Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Summary

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Abstract

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both "classic" regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Introduction

The observation of regeneration in a single hydra was the seminal event in the advent of biology as an experimental science^1,2. Regeneration remains a phenomenon of extraordinarily broad appeal to biologist and lay person alike. The potential for developmental biologists, clinicians, biomedical scientists and tissue engineers to understand and overcome the limits on human regeneration makes regeneration biology more than intrinsically interesting.

Now, with the use of emerging technologies such as genome sequencing and gain and loss of function tools, the field is poised to tease apart regenerative mechanisms and to ultimately understand how various species can regenerate while others cannot. The degree of commonality in molecular, cellular and morphological responses remains to be elucidated, but so far it appears that the basic responses among animals that can regenerate is more similar than would have been imagined only a decade ago³.

Cnidarians in particular are facile at regenerating almost all of their body parts amid a broad spectrum of morphological diversity. From the solitary fresh water polyp, Hydra along with the tiny marine polyps that build immense coral reefs, to the complex colonial siphonophores, such as the Portuguese Man-O-War, regeneration is often a mode of reproduction, in addition to a mechanism for repairing or reforming damaged or lost body parts resulting from injury and predation. Whether the diverse species of Cnidaria use similar or different mechanisms for regeneration is a fundamentally interesting question^4-6.

We, and others have been developing the anthozoan, Nematostella vectensis as a model for regeneration^7-17. We recently developed a staging system for describing regeneration of an entire body from a morphologically uniform piece of tissue bisected from the aboral end of the polyp¹⁰. While Nematostella polyps can regenerate when bisected at any level, we chose to cut adults at an aboral position in the most morphologically simple region, the physa, in part because this is close to the normal plane of natural asexual fission¹⁸, and also because it permits observation and molecular analyses of how an entire body is reassembled from the simplest morphological components.

The Nematostella Regeneration Staging System (NRSS) provides a relatively simple set of morphological benchmarks that could be used to score the progress of any aspect of regeneration by an amputated physa, under normal culture conditions or experimentally perturbed situations such as small molecule treatments, genetic manipulation, or environmental alteration. As anticipated, the NRSS is becoming adopted as a morphological scaffold on which the cellular and molecular events of regeneration can be referenced¹⁰.

Finally our method of cutting produces a gaping hole several orders of magnitude greater than the pin point puncture used in a recent study¹⁷, yet both wounds heal in around 6 hours. Documenting the visually arresting and distinct phases of wound closure should suggest experimental approaches to explain the apparent independence of the size of a wound and the time it takes to close. Thus, a deeper visual understanding of the aboral amputation process, provided by this protocol, will aid further investigations into this model regeneration system and broaden the application of this staging system using Nematostella vectensis.

Protocol

1. Conditioning of Animals for Temperature, Nutrition and Light/Dark Cycle

1. Obtain Nematostella vectensis adults from one of the numerous Nematostella labs worldwide, or a non-profit supplier (Table 1)
2. Maintain Nematostella at constant temperature (typically between 18 and 21 °C) in the dark, in "1/3x" Artificial Sea Water (ASW) at a salinity of 12 parts per thousand (ppt). Maintain cultures in simple soda-lime glass culture dishes, typically 250 mL or 1.5 L capacity¹¹.
   Note: These simple culture conditions are commonly used among labs that study Nematostella, but culture care can also be automated¹⁹.
3. Feed Nematostella freshly hatched Artemia nauplii 2 - 4x per week. Hatch Artemia cysts in full strength (36 ppt) or 1/3x ASW at 30 °C, in a shallow rectangular glass dish²⁰ or in any of a number of small scale, commercial or homemade brine shrimp hatcheries. If an incubator is not available, shrimp will hatch at RT but do so more slowly.
   Note: This often requires more than 24 h for completion.
4. Replace anemone culture water at least once a week. For best adult health, thoroughly clean (without soap) the culture bowls once a week of accumulated mucous secretions, which coat the bowl and can trap uneaten food and waste, and entangle the animals.

2. Selection and Relaxation of Nutritionally Conditioned Animals

1. Select size-matched polyps of approximately the same length (3 - 5 cm, when naturally relaxed) and place them in a bowl separated from the colony for three days prior to amputation.
   Note: The number of animals selected for cutting will be determined by the experiment being conducted, of course, but in general we recommend at least five animals per sample point with six replications. Thus, in a typical experiment a minimum of 30 animals would be preselected. In general, it is wise to select more than the minimum number (30) since amputations that are irregular (see below) can later affect scoring.
2. Remove the dish of selected animals from the incubator into room light at least one hour prior to amputation.
   Note: Exposure to room light and vibrations of handling will likely cause the animals to contract, so they need to be adapted or "relaxed" by incubation on the lab bench. Animals will become refractory to touch and light exposure and at that point can be moved by gentle pipetting.
3. Optional: Anesthetize the animals by adding 7.5% MgCl₂ (in 1/3x ASW). Gently add the MgCl₂ solution to the bowl with a standard plastic 5 mL pipette.
   Note: Although animals will eventually become habituated to the light and to physical manipulation, it may be advantageous to anesthetize animals to maintain or "fix" the relaxed state after they have become elongated^16,21,22.
4. Use a wide bore (>0.5 cm) plastic pipette to transfer (in 1/3x ASW) five animals from the pool to be amputated, into the bottom of a sterile glass cutting dish of 100 mm diameter containing 12 ppt ASW. Place the dish on to the stage of a stereomicroscope with variable magnification between 10 - 40X.
   ​Note: If the animals have not been anesthetized and relaxed for cutting, they may still respond to touch and stereoscope illumination and thus may need a few minutes to become relaxed again.

3. Amputation

1. Using a sterile scalpel, amputate the aboral physa from each polyp, with the goal to obtain a section of the physa that is approximately as long as it is wide and containing no mesentery.
   Note: The ideal cut site is just aboral to the termination of the mesentery. At the plane of cutting there is a transition from mesentery proper to thin lines corresponding to each mesenterial insertion (see Figure 1, arrows). Absence of mesentery is critical because it produces mucous that may facilitate 'plugging' the hole^17,30.
   1. Place the scalpel blade in contact with the animal at the desired site of amputation. Do this either unassisted (freehand), or by gently grasping the animal's body with a #5 forceps (Dumont style or similar).
   2. Cut through the tissue by leveraging the curved blade of the scalpel in a 'rocking' motion across the body.
      Note: The tissue should sever cleanly as the scalpel is rocked and liberate the desired section of physa from the donor. However, if a small piece of tissue still connects the body and the physa, cut it with the scalpel. Do not attempt to separate the connected pieces by pulling, as this may damage the physa.
2. Remove each amputated 'donor' polyp from the dish and return it to a separate bowl labeled 'pooled amputees'; date the bowl and return it to stock culture.
   Note: Amputated polyps will heal the aboral wound within a day and then can be fed normally. They will regenerate a normal looking physa within two weeks at which point the physa can be amputated again if desired.
3. Rinse the excised physa that remain in the cutting dish in 12 ppt ASW, then transfer each physa to a separate sterile well in a multi-well cell culture plate that already has 10 mL of 12 ppt ASW in each well.
   Note: This example uses a six well plate, with each well holding 10 mL of seawater and five excised physa. In general seawater should cover the physa sufficiently to avoid exposure to air due to movement in handling and potential evaporation. The plate or wells should have a lid.
4. Repeat steps 3.1 - 3.3 to collect at least 5 physa in each well reserved for each experimental treatment.
5. Incubate the physa at a temperature that will provide the best rate of regeneration for the planned experimental interrogations. Place the plate containing the physa into a temperature regulated incubator, at a fixed temperature determined by the desired rate of regeneration.
   ​Note: The physa will regenerate missing tissues and form a full polyp when incubated at temperatures between 15 and 27 °C. The rate of regeneration is temperature dependent except for the first two stages. The average day for reaching Stage 4 for all temperatures is 7 d after cutting and this also coincides with regeneration at 21 °C. At 27 °C, Stage 4 is reached about 3 days earlier and at 15 °C, Stage 4 is delayed by about 3 d compared to regeneration at 21 °C (also see Reference 10).

4. Assessing Regeneration with the Nematostella Regeneration Staging System (NRSS)

1. Score the physa using a stereo-compound microscope with variable magnification (10 - 80X). Score the freshly cut Nematostella physa as Stage 0 and continue scoring at the same time each day post amputation (dpa) using the NRSS¹⁰.
   Note: For key staging criteria and details refer Reference 10.
   1. Score physa as Stage 0 (Open Wound) if a freshly cut physa appears as a cup- shaped mass resembling a flaccid balloon, with an open wound site is likely visible.
      ​Note: The wound edges might also stick together from the outset, but the tissue will still be collapsed and lack rigidity. The edges of the open wound may be observed undergoing radial contraction as the wound heals.
   2. Score physa as Stage 1 (Wound Closed) if the amputation wound appears closed.
      Note: Wound location will correspond to the future oral pole. The outer surface around the future oral pole may begin to display distinct arches corresponding to the underlying radially symmetric endodermal mesenterial insertions.
   3. Score physa as Stage 2 (Radial Arches) if the surface of the oral pole appears inflated, revealing eight raised arches arranged in a radially symmetric pattern and separated by grooves. Observe small, hemispherical blebs at the apex of the arches. They will be about as tall as wide, likely transient, and initially comprised by a single ectodermal cell layer.
      Note: In some cases double-layered blebs may stabilize. Note: At this or later stages a mucous layer may appear to encapsulate the physa (Figure 2) in a membranous 'sheath'. This encapsulating material should be removed to facilitate scoring.
   4. Score physa as Stage 3 (Tentacle) if the buds of the tentacles containing endodermal and ectodermal tissue layers are stably formed at the oral end of at least some radial arches.
      Note: The tentacles are longer than they are wide and are minimally motile. The physa will show increased, but variable inflation so that mesentery rudiments may become visible extending from the mesenterial insertion into the body cavity (coelenteron).
   5. Score physa as Stage 4 (Linear Mesenteries) if the physa contains eight distinct, visible mesenteries that extend into the coelenteron from insertions in the body wall, with oral-aboral lengths that are more than twice their radial width measured from where they appear to connect to the pharynx at its aboral end (enterostome).
      Note: Four or fewer mesenteries have "pleated" internal free edges. The pharynx is visible. More than eight tentacles are visible, motile and sometimes they contract into the body.
   6. Score physa as Stage 5 (Predominantly Pleated Mesenteries) if the physa has more than four mesenteries with pleating, and the pleating is more full and sinuous than at Stage 4. The animal has an almost "normal" adult appearance, but there are no visible gonadal cells.

Results

The progression of morphological events during regeneration in severed physa is shown in Figure 1A, which includes representative views of physa at each NRSS stage. The typical physa cut site is indicated on the adult (arrowheads). The photographs in Figure 1A show progressive regeneration of oral and body structures from freshly cut physa through fully formed polyp. Figure 1B, C show the arrangement of internal septa, the mesenteries, at...

Discussion

Use of Nematostella as a model of wound healing and regeneration is becoming increasingly popular. Thus, it is important to be able to visualize the morphological patterns of a particular protocol before effective cellular and molecular analyses can be assigned and compared. Nematostella have a high degree of regenerative "flexibility", being able to reform almost any missing structure amputated at any location, at post-planula stages of life. Thus, various investigators have examined regenerati...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nematostella vectensis, adults	Marine Biological Lab (MBL)		non-profit supplier
Glass Culture Dish, 250 mL	Carolina Biological Supply	741004	250 mL
Glass Culture Dish, 1,500 mL	Carolina Biological Supply	741006	1,500 mL
Polyethylene transfer pipette, 5 mL	USA Scientific	1022-2500	narrow bore, graduated
Polyethylene transfer pipet, tapered	Samco	202-205	cut off 1 inch of tip to make wide bore
Disposable Scalpel	Feather Safety Razor Co. Ltd	no. 10	blade should be curved
#5 Dumont Fine point tweezers	Roboz	RS5045	alternative suppliers available
Pyrex Petri dish, 100 mm diameter	Corning	3160	can substitute other glass Petri plates
Sterile 6-well plate	Corning Falcon	353046	or similar from other manufacturer
Sterile 12-well plate	Nunc	150628	or similar from other manufacturer
Sterile 24-well plate	Cellstar, Greiner bio-one	662-160	or similar from other manufacturer
Brine shrimp hathery kit	San Francisco Bay; drsfostersmith.com	CD-154005	option for growing brine shrimp
pyrex baking dish	common in grocery stores		option for growing brine shrimp
artificial seawater mix 50 gal or more	Instant Ocean; drsfoster-smith.com	CD-116528	others brands may suffice
Plastic tub for stock ASW preparation	various		common 25 gallon plastic trash can OK
Polypropylene Carboy	Carolina Biological Supply	716391	For working stock of ASW @ 12 ppt
Beaker, Graduated, 4,000 mL	PhytoTechnology Laboratories	B199	For dilution of 36 ppt ASW to 12 ppt
Stereomicroscope and light source	various		with continuous 1 - 40X magnification