wait for the next pipetting step until specimens have sunken to the bottom again.

• 13. The protease step during in situ hybridizations is a critical step and has to be carefully timed. If the protocol is not successful, first try making fresh FA, and allow for different batches with slightly differently treated animals regarding protease incubation duration, temperature and concentration. The maximum activity of proteinase K is at a temperature of about 37 °C. Take special care if working with juveniles, as they are more susceptible to protease.

• 14. The length of hybridisation can vary considerably for different probes, probably linked to gene expression levels in the animals. As a rule of thumb, shorter incubation times lead to lower background, so it is not advisable to always use a very long incubation time. Too much background can be addressed with treating the specimens with RNase A after the stringency washes, as the enzyme only digests single stranded RNA.

• 15. All washing steps in PBS can be extended for potentially even better stainings. Wash often, wash long, with the exception of SSC washing steps during in situ hybridizations.

• 16. Animals can be maintained in 24-well plates or in embryo dishes for RNAi treatment; smaller containers are preferable due to the amount of required dsRNA. Feeding animals with algae during treatment is not detrimental to the effects of RNAi treatment [6]. Antibiotics may be used to prevent premature degradation of dsRNA.

• 17. The presented protocol describes a BrdU and EdU pulse staining , that is, animals are killed after the BrdU/EdU pulse. For a pulse-chase staining , animals are kept alive for a period of time after the BrdU/EdU pulse.

• 18. For BrdU stainings, the protease step is the most critical step. Carefully observe the epidermis before adding protease, and only stop the protease if a difference in the epidermal surface can be seen. EdU labeling is much less critical regarding the protease treatment and generally gives more consistent results than BrdU labeling—however, EdU labeling is much more expensive.

• 19. This step ensures that the DNA strands become denatured for the BrdU antibody to be able to bind.

• 20. For the binding affinity of the antibodies, it is important that the pH is close to neutral.

• 21. Recycle primary antibodies to save money and to obtain better stainings with less background in subsequent stainings. Store recycled antibodies at 4 °C. Typically, antibodies can be reused 2–3 times. If available, use a slow shaker for incubation and washing steps.

• 22. Use 20 μL mounting medium for coverslips measuring 21 × 26 mm, for small animals like Isodiametra it will be the exact amount of liquid needed for coverslipping.

• 23. Also animals stored in an RNA preservation buffer can be used, but we have better results with live animals.

• 24. It is advisable to use more expensive microcentrifuge tubes for extracting RNA or DNA, as to avoid leaky tubes during shaking steps.

• 25. In case of visible contaminations, repeat the chloroform step.

• 26. Take care to not let the RNA pellet in RNA extractions air dry for longer than ca. 30 min, as hardened RNA pellets are very difficult to elute. This problem does not exist for DNA pellets.

• 27. Also animals stored in 70% or higher ethanol can be used.

• 28. In case of visible contaminations, repeat the phenol–chloroform–isoamyl alcohol step.