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Citation: Huysseune, A.; Larsen,
U.G.; Larionova, D.; Matthiesen, C.L.;
Petersen, S.V.; Muller, M.; Witten, P.E.
Bone Formation in Zebrafish: The
Significance of DAF-FM DA Staining
for Nitric Oxide Detection.
Biomolecules 2023,13, 1780. https://
doi.org/10.3390/biom13121780
Academic Editor: Enrico Moro
Received: 25 October 2023
Revised: 2 December 2023
Accepted: 5 December 2023
Published: 12 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomolecules
Article
Bone Formation in Zebrafish: The Significance of DAF-FM DA
Staining for Nitric Oxide Detection
Ann Huysseune 1, 2, *, Ulrike G. Larsen 3, Daria Larionova 1, Cecilie L. Matthiesen 3, Steen V. Petersen 3,
Marc Muller 4and P. Eckhard Witten 1
1Research Group Evolutionary Developmental Biology, Biology Department, Ghent University,
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium; daria.larionova@ugent.be (D.L.);
peckhardwitten@aol.com (P.E.W.)
2Department of Zoology, Faculty of Science, Charles University, Vinicna 7, 128 44 Prague, Czech Republic
3Department for Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10, 8000 Aarhus, Denmark;
ul@biomed.au.dk (U.G.L.); cl@biomed.au.dk (C.L.M.); svp@biomed.au.dk (S.V.P.)
4Laboratoire d’Organogenèse et Régénération, GIGA-R 1, Avenue de l’Hôpital, B34 Sart Tilman,
4000 Liège, Belgium; m.muller@uliege.be
*Correspondence: ann.huysseune@ugent.be or huysseua@natur.cuni.cz
Abstract:
DAF-FM DA is widely used as a live staining compound to show the presence of nitric
oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers
of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed.
Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the
fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were
observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus.
Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von
Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both
the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds
non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is
nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS
enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA
is appropriate to reveal bone formation
in vivo
, independent of mineralization of the bone matrix,
but it does not demonstrate intracellular NO.
Keywords: zebrafish; osteoblasts; ossification; nitric oxide; notochord sheath; bulbus arteriosus
1. Introduction
Since its discovery in 1987 as a signaling molecule in the cardiovascular system, nitric
oxide (NO) has been identified as a major player in a wide range of physiological processes,
including neurotransmission and immune responses (recently reviewed in, e.g., [1,2]).
Because NO is a gaseous, free radical molecule with a short lifetime, its demonstration
most often depends on localizing the enzyme that catalyzes the production of NO from
L-arginine, nitric oxide synthase, or NOS. For example, in skeletal tissues, osteoblasts,
osteocytes, and osteoclasts, cells that moderate aspects of bone formation and bone re-
sorption, express different isoforms of NOS [
3
,
4
]. Furthermore, NO has been found to
have a regulatory role in bone with complex actions in both osteoblastic and osteoclastic
lineages [5–9].
Over time, methods have also been developed to directly reveal the presence of NO.
Kojima et al. [
10
] designed several diaminofluoresceins (DAFs) including DAF-2. These
DAFs are converted to the triazole form by a reaction with NO, thereby greatly enhancing
their fluorescence. The diacetate ester derivative (DAF-2 DA) allows NO imaging in living
cells as it easily enters the cells to be converted into DAF-2 by intracellular esterases. DAF-2
Biomolecules 2023,13, 1780. https://doi.org/10.3390/biom13121780 https://www.mdpi.com/journal/biomolecules
Biomolecules 2023,13, 1780 2 of 14
then reacts with NO to form DAF-2T (the triazole form of DAF-2), which becomes trapped
in the cytosol. Kojima et al. [
11
] next designed DAF-FM by introducing a methyl group and
replacing chlorine with fluorine atoms in DAF-4. This derivative (4-amino-5-methylamino-
2
0
-7
0
-difluorofluorescein, or DAF-FM) and its diacetate ester (DAF-FM DA) have found
more general usage. Its triazole form, DAF-FM T, is strongly fluorescent, less susceptible to
photobleaching, and the fluorescence intensity is stable above pH 5.8 [
11
]. Therefore, DAF-
FM can detect lower levels of NO. Like DAF-2 DA, DAF-FM DA spontaneously crosses
the plasma membrane and is subsequently cleaved by esterases to generate intracellular
DAF-FM. The latter is then oxidized by NO to form a triazole product. When measured in
a buffer, this reaction increases the fluorescence quantum yield by about 160-fold [11].
The zebrafish, a small vertebrate that is particularly amenable to experimentation and
live imaging, has been a model of choice in revealing NO in the skeletal system via live
staining. Several authors have reported that DAF-FM DA, applied as live staining on em-
bryos and early postembryonic stages, is a reliable method to label early ossification centers
in developing zebrafish [
12
–
14
]. However, in these studies, the method was employed to
detect NO on whole-mount embryos but not to detect NO at a cellular level.
Because of the growing field of biomedical research using zebrafish to develop skeletal
disease models, as well as the interest of the NO field, we set out to refine these observations
and to assess the precise (cellular) localization of DAF-FM DA signals in the developing
zebrafish embryo. We found that DAF-FM DA stains the extracellular matrix in early
centers of bone formation, independent of mineralization of the bone matrix, as well as in
the notochord sheath and the bulbus arteriosus. The significance of NO for the function of
osteoblasts is nevertheless revealed by the absence of bones and of DAF-FM DA staining
after the inhibition of NO production.
2. Materials and Methods
2.1. Sample Collection
Adult wildtype (WT) zebrafish (Danio rerio) were maintained and embryos were raised
under standard conditions at 28.5 ◦C and a 14/10 h light/dark cycle, according to [15].
2.2. NO Staining on Live Material
Live staining was performed both on manually dechorionated zebrafish embryos (30,
36, 42 h post-fertilization, hpf) as well as on early postembryonic stages of zebrafish up to
the first independent feeding stage (5 days post-fertilization, dpf, for zebrafish [16]).
DAF-FM DA was used as a live stain according to a published protocol [
12
]. Briefly,
specimens were soaked for up to 3 h (times as indicated) in a freshly made solution of
5
µ
M DAF-FM DA in 0.1% DMSO, shielded from light. The controls were simultaneously
soaked in 0.1% DMSO. To test the specificity of DAF-FM DA staining for NO, staining
with DAF-FM DA was also carried out after a treatment of 30 min with NO scavenger
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (c-PTIO,
EMD Millipore, Burlington, MA, USA) at a concentration of 500
µ
M in 0.05% DMSO. After
staining, specimens were rinsed in PBS and sedated using MS222. Unless stated otherwise,
images of live, sedated animals were taken using a Zeiss Axio Zoom V16 Fluorescence
Stereo Zoom microscope equipped with a 5 MP CCD camera for imaging under standard
magnification (20 and 80
×
) and standard illumination (50,000 and 110,000 ms, resp.).
Particular images were recorded using a Zeiss Axio Observer Z1 microscope equipped
with an apotome 2 unit (inverted setup). After imaging, animals were euthanized using an
overdose of MS222 and fixed in 4% PFA for processing in glycol methacrylate (GMA).
2.3. NO Staining on Fixed Material
WT zebrafish of 8 dpf were euthanized using an overdose of MS222 and fixed either in
4% paraformaldehyde (PFA), in acetone, or in 70% ethanol and exposed to 5
µ
M DAF-FM
DA in 0.1% DMSO or to the control vehicle for 3 h in the dark. They were next immediately
observed and photographed.
Biomolecules 2023,13, 1780 3 of 14
2.4. NO Staining after Inhibition of NOS
DAF-FM DA staining was also performed after the inhibition of NO production using
1-[2-(trifluoromethyl)phenyl]imidazole (TRIM, Sigma-Aldrich, St. Louis, MO, USA). TRIM
specifically inhibits the function of the enzymes catalyzing the formation of NO, nitric
oxide synthases (NOS), in particular nNOS and iNOS, and eNOS with a lower efficiency.
The latter is, however, not present in zebrafish [
17
]. TRIM was used as a 100
µ
M solution in
the embryo medium, prepared from a 10 mM stock solution in 10% DMSO, and applied in
darkness uninterruptedly on WT zebrafish from 30 hpf to 5 dpf. After rinsing, DAF-FM
DA staining was performed for 3 h, followed by live imaging, as described above. Controls
of the inhibition were carried out using DMSO at a similar concentration as the inhibitor
(0.1%).
2.5. Processing for GMA Sections
All specimens live-stained with DAF-FM DA, as well as their controls, were embedded
in GMA according to a published protocol [
18
]. Briefly, they were dehydrated shortly in
acetone and transferred to the GMA monomer for 24 h. Specimens were next placed into
the monomer with an added catalyst, and blocks were allowed to polymerize over two
days. Serial 3
µ
m sections were made using a Prosan HM350 microtome. Sections were
temporarily mounted with PBS or permanently with Vectashield containing DAPI. During
all processing steps, samples, as well as the sections obtained from them, were protected
from light.
2.6. Demonstration of Mineralized Tissues
For the detection of mineral salts, both Von Kossa and Alizarin red S staining were
used. The selected sections of specimens previously live-stained with DAF-FM DA were
processed for Von Kossa staining, according to [
19
]. Briefly, after rinsing in PBS and
distilled H
2
O, sections were immersed for 45 min in 1% AgNO
3
under UV light, rinsed,
soaked for 5 min in 3% Na
2
S
2
O
3
, rinsed, soaked for 5 min in Von Gieson stain solution,
followed by a short differentiation in 96% ethanol, and air-dried. For observation, sections
were temporarily coverslipped with PBS, and the same areas, photographed prior to Von
Kossa staining with the GFP filter for the DAF-FM DA signal, were photographed using
brightfield illumination. Likewise, Alizarin red S staining (0.5% solution, pH 9, 1 min) was
applied onto GMA sections of specimens live-stained with DAF-FM DA and observed with
a rhodamine filter for the Alizarin red S signal. Overlay pictures were produced using
Photoshop version 6.0.
2.7. Elastin Staining
WT zebrafish of 5 dpf were euthanized using an overdose of MS222, fixed in PFA,
and processed for paraffin embedding according to standard procedures. Serial 5
µ
m
cross sections were made using a Prosan HM350 microtome. Elastin was demonstrated
using an adapted Verhoeff’s protocol (without Von Gieson counterstain) [
20
]. Briefly,
after deparaffinizing and hydration, sections were stained for 30 min in Verhoeff’s iodine
solution, rinsed, and differentiated in 2% FeCl
2
for time intervals of 15 s, alternating with
rinsing in distilled water, observation and microphotography.
2.8. Processing for Epon Sections and Transmission Electron Microscopy
Zebrafish embryos of 5 dpf, treated with TRIM, as well as control embryos, were
euthanized using an overdose of MS222, fixed in a mixture of 1.5% glutaraldehyde and 1.5%
paraformaldehyde (PG), postfixed in OsO
4
and processed for epon embedding according
to a published protocol [
21
]. Semithin (1
µ
m) sections were stained with Toluidine blue
for 2 min (0.2% Toluidine Blue, 2% Na
2
B
4
O
7
), rinsed with water, air-dried, and mounted
with DPX (Fluka, Buchs, Switzerland). Ultrathin sections (70 nm) were cut with a Reichert
Ultracut E, were contrasted with uranyl acetate and lead citrate, and examined under a Jeol
Biomolecules 2023,13, 1780 4 of 14
JEM 1010 transmission electron microscope (Jeol Ltd., Tokyo, Japan) operating at 60 kV.
Microphotographs were taken with a Veleta camera (Emsis, Muenster, Germany).
2.9. Observations and Microphotography
Observations and microphotography of GMA and semithin epon sections were
performed using a Zeiss Axio Imager Z1 compound microscope equipped with epi-
fluorescence. Photographs were taken with an Axiocam 503 color camera (Carl Zeiss,
Oberkochen, Germany).
3. Results
Soaking early postembryonic zebrafish in DAF-FM DA according to published pro-
tocols specifically stained different bone elements, which is consistent with previous re-
ports [
12
–
14
] (Figure 1A,B). In addition, a strong signal was observed around the notochord
and anterior to the heart, similar to what has been reported in the literature cited above.
In contrast, cartilage gave a signal hardly detectable against the background. Although
the irregular ventral border of the opercular bone may suggest the presence of labeled
osteoblasts (Figure 1B), the signal associated with bony structures, as well as with the teeth,
appeared, in sections, to be located in the extracellular matrix and not in the bone-forming
cells, the osteoblasts (Figures 1C and S1). Likewise, the signal associated with the noto-
chord was located in the notochord sheath (Figure 1D); the signal associated with the heart
was limited to the bulbus arteriosus and appeared to label intercellularly located fibrous
material (Figure 1E).
Biomolecules 2023, 13, x FOR PEER REVIEW 4 of 14
1.5% paraformaldehyde (PG), postxed in OsO4 and processed for epon embedding
according to a published protocol [21]. Semithin (1 µm) sections were stained with
Toluidine blue for 2 min (0.2% Toluidine Blue, 2% Na2B4O7), rinsed with water, air-dried,
and mounted with DPX (Fluka, Buchs, Swierland). Ultrathin sections (70 nm) were cut
with a Reichert Ultracut E, were contrasted with uranyl acetate and lead citrate, and
examined under a Jeol JEM 1010 transmission electron microscope (Jeol Ltd., Tokyo,
Japan) operating at 60 kV. Microphotographs were taken with a Veleta camera (Emsis,
Muenster, Germany).
2.9. Observations and Microphotography
Observations and microphotography of GMA and semithin epon sections were
performed using a Zeiss Axio Imager Z1 compound microscope equipped with
epiuorescence. Photographs were taken with an Axiocam 503 color camera (Carl Zeiss,
Oberkochen, Germany).
3. Results
Soaking early postembryonic zebrash in DAF-FM DA according to published
protocols specically stained dierent bone elements, which is consistent with previous
reports [12–14] (Figure 1A,B). In addition, a strong signal was observed around the
notochord and anterior to the heart, similar to what has been reported in the literature
cited above. In contrast, cartilage gave a signal hardly detectable against the background.
Although the irregular ventral border of the opercular bone may suggest the presence of
labeled osteoblasts (Figure 1B), the signal associated with bony structures, as well as with
the teeth, appeared, in sections, to be located in the extracellular matrix and not in the
bone-forming cells, the osteoblasts (Figures 1C and S1). Likewise, the signal associated
with the notochord was located in the notochord sheath (Figure 1D); the signal associated
with the heart was limited to the bulbus arteriosus and appeared to label intercellularly
located brous material (Figure 1E).
Figure 1.
Live staining of early postembryonic zebrafish embryos with DAF-FM DA. (
A
,
B
). Live
staining with DAF-FM DA, according to published protocols, reveals signals in bony structures
(opercular, arrow; cleithrum, arrowhead), as well as in the heart (asterisk) and around the notochord,
which is consistent with earlier reports. Axio Observer image. (
C
–
E
). Three
µ
m GMA sections of the
specimen shown in (
A
,
B
), counterstained with DAPI, showing the opercular bone (
C
), notochord
(
D
), and bulbus arteriosus (
E
). Note that nuclei lie outside fluorescent domains. Scale bars in
(A,B) = 200 µm, in (C,E) = 20 µm, in (D) = 10 µm.
Biomolecules 2023,13, 1780 5 of 14
We reasoned that three hours of staining might allow non-specific reactions to build
up in the extracellular matrix, masking or perhaps abolishing an earlier intracellular signal.
Thus, we soaked 5 dpf embryos in DAF-FM DA according to the recommended procedure
but observed and sacrificed embryos every 20 min for the 3 h duration of staining (Figure 2).
However, even after the short-term interval of 20 min, the staining pattern was comparable
to that after 3 h, albeit slightly weaker: fluorescent signals were observed in the bulbus
arteriosus of the heart, the notochord sheath, the matrix of the forming bones, and the
dentine and attachment bone of the teeth (Figures 2and S1).
Biomolecules 2023, 13, x FOR PEER REVIEW 5 of 14
Figure 1. Live staining of early postembryonic zebrash embryos with DAF-FM DA. (A,B). Live
staining with DAF-FM DA, according to published protocols, reveals signals in bony structures
(opercular, arrow; cleithrum, arrowhead), as well as in the heart (asterisk) and around the
notochord, which is consistent with earlier reports. Axio Observer image. (C–E). Three µm GMA
sections of the specimen shown in (A,B), counterstained with DAPI, showing the opercular bone
(C), notochord (D), and bulbus arteriosus (E). Note that nuclei lie outside uorescent domains. Scale
bars in (A,B) = 200 µm, in (C,E) = 20 µm, in (D) = 10 µm.
We reasoned that three hours of staining might allow non-specic reactions to build
up in the extracellular matrix, masking or perhaps abolishing an earlier intracellular
signal. Thus, we soaked 5 dpf embryos in DAF-FM DA according to the recommended
procedure but observed and sacriced embryos every 20 min for the 3 h duration of
staining (Figure 2). However, even after the short-term interval of 20 min, the staining
paern was comparable to that after 3 h, albeit slightly weaker: uorescent signals were
observed in the bulbus arteriosus of the heart, the notochord sheath, the matrix of the
forming bones, and the dentine and aachment bone of the teeth (Figures 2 and S1).
Figure 2.
Live staining of 5 dpf zebrafish embryos with DAF-FM DA for short intervals. Column (
A
):
live imaging of the head region at the time indicated on the left; inset: magnification of the opercular
bone. The strong blurred signal visible ventrally is the bulbus arteriosus. Scale
bars = 200 µm
.
Columns (
B
–
D
): 3
µ
m GMA sections of the corresponding embryos. Column (
B
): bulbus arteriosus.
Scale bars = 10
µ
m. (
C
): notochord in the head region. Scale bars = 20
µ
m except for 60 min: 10
µ
m.
(
D
): opercular bone. Scale bars = 10
µ
m except for 40 min: 20
µ
m. Column (
A
): anterior to the left;
(B,C): dorsal to the top; (D): lateral to the left.
Biomolecules 2023,13, 1780 6 of 14
To exclude the possibility that the signal first appears inside the cells (e.g., osteoblasts)
and subsequently leaks out into the ECM, we performed the same experiment, using
5 dpf embryos but exposing them for even shorter time intervals, with live observations
after 1, 3, 5, 10 and 20 min. Under these conditions, the notochord sheath and the bulbus
arteriosus were the first structures to give a signal after 3 min. A slight signal appeared
in the opercular bone after 5 min. Although very weak after these short time intervals of
staining, the signal in the bones was again clearly limited to the matrix (Figure S2).
To assess whether the staining pattern observed could simply be an artifact unrelated
to the live uptake of the compound, we also performed DAF-FM DA staining on fixed
material. The three different fixatives used (4% PFA, acetone, or 70% ethanol) showed
different patterns of fluorescence, with PFA being the strongest, including a dotted pattern
in the skin as well as a signal in axial structures, but not the notochord. Control zebrafish
that were treated with DMSO showed hardly any fluorescence. This shows that staining
after fixation can indeed yield a specific pattern of fluorescence, which is, however, different
from that obtained after live staining (Figure S3).
Next, we compared live DAF-FM DA staining with or without pretreatment with the
NO scavenger c-PTIO (Figure 3). Staining with DAF-FM DA after treatment with the NO
scavenger c-PTIO weakened but did not abolish the signal.
Biomolecules 2023, 13, x FOR PEER REVIEW 6 of 14
Figure 2. Live staining of 5 dpf zebrash embryos with DAF-FM DA for short intervals. Column
(A): live imaging of the head region at the time indicated on the left; inset: magnication of the
opercular bone. The strong blurred signal visible ventrally is the bulbus arteriosus. Scale bars = 200
µm. Columns (B–D): 3 µm GMA sections of the corresponding embryos. Column (B): bulbus
arteriosus. Scale bars = 10 µm. (C): notochord in the head region. Scale bars = 20 µm except for 60
min: 10 µm. (D): opercular bone. Scale bars = 10 µm except for 40 min: 20 µm. Column (A): anterior
to the left; (B,C): dorsal to the top; (D): lateral to the left.
To exclude the possibility that the signal rst appears inside the cells (e.g.,
osteoblasts) and subsequently leaks out into the ECM, we performed the same
experiment, using 5 dpf embryos but exposing them for even shorter time intervals, with
live observations after 1, 3, 5, 10 and 20 min. Under these conditions, the notochord sheath
and the bulbus arteriosus were the rst structures to give a signal after 3 min. A slight
signal appeared in the opercular bone after 5 min. Although very weak after these short
time intervals of staining, the signal in the bones was again clearly limited to the matrix
(Figure S2).
To assess whether the staining paern observed could simply be an artifact unrelated
to the live uptake of the compound, we also performed DAF-FM DA staining on xed
material. The three dierent xatives used (4% PFA, acetone, or 70% ethanol) showed
dierent paerns of uorescence, with PFA being the strongest, including a doed paern
in the skin as well as a signal in axial structures, but not the notochord. Control zebrash
that were treated with DMSO showed hardly any uorescence. This shows that staining
after xation can indeed yield a specic paern of uorescence, which is, however,
dierent from that obtained after live staining (Figure S3).
Next, we compared live DAF-FM DA staining with or without pretreatment with the
NO scavenger c-PTIO (Figure 3). Staining with DAF-FM DA after treatment with the NO
scavenger c-PTIO weakened but did not abolish the signal.
Figure 3. Live staining of 5 dpf zebrash with DAF-FM DA after the use of the NO scavenger c-
PTIO. (A–D). Live staining of 5 dpf zebrash for 20 min (A,B) or 3 h (C,D) with DAF-FM DA either
without c-PTIO (A,C) or after 30 min treatment with the NO scavenger c-PTIO (B,D). All images
were taken strictly under the same illumination. Scale bars = 200 µm.
Figure 3.
Live staining of 5 dpf zebrafish with DAF-FM DA after the use of the NO scavenger c-PTIO.
(
A
–
D
). Live staining of 5 dpf zebrafish for 20 min (
A
,
B
)or3h(
C
,
D
) with DAF-FM DA either without
c-PTIO (
A
,
C
) or after 30 min treatment with the NO scavenger c-PTIO (
B
,
D
). All images were taken
strictly under the same illumination. Scale bars = 200 µm.
Because staining with DAF-FM DA appeared to be associated most with mineralized
structures (bones and teeth–the notochord sheath mineralizes later), we asked whether the
mineral in these structures could cause non-specific staining, i.e., whether the fluorescent
signal could coincide with mineralized areas. To address this question, we selected a
number of sections with a strong fluorescent signal from the live staining experiment and
reutilized these for Von Kossa staining. The overlay of the fluorescent DAF-FM DA and
Biomolecules 2023,13, 1780 7 of 14
brightfield Von Kossa images clearly showed more extensive fluorescent staining than that
revealed by Von Kossa staining for mineralized areas (Figure 4). Thus, some (mostly smaller,
thinner) bones that were clearly stained using DAF-FM DA did not show any staining for
minerals (Figure 4A–A
00
). Other (larger) bones consistently showed a rim of fluorescent
staining that was negative with Von Kossa, likely corresponding to the osteoid layer of the
bone (Figure 4C–C
00
). Staining with Alizarin red S exactly replicated the results obtained
with Von Kossa staining: DAF-FM DA labeled small bones that were left unstained with
Alizarin red S (i.e., not yet mineralized) (Figure 4B–B
00
), as well as non-mineralized and
mineralized (Alizarin red S-positive) areas of larger bones (Figure 4D–D
00
). This indicates
that DAF-FM DA stains the organic extracellular matrix and not its mineral component.
Biomolecules 2023, 13, x FOR PEER REVIEW 7 of 14
Because staining with DAF-FM DA appeared to be associated most with mineralized
structures (bones and teeth–the notochord sheath mineralizes later), we asked whether
the mineral in these structures could cause non-specic staining, i.e., whether the
uorescent signal could coincide with mineralized areas. To address this question, we
selected a number of sections with a strong uorescent signal from the live staining
experiment and reutilized these for Von Kossa staining. The overlay of the uorescent
DAF-FM DA and brighteld Von Kossa images clearly showed more extensive uorescent
staining than that revealed by Von Kossa staining for mineralized areas (Figure 4). Thus,
some (mostly smaller, thinner) bones that were clearly stained using DAF-FM DA did not
show any staining for minerals (Figure 4A–A″). Other (larger) bones consistently showed
a rim of uorescent staining that was negative with Von Kossa, likely corresponding to
the osteoid layer of the bone (Figure 4C–C″). Staining with Alizarin red S exactly
replicated the results obtained with Von Kossa staining: DAF-FM DA labeled small bones
that were left unstained with Alizarin red S (i.e., not yet mineralized) (Figure 4B–B″), as
well as non-mineralized and mineralized (Alizarin red S-positive) areas of larger bones
(Figure 4D–D″). This indicates that DAF-FM DA stains the organic extracellular matrix
and not its mineral component.
Figure 4.
Comparison of DAF-FM DA-stained structures with staining for mineralized tissue.
(A–A00)
.
Overview section of DAF-FM DA live-stained 5 dpf zebrafish (
A
) stained with Von Kossa for mineral-
ized structures (
A0
) and overlay (
A00
). Note the absence of Von Kossa staining in the para-sphenoid
(arrow) and entopterygoid bones (arrowheads). (
B
–
B00
). Overview section of a DAF-FM DA live-
stained 5 dpf zebrafish (
B
) stained with Alizarin red S for mineralized structures (
B0
) and overlay (
B00
).
The image is dark because of the complete absence of Alizarin red S staining in the parasphenoid
(arrow) and entopterygoid bones (arrowheads) (compared with the positive staining of mineralized
Biomolecules 2023,13, 1780 8 of 14
bone in
D0
). (
C
–
C00
). Details of the opercular bone in a section of a DAF-FM DA live-stained 5 dpf
zebrafish (
C
) stained with Von Kossa for mineralized structures (
C0
) and overlay (
C00
). Note the
distinct zone of DAF-FM DA-positive staining around the area positive for minerals using Von Kossa
(arrowheads). (
D
–
D00
). Details of the opercular bone in a section of a DAF-FM DA live-stained 5 dpf
zebrafish (
D
) stained with Alizarin red S for mineralized structures (
D0
) and overlay (
D00
). Note that
the DAF-FM DA-positive area is clearly larger than the area marked with Alizarin red S. Scale bar for
(A–A00) = 50 µm and for (B–B0 0 ) to (D–D00 ) = 20 µm.
To assess whether the signal in the bulbus arteriosus (Figure 5A) corresponds to elastin,
we compared the distribution of the signal with elastin, as revealed by Verhoeff’s elastin
stain. Since Verhoeff’s staining cannot be performed on GMA-embedded sections, we
necessarily had to compare images from different animals. However, abundant and distinct
elastic fibers were demonstrated in the bulbus arteriosus (Figure 5C), which were similar
in distribution and morphology to the fluorescent signals observed after DAF-FM DA
staining (Figure 5B). Surprisingly, TEM pictures of the 5 dpf zebrafish bulbus (Figure 5D)
revealed only weak evidence of elastin fibers, suggesting that the fluorescent signal may
reveal components associated with the elastin-rich extracellular matrix.
Biomolecules 2023, 13, x FOR PEER REVIEW 8 of 14
Figure 4. Comparison of DAF-FM DA-stained structures with staining for mineralized tissue. (A–
A″). Overview section of DAF-FM DA live-stained 5 dpf zebrash (A) stained with Von Kossa for
mineralized structures (A′) and overlay (A″). Note the absence of Von Kossa staining in the para-
sphenoid (arrow) and entopterygoid bones (arrowheads). (B–B″). Overview section of a DAF-FM
DA live-stained 5 dpf zebrash (B) stained with Alizarin red S for mineralized structures (B′) and
overlay (B″). The image is dark because of the complete absence of Alizarin red S staining in the
parasphenoid (arrow) and entopterygoid bones (arrowheads) (compared with the positive staining
of mineralized bone in D′). (C–C″). Details of the opercular bone in a section of a DAF-FM DA live-
stained 5 dpf zebrash (C) stained with Von Kossa for mineralized structures (C′) and overlay (C″).
Note the distinct zone of DAF-FM DA-positive staining around the area positive for minerals using
Von Kossa (arrowheads). (D–D″). Details of the opercular bone in a section of a DAF-FM DA live-
stained 5 dpf zebrash (D) stained with Alizarin red S for mineralized structures (D′) and overlay
(D″). Note that the DAF-FM DA-positive area is clearly larger than the area marked with Alizarin
red S. Scale bar for (A–A″) = 50 µm and for (B–B″) to (D–D″) = 20 µm.
To assess whether the signal in the bulbus arteriosus (Figure 5A) corresponds to
elastin, we compared the distribution of the signal with elastin, as revealed by Verhoe’s
elastin stain. Since Verhoe’s staining cannot be performed on GMA-embedded sections,
we necessarily had to compare images from dierent animals. However, abundant and
distinct elastic bers were demonstrated in the bulbus arteriosus (Figure 5C), which were
similar in distribution and morphology to the uorescent signals observed after DAF-FM
DA staining (Figure 5B). Surprisingly, TEM pictures of the 5 dpf zebrash bulbus (Figure
5D) revealed only weak evidence of elastin bers, suggesting that the uorescent signal
may reveal components associated with the elastin-rich extracellular matrix.
Figure 5. Demonstration of elastic bers in the bulbus arteriosus. (A). Semithin Toluidine blue-
stained cross section of the bulbus arteriosus in a 5 dpf zebrash. (B). Section of the bulbus after live
staining of a 5 dpf zebrash with DAF-FM DA. (C). Elastin staining of the bulbus according to an
adapted Verhoe’s stain. (D). TEM image of the bulbus of a 5 dpf zebrash. Arrows indicate the
ECM (elastin bers in (C)). Scale bars in (A–C) = 20 µm, in (D) = 1 µm.
NO is assumed to play a role in bone cell metabolism [22]. To assess whether the
surprising absence of staining in the osteoblasts is a failure due to technical or chemical
limitations, we applied TRIM, an inhibitor of all NOS enzymes that catalyze the
production of NO, and subsequently stained the treated embryos with DAF-FM DA. Live
Figure 5.
Demonstration of elastic fibers in the bulbus arteriosus. (
A
). Semithin Toluidine blue-stained
cross section of the bulbus arteriosus in a 5 dpf zebrafish. (
B
). Section of the bulbus after live staining
of a 5 dpf zebrafish with DAF-FM DA. (
C
). Elastin staining of the bulbus according to an adapted
Verhoeff’s stain. (
D
). TEM image of the bulbus of a 5 dpf zebrafish. Arrows indicate the ECM (elastin
fibers in (C)). Scale bars in (A–C) = 20 µm, in (D)=1µm.
NO is assumed to play a role in bone cell metabolism [
22
]. To assess whether the
surprising absence of staining in the osteoblasts is a failure due to technical or chemical
limitations, we applied TRIM, an inhibitor of all NOS enzymes that catalyze the pro-
duction of NO, and subsequently stained the treated embryos with DAF-FM DA. Live
observations from TRIM-treated and DAF-FM DA-stained embryos (Figure 6A,B), as well
as whole mount staining (Figure 6C,D) and sections prepared from TRIM-treated em-
bryos (
Figure 6E–H0
), clearly revealed that bone structures (opercular, cleithrum) were
absent (compare Figure 6E,E
0
with Figure 6G,G
0
for the opercular, and Figure 6F,F
0
with
Figure 6H,H0for the cleithrum).
Biomolecules 2023,13, 1780 9 of 14
Biomolecules 2023, 13, x FOR PEER REVIEW 9 of 14
observations from TRIM-treated and DAF-FM DA-stained embryos (Figure 6A,B), as well
as whole mount staining (Figure 6C,D) and sections prepared from TRIM-treated embryos
(Figure 6E–H′), clearly revealed that bone structures (opercular, cleithrum) were absent
(compare Figure 6E,E′ with Figure 6G,G′ for the opercular, and Figure 6F,F′ with Figure
6H,H′ for the cleithrum).
Figure 6. Absence of bone after inhibition of NO formation. (A,B). Live staining of 5 dpf zebrash
with DAF-FM DA for 3 h in the dark after 90 h of treatment with the NOS inhibitor TRIM (A) or its
solvens (0.1% DMSO) (B). Note the presence of opercular bone (arrow) and cleithrum (arrowhead)
in the control, which are clearly absent in the TRIM-treated specimen. (C,D). Alizarin red S whole
mount staining of TRIM-treated embryos likewise shows the complete absence of bones (C)
compared to their presence in control embryos (D). Arrow: opercular bone; arrowhead: cleithrum.
(E–H′). Semithin sections at comparative cross-sectional levels show the absence of opercular bone
and cleithrum ((E,F), and higher magnication in (E′,F′)) compared to their distinct presence in
control embryos ((G,H), and their higher magnication in (G′,H′)). Opercular bone: arrow;
cleithrum: arrowhead; asterisks indicate the location of absent bones. Scale bars in (A,B) = 200 µm,
in (E–H) = 100 µm, in (E′–H′) = 50 µm.
This result supports the implication of NO production in bone formation and
challenges DAF-FM DA as a marker of intracellular NO production in osteoblasts.
Figure 6.
Absence of bone after inhibition of NO formation. (
A
,
B
). Live staining of 5 dpf zebrafish
with DAF-FM DA for 3 h in the dark after 90 h of treatment with the NOS inhibitor TRIM (
A
) or its
solvens (0.1% DMSO) (
B
). Note the presence of opercular bone (arrow) and cleithrum (arrowhead) in
the control, which are clearly absent in the TRIM-treated specimen. (
C
,
D
). Alizarin red S whole mount
staining of TRIM-treated embryos likewise shows the complete absence of bones (
C
) compared to their
presence in control embryos (
D
). Arrow: opercular bone; arrowhead: cleithrum. (
E
–
H0
). Semithin
sections at comparative cross-sectional levels show the absence of opercular bone and cleithrum
((
E
,
F
), and higher magnification in (
E0
,
F0
)) compared to their distinct presence in control embryos
((
G
,
H
), and their higher magnification in (
G0
,
H0
)). Opercular bone: arrow; cleithrum: arrowhead;
asterisks indicate the location of absent bones. Scale bars in (
A
,
B
) = 200
µ
m, in
(E–H) = 100 µm
, in
(E0–H0) = 50 µm.
This result supports the implication of NO production in bone formation and chal-
lenges DAF-FM DA as a marker of intracellular NO production in osteoblasts.
Biomolecules 2023,13, 1780 10 of 14
4. Discussion
A clear and consistent result of live staining zebrafish embryos with DAF-FM DA is
the strong signal in the extracellular matrix, whether from the bone or from teeth (dentine,
bone of attachment). The lack of signal in bone-related cells is especially surprising,
given the regulatory role of NO in bone formation [
5
–
9
,
22
], as supported by our inhibition
experiments. To explain this rather puzzling result, we are hindered by the fact that staining
must be carried out on live specimens. This limits the use of relevant controls prior to or
during staining, such as decalcification or enzymatic digestion of extracellular components.
On the other hand, the results may not be so surprising given that the vast majority of
studies that have utilized DAF-FM DA to demonstrate NO have been performed on isolated
cells in culture or on organ fragments, i.e., in vitro or ex vivo (e.g., [23–25]).
A few studies have reported on the live staining of early postembryonic zebrafish using
DAF-FM DA. Although some of these papers used DAF-FM DA specifically for skeletal
studies [
13
,
14
], they mostly refrained from attributing the signals directly to osteoblasts
and referred to the signals as issuing from ‘forming bones’ [
12
] or ‘ossified structures’ [
13
].
Here, we show that the compound, which was developed to reveal intracellular
NO [
11
], is exclusively labeling the extracellular matrix. Renn et al. [
13
] noticed already
that, at 6 dpf, the signal labeled the opercular bone but not the osteoblasts lining the
bone and attributed this to an altered NO production status of the cells. In the present
study, we show that the lack of DAF-FM DA signal in the osteoblasts is not limited to later
postembryonic stages; rather, it is the matrix that is labeled throughout early postembryonic
development. Our experiments targeting the inhibition of NO production using TRIM
nevertheless clearly show the absence of bone structures and, thus, are a strong indication
of the role of NO in bone formation.
Different explanations can be proposed for the lack of intracellular staining of the
osteoblasts. One possibility is that in an
in vivo
context, NO diffuses out of osteoblasts
within seconds after its production and reacts with DAF-FM DA even before this is able to
penetrate into the cells. Under physiological conditions, NO, as an uncharged molecule,
is readily diffusible, with a range of approximately 150–300
µ
m for a time of 4–15 s. This
distance does not correspond to a straight-line trajectory (since the process is random) but
rather the radius of a sphere [
26
]. Under certain conditions, the half-life of NO can reach
10 min in a solution [
27
]. The above scenario raises the question of where the conversion
occurs into the triazole form (which is necessary to produce a strongly fluorescent molecule);
does this occur in the extracellular space or within the bone matrix itself? The second option
requires DAF-FM DA to have spread within the matrix before NO. In a recent study, where
anosteocytic (medaka) and osteocytic (zebrafish) bone was compared, it was observed that
water can easily diffuse and exchange within the extracellular matrix in teleost bones, and
more so in medaka than in zebrafish [
28
]. These results are relevant since the bones in
early postembryonic zebrafish are virtually anosteocytic [
29
], making them more similar
to medaka bone. Thus, one may hypothesize an easy access of intercollagenous spaces
for diffusion. Still, this scenario also requires that the diacetate in DAF-FM DA does not
interfere with the formation of a triazole. The observation that no staining is ever observed
in the extracellular space surrounding the bone suggests that the reactions turning DAF-FM
DA into a fluorescent derivative take place within the matrix itself.
The weak effect on staining after the use of the NO scavenger c-PTIO, in contrast with
the dramatic loss of staining after the use of the NOS inhibitor TRIM, can be explained
by the different chemical actions of both compounds: c-PTIO oxidizes the NO that has
been produced; TRIM blocks the enzymes necessary for NO production. In addition,
many pitfalls have been identified regarding the use of c-PTIO, at least in plants [
30
]. For
example, Arita et al. [
31
] showed that, contrary to the widely held presumption, the NO
scavenger c-PTIO does not suppress but actually enhances the conversion of DAF into the
DAF-2T fluorophore.
It is important to point out that the conversion from DAF-FM to its triazole product is
probably not direct. DAF-FM is weakly fluorescent and is likely to be first non-specifically
Biomolecules 2023,13, 1780 11 of 14
oxidized to an anilinyl radical, which then reacts with NO to form the fluorescent triazole
product [
32
]. It was reported that DAF-2 rapidly reacts with dehydroascorbic acid (DHA)
under physiologically relevant conditions to generate compounds (DAF-2-DHAs) that have
fluorescence emission profiles similar to that of DAF-2 triazole (DAF-2T) [
33
]. Likewise,
Wardman [
32
] describes how DAF-2 can be non-specifically oxidized to yield a fluorescent
molecule. Whether the same is true for DAF-FM, which is a DAF-4 derivative, is unclear
to us. Moreover, according to sources cited in [
34
], DAF-2 reacts with peroxynitrite rather
than nitric oxide.
An entirely different explanation for the fluorescent signal in the bone and dentine
matrix may relate to their calcium content. It has been both reported and rebutted that DAF-
2 is sensitive to the presence of divalent cations, especially calcium, in the medium [
34
].
However, our observations that DAF-FM DA stains small, not-yet mineralized bones, as
well as the osteoid layer around mineralized bones, argue against the calcium sensitivity
of DAF-FM DA. Likewise, Renn et al. [
13
] observed that DAF-FM DA labels the non-
mineralized osteoid of the early cleithrum.
In the bulbus arteriosus, the staining is also extracellular, as revealed by DAPI staining,
and is, thus, excluded both from endothelial and smooth muscle cells. Instead, the intensive
staining with DAF-FM DA matches the distribution of elastic fibers, as demonstrated by Ver-
hoeff’s staining, although direct superimposition with the fluorescent signal is not possible
using this technique. Ultrastructural observations suggest that the signal might derive from
elastin-associated material rather than from elastin fibers proper. The bulbus arteriosus in
teleosts is known to have a multilayered structure, with the largest, middle layer containing
abundant elastic fibers [
35
,
36
]. In sticklebacks, the elastic fibers are reported to be 15 nm
in diameter, associated with amorphous material and completely fill the space between
the adjacent rows of smooth muscle cells [
35
]. Interestingly,
Rodriguez et al.
[
37
] already
reported the strong staining of rat aortic tissue with DAF-2T DA. Similar to our conclusion
that the signal is located in the elastic fiber-rich extracellular space, they suggested that the
emission of DAF-2 T from aortic tissue originates predominantly from the elastic laminae.
They also proposed several mechanisms that could account for the localization of this
signal, drawn from earlier studies, such as enhanced N
2
O
3
production occurring within the
hydrophobic environment of the lamina [
37
]. Alternatively, it may reflect the preferential
accumulation of the fluorophore within elastic fibers. This explanation is supported by
the reported interaction between fluorescein and collagen, suggesting that this type of
fluorophore binds strongly to these proteins [
37
]. According to McCarthy [
38
], reactive
dyes such as the succinimidyl ester of carboxyfluorescein diacetate [CFDA] bind covalently
and predominantly to proteins.
Finally, sections show that in the notochord, the NO signal is clearly limited to the
notochord sheath and does not label the cytoplasm of the vacuolated cells [
12
]. The
notochord sheath is an extracellular matrix containing collagens and elastin (reviewed
in [39]).
Interestingly, the bone and dentine matrix, notochord sheath, and bulbus elastic
laminae are not the only extracellular compartments that supposedly reveal the presence
of NO. NO is also found in saliva, plasma, and blood [
40
]. Lundberg et al. [
41
] observed
elevated NO levels in the gastric lumen, probably resulting, in the authors’ view, from non-
enzymatic NO production and requiring an acidic environment. The latter was justified by
the observation that NO in expelled air was reduced by 95% after pretreatment with the
proton pump inhibitor omeprazole. The substrate for intragastric NO formation is probably
nitrite, given that nitrite is reduced in an acidic environment, thus forming NO [
42
]. More
recently, Wang et al. [
43
] confirmed that the reaction of DAF-FM DA with nitrite in an
acidic medium results in the formation of triazolofluorescein (DAF-FM T). A reaction with
nitrite in the bone matrix of zebrafish embryos is nevertheless unlikely based on the pH of
bone matrix (for rodent calvariae, around pH 7.4 [
44
]) as well as on the toxicity of nitrite.
Moreover, NO would still be the most likely source of nitrite.
Biomolecules 2023,13, 1780 12 of 14
5. Conclusions
In conclusion, the fluorescence patterns observed when using DAF-FM DA as a live
stain for zebrafish embryos may not necessarily correlate with local NO production, nor
do these patterns exclude the concept that cells responsible for the deposition of this
matrix can produce NO. Indeed, the inhibition of NO production at early embryonic stages
completely abolishes all bone formation, suggesting an important role for NO in osteoblast
function. However, DAF-FM DA is not the appropriate technique to demonstrate NO
production in osteoblasts, chordoblasts, or smooth muscle cells in the
in vivo
context of
zebrafish embryos. Additional controls must be carried out; not least, the analysis of
these signals should be performed at a sufficient resolution to assert whether the signal
is found intra- or extracellularly. Irrespective of these caveats, the compound can be used
in zebrafish research to visualize specific anatomical structures, including the notochord,
the bone matrix in early ossification centers, the teeth, and the elastic fibers in the bulbus
arteriosus. Because many studies that use zebrafish to model human bone diseases rely
on demonstrating mineralized structures with Alizarin red S, DAF-FM DA staining can
become an easy-to-perform and quick evaluation method to assess the presence of the
earliest osteoid. Likewise, DAF-FM DA staining could be a valuable tool for the live
imaging of heart function.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/biom13121780/s1, Figure S1. Semithin cross sections through 5 dpf
zebrafish live stained with DAF-FM DA showing labeling in the teeth. A: overview section after
120 min of staining. B, C: magnifications of teeth after 20 min (B) and 3 h of staining (C). Teeth are
indicated by arrows, and the attachment pedicels by arrowheads. Note also staining of the bone
around the tooth-bearing ceratobranchial 5 (cb) as well as of the notochord sheath (ns). Scale bar
in
A = 50 µm
, in B & C = 20
µ
m. Figure S2. Live staining of 5 dpf zebrafish with DAF-FM DA for
very short intervals. Live imaging of the head region at the time indicated on the left. The bulbus
arteriosus is visible already after 3 min, and the opercular bone after 5 min. Scale bars = 200
µ
m.
Figure S3. Staining with DAF-FM DA after fixation of 8 dpf zebrafish. A–C: 3 h staining with DAF-FM
DA after fixation as indicated. D: 3 h treatment of DMSO 0.1% after fixation in 4% PFA (compare
with A). All images were taken strictly under the same illumination. Scale bars = 1 mm.
Author Contributions:
Conceptualization, A.H., S.V.P. and P.E.W.; methodology, A.H., U.G.L.,
C.L.M., D.L., S.V.P. and P.E.W.; formal analysis, A.H., U.G.L., D.L. and P.E.W.; writing—original draft
preparation, A.H. and U.G.L.; writing—review and editing, A.H., M.M. and P.E.W.; visualization,
A.H., D.L., C.L.M. and P.E.W.; funding acquisition, A.H., M.M. and P.E.W. All authors have read and
agreed to the published version of the manuscript.
Funding:
AH was funded by the Czech Science Foundation, GACR n
◦
22-25061S. C.L.M. was
supported by Aarhus University Graduate School. S.V.P. was funded by a grant from Aarhus
University Research Foundation (AUFF-E-2021-9-17). M.M. and P.E.W. were funded by the European
Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie
grant agreement No 766347. M.M. is a Maître de Recherche at the FNRS. P.E.W. acknowledges
Ghent University for funding the International Network on Ectopic Calcification (INTEC–https://www.
itnintec.com (accessed on 22 October 2023).
Institutional Review Board Statement:
Ethical review and approval were waived for this study due
to the use of developmental stages prior to the stage of independently feeding the larval form in
accordance with the EU Directive. 2010/63/EU on the protection of animals used for scientific purposes.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The sections pictured in this paper are kept in the slide collection of
the Research Group Evolutionary Developmental Biology of Ghent University and are available for
inspection upon request.
Acknowledgments: The technical help provided by M. Soenens is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
Biomolecules 2023,13, 1780 13 of 14
References
1. Lundberg, J.O.; Weitzberg, E. Nitric oxide signaling in health and disease. Cell 2022,185, 2853–2878. [CrossRef]
2.
Piacenza, L.; Zeida, A.; Trujillo, M.; Radi, R. The superoxide radical switch in the biology of nitric oxide and peroxynitrite. Physiol.
Rev. 2022,102, 1881–1906. [CrossRef] [PubMed]
3.
Brandi, M.L.; Hukkanen, M.; Umeda, T.; Moradi-Bidhendi, N.; Bianchi, S.; Gross, S.S.; Polak, J.M.; MacIntyre, I. Bidirectional
regulation of osteoclast function by nitric oxide synthase isoforms. Proc. Natl. Acad. Sci. USA
1995
,92, 2954–2958. [CrossRef]
[PubMed]
4.
Helfrich, M.H.; Evans, D.E.; Grabowski, P.S.; Pollock, J.S.; Ohshima, H.; Ralston, S.H. Expression of nitric oxide synthase isoforms
in bone and bone cell cultures. J. Bone Miner. Res. 1997,12, 1108–1115. [CrossRef] [PubMed]
5. Evans, D.M.; Ralston, S.H. Nitric oxide and bone. J. Bone Miner. Res. 1996,11, 300–305. [CrossRef] [PubMed]
6.
Burger, E.H.; Klein-Nulend, J.; Smit, T.H. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodeling
osteon—A proposal. J. Biomech. 2003,36, 1453–1459. [CrossRef] [PubMed]
7.
Saura, M.; Tarin, C.; Zaragoza, C. Recent insights into the implication of nitric oxide in osteoblast differentiation and proliferation
during bone development. Sci. World J. 2010,10, 624–632. [CrossRef] [PubMed]
8. Moharrer, Y.; Boerckel, J.D. Tunnels in the rock: Dynamics of osteocyte morphogenesis. Bone 2021,153, 116104. [CrossRef]
9.
Yan, T.; Xie, Y.; He, H.; Fan, W.; Huang, F. Role of nitric oxide in orthodontic tooth movement. Int. J. Molec. Med.
2021
,48, 168.
[CrossRef]
10.
Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Detection and imaging of
nitric oxide with novel fluorescent indicators: Diaminofluoresceins. Anal. Chem. 1998,70, 2446–2453. [CrossRef]
11.
Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Hirata, Y.; Nagano, T. Fluorescent indicators for imaging nitric oxide production.
Angew. Chem. Int. Ed. 1999,38, 3209–3212. [CrossRef]
12.
Lepiller, S.; Laurens, V.; Bouchot, A.; Herbomel, P.; Solary, E.; Chlub, J. Imaging of nitric oxide in a living vertebrate using a
diaminofluorescein probe. Free Radic. Biol. Med. 2007,43, 619–627. [CrossRef] [PubMed]
13.
Renn, J.; Pruvot, B.; Muller, M. Detection of nitric oxide by diaminofluorescein visualizes the skeleton in living zebrafish. J. Appl.
Ichthyol. 2014,30, 701–706. [CrossRef]
14.
Windhausen, T.; Squifflet, S.; Renn, J.; Muller, M. BMP signaling regulates bone morphogenesis in zebrafish through promoting
osteoblast function as assessed by their nitric oxide production. Molecules 2015,20, 7586–7601. [CrossRef] [PubMed]
15.
Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th ed.; University of Oregon Press:
Eugene, ON, USA, 2000.
16.
Strähle, U.; Scholz, S.; Geisler, R.; Greiner, P.; Hollert, H.; Rastegar, S.; Schumacher, A.; Selderslaghs, I.; Weiss, C.; Witters, H.; et al.
Zebrafish embryos as an alternative to animal experiments—A commentary on the definition of the onset of protected life stages
in animal welfare regulations. Reprod. Toxicol. 2012,33, 128–132. [CrossRef] [PubMed]
17.
Annona, G.; Sato, I.; Pascual-Anaya, J.; Osca, D.; Braasch, I.; Voss, R.; Stundl, J.; Soukup, V.; Ferrara, A.; Fontenot, Q.; et al.
Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development. Proc. R. Soc. B
2022
,
289, 20220667. [CrossRef]
18.
Oralová, V.; Rosa, J.T.; Soenens, M.; Bek, J.W.; Willaert, A.; Witten, P.E.; Huysseune, A. Beyond the whole-mount phenotype:
High-resolution imaging in fluorescence-based applications on zebrafish. Biol. Open 2019,8, bio042374. [CrossRef] [PubMed]
19. Sheehan, D.; Hrapchak, B. Theory and Practice of Histotechnology, 2nd ed.; Battelle Press: Columbus, OH, USA, 1987; p. 481.
20.
Presnell, J.K.; Schreibman, M.P. Humason’s Animal Tissue Techniques, 5th ed.; The Johns Hopkins University Press: Baltimore, MD,
USA, 1997; p. 600.
21.
Huysseune, A.; Soenens, M.; Sire, J.-Y.; Witten, P.E. High resolution histology for craniofacial studies on zebrafish and other
teleost models. Meth. Molec. Biol. 2022,2403, 249–262.
22. Hauschka, P.V.; Damoulis, P.D. Functions of nitric oxide in bone. Biochem. Soc. Trans. 1998,26, 39–44. [CrossRef]
23.
Li, Y.; Tan, Y.; Yang, B.; Zhang, G.; Xiao, X.; Zheng, L. Effect of calcitonin gene-related peptide on nitric oxide production in
osteoblasts: An experimental study. Cell Biol. Int. 2011,35, 757–765. [CrossRef]
24.
Suh, K.S.; Chon, S.; Choi, E.M. Limonene protects osteoblasts against methylglyoxal-derived adduct formation by regulating
glyoxalase, oxidative stress, and mitochondrial function. Chem. Biol. Interact. 2017,278, 15–21. [CrossRef] [PubMed]
25.
Jin, Z.; Kho, J.; Dawson, B.; Jiang, M.-M.; Chen, Y.; Ali, S.; Burrage, L.C.; Grover, M.; Palmer, D.J.; Turner, D.L.; et al. Nitric oxide
modulates bone anabolism through regulation of osteoblast glycolysis and differentiation. J Clin. Investig.
2021
,131, e138935.
[CrossRef]
26.
Lancaster, J.R. A tutorial on the diffusibility and reactivity of free nitric oxide. Nitric Oxide—Biol. Chem.
1997
,1, 18–30. [CrossRef]
[PubMed]
27.
Hilderbrand, S.A.; Lim, M.H.; Lippard, S.J. Fluorescence-based nitric oxide detection. In Topics in Fluorescence Spectroscopy;
Geddes, C.D., Lakowicz, J.R., Eds.; Springer: Boston, MA, USA, 2005; Volume 9, pp. 163–188.
28.
Silveira, A.; Kardjilov, N.; Markötte, H.; Longo, E.; Grevin, I.; Lasch, P.; Shahar, R.; Zaslansky, P. Water flow through bone: Neutron
tomography reveals differences in water permeability between osteocytic and anosteocytic bone material. Mater. Des.
2022
,
224, 111275. [CrossRef]
29. Huysseune, A. Skeletal system. In The Laboratory Fish; Ostrander, G., Ed.; Academic Press: London, UK, 2000; pp. 307–317.
Biomolecules 2023,13, 1780 14 of 14
30.
D’Alessandro, S.; Posocco, B.; Costa, A.; Zahariou, G.; Lo Schiavo, F.; Carbonera, D.; Zottini, M. Limits in the use of cPTIO as
nitric oxide scavenger and EPR probe in plant cells and seedlings. Front. Plant Sci. 2013,4, 340. [CrossRef]
31.
Arita, N.O.; Cohen, M.F.; Tokuda, G.; Yamasaki, H. Fluorometric detection of nitric oxide with diaminofluoresceins (DAFs):
Applications and limitations for plant NO research. Plant Cell Monogr. 2006,5, 269–280. [CrossRef]
32.
Wardman, P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues:
Progress, pitfalls, and prospects. Free Radic. Biol. Med. 2007,43, 995–1022. [CrossRef]
33.
Kim, W.-S.; Ye, X.; Rubakhin, S.S.; Sweedler, J.V. Measuring nitric oxide in single neurons by capillary electrophoresis with
laser-induced fluorescence: Use of ascorbate oxidase in diaminofluorescein measurements. Anal. Chem.
2006
,78, 1859–1865.
[CrossRef]
34.
Balcerczyk, A.; Soszynski, M.; Bartosz, G. On the specificity of 4-amino-5-methylamino-2
0
,7
0
-difluorofluorescein as a probe for
nitric oxide. Free Radic. Biol. Med. 2005,39, 327–335. [CrossRef]
35.
Benjamin, M.; Norman, D.; Santer, R.M.; Scarborough, D. Histological, histochemical and ultrastructural studies on the bulbus
arteriosus of the sticklebacks, Gasterosteus aculeatus and Pungitius pungitius (Pisces: Teleostei). J. Zool.
1983
,200, 325–346.
[CrossRef]
36.
Icardo, J.M. The teleost heart: A morphological approach. In Ontogeny and Phylogeny of the Vertebrate Heart; Sedmera, D., Wang, T.,
Eds.; Springer: New York, NY, USA, 2012; pp. 35–53. [CrossRef]
37.
Rodriguez, J.; Specian, V.; Maloney, R.; Jourd’heuil, D.; Feelisch, M. Performance of diamino fluorophores for the localization of
sources and targets of nitric oxide. Free Radic. Biol. Med. 2005,38, 356–368. [CrossRef] [PubMed]
38.
McCarthy, D.A. Fluorochromes and fluorescence. In Flow Cytometry: Principles and Applications; Macey, M.G., Ed.; Humana Press
Inc.: Totowa, NJ, USA, 2007; pp. 59–112.
39.
Witten, P.E.; Hall, B.K. The Notochord. Development, Evolution and Contributions to the Vertebral Column; CRC Press: Boca Raton, FL,
USA, 2022; pp. 1–250.
40.
Lundberg, J.O.; Weitzberg, E.; Gladwin, M.T. The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev.
Drug Discov. 2008,7, 156–167. [CrossRef] [PubMed]
41.
Lundberg, J.O.N.; Weitzberg, E.; Lundberg, J.M.; Alving, K. Intragastric nitric oxide production in humans: Measurements in
expelled air. Gut 1994,35, 1543–1546. [CrossRef] [PubMed]
42. Archer, S. Measurement of nitric oxide in biological models. FASEB J. 1993,7, 349–360. [CrossRef]
43.
Wang, Q.; Huang, H.; Ning, B.; Li, M.; He, L. A Highly sensitive and selective spectrofluorimetric method for the determination
of nitrite in food products. Food Anal. Methods 2016,9, 1293–1300. [CrossRef]
44.
Neuman, M.W.; Neuman, W.F. On the measurement of water compartments, pH, and gradients in calvaria. Calcif. Tissue Int.
1980
,
31, 135–145. [CrossRef]
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