1. Introduction
To varying degrees, four-limbed vertebrates (tetrapods), including humans, generally have the ability to regenerate lost complex tissues or body parts after trauma early in development, but as they grow and become adults, this ability is reduced or lost, and the deficient areas heal instead by being covered with fibrotic tissue [
1,
2]. Contrary to this general rule, newts, which belong to a group of the family Salamandridae in urodele amphibians, have the ability to repeatedly regenerate lost body parts, regardless of their age, even after reaching adulthood beyond metamorphosis [
3,
4,
5,
6,
7,
8,
9,
10,
11,
12,
13,
14]. It is believed that this outstanding regenerative ability of adult newts is based on a mechanism of cellular reprogramming/dedifferentiation that is unique to newts: in adult newts, terminally differentiated somatic cells, which have already lost premature traits such as multipotency factor expression and proliferative activity and have become highly specialized for specific physiological functions, are reprogrammed/dedifferentiate into stem/progenitor-like cells upon trauma. For example, retinal pigment epithelium (RPE) cells in the eyes are reprogrammed into RPE stem cells for retinal regeneration [
1,
6,
7,
9] while striated muscle fibers (tubular, multinucleated cells with sarcomeres) in the limbs dedifferentiate into mononucleated myogenic cells for muscle regeneration [
10,
14]. This ability could be interpreted as allowing adult newts to mobilize stem/progenitor-like cells from terminally differentiated somatic cells as regenerative material to complement resident somatic stem/progenitor cells that have reduced their contribution to regeneration as adults [
14]. Therefore, body part regeneration in adult newts is a useful model system for basic research toward new regenerative medicine combining stem cells and dedifferentiation. We have been studying the mechanism of the regeneration of body parts using adult fire-bellied newts,
Cynops pyrrhogaster.
In a previous study, using an adult
C. pyrrhogaster limb regeneration system, we comprehensively searched for unique genes whose number of transcripts was significantly elevated in the blastema, a cell mass that appears at the tip of an amputated limb, and discovered an orphan gene,
Newtic1, which is found only in urodele amphibians [
15]. That study [
15] revealed the following.
Newtic1 theoretically encodes a 40.7 kD protein that has a transmembrane domain at the N-terminus followed by a cytoplasmic domain. The Newtic1 protein is expressed on a subset of erythrocytes, namely polychromatic normoblasts (PcNobs), premature erythrocytes that make up 80–90% of circulating erythrocytes. PcNobs are transparent or light pink in color, suggesting that hemoglobin synthesis has not started or is in progress. Newtic1 protein is localized along the equator of PcNobs. It must be noted that in adult newts, normoblasts are produced in the spleen and mature in blood vessels during circulation and that fully mature erythrocytes in adult newts are red due to their large amount of hemoglobin but still retain nuclei, corresponding to human orthochromatic normoblasts (OcNob). OcNobs of adult newts never express Newtic1 protein.
Newtic1-positive (+) PcNobs are observed in at least two situations [
15]. The first is in normal circulating blood, in which only about 20% of PcNobs express Newtic1 protein, which gathers around monocytes, forming erythrocyte clump (EryC)-monocyte complexes. The minimum size of an EryC-monocyte complex is comprised of 1–2 monocytes and 6–10 PcNobs. The second is in regenerating limbs, as predicted at the time of
Newtic1 gene isolation. At Stage I (5 post-operative days (pod)), when the amputation surface of the limb is covered by the wound epidermis and lymphocytes, including monocytes, accumulate between the amputation surface and the wound epidermis, PcNobs retained in the vessels dilated by the inflammatory reaction just below the amputation surface begin to express Newtic1 protein. At Stage II (14 pod), when the blastema begins to protrude, emerging Newtic1(+) PcNobs enter the blastema along the inside of extending vessels, and their number and expression of the Newtic1 protein increases. As the blastema grows further and reaches Stage III (27 pod), when the entire surface of the blastema is covered by pigmented epithelium and capillaries extending beneath the epidermis, the density of Newtic1(+) PcNobs increases in the distal part of the blastema. Later, as the cartilage begins to differentiate in the regenerating limb, elongated blood vessels fuse to form loops, and as circulation resumes, Newtic1(+) PcNobs are cleared by normal peripheral blood.
We further showed in our previous study that erythrocytes of adult newts contain transcripts of numerous secreted factors, including growth factors (TGFβ1, IGFII, BMP2, PDGFC, VEGEC, and nsCCN) and matrix metalloproteinases (Col-a, Col-b, MMP3/10, MMP9, and MMP21) [
15]. We also demonstrated by immunocytochemistry that TGFβ1 and BMP2 are present in erythrocytes at the protein level. Focusing on Newtic1(+) PcNobs in the growing blastema, we further demonstrated that these cells lost immunoreactivity to TGFβ1 and BMP2 from the cytoplasm when they were translocated into the blastema, suggesting the possibility of growth factor secretion by Newtic1(+) PcNobs during blastema growth. However, it remains elusive how the expression of Newtic1, a putative membrane protein, is linked to the secretion of growth factors from PcNobs.
In this study, we addressed the above issue using morphological techniques as a basis for investigating the physiological roles of Newtic1 and Newtic1(+)PcNobs in limb regeneration of adult newts. We found that the Newtic1 protein localizes to cytoplasmic globular structures, which accumulate along the marginal band of PcNob in the limb blastema. Our observations provide insights leading to a hypothesis that Newtic1 protein might be a component of membrane vesicles containing growth factors such as TGFβ1 and may contribute to the transport of these factors by tethering membrane vesicles to tubulin fibers that become aligned just below the equatorial plane of PcNob during blastema formation.
2. Materials and Methods
Experiments presented herein were performed at Kitasato University, Yokohama City University, and the University of Tsukuba. All methods were carried out in accordance with the ARRIVE guidelines as well as the Regulations for the Handling of Animal Experiments in each university. Experiments using live animals were conducted only at the University of Tsukuba. All experimental protocols for live animals were approved by the Animal Care and Use Committee of the University of Tsukuba (170110).
2.1. Animals
Adult Japanese
C. pyrrhogaster (total body length: male, about 9 cm; female, 11–12 cm) was used. Newts were captured from Aichi, Fukui, Fukushima, and Hyogo prefectures by a supplier (Aqua Grace, Yokohama, Japan) and stored at the University of Tsukuba. A strain of
C. pyrrhogaster, Toride-Imori, was also used [
16]. The animals were reared at 18–22 °C under natural light conditions until experiments as described previously [
12,
13,
14,
15].
2.2. Anesthesia
Animals were anesthetized in 0.1% FA100 (4-allyl-2-methoxyphenol; LF28C054; DS Pharma Animal Health, Osaka, Japan) dissolved in water at room temperature (RT: 22 °C) for 45 min, as described previously [
12,
13,
14,
15].
2.3. Limb Amputation
After anesthesia, animals were rinsed in Elix water (Merk Millipore, Sigma-Aldrich, Tokyo, Japan) and dried on a paper towel (Elleair Prowipe, Soft High Towel, Unbleached, 4P; Dio Paper Corporation, Tokyo, Japan). After tying a tourniquet—a twisted string made of a piece of soft paper (Elleair Prowipe, Soft Wiper S200; Dio Paper Corporation)—just below the shoulder on the right forelimb, the forearm was amputated in the middle (at the mid-zeugopod region) by a razor blade (FA-10; FEATHER Safety Razor Co., Ltd., Osaka, Japan) as described previously [
12,
14]. Amputees were momentarily placed on dry paper towels until the bleeding stopped. After the tourniquet was removed, they were transferred to moist containers with a lid containing air vents (up to three newts per container of length 200 mm × width 150 mm × height 55 mm) and allowed to recover. The moist container was always kept in a semi-dry condition in which the bottom was covered by a moist paper towel (Elleair Prowipe, Soft High Towel, Unbleached, 4P) that was tightly wrung [
12,
13]. Paper towels were replaced with new ones every other day. The stages of limb regeneration were determined according to previous criteria [
15].
2.4. Tissue Preparation
For immunohistochemistry of limb blastema, forelimbs were re-amputated in the middle of the upper arm under anesthesia and fixed in a modified Zambony’s fixative, which was prepared by dissolving 0.2% picric acid in 2% paraformaldehyde (PFA)-containing phosphate-buffered saline solution (PBS; pH 7.5), at 4 °C for 6 h [
15]. Limb samples were washed thoroughly with PBS at 4 °C (5 min × 2, 10 min × 2, 15 min × 2, 30 min × 2, and 1 h × 2) and then allowed to soak in 30% sucrose in PBS at 4 °C. They were embedded into Tissue-Tek O.C.T. Compound (4583; Sakura Finetek USA, Inc., Torrance, CA, USA), frozen at about −30 °C in a cryostat (CM1860; Leica Biosystems, Tokyo, Japan) and sectioned at about 20 μm thickness. Tissue sections were attached to gelatin-coated coverslips (18 × 18 mm
2, Thickness No. 1; Matsunami, Osaka, Japan), air-dried, and stored at −20 °C until use. After blastemal samples were collected, animals were kept in their original moist containers and allowed to regenerate for use in other studies.
2.5. Blood Cell Preparation
Normal circulating blood (named ‘intact blood’ hereafter) was collected from intact limbs immediately after amputation. Blastemal blood was collected from regenerating limbs at 3–4 weeks post amputation by making a small slit on the top of the blastema with the tip of an FA-10 razor blade (see
Figure 1D). Blood was harvested from 2–5 animals in 2 mL PBS (pH 7.5) in a plastic dish (inner diameter: 35 mm; PS-30; MonotaRO Co., Ltd., Hyogo, Japan) on ice. About 80–100 µL of intact blood and about 10–15 µL of blastemal blood could be collected per animal. As for the blastemal blood, further collection should be avoided in order to minimize contamination of intact blood. About half of those volumes were blood cell components, which were collected into one 1.5 mL protein low-binding tube (Proten LoBind Tube, 022431081; Eppendorf AG, Hamburg, Germany) by two centrifugations (80 g, 1 min), and rinsing with 1 mL of chilled PBS, followed by centrifugation (80 g, 1 min). These cell samples were used for the following experiments. Developmental stages of erythrocytes were determined according to previous criteria [
15]. After the collection of blood samples, animals were kept in their original moist containers and allowed to regenerate for use in other studies.
2.7. Immunostaining of Tissue Sections and Blood Cells
Immunofluorescence staining of tissue sections was carried out as described previously [
15]. Briefly, tissue sections were washed thoroughly (PBS, 0.2% TritonX-100 in PBS, PBS; 15 min each), incubated in blocking solution (5% bovine serum albumin (BSA, 050 M 1599; Sigma-Aldrich in Merck, Tokyo, Japan)/2% normal goat serum (S-1000; Vector Laboratories, Burlingame, CA, USA)/0.2% TritonX-100 in PBS) for 2 h, washed as previously indicated, then incubated in primary antibody diluted with blocking solution at 4 °C for 15 h. After washing thoroughly, sections were incubated in Alexa Fluor 488- or rhodamine-conjugated secondary antibody diluted with blocking solution for 4 h and washed thoroughly. For double staining of TGFβ1 and Newtic1, or BMP2 and Newtic1, whose antibodies were produced in rabbits, tissue sections were first stained with primary antibody for TGFβ or BMP2 and secondary antibody conjugated with Alexa Fluor 488 and then stained with primary antibody for Newtic1 and secondary antibody conjugated with rhodamine. After the stained tissue sections were washed, the nuclei of cells were counterstained with DAPI (1:50,000, D1306; Thermo Fisher Scientific, Tokyo, Japan) or TO-PRO-3 Iodide (1:50,000, T3605; Thermo Fisher Scientific). Sections were mounted on a glass slide with 90% glycerol in PBS.
For immunostaining of blood cells, at the initial stage of the study (
Figure 1E–H), the collected cells were re-suspended with 1 mL of 4% PFA in PBS (pH = 7.4; RT) and fixed at RT for 2 h. The cells were washed and immunostained in the same way as was performed with tissue sections, except for the use of 1.5 mL tubes and centrifugation for washing. In all other experiments presented in this paper, the collected cells were re-suspended in 1 mL of chilled newt normal saline (115 mM NaCl, 3.7 mM KCl, 3 mM CaCl
2, 1 mM MgCl
2, 18 mM D-glucose, 5 mM HEPES; pH = 7.5 [
4]), and then 170–190 µL of cell suspension was plated on each of the poly-
d-lysine coated 12 mm round coverslips (354086, Corning BioCoat, Poly-
d-Lysine Cellware; Discover Laboratory Inc., Bedford, MA, USA) placed in 35 mm plastic dishes (one coverslip per dish). For immunostaining, after the cells were allowed to attach to the cover slip for 20 min at RT, the dishes were carefully filled with 4% PFA in PBS (pH = 7.4; RT), and the cells were fixed for 30 min at RT. Then, the coverslips on which the cells were immobilized were carefully transferred into other 35 mm plastic dishes filled with fresh 4% PFA in PBS (one coverslip per dish), and the cells were fixed for an additional 1 h at RT. After fixation, the coverslips with cells were transferred into a plastic cup (φ86 × 40 mm) containing 50 mL PBS (up to two coverslips per dish) and washed thoroughly (PBS, 0.2% TritonX-100 in PBS, PBS; 15 min each). The cells on coverslips were immunostained in the same way as was performed with tissue sections, except for the process following the use of a gold nanoparticle-conjugated secondary antibody (Goat anti-Rabbit IgG (H&L) Ultra Small; AURION Immuno Gold Reagents & Accessories, Wageningen, The Netherlands) for Newtic1 immunoelectron microscopy.
For Newtic1 immunoelectron microscopy, after the secondary antibody was thoroughly washed, cells were fixed with 2% glutaraldehyde in PBS for 30 min at RT. After washing in PBS (15 min × 3), cells were fixed with 1% OsO4 in PBS for 30 min on ice. After washing in chilled MilliQ water (10 min × 6; Merk Millipore), the cells were incubated with R-Gent SE-LM silver enhancement reagent (500011; AURION Immuno Gold Reagents & Accessories) for 22 min at RT and washed in chilled MilliQ water (10 min × 6). The incubation time for silver enhancement was determined by observing reactions under a microscope (BX50; Olympus, Tokyo, Japan). The stained blood cell samples were stored in MilliQ water at 4 °C.
2.8. Preparation of Ultrathin Sections of PcNobs for Transmission Electron Microscopy (TEM)
In experiments to examine the presence of an endomembrane system in PcNobs, blood cells attached to coverslips were fixed with 2% PFA/2.5% glutaraldehyde in PBS for 30 min × 2 at RT in the same manner as was conducted with 4% PFA in PBS. After washing in PBS for 5 min × 5 at RT, the cells were fixed with 1% OsO4 in PBS for 1 h on ice. The cells were thoroughly washed in chilled PBS for 3 min × 3, followed by washing in chilled MilliQ water for 15 min × 3. The fixed blood cell samples were stored in MilliQ water at 4 °C.
The blood cell samples for immunoelectron microscopy and for observation of an endomembrane system were en bloc stained with 1% uranyl acetate in 50% ethanol overnight at RT, then washed in distilled water. Then, the specimens were dehydrated with a graded series of ethanol (50, 60, 70, 80, 90, 95, and 99.5% for 5 min each and 3 times in 100% for 10 min each) and propylene oxide (twice for 10 min each), then followed by infiltration and embedding in Epon 812 (TAAB Laboratories Equipment Ltd., Berks, UK). Prior to making ultrathin sections, semithin sections (300–500 nm thick) were prepared from the epoxy block containing the sample with an ultramicrotome (ULTRACUT R, Leica Biosystems), stained with toluidine blue, and examined for Newtic1(+) PcNobs under an optical microscope. The epoxy block, whose semithin sections confirmed the existence of Newtic1(+) PcNobs, was further sliced into ultrathin sections (80–90 nm thick) by an ultramicrotome equipped with a diamond knife (Diatome Ltd., Nidau, Switzerland). Sections were doubly stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (JEM-1400 flash, JEOL, Tokyo, Japan) operated at 80 kV.
2.9. Organelle Staining in Live Blood Cells
The Golgi apparatus and endoplasmic reticulum (ER) of live blood cells were visualized using the Organelle-ID RGB III Assay Kit (ENZ-51032-K100; Enzo Life Sciences Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions. Briefly, blood cell samples collected in a protein low-binding 1.5 mL tube, as mentioned above, were immediately re-suspended in 500 µL 1× Organelle-ID Reagent III and incubated for 30 min on ice. After the reagent was removed from the tube following centrifugation (80 g, 1 min), the cells were re-suspended in 1 mL of 80% Leibovitz’s L-15 medium (41300-039; Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Lot#: AJF10577; Cat#: SH30071.03; Hyclone, Cytiva, Tokyo, Japan), and incubated for 60 min at 25 °C in an incubator (HB-100; TAITEC, Saitama, Japan). After the medium was removed from the tube following centrifugation (80 g, 1 min), the cells were re-suspended in 500 µL of a 1× Assay solution in the kit. After the solution was removed from the tube following centrifugation (80 g, 1 min), the cells were re-suspended with 400 µL of the 1× Assay solution. For imaging, the cell suspension was transferred to glass bottom dishes (35 mm Glass Base Dish; IWAKI, AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan) or placed on glass slides (about 30 µL per slide) and overlaid with coverslips (18 × 18 mm2, Thickness No.1; Matsunami).
The lysosomes and mitochondria of live blood cells were visualized using the Organelle-ID-RGB I KIT (ENZ-53007-C200; Enzo Life Sciences Inc.) according to the manufacturer’s instructions. Processing of blood cell samples was basically the same as for the Organelle-ID RGB III Assay Kit described above, except that cells were incubated in 0.2% Reagent I diluted in 1 mL of 80% L-15 medium containing 10% FBS for 30 min at 25 °C, and then washed in PBS.
2.10. Immunoblotting
Blood cell samples collected from five animals in a protein low-binding 1.5 mL tube, as mentioned above, were further washed three times with 1 mL of chilled PBS via centrifugation (80 g, 1 min) and then used for immunoblotting. After centrifugation and removal of PBS, cells were dissolved in 500 µL cell lysis buffer (0.1% TritonX-100 in PBS containing 1% protease inhibitor cocktail (P8340; Merck Sigma-Aldrich, Tokyo, Japan)) by tapping. After spinning down the content (7740 g, 30 s), the supernatant containing soluble cytoplasmic and plasma membrane proteins was transferred into a fresh protein low-binding 1.5 mL tube (sample 1) and kept on ice. The deposit on the bottom of the tube, which contained nuclei and cytoskeletons, was washed with 500 µL of cell lysis buffer and then with 1 mL of PBS by tapping and spinning down. After PBS was removed, the deposit was mixed with DNase I solution (RQ1 RNase-Free DNase, M6101; Promega, Madison, WI, USA) and incubated for 20 min at 37 °C to degrade DNA. After washing three times in 1 mL PBS by tapping and spinning down, the pellet of cytoskeletal proteins was kept on ice (sample 2).
Prior to denaturing proteins by heat, 100 µL of sample 1 was transferred to a fresh protein low-binding 1.5 mL tube and mixed with 100 µL of 2× Laemmli Sample Buffer (161-0737; Bio-Rad, Hercules, CA, USA) containing 10% 2-mercaptoethanol. For sample 2, the pellet in the tube was immersed in 100 µL of 1× Laemmli Sample Buffer (diluted with PBS) containing 10% 2-mercaptoethanol and dissociated by sonication (46 kH, VS-70U; Iuchi/AS ONE, Osaka, Japan) for a few min. Subsequently, the proteins in the tubes were heat denatured by placing them in boiling water for 5 min. These protein samples were stored at −80 °C until use. Immediately before use, protein samples were thawed, heat denatured, spun down, and placed at RT.
Proteins in samples 1 and 2 (each 20 μL/well) and those of the molecular weight marker (All Blue Prestained Protein Standards, 1610373; Bio-Rad; 10 μL/well) were separated on a 4–15% gradient gel (4561083, Mini-PROTEAN TGX Precast Gels; Bio-Rad) by SDS-PAGE, and transferred to an activated Immun-Blot® PVDF membrane (1620–174; Bio-Rad). The membrane was cut into strips, including lanes for the protein sample of interest and the molecular weight marker. Membrane strips were individually washed in TBST (100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20) for 10 min, incubated in blocking solution (5% bovine serum albumin and 2% goat normal serum in TBST) containing 2% avidin D (Avidin/Biotin Blocking kit, SP-2001; Vector Laboratories) for 1 h, washed in TBST (1, 10, 20 min), and then incubated with primary antibody diluted in blocking solution containing 2% biotin (Avidin/Biotin Blocking kit) at 4 °C for 15 h. After washing in TBST thoroughly (1 min, 15 min × 3), they were incubated with biotinylated secondary antibody diluted in blocking solution for 90 min at RT. After a thorough wash in TBST, they were incubated in AB complex (Vectastain ABC Elite kit, PK-6100; Vector Laboratories) prepared with TBST for 90 min at RT. After washing thoroughly in TBST, they were incubated in DAB (SK-4100; Vector Laboratories) for up to 4 h at RT. Once protein bands became visible, they were washed in distilled water (5 min × 6) to stop the reaction.
2.11. Image Acquisition and Analysis
Fluorescence images of blood cell samples at the initial stage of the study (
Supplementary Figure S2A,C,E) were acquired by a charge-coupled device (CCD) camera system (DP73; cellSens Standard 1.6; Olympus) attached to a fluorescence microscope (BX50; Olympus) as described previously [
15]. In all other experiments presented in this paper, transmitted light and fluorescence images of blood cell samples were acquired by an all-in-one fluorescence inverted microscope system (BZ-X800; KEYENCE, Osaka, Japan) with filter sets for TRITC (OP-87764; exciter: 545/25 nm; emitter: 605/70 nm), GFP (OP-87763; exciter: 470/40 nm; emitter: 525/50 nm) and DAPI (OP-87762; exciter: 360/40 nm; emitter: 460/50 nm).
Confocal images of tissue sections and blood cells at the initial stage of the study (
Figure 1I,
Supplementary Figures S2B,D and S3) were acquired through an LSM510 microscope system (LSM 5.0 Image Browser software; Carl Zeiss, Jena, Germany) as described previously [
15]. In all other experiments presented in this paper, confocal images were acquired through a LSM700 microscope system (ZEN 2009, ver. 6.0.0.303; Carl Zeiss, Oberkochen, Germany) with filter sets for rhodamine (Diode 555 Laser; emitter: BP 575–640 nm), Alexa Fluor 488 (Diode 488 Laser; emitter: BP 515–565 nm) and DAPI (Diode 405-5 Laser; emitter: BP 445/50 nm). In experiments to analyze the size of immunofluorescent dots and their positional relationships at a limitation of optical microscope resolution, a 100× oil objective lens was used in combination with a 10× digital zoom. An averaging function for multiple images at the same optical plane (optical section: 300 nm) was also applied to minimize noise, except for taking serial images along the Z-axis.
TEM images were acquired with a digital camera, Phurona, controlled with the software of RADIUS (EMSIS GmbH, Munich, Germany).
Microscope images were analyzed by software for image acquisition systems and by Adobe Photoshop 2022 (San Jose, CA, USA). The area size of immunofluorescence was measured using NIH ImageJ 1.53t (
https://imagej.nih.gov/ij/ (accessed on 29 September 2022)). Images of immunoblotted membranes were obtained by a scanner (TS8430; Canon, Tokyo, Japan). Figures were prepared using Adobe Photoshop 2022. Image brightness, contrast, and sharpness were adjusted according to the journal’s guidelines.
2.12. Statistics
In all experiments, we obtained data from three or more rounds of independent trials. Statistical analysis was made using BellCurve for Excel (version 3.23 Social Survey Research Information, Tokyo, Japan). Data in the text are presented as the mean ± SE.
4. Discussion
As a fundamental basis for studying the physiological function of Newtic1, a membrane protein expressed in PcNobs that accumulate in the blastema of the limbs of adult
C. pyrrhogaster, we investigated the relationship between Newtic1 and factor secretion using morphological techniques. We found that Newtic1 localizes to globular structures that accumulate in the marginal band, rather than on the cell membrane, along the equator of PcNobs, and that Newtic1 globular structures are associated with microtubules. Newtic1 immunoreactivity along the equator was found only in PcNobs with a well-developed marginal band. Considering these results with reference to the kinetics of Newtic1 expression during blastema formation in the limb [
15], Newtic1(+) globular structures are thought to accumulate in the marginal bands of PcNobs, that develop in blood vessels which extend into the blastema as it grows.
PcNobs have a well-developed endomembrane system and are capable of transporting secretory vesicles from the cytoplasm to the cell membrane, and since the immunofluorescence of Newtic1 encompasses that of TGFβ1, it is reasonable to assume that Newtic1(+) globular structures are secretory vesicles. It is generally known that secretory vesicles are transported along microtubules to the cell membrane [
24]. Therefore, it is hypothesized that the Newtic1 protein is involved in intracellular secretory vesicle trafficking by localizing to the membrane of secretory vesicles containing primarily TGFβ1 and binding directly or indirectly to microtubules via some proteins such as microtubule-associated proteins (MAPs) [
24]. Of course, the possibility that the Newtic1(+) globular structures and TGFb1-containing secretory vesicles just happen to be in close proximity cannot be completely ruled out due to the limitations of resolution in optical microscopy. However, the fact that the single dots of Newtic1 immunoreactivity appear to encapsulate TGFb1 immunoreactivity would support this hypothesis.
It is possible that Newtic1(+) secretory vesicles carry other factors as well since there are also Newtic1(+) globular structures that are not immunoreactive for TGFβ1. However, BMP2 does not appear to be associated with Newtic1, at least during blastema growth, and another mechanism may underlie BMP2 secretion. The fact that Newtic1 immunoreactivity was not observed on the cell membrane suggests that the vesicles that fuse to the cell membrane may separate from the marginal band, leaving the Newtic1(+) membrane component behind. In PcNobs of intact blood, Newtic1(+) granules along the equator appeared to contain neither TGFβ1 nor BMP2. This may be because PcNobs have finished secreting and have returned to circulation, or there may be some other function for Newtic1(+) granules in PcNobs of intact blood.
In this study, the influence of detergent may be a concern. Note that the low density of Newtic1(+) globular structures in immunoelectron microscopy compared to confocal microscopy is not due to the detergent since the procedure up to the secondary antibody reaction was essentially the same in both techniques (see
Section 2). This is primarily because the section used in immunoelectron microscopy was 80–90 nm thick, in which only one globular structure could be contained, while the optical section in confocal microscopy was at least 300 nm thick, so in areas of a high density of globular structures in the marginal band, the fluorescence of three or more globular structures would overlap.
It is likely that Newtic1 is a microtubule-associated protein and constitutes a secretory vesicle, which is why Newtic1 was able to remain tightly associated with the marginal band and TGFβ1 was able to exist in a positional association with Newtic1 even when the intracellular membrane structures were mostly lysed out by detergent. However, the exact distribution of these proteins in the endomembrane system was not visible under the current conditions. In the future, this problem should be solved by producing antibodies compatible with electron microscopy. Technology to express fluorescent-tagged Newtic1 protein in living PcNobs would enable studies of the regulation of Newtic1 expression and time-lapse analysis of the intracellular dynamics of secretory vesicles.
In mammals, including humans, TGFβ1 acts to suppress the inflammation during wound healing but also actively contributes to fibrosis and scar formation [
25]. Newts, on the other hand, do not exhibit fibrosis or scar formation [
1,
6,
9,
12,
13]. Our current study reiterates the possibility that TGFβ1 is secreted into the tissues during blastema formation. This suggests that in newts, TGFβ1 may contribute to scarless regeneration rather than to scar healing. Newtic1 and Newtic1(+) PcNobs appear to have a role in delivering TGFβ1, or even other factors, into tissues during blastema formation. To prove these hypotheses, further studies should be conducted in the future using genetically modified newts and in vitro experimental systems.