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. Author manuscript; available in PMC: 2019 Dec 3.
Published in final edited form as: Dev Dyn. 2013 Oct 7;242(11):1320–1331. doi: 10.1002/dvdy.24021

A Staging System for the Regeneration of a polyp from the aboral physa of the Anthozoan Cnidarian Nematostella vectensis

Patricia E Bossert 1,*, Matthew P Dunn 1,*, Gerald H Thomsen 1
PMCID: PMC6889817  NIHMSID: NIHMS734722  PMID: 23913838

Abstract

Background

As the sea anemone Nematostella vectensis emerges as a model for studying regeneration, new tools will be needed to assess its regenerative processes and describe perturbations resulting from experimental investigation. Chief among these is the need for a universal set of staging criteria to establish morphological landmarks that will provide a common format for discussion among investigators.

Results

We have established morphological criteria to describe ‘stages’ for rapidly assessing regeneration of the aboral physa of Nematostella. Using this staging system, we observed rates of regeneration that are temperature independent during wound healing and temperature dependent afterwards. Treatment with 25 µM lipoic acid delays the progression through wound healing without significantly affecting the subsequent rate of regeneration. Also, while an 11-day starvation prior to amputation causes only a minimal delay in regeneration, this delay is exacerbated by lipoic acid treatment.

Conclusions

A system for staging the progression of regeneration in amputated Nematostella physa based on easily discernable morphological features, provides a standard for the field. This system has allowed us to identify both temperature-dependent and independent phases of regeneration, as well as a nutritional requirement for normal regenerative progression that is exacerbated by lipoic acid.

Keywords: Regeneration, Cnidarian, Nematostella, staging system, lipoic acid, Anthozoa

Introduction

It is becoming clear that the facility of integrating molecular, organismal and ecological studies makes the basal-branching Cnidarian Nematostella vectensis a powerful model to address fundamental and far reaching questions in biology (Darling et al., 2005; Reitzel et al., 2008; Burton and Finnerty, 2009). A great deal of effort has recently been directed towards characterizing evolutionarily conserved Cnidarian genes known to play critical developmental roles in bilaterians (e.g. Galliot, 2000; Steele, 2002; Scholz and Technau, 2003; Ball et al., 2004; Finnerty et al., 2004; Martindale et al., 2004; Broun et al., 2005;Matus et al., 2008), and there is now a growing interest in regeneration of Nematostella vectensis as well (Darling et al., 2005; Reitzel et al., 2007; Burton and Finnerty, 2009; Tucker et al., 2011; Passamaneck and Martindale, 2012; Trevino et al., 2012; Stefanik et al., 2013; Tucker et al., 2013). Despite the facility with which it can be cultured in the laboratory, its ease of field collection (Hand and Uhlinger, 1992; Hand and Uhlinger, 1994) and its robust ability to regenerate (Reitzel et al., 2007), our understanding of the biology of regeneration in Nematostella is in its infancy compared to Hydra.

Unlike the Hydra, whose polyp is composed of a morphologically uniform epithelial body column capped by a head or foot at either terminus, Nematostella exhibits a complex morphological organization with specialized organs patterned in perpendicular axes; the oral/aboral (longitudinal) axis and the directive (radial) axis (Figure 1). At the oral pole, a slit-like mouth (called an aperture) is contiguous with a hollow wedge shaped organ, the pharynx, which opens into to the body cavity, or coelenteron, at the enterostome (Figure 1B). Surrounding the mouth is a double array of motile, stinging, grasping tentacles that can be indrawn, along with the mouth, forming an eversible chamber that displaces the pharynx aborally when the tentacles are withdrawn. The body column, or scapus, of Nematostella is composed of two muscular epithelial sheets, an ectodermal layer on the external surface of the animal, and an endodermal cell layer facing the coelenteron. The muscle fibers of the endodermal sheet are oriented horizontally in a circular muscle layer that allows radial constriction of the polyp. These layers are separated by a thin nearly acellular matrix, the mesoglea. Lining the coelenteron are eight mesenteries; muscular endodermal organs that extend radially from the column wall towards the center of the body cavity (Stephenson, 1928b; Stephenson, 1928a) (Figure 1C). The mesenteries, observed along their radial axis, comprise muscle tissue nearest the body wall, gonads in an intermediate position, and digestive tissue at the loose internal end that terminates in a clover-leafed trefoil tract called the mesenterial filament (Figure 1E). At the aboral pole of the animal is the physa, a digging organ with which it burrows into mud, allowing it to hide from prey (Hand and Uhlinger, 1994). The capitulum and the physa of Nematostella are analogous to the head and foot of Hydra, respectively. With its complex anatomy and bidirectional asymmetry, regeneration in Nematostella requires cells to respond to positional information more like the bilateria than Hydra, yet it is more readily amenable to extensive experimental manipulation than most bilateria.

Figure 1. Nematostella Anatomy.

Figure 1

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(A) Photograph showing a Nematostella vectensis polyp. Dotted red line indicates amputation level. Scale bar is 0.5 mm. (B) Cartoon illustrating several anatomical structures of the polyp. Capitulum, scapus, and physa are distinguished, in addition to axial location, by ectodermal thickness and organization. Wavy light gray lines correspond to tentacles, dark gray spots are nematocysts, the orange trapezoid represents the pharynx, thick black lines are the mesenterial insertions, the dark purple bars are the mesenteries. (C) Cartoon cross section of the dotted oval in B, illustrating the radial projection of the eight mesenteries. (D) High magnification cartoon of the area in the dotted rectangle in (B) illustrating pleated mesentery as seen from an oblique angle. (E) High magnification cartoon of a mesentery viewed in cross section. Muscle (diagonal lines) tissue is located closest to the body wall, gonads (white circles) are positioned medially, and the digestive region, including the trefoil-shaped mesenterial filament, is located at the free edge of the mesentery, within the coelenteron (Stephenson, 1928a). (apt) aperture, (cap) capitulum, (ent) enterostome, (mf) mesenterial filament, (mi) mesenterial insertion, (ph) physa, (sc) scapus.

Nematostella has the ability to regenerate its entire body from a small stump of amputated physal tissue, but this remarkable ability has yet to be examined in detail. Before such work can be undertaken, additional experimental tools are needed to facilitate the use of this regenerative model. For example, to understand the events that occur during anemone regeneration in a morphogenetic context, it will be useful to have anatomical markers that describe discrete stages of regeneration over a defined time course.

We present simple criteria, based on easily observed gross morphologies, to define a Nematostella Regeneration Staging System (NRSS). This method, adapted from one used in the Galliot laboratory with Hydra spp. (Bossert and Galliot, 2012), is a fast and effective way to codify perturbations of regeneration that permit formation of testable hypotheses. We also present an examination of the reliability of this system in establishing a common understanding among independent scientists, and two simple experiments to demonstrate the efficacy of the staging criteria in evaluating the kinetics of regeneration after experimental manipulations.

Results and Discussion

Regeneration Staging System

To date, the limited literature on Nematostella regeneration (Reitzel et al., 2007; Burton and Finnerty, 2009; Tucker et al., 2011; Passamaneck and Martindale, 2012; Trevino et al., 2012; Tucker et al., 2013) makes evaluation of experimentally induced changes in regeneration difficult, if not impossible. For example, when no aberrant morphology is manifest, subtle changes in the time course of events are likely to be overlooked, and comparisons between, or even within, investigator labs may be hampered by inconsistencies due to a lack of a “normal” benchmark for regeneration. We, therefore, developed the NRSS for sea anemone physa, a piece of relatively simple, distal tissue amputated from mature adults by transverse bisection, at the terminus of the mesenteries (Figure 1). With this NRSS, researchers will now have a common language to refer to different aspects of Nematostella regeneration, aside from simply measuring the time post-amputation (e.g. days postamputation, dpa; hours postamputation, hpa), which is an arbitrary and variable marker of regenerative progression. In the following section, we characterize the progression of a physal piece from an initial 'empty sac' of morphologically near-uniform tissue, through discrete stages to a wholly regenerated individual, indistinguishable from age-matched, non-manipulated animals.

Nematostella Regeneration Staging System (NRSS)

Stages are named and assigned to a number between 0 and 5. Within each integral stage descriptor, morphological landmarks that constitute definitive stage characteristic(s) are described (and italicized). Additional commonly observed morphological features are described that, while not constituting minimal criteria for subsequent stages, provide a more complete picture of the regenerative process, and can be considered 'additional morphologies.' Times are indicated with each stage description to provide a sense of the regeneration rate at a typical lab culture temperature, 18°C. A formal evaluation of temperature on the rate of regeneration is presented in Figure 6.

Figure 6. Effects of Temperature on Regeneration.

Figure 6

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(A) Initial wound healing stages of regeneration appear temperature independent, while the rate of regeneration through later stages varies with culture temperature. Error bars correspond to standard error of the mean, n=4. (B) The number of regenegesta observed throughout regeneration varied with temperature. Note that the y-axis corresponds to the number of regenegesta observed each day and is not cumulative.

Overview: Stages 0 – 2. (Figure 2)

Figure 2. NRSS Stages 0 through 2.

Figure 2

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(A) Stage 0, Open Wound. The wound site may be visibly expanding and contracting, and is seen here in a contracted position. (B) Stage 1, Closed Wound. The wound has closed, although the mesenterial insertions may not meet in an organized fashion at the new oral pole. (C) Stage 2, Radial Arches. The puffed arches, appearing constrained at the mesenterial insertions, give the oral pole the appearance of a stalk-less pumpkin. (D) High magnification view of tissue blebs (dotted orange line). The bleb is nearly spherical in shape and is a single cell layer thick. (E) High magnification view of tentacle buds (dotted red line). Tenacle buds are wider at the base than tip, may appear mound-shaped or pointed, and are two cell layers thick. (A) and (B) are oriented with the oral pole facing out from the page. (C–E) are oriented with the oral pole to the right. Asterisks indicate site of amputation/oral pole. Scale bars are 0.5mm (A–C) and 0.1mm (D,E).

Early stages of regeneration are characterized by wound closure closely followed by a reorganization of the physa that likely involves wound healing prior to visible regeneration of tentacles, pharynx and mesentery. The ratio of the oral/aboral axis relative to the physal diameter is less than 1.5 in Stages 0 – 2. Physa are amputated to ensure this ratio is initially approximately 1.

Stage 0 - Open Wound: Immediately following amputation, the physa is a flaccid cup-shaped tissue mass, resembling a ruptured balloon with a clearly visible opening. Additional Morphologies: The diameter of the open wound decreases; thus it appears that the wound is closing or ‘healing’. The physa is more inflated and less flaccid than immediately after amputation. Contraction of the circular muscle layer, which spans the circumference of the endoderm (Stephenson, 1928a), may be reducing the size of the open wound, but at the site of amputation the wound nevertheless remains visible (Figure 2A). This closure response is dynamic as the physa can be seen to slowly expand and contract the size of the wound for up to eight hours after cutting (~ 0 – 8 hpa).

Stage 1 – Wound Closed: The wound at the amputation site has closed. The sealed wound site is now the new oral pole. If the angle of the amputation was not perpendicular to the oral-aboral axis, the closure point may be offset from the center of the oral pole and/or at an angle to the oral-aboral axis, and mesenterial insertions may not meet in a radially organized pattern at the new pole (Figure 2B). Additional Morphologies: Tissue at the oral surface may begin to reorganize such that the mesenterial insertions form a spoke-like radial array around the closed wound. The oral surface of the regenerate begins to arch, with the oral pole appearing to recede aborally (~ 8 – 48 hpa).

Stage 2 – Radial Arches: The oral surface of the regenerating physa has inflated, forming eight radially symmetric arches, giving the physa a superficial resemblance to the surface of a stalk-less pumpkin. (Figure 2C) Tissue blebs are often present at, or in a ring surrounding, the oral pole (Figure 2D, dotted orange line). These blebs are dynamic single cell-layered bubbles filled with rapidly circulating fluid, and likely do not correspond to tentacle buds. Additional Morphologies: Tentacle buds, characterized as bi-layered protrusions having a length to width ratio less than 1, are visible near the oral pole (Figure 2E, dotted red line). These buds are usually wider proximally than distally and are visually distinct from blebs. Tissue blebs may co-exist with tentacle buds. A denser, darker region at the oral end of the regenerating physa is sometimes visible and may correspond to the early stages of pharynx formation (~ 2 – 5 dpa).

Overview: Stages 3 – 5. (Figure 3)

Figure 3. NRSS Stages 3 through 5.

Figure 3

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(A–E) Stage 3, Tentacles. (A) Tentacle rudiments, longer than wide, are visible at the oral pole. Both pharynx (orange arrowhead) and regenegesta (green arrowhead) may be visible at this stage. (B) High magnification view of the box in (A) illustrating the tentacle bud. (C) The ratio of the oral-aboral axis to the directive axis is variable at this stage, as the animals begin to inflate or contract along their axes. (D) Early mesentery primordia may be visible in Stage 3, but recede into the mesenterial insertion at a distance of less than twice the height from enterostome to mesenterial insertion. Purple arrowhead indicates mesentery. Green and orange arrowheads are as in (A). (E) High magnification view of the box in (D) illustrating the recession of the mesenterial filament into the mesenterial insertion. (F–J) Stage 4, Linear Mesentery. (F) Recession of the mesenterial filament into the mesenterial insertion extends beyond twice the distance from the enterstome to the mesenterial insertion. Arrowheads are as in (D). (G) High magnification view of the box in (F) showing the increased length of the mesentery compared to (E). (H) Oral-aboral axis length, scapus opacity, and tentacle number and length are variable. (I) High magnification view of the box in (H) illustrating early pleating of mesenteries (green line) in the same animal with unpleated mesenteries (yellow line). (J) Aboral view of a Stage 4 animal with four pleated (green arrowheads) and four unpleated (yellow arrowheads) mesenteries. (K–M) Stage 5, Pleated Mesentery. (K) Aboral view of a Stage 5 animal with 8 pleated mesenteries (green arrowheads). Note that asymmetric inflation diminishes apparent pleating in one mesentery (black arrowhead). (L) The majority of the mesenteries demonstrate visible pleating. (M) High magnification view of the box in (L) illustrating the increased extent of pleating (green line). Scale bars are 0.5mm.

Stages 3 through 5 correspond to the regeneration of a miniature animal capable of feeding. The major landmarks are the emergence of tentacles and the appearance of mesenteries emanating from the aboral surface of the pharynx, the enterostome. Bi-layered tentacles elongate and become increasingly motile. The ratio of the oral/aboral axis relative to the directive axis is greater than 1.5 as the animal elongates and also becomes increasingly inflated and transparent. Since we find that temperature strongly modulates progression through Stages 2 to 5 (see below), all times are given for regeneration at 18°C.

In most regenerating physa, between Stage 3 and 4, a spherical mass between 0.5 and 2.0 mm in diameter forms inside the animal, most visibly by Stage 4, as the regenerating animal inflates (Figure 3, green arrowheads in A, D, and F). Usually solid, but occasionally hollow and ciliated, the mass rotates and traverses pole to pole in the actively circulating fluid of the coelenteron. We propose to name this mass the regenegesta (RGE) because we observe it during regeneration and it will be expelled by Stage 5 or shortly thereafter. The origin of these masses is uncertain, but they appear to arise from the enterostome and may represent cells sloughed off through tissue reorganization early in regeneration. Molecular and histological analysis should help identify the origins and composition of these regenegesta.

Stage 3 – Tentacle: True tentacle buds (i.e. definitively two cell layers) are present. They are longer than wide, minimally motile, and vary in number (Figure 3A, dotted red line in 3B). The pharynx may become visible at this stage (Figure 3A,D, orange arrowheads). It is most readily observed as a darkened internal mass near the oral pole when the animal is viewed in profile under the light microscope. Length of the oral-aboral axis relative to the diameter of the regenerating physa is highly variable (Figure 3, compare A to C). Additional Morphologies: The animal shows increasing inflation and transparency. Tentacles are increasing in number, length, and motility, and though present, are often retracted internally. Nematocysts are sometimes visible. Rudimentary mesenteries may become visible when the animal inflates. If mesenteries are visible, the surface of the mesentery facing the body cavity (the mesenterial filament) is smooth or un-pleated. The length of the mesentery along the oral-aboral axis is less than double the height of the mesentery (the distance from the mesenterial insertion to the endodermal surface of the pharynx) at the enterostome (Figure 3E) (~ 5 – 8 dpa).

Stage 4 – Linear Mesenteries: Easily visible mesenteries, protruding radially from the mesenterial insertions toward the coelenteron, are linear and unpleated even upon contraction of the regenerate. These often appear initially in the pharynx-adjacent region, and extend radially into the coelenteron, diminishing in height at progressively aboral locations (Figure 3F, G). Mesenteries become indistinguishable from the mesenterial insertion at a distance no less than twice the height from the insertion to the enterostome (compare Figure 3E to G). When viewed along the oral-aboral axis, with the aboral pole closest to the viewer, the enterostome may be seen within the body cavity with the rudiments of the eight mesenteries radiating outward (Figure 3J). Tentacle number varies. Additional Morphologies: Tentacles may be elongated, fully motile, and/or contain nematocysts. Pharynx may be clearly visible and well defined. Although a well-defined pharynx and elongated tentacles are not definitive criteria for Stage 4 (Figure 3, compare F and H), they may occur concurrently with, or even prior to, mesenteries. Their inclusion as "Additional Morphologies" is not meant to reflect a sequential origin. Pleating is readily visible along the mesenterial filament of no more than four mesenteries (Figure 3I, compare yellow to green lines), usually two opposing couples. This pleating typically arises from the pharynx-adjacent region, where the elaboration of pleating is seen more extensively, and terminates aborally in a smooth filament that becomes indistinguishable from the mesenterial insertion. The sequence of pleating appears to follow the mesentery developmental sequence as described in Stephenson (1928a) (~ 8 – 12 dpa).

NOTE: Extent of pleating, or folding, of the mesenterial filament defines transition to Stage 5. It can be overlooked if the animal is relaxed and fully extended. Gently touching the animal with a soft probe will cause it to contract and, if pleating is present, will become evident. The pleating does not appear to correlate to the elaboration of the trefoil tracts of the filament, but rather to a fullness of mesentery growth.

Stage 5 – Predominantly Pleated Mesenteries: More than four pleated mesenteries are visible, compare Figure 3K to J, green (pleated) to yellow (unpleated) arrowheads. As the regenerate progresses from Stage 4 to Stage 5, both the number of pleated mesenteries and the fullness of the pleating within each mesentery increases (Figure 3, compare L to H, and green lines in M to I). (~ 12 – 14 dpa).

A summary model of the key morphological characteristics and transitional features described in the definition of the NRSS above is illustrated in Figure 4, and Table 1 summarizes these criteria.

Figure 4. Cartoon Illustrating Progression of NRSS Stages.

Figure 4

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(A) Stage 0, open wound. (B) Stage 0 - double-headed red arrows indicate the dynamic expansion and contraction of the wound edge (dotted circle). (C) Stage 1, wound closure (red arrows) at new oral pole (green arrowhead). (D) Stage 1 with additional morphologies: the oral pole appears to become recessed relative to the apex of the inflating arches. (E) Stage 2, arching of oral tissue results in pumpkinhead appearance (green arrowhead). (F) Stage 2 with additional morphologies: physa may begin to elongate and narrow (red arrows). Tentacle buds (red) and blebs (light blue) may appear at the oral ring. (G) Stage 3, tentacles (green arrowhead) appear longer than wide. (H) Stage 3 with additional morphologies: darkening of tissue (orange mass) corresponding to regenerating pharynx may be visible (green arrowhead). (I and I') Stage 4, linear and unpleated mesenteries appear at least twice as long as they are tall at the enterostome. Mesenteries of sufficient (check) and insufficient ('X') length are diagrammed. (J and J') Stage 4 with additional morphologies: mesenterial filaments acquire a pleated appearance in four or less mesenteries. Tentacles increase in number and length. (K) Stage 5, pleated mesenteries now number more than four. (K') Aboral view of Stage 5 mesenteries. All animals oriented with oral pole to the right, except in K'.

Table 1.

NRSS Stage Reference Table

Stage
Number		 Stage Name OA/D
ratio		 Tentacles Tentacle
L/W
ratio		 Pharynx Mesentery
L/H ratio		 Number of
pleated
mesenteries		 Additional Characteristics
0 Open Wound <= 1.5 - - - - - Immediately post-amputation
0.5 - - - - - Dynamic constriction visible around open wound
1 Wound Closed - - - - - Site of closed wound may appear disorganized
1.5 - - - - - Arches begin to inflate
2 Radial Arches - - - - - Radial array of inflated arches; +/− blebs
2.5 Variable Buds may be visible <= 1 a darkening may be visible sub-orally - - +/− blebs
3 Tentacle > 1.5 Clearly defined tentacles > 1 pharynx may be visible sub-orally - -
3.5 Increasing number and length > 2 < 2 0 Increased inflation and transparency
4 Linear Mesentery distinct visible pharynx > 2 No pleating visible, even upon contraction
4.5 <= 4 Increased stimulus reactivity, pleating possibly visible only upon contraction
5 Pleated Mesentery > 4 NRSS complete
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Reliability of the NRSS

With a set of definitions of regeneration stages in place, we sought to determine the efficacy of the NRSS as a means of communicating progression through regeneration between investigators, by assessing the inter-rater reliability of the NRSS.

To this end we performed a blind trial of the NRSS between two individuals scoring regenerating physa, simultaneously with the examination of temperature on regeneration rate described below. Authors MPD and PEB independently scored 100 regenerating physa, twenty at each of five different temperatures, over the course of 17 days, without discussing their findings. A comparison of the NRSS progression of animals at standard culture temperature (18 °C) as scored by these authors is shown in Figure 5. Several reliability statistics - the Pearson product-moment correlation coefficient (R), a weighted Cohen's kappa (κw) (Cohen, 1968), and Krippendorff's alpha (α) (Hayes and Krippendorff, 2007) - were determined for each temperature (Table 2). Values of reliability statistics approaching 1 indicate nearly perfect positive correlation; Fleiss (2003) suggests that κw values of "≥0.75 or so signifies excellent agreement", while Krippendorf (2004) endorses α values of ≥0.80 as reliable. As shown in Table 2, all R values were above 0.98, κw above 0.80, and α above 0.92, suggesting that the NRSS permitted high reliability across raters in identifying the stage of an anemone's regeneration.

Figure 5. Inter-Rater Reliability of the NRSS.

Figure 5

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Graph comparing Average Stage Regeneration of four sets of five amputated physa at 18 °C, as scored by MPD and PEB. Investigators were scoring the same animals independently without communication.

Table 2.

Inter-rater Reliability of the NRSS at Different Temperatures

Temperature R κw α
15 °C 0.983 0.916 0.926
18 °C 0.991 0.896 0.963
21 °C 0.992 0.801 0.929
24 °C 0.988 0.898 0.963
27 °C 0.998 0.813 0.943
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Effect of Culture Temperature on Regeneration Rate

To examine whether the rate of regeneration was subject to fluctuations in culture conditions, we examined the rate of progression through the staging system described above at five different culture temperatures, 15, 18, 21, 24, and 27°C. Four sets of five freshly amputated physa were cultured at each temperature, for a total of 100 physa. Each animal was observed every 24 hours following amputation and staged according to the NRSS outlined above. As seen in Figure 6A, while all the animals proceeded from Stages 0 through 2 rather synchronously, progression through later stages was temperature dependent. Animals cultured at higher temperatures proceeded through the regeneration stages at a faster rate than those maintained at lower temperatures. Nematostella cultured at 27°C regenerated at the fastest rate, progressing from Stage 2 to Stage 4.5 in under 4 days, with each group of animals taking longer at progressively lower temperatures.

The two distinct phases of regeneration, the first being temperature independent (Stages 0–2) and the other being temperature dependent (Stages 2–5), suggest that the metabolic demands on wound closure are low, compared to the metabolically intensive morphological restructuring occurring at the later regeneration stages. Previous studies on Hydra (Peebles, 1898), planarians (Brøndsted and Brøndsted, 1961), fiddler crabs (Weis, 1976), and urodele amphibians (Schauble and Nentwig, 1974; Young et al., 1983; Tattersall et al., 2012) have also described a temperature-based influence on the rate of regeneration. Similarly, the separation of regeneration into 'wound healing' and 'regenerating' phases was seen in newts (Schauble and Nentwig, 1974; Tattersall et al., 2012), salamanders (Bryant et al., 2002; Gardiner et al., 2002), and planarians (Brøndsted and Brøndsted, 1961), however while the wound healing phase in newts also appeared to be temperature independent (Schauble and Nentwig, 1974; Tattersall et al., 2012), that of the planarians appeared to vary with temperature, although this is difficult to assess from the published data (Brøndsted and Brøndsted, 1961). Recent work in Nematostella has shown that, in bisection of juvenile polyps (5–10 mm in length), cell proliferation begins to increase roughly 24 hpa (at 22°C), reaching a maximum at roughly 48 hpa (Passamaneck and Martindale, 2012). This maximal cell proliferation level corresponds well with the transition from wound healing to regeneration at NRSS Stage 2, although the NRSS is characterizing regeneration of older, larger polyps.

The number of tissue masses, or regenegesta, formed in regenerating physa is correlated with temperature. The total number of regenegesta observed (both egested and yet to be egested) was tallied at each temperature, and the cumulative number of regenegesta per day was plotted as a function of temperature (Figure 6B). Higher temperatures correlated with larger numbers of total regenegesta produced. A photomicrograph of a Stage 3 regenerating polyp egesting a regenegesta is shown (Figure 6B, inset).

Lipoic Acid and Starvation Both Affect Regeneration

We next evaluated the efficacy of our staging system in an hypothesis-driven experiment. Based on studies in Hydra that demonstrated that lipoic acid permanently arrests regeneration (Ham and Eakin, 1958; Eakin, 1961), we hypothesized a similar arrest would occur in Nematostella. We therefore pulsed Nematostella physa in 25 µM lipoic acid for 24 hours immediately following amputation (dpa 0 – 1). Initial experiments revealed no clear arrest of regeneration nor did we observe any aberrant phenotype resulting from drug treatment.

We then repeated the experiment, and assessed the kinetics of regeneration of these physa using the NRSS (Figure 7). We found that the experimental and control physa completed regeneration simultaneously, however, the lipoic acid treated physa exhibited a lag in regeneration. This lag was undetectable in preliminary experiments prior to utilizing the NRSS.

Figure 7. Effects of Lipoic Acid and Starvation on Regeneration.

Figure 7

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Physa treated with lipoic acid, prior starvation, or both, exhibited delays in regeneration compared to untreated control physa. Lipoic acid treatment, regardless of prior nutritional state, results in a lag in progression from Stage 1 to Stage 2. Prior starvation alone causes a 6–18 hour delay in stage progression beginning at Stage 2, while combination of both lipoic acid and prior starvation exacerbates this lag. Error bars correspond to standard error of the mean, n=4.

All regenerating physa progressed from open wound (Stage 0) through wound closure (Stage 1) and into the characteristic transitional reorganization of tissue at the same rate. In contrast, physa pulsed with lipoic acid reached Stage 2 after a lag of approximately 1 day compared to physa not pulsed with the drug (Figure 7, compare blue to red lines).

We next assessed the effects of nutritional state on regeneration, hypothesizing that the ability of the physa to meet the energy demands of regenerating complex structures would be negatively affected by prior starvation of the polyp. Starting 11 days prior to amputation, animals were starved as described in the Experimental Procedures. Following amputation, the progression of regenerating physa was observed and scored using the NRSS. We performed this starvation experiment in parallel on physa treated with lipoic acid, as above, and on un-drugged physa amputated from starved polyps.

Contrary to our hypothesis, starvation alone had a relatively minor effect on subsequent physal regeneration over the 10-day period of observation (Figure 7, compare blue to green lines). After reaching Stage 2, physa from starved animals lagged approximately 6 to 18 hours behind physa from fed animals in reaching each benchmark of the NRSS until regeneration was almost complete by dpa 9.

After regenerating physa reach Stage 2, there appears to be an interaction between drug pulse and the nutritional condition of the animal from which the physa was removed. Physa from starved donors, regenerating after a pulse of lipoic acid, lag at least 1 day behind physa regenerating subsequent to all other treatments until regeneration is almost complete (Figure 7, compare red to purple lines). This is in contrast to the 6 to 18 hour delay in undrugged starved physa described above.

Taken in conjunction with the temperature dependency illustrated above, these experiments suggest a metabolic component to regeneration manifests only after wound healing and the reorganization of the oral pole completes. Also, in these experiments, the effect of a lipoic acid pulse on regeneration appears to have an ‘early’ vs. ‘late’ component. That is, before Stage 2 the drug effect is independent of prior nutritional conditioning but after Stage 2 the effect of the drug appears to be mediated by the underlying metabolic state of the physa. Given the role of lipoic acid in the regulation of cellular energetics, redox regulation of transcription and cellular signaling (Packer and Cadenas, 2011) it will be interesting to examine the mechanism(s) underlying the phenomena we find using the NRSS.

On Nematostella Regeneration

In establishing the NRSS above, we observed that following wound healing, structures characteristic of the oral end, the mouth and tentacles, regenerated prior to the mesenteries, which are more proximal to the aboral end. The case of the pharynx is slightly complicated, however by the opacity of the regenerate in Stages 2 through 4. While the tentacles are observed before a defined pharynx is clearly visible, the timing of tentacle relative to pharynx regeneration is not readily apparent from either the NRSS as described or from morphological observation.

Metazoan regeneration has long been classified into two types: morphallaxis, in which cells from throughout the organism contribute to the regenerating region in the absence of cell division or blastema formation, and epimorphosis, where a local blastema arises at the wound site and regenerates lost tissue through proliferation (Morgan, 1901). Classically, Hydra regeneration was seen as an example of the former while amphibian limb and planarian regeneration demonstrated the latter. A recent Nematostella study demonstrating the necessity of cell proliferation for proper regeneration concluded therefore that Nematostella regenerated via epimorphosis (Passamaneck and Martindale, 2012).

However, the utility of these classical terms, based on gross morphological studies over a century old, has been called into question in light of advances in scientific techniques that allow closer examination of the process at the cellular and molecular levels. A variety of investigations have revealed that aspects of both epimorphosis and morphallaxis are present in many regenerating groups (e.g., Reddien and Sánchez Alvarado, 2004; Agata et al., 2007; Galliot and Chera, 2010). Based on observations in urodeles, cockroaches, and planarians (Iten and Bryant, 1975; French, 1976; Agata et al., 2003), Agata et al. (2007) suggested a unifying trait across metazoans was the initial regeneration at the wound site of the most distal structure, which they termed 'distalizaton'. Following distalization, the proximal and newly distal structures interacted to direct regeneration of progressively intermediate structures, which they termed 'intercalation'. Based on the ubiquity of this process, as opposed to the more variable distinction between epimorphosis and morphallaxis, Agata et al. (2007) therefore proposed replacing the equivocal morphallaxis and epimorphosis terminology with the more biologically relevant distalization and intercalary regeneration.

The progression we observed of mouth and tentacles followed by mesenteries suggests distalization and intercalary regeneration as discussed by Agata et al. (2007), but since we are unable to establish exactly when the pharynx forms this remains an open question. True distalization requires that the regenerating physa establishes the distal-most structure first, which in Nematostella is the enterostome. Given the typical opacity of the animal at early NRSS stages, use of the NRSS alone cannot address this issue. However, we look forward to a closer, cellular and molecular examination of regenerating structures in the future to resolve this issue, which we have not evaluated here because we want to present a simple and rapid scoring system applicable to live animals.

Conclusions

The system described here is a method that permits analysis of the kinetics of regeneration of a complete polyp from the physa of Nematostella vectensis and can be used to score normal regenerative processes and responses to experimental manipulation. The NRSS uses gross morphological criteria that can be seen with simple optics so that relatively large numbers of animals can be staged rapidly. While a thorough histological and molecular analysis of Nematostella regeneration would be a welcome addition to our knowledge both of this animal's biology and of the process of regeneration in general, our goal was to provide a tool for simple, rapid assessment of regenerating animals that would provide a common set of standards for the field, ones that could be easily learned and readily deployed by investigators.

In addition to describing regeneration from physa, the NRSS could, with minimal modification, be adapted to score regeneration of structures from aboral pieces bisected within the scapus. For example, while some earlier regeneration studies (Reitzel et al., 2007; Burton and Finnerty, 2009; Tucker et al., 2011; Trevino et al., 2012; Tucker et al., 2013) utilized mid-scapus bisections instead of the physal amputations described here, examination of the published figures and times post amputation specified suggests that the majority of gene expression patterns described take place during the temperature-independent wound-healing phase of regeneration. Upon examination of regenerating aboral fragments of mid-scapus bisections, we have observed that these animals progress through Stages 0 – 3 (Open Wound through Tentacles) of the NRSS as described. How (or whether) the preexisting mesentery is incorporated into the regenerating oral pole of the animal, and thus the relevance of Stage 4, is unclear from a cursory examination. A closer examination of this process will allow adaptation of the NRSS for studies utilizing mid-scapus bisection.

We envision that the NRSS will provide a base for layering on more detailed information about the molecular, cellular and tissue biology of regeneration, as well as be useful for fast, scalable screens (e.g. mutagenesis, small molecules). Our finding that lipoic acid affects the regenerative kinetics of nutritionally deprived physa was not observable in our experiments prior to development of the NRSS, but was revealed after applying this scoring system. How nutritional reserves are mobilized during regeneration, in the absence of feeding, is a generally important question raised by this study, and is now under investigation.

Experimental Procedures

Anemone Culture

Nematostella were maintained between 18 and 24°C, unless otherwise indicated, in non-circulating 1/3x Artificial Sea Water (ASW, 12ppt) in the dark and fed freshly hatched Artemia nauplii twice per week. Culture bowls were cleaned once a week. For starvation experiments, starved animals were deprived of Artemia for at least ten days prior to amputation. To examine the effects of temperature on regeneration rate, animals were maintained at 21°C prior to amputation, and then maintained at the indicated culture temperature until all experimental animals reached Stage 5.

Amputation

Size-matched polyps (approximately 3–5cm long) were selected based on length of physa aboral to the site of mesentery termination (at least the length of the diameter). Selected animals were maintained separate from the colony for three days prior to amputation. Animals were relaxed by adding menthol crystals to the ASW until no longer responsive to tactile stimulation. A length of physa approximately as wide as long and containing no mesentery was amputated from the animal using a sterile scalpel. Amputated physa were rinsed once in fresh ASW, and then maintained in 2 mL ASW per physa. Recovery from menthol relaxation was not found to have any effect on the progression through the NRSS when compared to physa amputated without menthol treatment.

Lipoic Acid Treatment

Lipoic acid (Catalogue Number: 462-20-4, Sigma), a kind gift from Dr. Zuzana Zachar, was dissolved in dimethylformamide (DMF) to produce a 1M stock solution. This was then diluted to 25 µM with ASW. Amputated physa were pulsed with lipoic acid for 24 hrs immediately subsequent to amputation, and removed to ASW thereafter. Drug-free controls were pulsed with an equivalent volume of DMF. Animals were maintained at 21 °C throughout the experiment.

Temperature Experiment and Statistics

At each temperature tested, four sets of five animals each were amputated and cultured in 10 mL of ASW (2 mL per physa), and scored every 24 hours. The NRSS scores of each set of five animals were averaged and plotted as a function of dpa. Animals exhibiting 'Additional Morphologies' were scored half of a stage above those without. Error bars correspond to the Standard Error of the Mean, n=4.

Inter-rater Reliability Calculations

Average Stage Regeneration of 20 animals per temperature over 17 days as scored by authors MPD and PEB were compared to determine the Pearson product-moment correlation coefficient (R), Cohen's weighted kappa (κw), and Krippendorff's alpha (α) for ordinal data. The Pearson product-moment correlation coefficient (R) values were calculated using Microsoft Excel's PEARSON function. Cohen's weighted kappa was calculated as described in Fleiss, et al. (2003). The Krippendorff's alpha (α) for ordinal data was calculated using the ReCal OIR web application (Freelon, 2013).

Bullet Points.

  • *

    We have established a staging system for regeneration in Nematostella vectensis

  • *

    Permits rapid staging based on easily discernable morphological criteria

  • *

    Allowed us to identify both delays in and reduced rates of regeneration

Acknowledgements

We would like to thank Drs. William Q. Gillis, Dianella G. Howarth, and Zuzana Zachar, and members of the Thomsen, Ballas, Holdener, Martin, and Sorotkin laboratories for helpful comments on the staging criteria and the manuscript. We also wish to thank Frank Howarth for generating the diagram in Figure 1D. This research was supported by a Stony Brook University School of Medicine Targeted Research Opportunity (TRO) grant # 2008-TRO and NYSTEM grant # C028107 to GHT. MPD was supported by USPHS Institutional National Research Service Award 5T32DK007521 from the NIDDKD, NIH.

References

  1. Agata K, Saito Y, Nakajima E. Unifying principles of regeneration I: Epimorphosis versus morphallaxis. Develop. Growth Differ. 2007;49:73–78. doi: 10.1111/j.1440-169X.2007.00919.x. [DOI] [PubMed] [Google Scholar]
  2. Agata K, Tanaka T, Kobayashi C, Kato K, Saitoh Y. Intercalary Regeneration in Planarians. Developmental Dynamics. 2003;226:308–316. doi: 10.1002/dvdy.10249. [DOI] [PubMed] [Google Scholar]
  3. Ball EE, Hayward DC, Saint R, Miller DJ. A simple plan - cnidarians and the origins of developmental mechanisms. Nature Reviews Genetics. 2004;5:567–577. doi: 10.1038/nrg1402. [DOI] [PubMed] [Google Scholar]
  4. Bossert P, Galliot B. How to use Hydra as a model system to teach biology in the classroom. International Journal of Developmental Biology. 2012;56:637–652. doi: 10.1387/ijdb.123523pb. [DOI] [PubMed] [Google Scholar]
  5. Brøndsted A, Brøndsted HV. Influence of Temperature on Rate of Regeneration in the Time-graded Regeneration Field in Planarians. Journal of Embryology and Experimental Morphology. 1961;9:159–166. [Google Scholar]
  6. Broun M, Gee L, Reinhardt B, Bode HR. Formation of the head organizer in hydra involves the canonical Wnt pathway. Development. 2005;132:2908–2916. doi: 10.1242/dev.01848. [DOI] [PubMed] [Google Scholar]
  7. Bryant SV, Endo T, Gardiner DM. Vertebrate limb regeneration and the origin of limb stem cells. International Journal of Developmental Biology. 2002;46:887–896. [PubMed] [Google Scholar]
  8. Burton PM, Finnerty JR. Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis. Dev Genes Evol. 2009;219:79–87. doi: 10.1007/s00427-009-0271-2. [DOI] [PubMed] [Google Scholar]
  9. Cohen J. Weighted kappa: Nominal scale agreement provision for scaled disagreement or partial credit. Psychological Bulletin. 1968;70:213–220. doi: 10.1037/h0026256. [DOI] [PubMed] [Google Scholar]
  10. Darling JA, Reitzel AM, Burton PM, Mazza ME, Ryan JF, Sullivan JC, Finnerty JR. Rising starlet: the starlet sea anemone, Nematostella vectensis. BioEssays. 2005;27:211–221. doi: 10.1002/bies.20181. [DOI] [PubMed] [Google Scholar]
  11. Eakin RE. Studies on Chemical Inhibition of Regeneration in Hydra. In: Lenhoff HM, Loomis WF, editors. The Biology of Hydra and some other Coelenterates. Coral Gables, Florida: University of Miami Press; 1961. [Google Scholar]
  12. Finnerty JR, Pang K, Burton PM, Paulson D, Martindale MQ. Origins of Bilateral Symmetry: Hox and Dpp Expression in a Sea Anemone. Science. 2004;304:1335–1337. doi: 10.1126/science.1091946. [DOI] [PubMed] [Google Scholar]
  13. Fleiss JL, Levin B, Cho Paik M. Statisical Methods for Rates and Proportions. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2003. p. 760. [Google Scholar]
  14. Freelon D. ReCal OIR: Ordinal, Interval, and Ratio Intercoder Reliability as a Web Service. International Journal of Internet Science in press. 2013 [Google Scholar]
  15. French V. Leg regeneration in the cockroach, Blatella germanica: II. Regeneration from a non-congruent tibial graft/host junction. Journal of Embryology and Experimental Morphology. 1976;35:267–301. [PubMed] [Google Scholar]
  16. Galliot B. Conserved and divergent genes in apex and axis development of cnidarians. Curr. Opin. Genet. Dev. 2000;10:629–637. doi: 10.1016/s0959-437x(00)00141-6. [DOI] [PubMed] [Google Scholar]
  17. Galliot B, Chera S. The Hydra model: disclosing an apoptosis-driven generator of Wnt-based regeneration. Trends in Cell Biology. 2010;20:514–523. doi: 10.1016/j.tcb.2010.05.006. [DOI] [PubMed] [Google Scholar]
  18. Gardiner DM, Endo T, Bryant SV. The molecular basis of amphibian limb regeneration: integrating the old with the new. Seminars in Cell & Developmental Biology. 2002;13:345–352. doi: 10.1016/s1084952102000903. [DOI] [PubMed] [Google Scholar]
  19. Ham RG, Eakin RE. Loss of regenerative capacity in Hydra treated with lipoic acid. Journal of Experimental Zoology. 1958;139:55–68. doi: 10.1002/jez.1401390105. [DOI] [PubMed] [Google Scholar]
  20. Hand C, Uhlinger KR. The Culture, Sexual and Asexual Reproduction, and Growth of the Sea Anemone Nematostella vectensis. Biological Bulletin. 1992;182:169–176. doi: 10.2307/1542110. [DOI] [PubMed] [Google Scholar]
  21. Hand C, Uhlinger KR. The Unique, Widely Distributed, Estuarine Sea Anemone, Nematostella vectensis Stephenson: A Review, New Facts, and Questions. Estuaries. 1994;17:501–508. [Google Scholar]
  22. Hayes AF, Krippendorff K. Answering the Call for a Standard Reliability Measure for Coding Data. Communication Methods and Measures. 2007;1:77–89. [Google Scholar]
  23. Iten LE, Bryant SV. The interaction between the blastema and stump in the establishment of the anterior-posterior and proximal-distal organization of the limb regenerate. Developmental Biology. 1975;44:119–147. doi: 10.1016/0012-1606(75)90381-4. [DOI] [PubMed] [Google Scholar]
  24. Krippendorff K. Content analysis: an introduction to its methodology. Thousand Oaks, California: Sage Publications, Inc.; 2004. p. 411. [Google Scholar]
  25. Martindale MQ, Pang K, Finnerty JR. Investigating the origins of troploblasty: 'mesodermal' gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa) Development. 2004;131:2463–2474. doi: 10.1242/dev.01119. [DOI] [PubMed] [Google Scholar]
  26. Matus DQ, Magie CR, Pang K, Martindale MQ, Thomsen GH. The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution. Dev Biol. 2008;313:501–518. doi: 10.1016/j.ydbio.2007.09.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morgan TH. Regeneration. New York: The Macmillan Company; 1901. p. 316. [Google Scholar]
  28. Packer L, Cadenas E. Lipoic acid: energy metabolism and redox regulation of transcription and cell signaling. Journal of Clinical Biochemistry and Nutrition. 2011;48:26–32. doi: 10.3164/jcbn.11-005FR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Passamaneck YJ, Martindale MQ. Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis. BMC Developmental Biology. 2012;12:34. doi: 10.1186/1471-213X-12-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Peebles F. The Effect of Temperature on the Regeneration of Hydra. Zoological Bulletin. 1898;2:125–128. [Google Scholar]
  31. Reddien PW, Sánchez Alvarado A. Fundamentals of Planarian Regeneration. Annual Review of Cell and Developmental Biology. 2004;20:725–757. doi: 10.1146/annurev.cellbio.20.010403.095114. [DOI] [PubMed] [Google Scholar]
  32. Reitzel AM, Burton PM, Krone C, Finnerty JR. Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission. Invertebrate Biology. 2007;126:99–112. [Google Scholar]
  33. Reitzel AM, Sullivan JC, Traylor-Knowles N, Finnerty JR. Genomic Survey of Candidate Stress-Response Genes in the Estuarine Anemone Nematostella vectensis. Biological Bulletin. 2008;214:233–254. doi: 10.2307/25470666. [DOI] [PubMed] [Google Scholar]
  34. Schauble MK, Nentwig MR. Temperature and Prolactin as Control Factors in Newt Forelimb Regeneration. Journal of Experimental Zoology. 1974;187:335–344. doi: 10.1002/jez.1401870304. [DOI] [PubMed] [Google Scholar]
  35. Scholz CB, Technau U. The ancestral role of Brachyury: expression of NemBra1 in the basal cnidarian Nematostella vectensis (Anthozoa) Development Genes and Evolution. 2003;212:563–570. doi: 10.1007/s00427-002-0272-x. [DOI] [PubMed] [Google Scholar]
  36. Steele RE. Developmental Signaling in Hydra: What Does It Take to Build a "Simple" Animal? Developmental Biology. 2002;248:199–219. doi: 10.1006/dbio.2002.0744. [DOI] [PubMed] [Google Scholar]
  37. Stefanik DJ, Friedman LE, Finnerty JR. Collecting, reaaring, spawning and inducing regeneration of the starlet sea anenome, Nematostella vectensis. Nature Protocols. 2013;8:916–923. doi: 10.1038/nprot.2013.044. [DOI] [PubMed] [Google Scholar]
  38. Stephenson TA. The British Sea Anemones. London: Dulau & Co., Ltd.; 1928a. p. 176. [Google Scholar]
  39. Stephenson TA. The British Sea Anemones. London: Dulau & Co., Ltd.; 1928b. p. 464. [Google Scholar]
  40. Tattersall GJ, Tyson TM, Lenchyshyn JR, Carlone RL. Temperature Preference During Forelimb Regeneration in the Red-Spotted Newt Notophthalmus viridescens. Journal of Experimental Zoology. 2012;317:248–258. doi: 10.1002/jez.1719. [DOI] [PubMed] [Google Scholar]
  41. Trevino M, Stefanik DJ, Rodriguez R, Harmon S, Burton PM. Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis. Developmental Dynamics. 2012;240:2673–2679. doi: 10.1002/dvdy.22774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tucker RP, Hess JF, Gong Q, Garvey K, Shibata B, Adams JC. A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration. Biology Open. 2013;2:217–226. doi: 10.1242/bio.20123103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tucker RP, Shibata B, Blankenship TN. Ultrastructure of the mesoglea of the sea anenome Nematostella vectensis (Edwardsiidae) Invertebrate Biology. 2011;130:11–24. [Google Scholar]
  44. Weis JS. Effects of Environmental Factors on Regeneration and Molting in Fiddler Crabs. Biological Bulletin. 1976;150:152–162. doi: 10.2307/1540596. [DOI] [PubMed] [Google Scholar]
  45. Young HE, Bailey CF, Dalley BK. Environmental conditions prerequisite for complete limb regeneration in the postmetamorphin adult land-phase salamander, Ambystoma. Anatomical Record. 1983;206:289–294. doi: 10.1002/ar.1092060307. [DOI] [PubMed] [Google Scholar]

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