A Proteoglycan-Like Molecule Offers Insights Into Ground Substance Changes During Holothurian Intestinal Regeneration

Abstract

Extracellular matrix remodeling is an essential component of regenerative processes in metazoans. Among these animals, holothurians (sea cucumbers) are distinguished by their great regenerative capacities. We have previously shown that fibrous collagen as well as other fibrous components disappear from the connective tissue (CT) early during intestinal regeneration, and later return as the organ primordia form. We now report on changes of the nonfibrous component of the CT. We have used Alcian Blue staining and an antibody, Proteoglycan Like-1 (PGL-1), that recognizes a proteoglycan-like antigen to identify the presence of proteoglycans in normal and regenerating intestines. Our results show that early in regeneration, the ground substance resembles that of the mesentery, the structure from where the new intestine originates. As regeneration proceeds, Alcian Blue staining and PGL-1 labeling reorganize, so that by 4 weeks the normal intestinal CT pattern is achieved. Together with our previous findings, the data suggest that CT components that might be detrimental to regeneration disappear early on, while those that might be beneficial to regeneration, such as proteoglycans, are present throughout the regenerative process.

Introduction

With a few exceptions, most mammalian tissues do not regenerate very well.¹ Model organisms used for regeneration studies encompass species of various phyla. Echinoderms hold the distinction of both being closely related to vertebrates and having extensive regenerative capacities. Among echinoderms, holothurians or sea cucumbers are known for their capacity to eviscerate their internal organs, and then regenerate them later on.² We use the species Holothuria glaberrima, as a model system for studying intestinal regeneration.³ In this model, we can induce an autotomy event where the intestines, along with other organs, are eviscerated through the cloaca. Following evisceration, a regenerative process begins with the thickening of the mesentery edges, which eventually form a solid primordium with blastema-like morphology by the end of the first week. Then, the lumen of the intestinal primordium forms by the migration of luminal epithelial cells from the esophagus and cloaca. Eventually, cells differentiate and the different layers of the intestine are formed, giving rise to an intestine with all the normal layers (albeit smaller in size) in about a month.^2–4

It is well known that remodeling of the extracellular matrix (ECM) is essential for regenerative processes. A classic example of this is mammalian skin wound healing. During this process, the prewound ECM is first replaced by a fibrin clot, and then by a temporary granular matrix, which is then followed by the formation of a collagen type 1 rich scar. Interestingly, mammalian early gestation fetuses have a much greater capacity for wound healing than their adult counterparts.^5,6 The differences in regenerative capacities between adult and fetal skin have been ascribed to differences in ECM components. Fetal wound healing is characterized by a greater deposition of collagen type III (reticular collagen) and high molecular weight hyaluronic acid. In contrast, adult healing features type I collagen deposition and a transient expression of low molecular weight hyaluronic acid. They also differ in the composition of proteoglycans.^6,7

We have previously shown that ECM remodeling is important for intestinal regeneration in H. glaberrima.⁸ In particular, fibrous collagen was shown to disappear from the connective tissue (CT) of the regenerate, concomitant with an increase in metalloprotease activity.^8,9 Although previous studies provided important information on the remodeling of major fibrous components of the holothurian intestinal ECM during regeneration, nonfibrous components, such as proteoglycans, were not examined. Proteoglycans are known to be essential for regenerative processes. For example, the loss of Syndecan-1 (Sdc-1), a membrane bound proteoglycan, has been correlated with poor healing of colonic ulcers in humans, and knock out of the Sdc-1 gene has been reported to slow down the regeneration of intestinal mucosa in dextran sodium sulfate (DSS)–induced murine ulcerative colitis.¹⁰

We have now focused on the nonfibrous components, specifically the ground substance, of the holothurian ECM. The ground substance is the gelatinous substrate that lies between cells and fibers of the CT and is made by glycosaminoglycans. To study the ground substance, we have used Alcian Blue staining, the classical stain for proteoglycans in ECM. In addition, we have generated a monoclonal antibody that labels a nonfibrous component of the ECM in the holothurian intestinal CT, Proteoglycan Like-1 (PGL-1). Our results show that both Alcian Blue and PGL-1 strongly stain the ECM of the mesentery of normal and regenerating animals. Moreover, we describe how proteoglycan staining remains homogeneously distributed during the formation of the new intestine and eventually acquires the pattern observed in the normal organ. The data suggest that while fibrous ECM components, such as collagen, deter regeneration, nonfibrous components, or at least proteoglycans, appear to favor the regenerative process.

Materials and Methods

Animals

Adult sea cucumbers (H. glaberrima) were collected from the northern coast of Puerto Rico and kept in seawater aquaria at 20C to 24C. Noneviscerated animals were stored in aquaria for a day before being sacrificed. Evisceration was induced by injecting 3 to 5 ml of 35 mM potassium chloride (KCl) (Sigma-Aldrich; St. Louis, MO) intracoelomically. All procedures were followed in accordance with the University of Puerto Rico and the National Institutes of Health (NIH) guidelines.

Antibody Production

Mice were immunized with regenerating sea cucumber tissue, prepared as follows. Intestinal rudiments from sea cucumbers at 5 days post evisceration (dpe) were dissected and homogenized manually in 50% ethanol using a glass homogenizer. After centrifugation for 2 min at 12,000 rpm, the supernatant was discarded and the pellet resuspended in phosphate-buffered saline (PBS), aliquoted, and stored at −20C. Two mice each were immunized intraperitoneally with 100 µl of a mixture of 5 dpe homogenate and Titermax adjuvant (Sigma-Aldrich) in a 1:1 proportion; a boost was performed 3 to 4 weeks after the initial immunization using a same mixture and volume. Periorbital blood collection was done 7 to 10 days after the boost for detection of antibodies using immunohistochemistry (IHC) as described below. Once the tested antisera resulted in the expected labeling, a final boost without adjuvant was performed to the selected mouse 3 days prior to the cell fusion. The fusion was performed as described previously¹¹ using splenocytes from the immunized mouse and myeloma cells from American Type Culture Collection (ATCC) (CRL-1581). After identifying positive antibody-producing hybridomas through IHC, the cells were cloned twice by limiting dilution in 96-well culture plates.

PGL-1 ascites was produced as follows. Mice were primed with an intraperitoneal injection of 0.2 ml Pristane (Sigma-Aldrich), 10 days prior to the implantation of hybridoma cells, at 1 × 10⁶ cells in 0.5 ml total volume prepared in sterile 0.85% sodium chloride (NaCl). Animals were monitored daily and euthanized to collect the ascitic fluid before identifying signs of distress.

Histology

Sea cucumbers were removed from aquaria and placed in anesthetic (1,1,1-Trichloro-2-methyl-2-propanol hydrate 98% [Sigma-Aldrich] diluted in seawater in a 2:1 ratio) for 30 min. Intestinal tissues, including the adjacent body wall, were dissected and fixed in 4% paraformaldehyde (Sigma-Aldrich) solution overnight at 4C. To wash off the paraformaldehyde, three 15 min 0.1M PBS (pH = 7.4) washes were done. The tissues were then stored in 30% sucrose (Sigma-Aldrich)/PBS solution at 4C until they were sectioned.

For sectioning, the tissue was placed in Optimal Cutting Temperature Compound (Tissue-tek OCT Compound, Electron Microscopy Sciences; Hatfield, PA) and sectioned in a Leica CM1850 Cryostat (Nussloch, Germany) at −30C to −35C. Cryosections (20 µm) were placed on Poly-Lysine (Sigma-Aldrich) coated slides and dried off.

IHC

IHC techniques have been described before.¹² Sections were obtained from small ascendant, small descendant, and large intestines, including the associated mesenteries as well as from anterior and posterior regions of regenerating intestines. The anterior section of the regenerate is located near the remaining esophagus and will give rise to the small intestine. The posterior portion of the regenerate is located near the cloaca and will give rise to the large intestine. Regenerating animals at stages 3, 5, 7, 14, 21, 28, and 35 dpe were studied. At least three animals were used per stage.

IHC experiments were carried out in the following manner. First, 50 µl of goat serum 1/50 was added to each tissue section and incubated in a humid chamber for 1 hr. They were then washed 3 times with 0.1M PBS (15 min each), and the primary antibody was added. Sections treated with primary antibody were incubated in the humid chamber overnight at room temperature. Negative controls included sections incubated without the antibody as well as sections incubated with other monoclonal antibodies that recognized cellular components.

The primary antibodies used were (1) PGL-1, produced in our laboratory (described in the “Antibody Production” section) both as undiluted hybridoma media or ascites (1/200–1/500 dilution), and (2) Holothuria glaberrima Collagen (HgCol) (1/10). This is a monoclonal antibody also made in our laboratory. It labels fibrous collagen as described previously by Quiñones et al.⁸

The next day, sections were washed with PBS and incubated with GAMCY3 (Jackson ImmunoResearch Laboratories; West Grove, PA) 1/1000 (diluted in radioimmunoassay [RIA] buffer), the secondary antibody, for 1 hr at room temperature. Finally, another set of PBS washes was done, and the slides were mounted using a glycerol buffered solution that contained 4′,6-diamidino-2-phenylindole (DAPI).

Alcian Blue Histology

Two methods of Alcian Blue histology were performed. These methods vary in the preparation of the Alcian Blue dye and in the steps that are performed in each of them. The first method used Alcian Blue at pH = 1 and the other at pH = 2.5. Both of these methods are described by Humason.¹³

Microscopy

Slides were observed in a Nikon Eclipse E600 (Tokyo, Japan) fluorescence microscope equipped with fluorescein isothiocyanate (FITC), rhodamine (R/DII), and DAPI filters. Sections were photographed using the SPOT-RT3 cameras and the accompanying SPOT basic software. The brightness and contrasts of the brightfield, and DAPI labeling, have been altered to reduce their obscuring effect on the primary antibody labeling. Using the SPOT basic software, color was added to differentiate the DAPI and antibody-specific labeling when the images were overlaid.

Biochemical Experiments

Protein Extraction

Dissected tissues were placed in a solution of radioimmunoprecipitation assay (RIPA) 1× buffer (20 mM Tris–Hydrochloric acid [Tris-HCl] at pH 7.5, 150 mM NaCl, 1 mM disodium ethylenediaminetetraacetate [Na₂EDTA], 1 mM ethylene glycol tetraacetic acid [EGTA], 1% Nonidet P-40 (octyl phenoxypolyethoxylethanol), 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate [Na₃VO₄]). Proteinase inhibitor cocktail 100× was added to the solution for a final concentration of 1%. The tissue was homogenized using a “PowerGen Model 125” Homogenizer (Fisher Scientific; Pittsburgh, PA) while in ice. This homogenate was then centrifuged at 1800 rpm for 10 min at 4C in a Centrifuge 5810 R 15 AMP version (Eppendorf North America; Hauppauge, NY), and the supernatant was kept at −40C until Western blotting. Proteins were also extracted using the RIPA IX solution plus sodium dodecyl sulfate (SDS) 1%.

RIPA-extracted proteins were quantified using the BCATM Protein Assay Kit (Thermo Scientific; Waltham, MA).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting

Protein samples were treated with loading buffer 1× and heated at 95C to 100C for 10 min prior to loading. The loading buffer (3×) consisted of 150 mM Tris-HCl, 100 mM dithiothreitol (DTT), 6% SDS, 0.3% bromophenol blue, and 30% glycerol in 22.5 ml of diluted water. The samples were loaded into an acrylamide gel consisting of a 3% stacking gel and a 8% to 12% running gel, and run at 200 V for approximately 50 min or until the loading buffer dye left the gel. Afterward, the samples were transferred to a polyvinylidene difluoride (PVDF) membrane at 200 V for 30 min. These membranes were then blocked with 5% nonfat dry milk (NFDM) for 2 hr. The 5% NFDM solution contained 10 mM of Tris-HCl (pH 7.4), 150 mM NaCl, and 5% NFDM. Excess NFDM was washed off with three 15 min long Tris-buffered saline with Tween-20 (TBST-20) 1× washes, and the membranes were incubated overnight with the primary antibody (PGL-1 ascite, 1/200) at 4C. The next day, three TBST-20 washes were done to remove excess primary antibody, and the secondary antibody (antimouse IgG-horse radish peroxidase-linked, GE Healthcare; Piscataway, NJ) was applied for 1 hr. After that, excess secondary antibody was washed off with three washes of TBST-20, and the membrane was treated with Luminol/Enhancer and Stable Peroxide Buffer in a 1:1 ratio (Pierce* ECL Western Blotting Substrate, Thermo Scientific; Piscataway, NJ), and revealed in a GelDoc XR+ System (Bio-Rad; Hercules, CA).

Alcian Blue and Silver Staining

Alcian Blue/Silver staining was performed as described by Møller.¹⁴

Results

PGL-1 Characterization

PGL-1 Immunolabeling

Prior to presenting the results obtained using the PGL-1 antibody, it is necessary to characterize its labeling and possible antigen. The fusion from where the PGL-1 antibody was obtained was done using intestinal tissue as an immunogen; thus, our first step in characterizing PGL-1 was to find out what structures are labeled in normal (noneviscerated) sea cucumber tissues. Sections of normal descending and ascending small intestines immunostained with PGL-1 showed a punctate labeling restricted to the CT of the submucosa and the mesentery. The labeling was not associated with cellular structures (as detected by their DAPI stained nuclei), suggesting that the PGL-1 antigen was a component of the ECM.

In the small intestine, PGL-1 labeling was distributed throughout the submucosa and concentrated in a more or less regular band in the center of the CT (Fig. 1A). In sections of large intestine, PGL-1 also produced a punctate labeling within the CT (Fig. 1C, E). However, the labeling pattern was less homogeneous than in the small intestine showing dense agglomerations in the dense CT adjacent to the mesothelium and in the CT adjacent to the luminal epithelium (LE).

Figure 1.

Normal intestine transverse sections of Holothuria glaberrima labeled with Proteoglycan Like-1 (PGL-1) and anticollagen (Holothuria glaberrima Collagen [HgCol]) using immunohistochemistry (IHC). In the small intestine, PGL-1 (A) shows punctate labeling of a band in the connective tissue (CT) present between the mesothelium (Me) and the luminal epithelium (LE). IHC with anticollagen (B) labels fibers that span the entire CT. In the large intestine, near the Me, PGL-1 (C) labels the dense CT (dCT) with more intensity than the loose CT, while anticollagen (D) labels fibers in both regions. In the large intestine, near the mucosa, PGL-1 (E) labeling is concentrated in a band (arrow) close to the LE. Labeling with anticollagen (F) is predominant in the basal lamina of the LE. In the mesentery, both PGL-1 (G) and anticollagen (H) label the CT. All figures show PGL-1 and HgCol (green) and 4′,6-diamidino-2-phenylindole (DAPI) labeling (red) of cell nuclei. Scale bar = 100 µm.

PGL-1 labeling pattern suggested that the antibody labeled a component of the ECM. As fibrous collagen is the best characterized ECM component in our model system,^8,9 we simultaneously labeled adjacent tissue sections with an anticollagen antibody (HgCol). (Double labeling was not possible because both antibodies were obtained from mouse hybridomas.)

The PGL-1 antigen and fibrous collagen shared some areas of the intestinal CT. However, in general terms, the distribution of collagen was more extensive than that of PGL-1. For example, in contrast to PGL-1 labeling that was restricted to a narrow band in the small intestine, collagen-labeled fibers spread throughout the entire CT (Fig. 1B). Similarly, in the large intestine, collagen fibers covered the entire CT (Fig. 1D, F), showing a different distribution than PGL-1. Thus, as previously described,⁸ fibrous collagen showed no preference for dense CT, in contrast to the labeling found for PGL-1 (FIG .1C). There were, however, important areas of apparent overlap. First, the narrow CT area of the mesentery showed both collagen fibers and the punctate PGL-1 labeling (Fig. 1G, H). Second, both fibrous collagen and the PGL-1 antigen labeling were concentrated in the CT adjacent to the LE (Fig. 1E, F).

As PGL-1 labeling was not restricted to the intestinal CT, we studied body wall sections that included nervous system structures, longitudinal and circular muscles, dermis, epidermis, and tissues associated with the holothurian tube feet. Wherever found, PGL-1 labeling was similar to that in the intestine; showing a punctate pattern and restricted to the CT components. In the muscles, PGL-1 labeled only the CT component (Supplemental Fig. 1A, C). In longitudinal sections, the most intense labeling was found adjacent to the coelomic epithelium and in between the two longitudinal muscle bundles. The body wall dermis contains the most extensive CT areas in the sea cucumber. PGL-1 labeling was present in the most external areas of the dermis, near the epidermis (Supplemental Fig. 1E); however, in other areas, the labeling was not strong enough to clearly determine the presence of the antigen.

The association between PGL-1 and collagen varied in extraintestinal tissues. For example, the distribution of collagen fibers in longitudinal muscles was similar to what was described for PGL-1 (Supplemental Fig. 1B, D). However, in the body wall, most of the collagen fibers were concentrated in the deeper areas of the dermis, within the CT proximal to the coelom and the radial nerve (data not shown), regions that were not labeled by PGL-1. Both PGL-1 and collagen labeling were found in the CT around the tube feet and external dermis (Supplemental Fig. 1E, F).

Taken together, these results suggest that PGL-1 labels a component of the ECM that can be found together with collagen, but that is also present in areas where collagen is less abundant or not present.

PGL-1 Western Blot

In an attempt to characterize the molecule(s) recognized by the PGL-1 antibody, we performed Western blot analyses. Initial experiments with proteins extracted with RIPA buffer revealed a thick blot of PGL-1 labeling in the region of the membrane corresponding to the stacking portion of the SDS-PAGE (Fig. 2A). Importantly, Coomassie and Alcian Blue/Silver stains were also performed. No similar band of protein was observed in this region with Coomassie (Fig. 2B). This stain, however, did show the 220 kDa myosin heavy chain band, suggesting a much larger kilodalton for the PGL-1 antigen. The Alcian Blue/Silver stain revealed a heavy “blot” of labeling in the same area as PGL-1 immunoblotting (Fig. 2C). The large size of the antigen, the lack of a Coomassie stain, and the presence of an Alcian Blue/Silver stain suggested that the PGL-1 antigen may be a proteoglycan^14,15 that may or may not be in a complex of proteins. The PGL-1 antigen remained in the stacking gel area of the gel even after treating the protein extracts with 1% SDS, sonication, or SDS-PAGE (data not shown).

Figure 2.

Gel electrophoresis analyses of Proteoglycan Like-1 (PGL-1) antigen. (A) PGL-1 Western blot shows a dense blot in the stacking gel. (B) Coomassie staining of proteins extracted from normal (noneviscerated) holothurian intestines fail to stain bands within the area of the gel where molecules with molecular weight >220 kDa should be present, nor within the stacking gel. The first lane shows labeling of myosin heavy chain (arrow) at 220 kDa. (C) Alcian Blue/Silver stains a strong smear in the stacking gel. Samples 1 to 3 are replicates.

Alcian Blue Staining for Proteoglycans in Normal Intestinal Tissues

Classical histological methods, in particular Alcian Blue stains, can be used to detect proteoglycans in tissue samples.¹³ According to Humanson, Alcian Blue at pH 2.5 stains all mucopolysaccharides, but at pH 1.0, only sulfated mucopolysaccharides are stained. Thus, we performed histological experiments using Alcian Blue staining at both pHs in normal small and large intestines.

In small intestines, staining at both pHs labeled a dense band of CT in the submucosa just underneath the mesothelium (data not shown). This coincided with our PGL-1 results. Even more striking was the similarity between the PGL-1 labeling pattern and Alcian Blue staining of normal large intestines. In both the histological and immunohistochemical methods, stronger labeling/staining was observed in the dense CT below the mesothelium (Fig. 3A–C). There was also a thin, but strong, band of labeling in the CT near the LE (Fig. 3D–E). In addition, both Alcian Blue pHs stained the CT of the mesenteries in the same regions labeled by PGL-1 and showed the CT of the mesentery to be continuous with the dense CT of the intestine (Fig. 3F, G).

Figure 3.

Immunohistochemical labeling with Proteoglycan Like-1 (PGL-1) and Alcian Blue staining of normal large intestines and mesenteries. Alcian Blue pH = 1 (A) and pH = 2.5 (B) stains the dense connective tissue (dCT) of the posterior intestine with a distribution that is similar to PGL-1 labeling (green) (C). Alcian Blue pH = 1 (D, F) and pH = 2.5 (E, G) labels the connective tissue (CT) adjacent to the luminal epithelium (arrows) (D, E), as well as the CT of the mesentery (F, G). Immunohistochemical section (C) is costained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei (red). Scale bar = 100 µm.

Proteoglycan Expression During Regeneration

Previous results from our group have shown that ECM remodeling occurs during intestinal regeneration in H. glaberrima.⁸ Specifically, collagen labeling disappears from the regenerating intestine while matrix metalloproteinases are concomitantly activated. Therefore, we decided to determine if and when proteoglycan labeling went through similar changes during intestinal regeneration. Observations were done in the anterior part of the regenerating intestine, adjacent to the esophagus and in the posterior part of the regenerating intestine adjacent to the cloaca.

At 5 dpe, staining with Alcian Blue at both pH = 1 (Fig. 4A) and pH = 2.5 (Fig. 4B) showed similar results, a pattern reminiscent of that in the normal mesentery where staining was present homogeneously throughout the CT of the regenerating blastema-like structure. Similarly, staining with PGL-1 showed a punctate labeling restricted to the CT that was continuous from the mesentery to the regenerating rudiment (Fig. 4C). Collagen fibers were also present in the regenerating rudiment at 5 dpe; however, they were disorganized and grouped into bundles in the rudiment adjacent to the mesentery and had disappeared from the distal tip of the rudiment (Fig. 4D).

Figure 4.

Immunohistochemical labeling with Proteoglycan Like-1 (PGL-1) and anticollagen (Holothuria glaberrima Collagen [HgCol]), and Alcian Blue staining of intestines during early regeneration (5 and 7 days post evisceration [dpe]). At 5 dpe, Alcian Blue pH = 1 (A) and pH = 2.5 (B) stains uniformly the regenerating rudiment connective tissue (CT). PGL-1 labels the entire CT with a punctate pattern (C), whereas anticollagen labels disorganized collagen fiber bundles (arrows) in the CT of the central area of the regenerate (D). At 7 dpe, Alcian Blue pH = 1 (E) and pH = 2.5 (F) also shows a uniform staining of the rudiment CT. At this stage, PGL-1 labels the CT of the regenerate with a punctate pattern although with an apparent decrease in labeling intensity (G). Labeling with anticollagen at 7 dpe shows a complete absence of collagen fibers in the CT of the regenerate (H). Immunohistochemical sections are labeled (green) for PGL-1 and HgCol, and are costained with 4′,6-diamidino-2-phenylindole (DAPI) (red) to visualize cell nuclei. Scale bar = 100 µm.

At 7 dpe, Alcian Blue staining at pH = 1 (4E) and pH = 2.5 (4F) as well as PGL-1 labeling (Fig. 4G) were still present in the CT of the growing rudiment. Although, in most cases, the labeling appeared to be slightly less intense than in the CT of the mesentery, it was contiguous and homogeneously distributed in the regenerating structure (Fig. 4G). Collagen labeling, however, was completely absent within the rudiment at this stage (Fig. 4H).

At 14 to 21 dpe, there was an increase in proteoglycan labeling in comparison with the 7 dpe stage. At this stage, most animals exhibited a disorganized, but CT-specific, proteoglycan labeling pattern. The results were variable and revealed individual differences in the rate of regeneration and ECM remodeling. While in most animals, the Alcian Blue and PGL-1 staining patterns lacked organization, some animals already showed narrow bands of proteoglycan labeling underneath the mesothelium of their anterior and posterior intestines. This was best observed with the PGL-1 immunoreactivity (Fig. 5A, B). Also at this stage, the proteoglycan labeling in some animals appeared to be cell associated, being found within cell bodies or close to the cells of the CT compartment, suggesting that the increase in labeling of proteoglycans is due to cellular synthesis of proteoglycans and its deposition in the surrounding space (Fig. 5C, D).

Figure 5.

Immunohistochemical labeling with Proteoglycan Like-1 (PGL-1) of regenerating intestines at 14 and 21 days post evisceration (dpe). PGL-1 punctate labeling (green) can be found at different densities in the connective tissue (CT) of the regenerating intestines at 14 dpe (A) and at 21 dpe (B). Labeling can be found in aggregates throughout the CT of the intestinal submucosa. Higher magnification of a 14 dpe intestinal section labeled with PGL-1 (C) and overlaid with transmitted light to define cellular outlines (D) clearly shows the presence of PGL-1 labeling within some cells (arrows) of the CT. A large number of these cells can be found adjacent to the mesothelium (Me) where an accumulation of immunoreactive material is eventually deposited forming the dense CT of the intestine. Immunohistochemical sections are labeled with PGL-1 (green) and stained with 4′,6-diamidino-2-phenylindole (DAPI) (red) to visualize cell nuclei. Scale bars A, B = 100 µm; Scale bars C, D = 50 µm.

At 28 to 35 dpe stages, although there was still some variability in the staining pattern and some animals showed stronger staining in certain CT areas than others, the labeling was better organized in different areas of the CT. At this stage, when labeled either with PGL-1 or Alcian Blue, most animals showed a large band of labeling underneath the mesothelium (Fig. 6A, C), dispersed labeling in the loose CT, and a narrow band of labeling underneath the LE (Fig. 6B, D). This labeling pattern is similar to what was found in normal (noneviscerated) animals (see Figs. 1 and 3).

Figure 6.

Immunohistochemical labeling with Proteoglycan Like-1 (PGL-1) and Alcian Blue staining of posterior intestines at late regeneration stages (28–35 days post evisceration [dpe]). The posterior regenerates of most animals showed an Alcian Blue pH = 1 (A, B) and PGL-1 (C, D) labeling pattern similar to that of the posterior (large) intestine of normal noneviscerated animals. In particular, both the histological stain and the antibody strongly labeled a band of connective tissue (CT) underlying the mesothelium (Me) (arrows on A and C) and another band underlying the luminal epithelium (LE) (arrowheads on B and D). Immunohistochemical sections are labeled with PGL-1 (green) and stained (red) with 4′,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. Scale bar = 100 µm.

Discussion

The PGL-1 Antigen Is Most Likely a Proteoglycan or a Proteoglycan-Associated Protein

PGL-1 labeling shows a punctate labeling pattern restricted to CTs present in various organs (intestine, mesentery, muscle, and body wall dermis). Our results suggest that the antigen recognized by PGL-1 is a basic structural nonfibrous component of the ground substance, most likely a proteoglycan. Importantly, a similar proteoglycan (Pisaster Matrix-1 [PM-1]) has been characterized from the gut of another echinoderm, the starfish (Pisaster ochraceus).¹⁶ This molecule was found in the gut during embryological development and showed a similar labeling pattern to PGL-1. The antigen was confirmed to be a proteoglycan and was also shown to label the ECM, mesenchymal cells, and endoderm.¹⁶ Moreover, PGL-1 immunoreactivity of intestinal sections showed a strong similarity with Alcian Blue pH = 1 and pH = 2.5. Importantly, the pH = 2.5 protocol is unspecific for sulfated and unsulfated proteoglycans, but the pH = 1 protocol is specific for sulfated proteoglycans.

Further evidence for the PGL-1 antigen identity comes from our Western blot experiments of intestinal protein extracts. PGL-1 labeling resulted in a smeared band at the top of the gel. This has been observed in the Western blotting of some individual proteoglycans. Two examples are the Chondroitin Sulfate Proteoglycan Sushi repeat protein X-linked 2 (SRPX2)¹⁷ and Sdc-1.¹⁸ In both cases, purified proteoglycans appear as smeared bands in Western blots. Similar smears have also been observed with antibodies against entire classes of proteoglycans like the Chondroitin Sulfate Proteoglycans,¹⁹ which could mean that PGL-1 labels an epitope shared by an entire class of proteoglycans. Another possibility is that the PGL-1 antigen might actually be part of a complex of proteoglycans. This is suggested by the fact that the PGL-1 antigen has a very high molecular weight band in Western blotting with both acrylamide and 1% agarose gels, and even when the protein samples were treated with 1% SDS. Importantly, the region labeled by PGL-1 was not stained with Coomassie blue. This is to be expected if the PGL-1 antigen is a proteoglycan because these molecules have been demonstrated to stain poorly with this stain.¹⁵ Because proteoglycans have negatively charged long glycosaminoglycan chains, they are more efficiently stained with cationic dyes such as Alcian Blue.^13,20 In fact, this was one of the methods used to confirm that PM-1 is a proteoglycan in the P. ochraceus study mentioned above.¹⁶ Similarly, staining our protein extracts with Alcian Blue/Silver produced a staining pattern that resembles PGL-1 labeling in Western blot, further strengthening our contention that PGL-1 identifies some type of proteoglycan.

Nonetheless, the possibility that other molecules might be the PGL-1 antigen must be discussed. Because of the labeling pattern described above, cell-associated molecules, such as integrins and cadherins, as well as fibrous ECM components, are unlikely to be the PGL-1 antigen. It is probably not a nonfibrous collagen either. Of the nonfibrous collagens, collagen type XXVI (a member of the short collagen family) has been found in the Strongylocentrotus purpuratus genome.²¹ However, the PGL-1 antigen is probably not one of these molecules because it appears to have a higher molecular weight than what is characteristic of these collagens.²² Importantly, however, fibril-associated collagens with interrupted triple helix (FACIT) collagens like collagen types XII and XIV are not in the sea urchin genome, and no mention of types VII and IX was reported in that study.²¹ Moreover, as fibrous collagen disappears from the CT during early regeneration (and FACIT collagens are associated with it in the case of mammals), FACIT collagens are probably not the PGL-1 antigen either.

Another major category of ECM molecules is that of the glycoproteins. In a previous study,⁸ our group studied the labeling of antibodies against two glycoproteins: laminin and fibronectin. As expected, the labeling of these molecules was concentrated on the basement membrane in normal tissues. The absence of PGL-1 labeling in basement membranes in normal or in regenerating animals strongly suggests that the PGL-1 antigen cannot be laminin, fibronectin, or other molecules that are restricted to these structures.

The genome of S. purpuratus contains other glycoproteins as potential candidates for the PGL-1 antigen such as thrombospondin-1 and -2, fibrillin, fibulin, and hemicentin.²¹ Of these, the thrombospondins and the fibulins are possible glycoprotein candidates for the PGL-1 antigen because they are both known to bind to proteoglycans.^23,24

In summary, although the molecular nature of the PGL-1 antigen remains undetermined, our data strongly suggest that it is a proteoglycan. Nonetheless, whatever the PGL-1 antigen turns out to be, it is important to emphasize that (1) it is a normal constituent of the ECM and in particular of the intestinal mesentery and submucosa, (2) it is found at all regeneration stages, and (3) its spatial distribution correlates with the organization of intestinal tissue layers into small and large intestine regions. In this respect, PGL-1 serves as an excellent marker for the presence of the ground substance in both normal and regenerating intestinal tissues.

The ECM in Regeneration of the Sea Cucumber Intestine

Alcian Blue staining and PGL-1 immunoreactivity confirmed that normal large intestine contains a dense layer of CT adjacent to the myoepithelial cells and a loose CT that extends all the way to the LE. None of the other ECM molecules that we have studied show this distribution.⁸ More importantly, the overall labeling of proteoglycans either by Alcian Blue or PGL-1 shows the least changes during the remodeling of the ECM that takes place in the regenerating intestine.

There is only one stage during regeneration in which the apparent intensity of the Alcian Blue and PGL-1 labeling appears to decrease: 7 dpe. It is possible that this is due to a decrease in proteoglycan production or an increase in its degradation. Importantly, however, we have previously shown that during the 7 dpe stage, the regenerating rudiment enters a growth spurt in preparation for the formation of the lumen that occurs later on.⁴ Thus, it is most likely that what is happening is that at 7 dpe, the growth of the rudiment is outpacing ground substance deposition.

Our data provide the following picture of intestinal CT remodeling: After evisceration, the mesentery remains attached at one end to the body wall with the ruptured end free in the coelomic cavity. During early regeneration, fibrous collagen and certain components of the ECM are degraded in a gradient beginning at the free end and moving toward the body wall. As the rudiment grows, its growth briefly outpaces deposition of new ground substance, which catches up soon afterward. Cells that enter the growing rudiment are responsible for the deposition of the new ground matrix and in particular the proteoglycans. This ECM remodeling appears to be important for the migration of the luminal epithelial cells and the formation of the lumen. After the lumen has formed, the basal lamina of the intestines is reestablished, and the ground substance begins to reorganize. Complete reorganization is achieved by the fourth week, and it is after this reorganization that the fibrous component of the CT regains its normal distribution. From this, it could be postulated that components that are lost during early regeneration, like fibrous collagen, are impediments for regeneration, which explains why they are degraded early on. On the contrary, proteoglycans as labeled by PGL-1 and histological stains seem to be necessary for intestinal regeneration because they remain during the entire process.

Proteoglycans play key roles in developmental processes, some of which are akin to what takes place during regeneration. For example, inhibition of sulfation by monochlorine trioxide (ClO₃) and selenate (SeO₄) has been shown to alter the formation of the oral and aboral axis in sea urchins by intervening with transforming growth factor (TGF) beta/Nodal signaling.²⁵ Similarly, heparan sulfate proteoglycans (HSPGs) have been found to play important roles in regulating Hedgehog and bone morphogenetic protein (BMP) in vertebrate development.^26,27

Proteoglycans are also known to be important mediators of wound healing and regeneration.^28,29 Examples of this are the Syndecans. Syndecans are a family of HSPGs that are usually bound to cell membranes and may also be secreted (shed). Sdc-1 deficient mice have defective proliferation and reepithelization after a wound. Interestingly, Sdc-4 has been found to both be overexpressed in human skin wound healing and coimmunoprecipitate with CT Growth Factor. Interestingly, skin wound healing in mice seems to be adversely affected by increasing the amount of shed (soluble) Sdc-1.²⁸ Other classes of proteoglycans, such as the chondroitin sulfate proteoglycans and keratan sulfate proteoglycans, have been found to be involved in regeneration. In this respect, it is interesting that the Alcian Blue pH = 1 labeling demonstrates the presence of sulfated proteoglycans during the regenerative process.