Studying Hemichordata WBR Using Ptychodera flava

Abstract

Hemichordates are benthic marine invertebrates closely related to chordates. Several species, including Ptychodera flava in the phylum Hemichordates, can undergo whole body regeneration from a small fragment. P. flava is widely distributed in the warm Indo-Pacific region and is easily collected in the lower tidal zone of a shallow beach with a coral reef. Here, we describe the methods for animal collection and preparation of regenerating tissues. The prepared tissues can be used for various molecular and/or histological experiments. We also demonstrate how to examine gene expression patterns in the tissues using whole mount in situ hybridization.

1 Introduction

Hemichordates, which are commonly known as acorn worms or pterobranchs, are benthic marine invertebrates. These animals belong to deuterostome and show morphological similarities to chordates such as branchial gills. Moreover, the orthology of some of these features is supported by gene expression and whole-genome analyses [1, 2]. In contrast to the limited capability of regeneration in solitary chordates, hemichordates can undergo whole body regeneration from a fragment of their body [3]. The regeneration of acorn worms tends to occur in anterior-posterior direction rather than other body axes, especially, two complete individuals are formed through regeneration if the body is split into two pieces behind the hepatic region. Although regeneration is observed in many hemichordates, asexual reproduction through the regenerative process or regeneration from a small piece occurs only in a few species [4, 5]. In addition, although acorn worms are found in the sea floor of various environments, only a few species can be easily collected. By combining these two advantages, Ptychodera flava is thus a suitable species for the study of regeneration (Fig. 1).

Fig. 1

External morphology of Ptychodera flava . This figure shows the dorsal view of the animal and left is the anterior tip. The body consists of three parts, proboscis, collar and trunk. The posterior end of trunk is terminated at the anus. The genital wings swell up in the reproductive season. The hepatic sacs are small projections in dark brown

Here, we demonstrate how to collect the animal and prepare regenerating tissues in the laboratory. P. flava is widely distributed in the Indo-Pacific region, and some populations have been reported in the temperate zone [6]. This species generally lives just under a sand flat in the intertidal zone of the coral reef with a relatively high population density [7]. These ecological characteristics allow easy access to the habitats and efficient collection of the worms. We also describe the techniques to handle the fragile worms. Although the body is very fragile, the small body size of P. flava reduces the difficulties of avoiding animal damage during collection on the beach. P. flava can be kept without any special aquarium equipment during regeneration. Physiological tolerance allows a high-density system for preparing regenerating tissues at a low cost.

Several hemichordates, including P. flava , have been used for studies using molecular techniques [8,9,10,11]. Extensive studies of gene expression patterns using embryonic and/or larval specimens have helped elucidate the mechanisms of animal evolution and development . However, visualization of gene expression in adult tissues is still challenging. In this chapter, we also describe the method of whole mount in situ hybridization of regenerating tissues. This method displays gene expression patterns without ambiguous staining .

2 Materials

All solutions should be prepared with ultrapure deionized water unless otherwise stated. RNase-free solutions and equipment are used for RNA probe synthesis and procedures of whole mount in situ hybridization before RNase treatment.

2.1 Animal Husbandry

• 1. Plastic sandwich bags.

• 2. 9 cm diameter plastic petri dishes.

• 3. 10 mL disposable plastic pipettes.

• 4. Filtered seawater (FSW): 5 μm filtered natural seawater.

• 5. Anesthetic seawater: 150 mM MgCl₂ in FSW.

• 6. Fixative: 4% (w/v) paraformaldehyde, 0.1 M NaCl, 0.1 M 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, pH 7.5. We recommend using freshly prepared fixative.

2.2 Preparation of RNA Probes

• 1. Heat stable DNA polymerase.

• 2. dNTPs.

• 3. Forward primer.

• 4. Reverse primer.

• 5. Plasmids containing gene of interest.

• 6. RNA polymerase.

• 7. Digoxigenin (DIG) RNA labeling mixture.

• 8. 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0.

• 9. 8 M LiCl.

• 10. RNase inhibitor.

• 11. Fluorometer supporting RNA-specific quantification.

2.3 Preparation of Preabsorbed Antibodies

• 1. Liquid nitrogen.

• 2. Pestle and mortar.

• 3. Acetone.

• 4. 10× maleic acid buffer: 1 M maleic acid, pH 7.5, 1.5 M NaCl. Adjust pH with NaOH.

• 5. MABT: 10% (v/v) 10× maleic acid buffer, 0.1% (v/v) Tween 20.

• 6. Blocking reagent A: 1% (w/v) blocking reagent in MABT. Store in aliquots of 1 mL at −20 °C.

• 7. 20 mg/mL bovine serum albumin.

• 8. Sheep serum.

• 9. Anti-digoxigenin-alkaline phosphatase Fab fragments from sheep.

2.4 Whole Mount In Situ Hybridization

• 1. PBST: 3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, 0.1% (v/v) Tween 20.

• 2. 20× saline sodium citrate (SSC) buffer: 3 M NaCl, 0.3 M sodium citrate.

• 3. Bleaching solution: 1% (v/v) hydrogen peroxide in PBST. Prepare just before use.

• 4. Permeabilization solution: 10 μg/mL proteinase K in PBST. Prepare just before use.

• 5. Postfixing solution: 4% (w/v) paraformaldehyde in PBST. Store in aliquots of 1 mL at −20 °C.

• 6. 0.1 M triethanolamine.

• 7. Acetylation solution: 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine. Prepare just before use.

• 8. Hybridization buffer: 50% (v/v) formamide, 30% (v/v) 20× SSC buffer, 0.1 mg/mL yeast total RNA, 0.1% (v/v) Tween 20, 0.1% (w/v) Ficoll 400, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) bovine serum albumin. Store at −20 °C.

• 9. Digoxigenin (DIG)-labeled RNA probes.

• 10. Washing buffer: 50% (v/v) formamide, 5% (v/v) 20× SSC buffer, 0.1% (v/v) Tween 20. Store in aliquots of 10 mL at −20 °C. Warm up to 65 °C before use.

• 11. Solution A: 500 mM NaCl, 10 mM Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.1% (v/v) Tween 20. Warm up to 37 °C before use.

• 12. RNase A solution: 20 μg/mL RNase A, 500 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, pH 8.0, 0.1% (v/v) Tween 20. Prepare just before use.

• 13. 2× SSCT: 10% (v/v) 20× SSC buffer, 0.1% (v/v) Tween 20. Warm up to 37 °C before use.

• 14. 1× SSCT: 5% (v/v) 20× SSC buffer, 0.1% (v/v) Tween 20. Warm up to 65 °C before use.

• 15. 0.2× SSCT: 1% (v/v) 20× SSC buffer, 0.1% (v/v) Tween 20. Warm up to 65 °C before use.

• 16. Blocking reagent B: 2 mg/mL bovine serum albumin, 10% (v/v) sheep serum in blocking reagent A. Store in aliquots of 1 mL at −20 °C.

• 17. Preabsorbed anti-digoxigenin-alkaline phosphatase Fab fragments.

• 18. TNT buffer: 0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% (v/v) Tween 20. Warm up to 37 °C before use.

• 19. TMNT buffer: 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl₂, 0.1% (v/v) Tween 20. Prepare just before use and warm up to 37 °C.

• 20. Nitroblue tetrazolium (NBT) stock solution: 100 mg/mL NBT in N, N-dimethylformamide (DMF). Store in aliquots of 100 μL in the dark at −20 °C.

• 21. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) stock solution: 50 mg/mL BCIP in DMF. Store in aliquots of 100 μL in the dark at −20 °C.

• 22. Staining solution: 1/4000 NBT, 1.75/1000 BCIP in TMNT buffer. Prepare just before use.

• 23. PBST–EDTA: 1 mM EDTA, pH 8.0 in PBST.

3 Methods

3.1 Animal Collection

• 1. Dig in sand using a trowel at the low tide zone to collect animals (see Fig. 2a and Note 1).

• 2. Pour seawater in the sand on the trowel to wash out the animals (Fig. 2b).

• 3. Transfer the collected animal from the trowel to a plastic sandwich bag filled with seawater (see Note 2).

• 4. Ten to fifteen animals can be kept in the same 1000 mL plastic bag during collection and transportation (see Note 3).

• 5. Avoid shaking the plastic bags during transportation to prevent shearing the animal body.

• 6. Transport the collected animals to the laboratory (see Note 4).

• 7. Treated FSW with bubble aeration for at least 30 min.

• 8. Add 30 mL of FSW in a 9 cm diameter plastic petri dish.

• 9. Transfer the collected animals into the dish one by one using a 10 mL disposable plastic pipette in which the conical tip is cut off (see Notes 5 and 6).

• 10. If the animals are tangled with each other in the plastic bag, dissociate the animals by gently pipetting up and down (Fig. 3a, b).

• 11. Remove sand attached to the surface of the animals using tweezers (Fig. 3c).

• 12. Pick up undamaged individuals after removing sand (see Note 7).

• 13. Keep the undamaged animals at 25 °C until the sand in the intestine of the animal has completely passed from the body (see Note 8).

• 14. Remove the excreted sand using a 1000 μL pipette with a wide-bore tip every day.

• 15. Loosen possible knots in the animal body using toothpicks (Fig. 3d).

Fig. 2

Comparison of the habitat and the sampling methods of Ptychodera flava . (a, b) Animal collection on a tidal flat in Okinawa, Japan. A trowel is used to dig the sand. The animals in the dug sand are exposed by addition of seawater. (c, d) The animals were collected by snorkeling in shallow water in Hawaii, USA. In this case, the sand was dug using a vigorous wave of the hand to find the animals

Fig. 3

Animal treatment and maintenance at the laboratory. (a, b) The tangled animals in the plastic bag after transportation and a dissociated individual in a pipette. (c) The sand with filmy mucus attached on the surface of the animal was removed using tweezers and a toothpick. (d) A knot made in the animal body was loosened by toothpicks

3.2 Preparation of Regenerating Tissues

• 1. Select undamaged individuals (see Note 9).

• 2. Remove the filmy mucus attached on the surface of the animals using tweezers (see Note 10).

• 3. Replace seawater in the dish with 30 mL of anesthetic seawater (see Note 11).

• 4. Incubate at 25 °C for 10 min (see Note 12).

• 5. Cut the body into two pieces using iris scissors (see Fig. 4a and Note 13).

• 6. Transfer each piece immediately to a new 9 cm diameter plastic petri dish filled with 30 mL of FSW (see Note 14).

• 7. Keep the fragments at 25 °C until a target stage in regeneration (see Fig. 5, Notes 15 and 16).

• 8. Transfer the regenerating tissue into a clean 1.5 mL plastic tube containing 1 mL of fixative (see Fig. 4c, and Note 17).

• 9. Incubate at 4 °C for 16 h.

• 10. Remove and discard all of the fixative from the tube.

• 11. Dehydrate the samples in 1 mL of an ethanol series (30% EtOH, 50% EtOH, 80% EtOH) at 4 °C for 30 min each step.

• 12. Replace the medium with 1 mL of 80% EtOH.

• 13. Incubate at 4 °C for 30 min (see Note 18).

Fig. 4

Preparation of regenerating tissues of Ptychodera flava . (a) An undamaged animal was cut into two pieces using iris scissors behind the hepatic sacs. The sand which are indicated arrowheads remaining in the intestine can be confirmed from the outside of the body as in Panel (b). (c) Collection of a regenerating tissue using iris scissors. The animal was not treated with anesthetic seawater. The arrowhead indicates the regenerating proboscis

Fig. 5

Anterior regeneration process of Ptychodera flava . (a) An undamaged individual before amputation. (b) A posterior piece just after amputation. (c) The wound is healing at 2 days postamputation (dpa). (d) The completion of wound healing at 3 dpa. (e) The regenerating tissue called blastema becomes visible at 4 dpa. (f) Two rudiments of collar are swelled on both lateral sides of the blastema at 5 dpa. (g) The collar rudiment surrounds the most prominent mass of the blastema at 7 dpa. The mouth opens at the ventral region between the regenerating proboscis and collar. (h) Complete function of proboscis and collar are recovered at 12 dpa. (i) At 17 dpa, the missing branchial region becomes visible. The process of gill regeneration continues for approximately 2 months. (Reprinted by permission from the Zoological Society of Japan: Zoological Science, Regeneration in the Hemichordate Ptychodera flava , Humphreys et al., 2010)

3.3 Preparation of RNA Probes

The method for preparation of digoxigenin-labeled RNA probes is modified from [12].

• 1. Amplify 10 ng of the template plasmid through PCR (see Note 19).

• 2. Mix 2.5 μL of the amplified solution and 7.5 uL of DIG labeling solution thoroughly using a 20 μL pipette (see Note 20).

• 3. Incubate at 37 °C for 16 h (see Note 21).

• 4. Adjust volume to 51.8 μL with RNase-free water.

• 5. Add 3.2 μL of 0.5 M EDTA and 25 μL of 8 M LiCl.

• 6. Mix thoroughly using a vortex mixer.

• 7. Incubate at −20 °C for 20 min.

• 8. Centrifuge the sample at 16,000 rcf at 4 °C for 20 min.

• 9. Discard the supernatant.

• 10. Add 200 μL of 80% EtOH.

• 11. Centrifuge the sample at 16,000 rcf at 4 °C for 5 min.

• 12. Discard the supernatant completely.

• 13. Vacuum dry the pellet for 10 min.

• 14. Add 50 μL of RNase-free water containing 10 units of RNase inhibitor (see Note 22).

• 15. Measure the concentration of the RNA probe using a fluorometer.

3.4 Preparation of Preabsorbed Antibodies

The method for preparation of preabsorbed antibodies is modified from [13].

• 1. Chill a pestle and mortar using liquid nitrogen.

• 2. Put a whole body of adult P. flava in the chilled mortar (see Note 23).

• 3. Immediately add liquid nitrogen to the mortar.

• 4. Grind the frozen sample to a fine powder using the pestle and mortar.

• 5. Transfer the powder to a clean, prechilled 50 mL plastic tube.

• 6. Add four volumes of the powder of prechilled acetone.

• 7. Incubate on ice for 30 min.

• 8. Centrifuge the sample at 10,000 rcf at 4 °C for 5 min.

• 9. Discard the supernatant.

• 10. Repeat steps 6 to 9 again.

• 11. Transfer the pellet to a clean 5 μm filter paper.

• 12. Dry up the pellet at room temperature (see Note 24).

• 13. Add 1.5 mg of the completely dried powder into a clean 1.5 mL plastic tube containing 400 μL of blocking reagent A.

• 14. Incubate at 70 °C for 30 min.

• 15. Add 50 μL of 20 mg/mL bovine serum albumin and 50 μL of sheep serum.

• 16. Cool down the solution to room temperature.

• 17. Add 0.5 μL of anti-digoxigenin-alkaline phosphatase Fab fragments.

• 18. Incubate at 4 °C for 16 h with gentle agitation.

• 19. Add 900 μL of blocking reagent A, 50 μL of 20 mg/mL bovine serum albumin and 50 μL of sheep serum.

• 20. Mix thoroughly using a 1000 μL pipette.

• 21. Centrifuge the sample at 10,000 rcf at 4 °C for 1 min.

• 22. Transfer the supernatant to clean 1.5 mL plastic tubes in 200 μL aliquots.

• 23. Store the aliquots at −20 °C.

3.5 Whole Mount In Situ Hybridization

• 1. Transfer dehydrated tissues to a clean 1.5 mL plastic tube with 100 μL of 80% EtOH using a 1000 μL pipette with a wide-bore tip (see Note 25).

• 2. Rehydrate tissues in an ethanol-PBST series (75% EtOH–25% PBST, 50% EtOH–50% PBST, 30% EtOH–70% PBST, 100% PBST) with 200 μL of each solution for 10 min.

• 3. Replace the medium with 200 μL of bleaching solution.

• 4. Incubate at room temperature for 10 min (see Note 26).

• 5. Rinse the sample twice in 200 μL of PBST.

• 6. Replace with 200 μL of permeabilization solution.

• 7. Incubate at 37 °C for 30 min.

• 8. Rinse the sample three times in 200 μL of PBST.

• 9. Perform postfixation in 200 μL of postfixing solution at room temperature for 1 h.

• 10. Rinse the sample twice in 200 μL of PBST.

• 11. Immerse the samples twice in 200 μL of 0.1 M triethanolamine at room temperature for 10 min.

• 12. Incubate in 200 μL of acetylation solution at room temperature for 10 min at room temperature.

• 13. Rinse the sample three times in 200 μL of PBST.

• 14. Replace PBST in the tube with 200 μL of 50% hybridization buffer/50% PBST.

• 15. Incubate at room temperature for 10 min.

• 16. Replace with 200 μL of hybridization buffer.

• 17. Incubate at room temperature for 10 min.

• 18. Replace the hybridization buffer with 200 μL of fresh hybridization buffer.

• 19. Incubate at 60 °C for 2 h (see Note 27).

• 20. Replace the buffer with 100 μL of hybridization buffer containing 0.1 ng/μL RNA probe (see Note 28).

• 21. Incubate at 60 °C for at least 16 h (see Note 29).

• 22. Remove the excess RNA probe in 200 μL of washing buffer at 65 °C for 20 min.

• 23. Repeat this wash for a total of three times (see Note 30).

• 24. Replace the buffer with 200 μL of solution A.

• 25. Incubate at 37 °C for 10 min.

• 26. Repeat this buffer replacement for a total of three times.

• 27. Replace with 200 μL of RNase A solution.

• 28. Incubate the sample at 37 °C for 30 min.

• 29. Rinse the sample three times in 200 μL of 2× SSCT at 37 °C.

• 30. Replace with 200 μL of 1× SSCT.

• 31. Incubate at 65 °C for 20 min.

• 32. Repeat steps 30 and 31 again.

• 33. Replace with 200 μL of 0.2× SSCT.

• 34. Incubate at 65 °C for 10 min.

• 35. Repeat steps 33 and 34 again.

• 36. Replace with 200 μL of MABT.

• 37. Incubate at room temperature for 10 min.

• 38. Repeat steps 36 and 37 for a total of three times.

• 39. Replace with 200 μL of blocking reagent A.

• 40. Incubate at room temperature for 20 min.

• 41. Replace with 200 μL of blocking reagent B.

• 42. Incubate at room temperature for 1 h.

• 43. Replace with 100 μL of preabsorbed antibody solution.

• 44. Incubate at room temperature for 2 h (see Note 31).

• 45. Rinse the sample twice in 200 μL of MABT at room temperature.

• 46. Remove the excess antibody in 1 mL of MABT at 37 °C for 16 h.

• 47. Replace the buffer with 1 mL of MABT (see Note 32).

• 48. Incubate at 37 °C for 20 min.

• 49. Repeat steps 47 and 48 for a total of four times.

• 50. Replace with 200 μL of TNT buffer.

• 51. Incubate at 37 °C for 10 min.

• 52. Repeat steps 50 and 51 again.

• 53. Replace with 200 μL of TMNT buffer.

• 54. Incubate at 37 °C for 10 min.

• 55. Repeat steps 53 and 54 again.

• 56. Replace with 200 μL of staining solution.

• 57. Incubate the samples at 37 °C (see Note 33).

• 58. Rinse the samples twice in 200 μL of PBST–EDTA at room temperature.

• 59. Replace with 200 μL of PBST twice.

• 60. Incubate at 37 °C for 12 h.

• 61. Replace with 1 mL of 30% EtOH–70% PBST.

• 62. Incubate at room temperature for 30 min.

• 63. Repeat incubation in fresh 30% EtOH–70% PBST for 90 min (see Note 34).

• 64. Rinse the sample twice in 200 μL of PBST (see Note 35).

• 65. Transfer the samples to a plastic petri dish filled with PBST using a 1000 μL pipette with a wide-bore tip.

• 66. Imaging on the samples using a camera mounted on a stereomicroscope (Fig. 6).

Fig. 6

Examples of whole mount in situ hybridization using regenerating tissues of Ptychodera flava . Gene expressions of soxb1 at 7 days postamputation were examined using an antisense probe (a) and a sense probe (b), respectively. The upper side is dorsal. The arrowheads show regenerating proboscis in each panel. The gene expressions of soxb1 were identified at regenerating rudiments of proboscis and collars located at the tip of anterior and surrounding region of them

4 Notes

• 1. The animals are commonly found in the low tide zone of a shallow beach with a coral reef (Fig. 2). Small individuals that are suitable for preparing regenerating tissues prefer a place where fine coral sand accumulates. These animals are abundant in sand less than 10 cm in depth and are rarely found in an anaerobic bottom layer. We generally collect small individuals with a 2–4 mm diameter of the trunk. Prominent fecal castings are accompanied by burrow systems of some large-sized acorn worms; however, P. flava does not form such a structure. The animals can also be collected in high tide conditions by snorkeling (Fig. 2c, d and [13]). Key points of species identification are shapes and colors of proboscis, genital wings and hepatic sacs. Any closely related species has not been reported in habitats of P. flava [6].

• 2. An animal should not be picked up with fingers to prevent shearing of the body by its own weight. The entire body of the animal should be held on a palm.

• 3. If a transportation longer than 3 h is expected, the plastic bags are put in a Styrofoam box with ice packs wrapped cushioning materials. The Styrofoam box has to be treated at temperature between 10 °C to 30 °C. This procedure can extend transportation period up to 2 days.

• 4. The color of animals sometimes changes from vivid yellow to light brown after transportation; however, this phenomenon does not affect the success rate of regeneration. All procedures were carried out at 20–25 °C after transportation. Cold conditions lower than 20 °C resulted in a lower success rate of regeneration.

• 5. We recommend using collected animals for experiments of regeneration as soon as possible. Five to ten animals can be kept in a 1 L tank with 800 mL of filtered seawater with gentle bubble aeration and fed 50 mL of the diatom Chaetoceros calcitrans once a week, however, the worms gradually become smaller and show a lower success rate of regeneration. The seawater in the tank should be replaced the next day of feeding. A 5 mm thick layer of fine coral sand in the tank suppresses autotomy. Collected animals can be maintained in the lab to prepare regenerating tissues using the compromise culture method up to 3 months.

• 6. Sharp tweezers should not be used to transfer the animals. The tweezers can easily damage the animal body. A conical tip of joining molded pipette (e.g., Corning) can be easily picked by hands, otherwise cut off using a razor blade. For transferring large animals, round disposable chopsticks are more suitable than pipettes. Artificial seawater is also acceptable for storage of the collected animals.

• 7. Any scale of damage is not allowed. A stereomicroscope can be used to assess the damage to animals smaller than 1 mm in diameter at the trunk. Damaged individuals will die or show signs of autotomy accompanying trunk constriction within a few days. A white or pale colored proboscis or posterior tip suggests that the individual is regenerating or has just completed regeneration. We do not use these individuals to collect regenerating tissues.

• 8. The treatment period is shorter than 3 days in many cases.

• 9. Animals changing to dark brown show a lower success rate of regeneration.

• 10. The presence of mucus layer on the surface of the animals can be distinguished by the refractive index differences. If a such layer is not be observed, this procedure can be omitted.

• 11. Anesthesia is not an essential treatment for amputation. Therefore, although the technical difficulties of amputation are increased, it is possible to prepare specimens for assessing the gene expression of early wound responses under natural conditions.

• 12. Increasing the incubation time to up to 30 min does not affect the rate of regeneration.

• 13. We usually cut an animal body behind the hepatic sacs (Fig. 4a, b). The body should be cut at once to avoid warping of the cut section. Although the success rate of regeneration depends on the cutting position [6, 14], the common success rate of regeneration is higher than 80% in our routine method.

• 14. Researchers should minimize carry-over of anesthetic seawater. Antibiotics do not significantly increase the success rate of regeneration.

• 15. The seawater in the dish should be replaced when its color changes to yellow or brown. The water change should be done with great care because a newly formed region can be broken by water current.

• 16. The stage of anterior regeneration can be identified based on the morphologies. On the other hand, it is difficult to distinguish the stage of posterior regeneration based on external structures. The difficulties come from the absence of characteristic structures in the posterior region even in undamaged individuals.

• 17. The dissected specimens should be smaller than a cube three millimeters on a side. Five to ten tissue specimens can be kept in the same 1.5 mL plastic tube. The composition of the fixative is suitable for in situ hybridization specimens. The dissected tissues for RNA extraction are dissolved in TRIzol reagent. We recommend a flash freezing method using liquid nitrogen rather than fixative for DNA extraction samples.

• 18. Dehydrated samples in 80% EtOH can be stored at −20 °C for at least 1 year.

• 19. The PCR is carried out in 20 μL scale. The composition of PCR solution and settings of thermal cycler follow the recommendation of manufacturer of employed DNA polymerase. Universal primers for the plasmid are suitable for the PCR. The specificity of PCR is confirmed using agarose gel electrophoresis.

• 20. The composition of DIG labeling solution containing RNA polymerase which recognizes promoter sequences on the plasmid follows manufacturer’s instruction.

• 21. The results of in vitro transcription are examined using native agarose gel electrophoresis.

• 22. The purified RNA probe can be stored at −80 °C.

• 23. Sands remaining in the intestine should be completely removed in advance of the antibody preparation.

• 24. Residual acetone deactivates antibodies. Breaking down the pellet using a clean medicine spoon helps to be shortened the incubation time. The completely dried powder can be stored at −20 °C.

• 25. Hydrophobic coating for plastic tubes is not necessary.

• 26. Hydrogen peroxide treatment reduces mucus secretion from tissues during high temperature incubation. The color of the samples is slightly bleached; however, it is still brownish after this step.

• 27. The viscosity of the solution is slightly increased after incubation. The solution in the 1.5 mL tube should be removed slowly using a 200 μL pipette.

• 28. The standard concentration of RNA probe in hybridization buffer is 0.1 ng/μL, however, the concentration should be optimized in the range of 0.01 to 1 ng/μL based on preliminary experiments.

• 29. If the viscosity of the solution is significantly increased than the prehybridization step, it suggests the failure of hydrogen peroxide treatment. The viscosity is gradually decreased by washing buffer and solution A. We do not reuse the hybridization buffer containing an RNA probe. The mucus released from the tissues denatures the integrity of the hybridization buffer.

• 30. The samples should be kept in warm conditions until step 36.

• 31. The incubation time should not be increased. A long incubation even at 4 °C gives rise to nonspecific staining .

• 32. The MABT is warmed to 37 °C before use.

• 33. The samples should be kept in dark conditions during incubation until step 63. The alkaline phosphatase binding to digoxigenin-labeled RNA probe produces purple or dark blue pigments. In contrast, light reddish-purple pigments are produced by the nonenzymatic reaction. The staining generally takes 30 min to 3 h. The samples should be rinsed using PBST–EDTA before changing the color of nonspecific staining to dark purple. Insufficient washing after RNA probe hybridization and/or antibody reaction induces false positive staining of grooves on the surface of the trunk region even in a negative control sample.

• 34. Although the standard procedure described in step 63 is effective to remove nonspecific staining , dehydration and rehydration using a series of 30% EtOH–70% PBST, 50% EtOH–50% PBST, and 80% EtOH–20% H₂O improve the contrast of the staining after postfixation (see Note 35).

• 35. Stained specimens can be stored at 4 °C for at least 1 month after performing postfixation again. The procedure is the same as step 9. The postfixation procedure also helps to fix stained pigments. In other words, the visibility of nonspecific staining is increased by postfixation if such staining remains.