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42
Tables
43
Antibody
Dilution
(IgG conc.)
Provider
Primary
Rabbit anti-Newtic1 polyclonal antibody
1:200
(2.1 μg/mL)
Custom made; Merck SigmaAldrich, Tokyo, Japan
Rabbit anti-TGFβ1 polyclonal antibody
1:500
(2.0 μg/mL)
LS-B14345; LifeSpan BioSciences,
Inc., Seattle, WA, USA
Rabbit anti-RFP polyclonal antibody
1:500
(2.0 μg/mL)
600-401-379; Rockland
Immunochemicals, city, PA, USA
Rabbit anti-BMP2 polyclonal antibody
1:500
(2.0 μg/mL)
LS-B13128; LifeSpan BioSciences,
InC., Seattle, WA, USA
Rabbit anti-alpha tubulin polyclonal
antibody
Figure 2
1:100-1:1000
(0.2-1.8 μg/mL)
Ab15246; abcam, Cambridge, UK
Figure 5
1:500
(0.4 μg/mL)
1:1000
(2.2 μg/mL)
T6793; Merck Sigma-Aldrich,
Tokyo, Japan
Rhodamine (TRITC)-conjugated AffiniPure
goat anti-rabbit IgG (H+L) polyclonal
antibody
1:500
(1.5 μg/mL)
111-025-003; Jackson
ImmunoResearch Laboratories,
West Grove, PA, USA
Alexa Fluor 488-conjugated goat anti-rabbit
IgG (H+L) polyclonal antibody
1:500
(4.0 μg/mL)
A11008; Thermo Fisher Scientific,
Tokyo, Japan
Alexa Fluor 488-conjugated AffiniPure Fab
Fragment goat anti-rabbit IgG (H+L)
polyclonal antibody
1:500
(1.4 μg/mL)
111-025-003; Jackson
ImmunoResearch Laboratories,
West Grove, PA, USA
Alexa Fluor 488-conjugated goat anti-mouse
IgG (H+L)
1:500
(4.0 μg/mL)
A11001; Thermo Fisher Scientific,
Tokyo, Japan
Biotinylated goat anti-rabbit IgG (H+L)
polyclonal antibody
1:500
(3.0 μg/mL)
BA-1000; Vector laboratories,
Newark, CA, USA
Goat anti-Rabbit IgG (H&L) Ultra Small
1:50
(1.2-1.6 μg/mL)
Mouse anti-acetylated tubulin monoclonal
antibody
Secondary
Table 1. Antibodies for immunostaining.
44
800011; AURION Immuno Gold
Reagents & Accessories,
Wageningen, the Netherlands
Figures
45
Figure 1. Adult newt limb regeneration. (A) The Japanese fire-bellied newt, Cynops
pyrrhogaster, exhibits a remarkable ability to regrow organs/tissues, e.g., eyes, brain,
jaw, heart, limbs, and tail. This organism contributes significantly to the understanding
of mechanism of body parts regeneration. (B) Stages of adult newt limb regeneration
(from amputation to newly regenerated limb). When a limb is amputated, the stump is
covered by the wound epithelium (WE). The cells dedifferentiate at the wound site into
progenitors to form the blastema. The blastema cells will undergo proliferation,
patterning, differentiation, and growth. Eventually, a new functional limb is
regeneratedKey stages in the early regenerative process: Stage I (wound healing and
early dedifferentiation), Stage II (late dedifferentitation, moderate early bud blastema,
and early bud blastema), Stage III (medium bud blastema) (Casco-Robles et al., 2018).
46
Figure 2. Blood cells of C. pyrrhogaster (modified from Young et al., 2013; R. M.
Casco-Robles et al. 2018). (A) Nucleated erythrocytes at different developmental stages.
They can be divided into BpNobs, PcNobs (subdivided into early, intermediate, and late
stages), and OcNobs. During the maturation of normoblasts, they change shape from
oblate spheroid to ellipsoid and gradually increase cytoplasmic space, with nucleus
becoming more compact (Casco-Robles et al., 2018). BpNobs were immature
normoblasts, have big round polychromatic nucleus and narrow cytoplasmic space.
PcNobs had a general ellipsoidal shape, relatively compact nucleus, and larger
cytoplasmic space. PcNobs are defined as normoblasts in a transitional to mature state,
which make up more than 97.8% of normoblasts. OcNobs, mature normoblasts,
synthesize and contain hemoglobin in cytoplasm. (B) Five types of leukocytes.
Leukocytes are traditionally categorized into granulocytes (neutrophils, eosinophils,
and basophils) and mononuclear leucocytes (lymphocytes and monocytes) based on
their nuclear shape and cytoplasmic granules. (C) Thrombocyte (platelet).
47
Figure 3. Double staining of TGFβ1 and Newtic1. Tissue sections are labeled with
TGFβ1 primary antibody and TGFβ1 secondary antibody conjugated with Alexa
Fluor488. Subsequently, the samples are washed thoroughly, labeled with Newtic1
primary antibody and Newtic1 secondary antibody conjugated with Rhodamine. At this
step, RFP primary antibody was introduced as negative control for Newtic1 primary
antibody. The nuclei of cells are stained before mounting coverslips and imaging.
48
Figure 4. Immunostaining of blastemal blood cells on coverslips. (A) Preparation of
blastemal blood cells. A small slit was made on the top of the blastema using the tip of
the blade. After collection of blastemal blood, cells were plated and attached on PolyD-Lysine coated coverslips. (B) Immunostaining of blood cells on coverslips. Samples
were fixed and performed immunocytochemistry, then observed under light microscope.
49
Figure 5. Workflow for the preparation of semithin and ultrathin sections of Newtic1
immunolabelled blood cells. After Newtic1 immunolabelling, samples were fixed,
stained, dehydrated, and embedded in epoxy block. Semithin sections were prepared
with an ultramicrotome using a glass knife, collected on glass slides, and confirmed by
light microscopy to find Newtic1(+) PcNobs. The area containing Newtic1(+) PcNobs
was chosen to be sectioned for TEM and the block face was re-trimmed. Finally,
ultrathin sections were prepared using a diamond knife. Specimens were collected on
TEM grides, stained with heavy metal solution, and observed under TEM to identify
Newtic1(+) dots.
50
Figure 6. Immunofluorescence labelling of Newtic1 in blastema. (A, B) Limb blastema
at 4 weeks post amputation. The forelimb was amputated at the midpoint between the
elbow and wrist. (C) Confocal image of a blastemal section. PcNobs with Newtic1
immunofluorescence (red) along the equator, i.e., Newtic1(+) PcNobs, had
accumulated in the blastema. Blue: TO-PRO-3 nuclear stain. we: wound epidermis.
Scale bars: 5 mm (A); 2 mm (B); 100 μm (C).
51
52
Figure 7. Immunofluorescence labelling of Newtic1 in polychromatic normoblasts
(PcNobs). (A) Cells in blastemal blood. Arrowheads indicate Newtic1(+) PcNobs. (B,
C) Diagonal side view of a Newtic1(+) PcNob. Fluorescent ring along the equator was
composed of small dots with immunoreactivity. (D) Magnified view of Newtic1(+)
PcNob with a 100× objective lens. In this cell, fluorescent dots were distributed not
only just beneath the plasma membrane along the equator, but also in the cytoplasm.
This cell is a relatively young PcNob compared to typical PcNobs like that in B, because
the nucleus was roundish-oval in shape and the cytoplasmic area was still narrow. (E)
Confocal image of fluorescent dots with a 100x objective lens. Optical section: 300 nm.
Arrows indicate typical fluorescent dots with a core of 200-300 nm in diameter. (F) The
percentage of Newtic1(+) PcNobs in total normoblasts of intact blood (n=6) and
blastemal blood (n=5). Data are presented as the mean ± SE. The proportion of
Newtic1(+) PcNobs in blastemal blood was significantly higher than that in intact blood
(Welch’s t-test, p=0.02). Scale bars: 40 μm (A); 20 μm (B, C); 5 μm (D); 500 nm (E).
53
Figure 8. α-Tubulin and Newtic1 double stain of PcNobs in blastemal blood. (A, B) αTubulin (1:100) and Newtic1. (C, D) α-Tubulin (1:100) and RFP. RFP antibody was
used as the control. (E, F) α-Tubulin (1:1000) and Newtic1. (G, H) α-Tubulin (1:1000)
and RFP. Note that the primary antibodies used here were generated by the same host
animal, and that Newtic1 or RFP was stained after α-tubulin staining. Arrowheads
indicate PcNobs with intense α-tubulin immunofluorescence along the marginal band.
Note that the immunoreactivity of Newtic1 was not recognized in the marginal band of
PcNobs, which had low or no immunoreactivity to α-tubulin (1:1000). Scale bars: 40
μm.
54
55
Figure 9. Acetylated tubulin and Newtic1 double stain of PcNobs in blastemal blood.
(A, B) Acetylated tubulin and Newtic1. The acetylated tubulin antibody stained
microtubules of the marginal band at various intensities. Arrowheads indicate PcNobs
with Newtic1 immunoreactivity along the marginal band. (C, D) Acetylated tubulin and
RFP. RFP antibody was used as the control. (E-J) A representative set of confocal
images showing the positional relationship between microtubules and Newtic1(+) dots
in the marginal band (n=7). (E, G, I) Magnified images of the upper part of the marginal
band of the PcNob (F, H, J), where the microtubules appear to be in the process of
assembling into a bundle with each other. Newtic1(+) dots were distributed in close
proximity to microtubules. Arrows indicate Newtic1(+) dots which localized in breaks
on microtubules or in small unacetylated regions. Scale bars: 40 μm (A-D); 1 μm (E,
G, I); 10 μm (F, H, J).
56
Figure 10. Correlative light-electron microscopy (CLEM) of Newtic1 immunostaining.
(A) Optical microscopy image of Newtic1(+) PcNobs obtained with AURION R-Gent
SE-LM. Cells were stained with toluidine blue. The arrowhead indicates a typical
Newtic1(+) PcNob with brown dots along the equator. The cytoplasm of those cells was
characteristically transparent, possibly because of less hemoglobin. (B) Enlargement of
the cell indicated by the arrowhead in A. (C) A transmission electron microscope image
of an ultrathin section (80-90 nm thick) of the cell in B. Scale bars: 40 μm (A); 20 μm
(B, C).
57
58
Figure 11. Newtic1 immunoelectron microscopy. (A) Enlargement of the image in
Figure 10C. mito: mitochondria. Asterisks indicate artifacts in the process of sample
preparation. (B, C) Enlargement of the images in boxes b and c in A. Reactants in the
form of black granules were distributed along the microtubules of the marginal band.
The granules were localized in close proximity on microtubules. (D, E) Magnified
images of typical granules. The images were obtained from a different PcNob. The
granules were globular structures with a diameter of about 100 nm, which appeared to
be composed of black reactant grains. Scale bars: 10 μm (A); 1 μm (B); 100 nm (C-E).
59
Figure 12. Newtic1 immunoblotting with blastemal blood cells. (A) Sample
preparation. The proteins of blastemal blood cells were fractionated into soluble
proteins in the cytoplasm and plasma membrane (sample 1) and insoluble proteins in
the cytoskeleton (sample 2). For details, see Methods. (B) Immunoblotting. In this case,
about 30 µL blood cells were collected from 5 limb blastemas (5 newts). A protein band
corresponding to Newtic1 (40.7 kD) was detected in both sample 1 and in sample 2.
Control: primary antibody omitted. α-Tubulin: 49.8 kD. For original images of the
blotted membranes, see Figure 13.
60
Figure 13. Originals of Figure 12B. (A) Raw data. (B) Contrast-enhanced images of
the raw data. Protein bands in the boxes in B were shown in Figure 12B. The 10-20kD,
25-37kD, 75kD, and 100-150kD bands were caused by non-specific reactions of the
secondary antibody (Casco-Robles et al., 2018). M: maker.
61
Figure 14. Live cell staining of endoplasmic reticulum (ER), Golgi apparatus, and
nucleus. Under normal fluorescence microscopy, ER (red) and Golgi apparatus (green)
almost overlapped; a basophilic normoblast (BpNob) is the precursor of erythrocytes,
OcNob represents mature erythrocytes, and PcNob represents erythrocytes in the
transition phase. ER and Golgi apparatus were more developed in immature
erythrocytes and became confined to narrow regions in OcNob. Blue: nucleus stained
with Hoechst 33342. Scale bars: 20 μm (A). Note that non-specific signals may be
caused by the staining and image acquisition condition.
62
Figure 15. Live cell staining of organelles in PcNobs. (A) Confocal images of live
PcNobs with ER and Golgi apparatus stained. Under this condition, in which the nuclei
(n) were not well stained with Hoechst 33342, the membrane structures surrounding
the nucleus (i.e., nuclear membrane) were clearly visible with ER markers (red). By
making thin optical sections, several Golgi apparatuses (green) were discerned as
distributed at the periphery of the ER. (B) Live cell staining of a lysosome (Lyso),
mitochondria (Mito), and nucleus. Mitochondria were, like ER/Golgi, more developed
in immature erythrocytes and became confined to narrow regions in OcNob. Scale bars:
20 μm (A, B).
63
Figure 16. TEM images showing endomembrane system in PcNobs. (A) TEM image
of Golgi apparatuses in PcNob. (B) TEM image of membrane vesicles in PcNob.
Arrowheads indicate vesicular structures. The arrow indicates microtubules of the
marginal band. (C) Enlargement of the membrane structure indicated by c in B. An Ωshaped membrane structure of approximately 100 nm in size was observed. Scale bars:
200 nm (A, B); 100 nm (C).
64
65
Figure 17. Immunoreactivity of BMP2 and TGFβ1 of PcNobs in blood. (A-C)
Fluorescence microscopy images showing immunoreactivity of BMP2, TGFβ1, and
RFP in PcNobs in intact blood. (D-F) Fluorescence microscopy images showing
immunoreactivity of BMP2, TGFβ1, and RFP in PcNobs in blastemal blood. In intact
blood, almost all PcNobs showed immunoreactivity to BMP2 and TGFβ1, although the
intensities were variable. Both reactivities were relatively intense around the nucleus
and low at the periphery (see Figure 19). However, for TGFβ1, a small number of cells
also showed immunoreactivity along the equator (arrowheads in B). In blastemal blood,
BMP2 immunoreactivity was reduced (D). For TGFβ1, there was an increase in PcNobs
showing immunoreactivity along the equator (arrowheads in E). Some of these cells
had decreased immunoreactivity in the cytoplasm. (G) The percentage of TGFβ1(+)
PcNobs in total normoblasts of intact blood (n=3) and blastemal blood (n=4). Data are
presented as the mean ± SE. The proportion of TGFβ1(+) PcNobs in blastemal blood
was significantly higher than that in intact blood (Student’s t-test, p=0.0026). Scale bars:
50 μm (A-F).
66
Figure 18. Immunoreactivity of TGFβ1 along the equator of PcNobs in blastemal blood.
(A-C) A representative set of confocal images of TGFβ1 and Newtic1 double stain of
PcNobs
in
blastemal
blood. Arrowheads
indicate
PcNobs
with
TGFβ1
immunoreactivity (green) along the equator. These cells were also stained with a
secondary antibody for Newtic1 immunostaining (red). (D-I) Magnified images of the
PcNob indicated by the magenta arrowhead in A-C. (D, F, H) Magnified images of the
upper left region of the immunoreactivity along the equator of the cell shown in E, G,
and I, respectively. Blue: nuclei stained with DAPI. Scale bars: 50 μm (A-C); 1 μm (D,
F, H); 20 μm (E, G, I).
67
68
Figure 19. Expression of BMP2 and TGFβ1 in normal circulating PcNobs. (A, B) A
representative set of images of BMP2 and Newtic1 double stain. (A) Normal
fluorescence microscopy images. Almost all PcNobs had BMP2 immunoreactivity
(green) in the cytoplasm, albeit of varying intensity. Their immunoreactivity was
intense around the nucleus and decreased toward the periphery. Arrowheads indicate
Newtic1(+) PcNobs, which showed red fluorescence in a ring along the equator. In the
optics here, nuclei stained with TO-PRO-3 (TP3) were detected in red. (B) Confocal
images of a typical Newtic1(+) PcNob. There was little overlap between the
immunoreactivity of Newtic1 (red) along the equator and that of BMP2 (green) in the
cytoplasm. Blue: TO-PRO-3 nuclear stain. (C, D) A representative set of images of
TGFβ1 and Newtic1 double stain. (C) Normal fluorescence microscopy images.
Almost all PcNobs had TGFβ1 immunoreactivity (green) in the cytoplasm, albeit of
varying intensity. In most cells, their immunoreactivity was intense around the nucleus
and decreased toward the periphery, as observed with BMP2, but in a small number of
cells, immunoreactivity was also observed along the equator. White arrowheads
indicate Newtic1(+)PcNobs, whose Newtic1 immunoreactivity along the equator did
not overlap with that of TGFβ1. Magenta arrowheads indicate Newtic1(+) PcNobs, in
which TGFβ1 immunoreactivity was observed along their equator. Nuclear stain with
TP3 is shown in red. (D) Confocal images of a typical Newtic1(+) PcNob without
TGFβ1 immunoreactivity along the equator. Blue: TO-PRO-3 nuclear stain. (E) A
representative set of normal fluorescence microscopy images of RFP and Newtic1
double stain. Note that all primary antibodies used here were produced by a rabbit.
Therefore, staining with Newtic1 antibody was preceded by staining with the other
primary antibodies. Scale bars: 100 μm (A, C, E); 5 μm (B, D).
69
70
Figure 20. Expression patterns of BMP2 and TGFβ1 in PcNobs in the limb blastema.
(A-C) A representative set of confocal images of BMP2 and Newtic1 double stain.
BMP2 immunoreactivity (green) was scattered throughout the tissue. In PcNobs, which
had accumulated in the vessels extending in the blastema, BMP2 immunoreactivity was
not observed in their cytoplasm. Slight reactions appeared to be distributed along their
equator, where Newtic1 immunoreactivity (red) was seen, but this could be due to the
thick optical sections. In fact, analysis of blood cells collected from the blastema
(Figure 17D) showed no BMP2 immunoreactivity at the margins of PcNobs. (D-F) A
representative set of confocal images of TGFβ1 and Newtic1 double stain. TGFβ1
immunoreactivity (green), most of which was observed in PcNobs that had accumulated
in blood vessels, was not detected in their cytoplasm and appeared to be distributed
granularly on the equator where Newtic1 immunoreactivity (red) was seen. This pattern
of immunoreactivity, unlike that of BMP2, was also observed in blood cells collected
from the blastema (Figure 17E). (G-I) A representative set of confocal images of RFP
and Newtic1 double stain for the control. Green: RFP; Red: Newtic1; Blue: TO-PRO3 nuclear stain. Note that all primary antibodies used here were produced by a rabbit.
Therefore, staining with Newtic1 antibody was preceded by staining with the other
primary antibodies. Scale bars: 40 μm.
71
Figure 21. Relationship of TGFβ1 immunoreactivity to Newtic1(+) dots. (A) Confocal
images along the equator of PcNob. This cell is different from those shown in Figure
18A-C. In this cell, multiple lines of immunoreactive granules were observed just
below the equator, presumably corresponding to the lines of microtubules. The
immunoreactivity of TGFβ1 was granular and mostly overlapped with the region of
Newtic1 immunoreactivity. However, the region of Newtic1 immunoreactivity was
wider than that of TGFβ1. Note that there were also Newtic1(+) dots that did not
overlap with TGFβ1 immunoreactivity. (B) Quanti ...
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