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Accepted Manuscript
Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like
(cart-like) gene during zebrafish embryogenesis
Atsuo Kawahara, Hitoshi Morita, Kanoko Yanagi, Hiroaki Suzuki, Takaaki Mori, Rie
Ohga, Kiyohito Taimatsu
PII:
S1567-133X(18)30068-1
DOI:
10.1016/j.gep.2018.08.002
Reference:
MODGEP 19031
To appear in:
Gene Expression Patterns
Received Date: 11 April 2018
Revised Date:
28 June 2018
Accepted Date: 15 August 2018
Please cite this article as: Kawahara, A., Morita, H., Yanagi, K., Suzuki, H., Mori, T., Ohga, R., Taimatsu,
K., Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like (cart-like) gene
during zebrafish embryogenesis, Gene Expression Patterns (2018), doi: 10.1016/j.gep.2018.08.002.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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Spatiotemporal
expression
of
the
cocaine-
and
amphetamine-regulated
transcript-like (cart-like) gene during zebrafish embryogenesis
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Atsuo Kawahara*, Hitoshi Morita, Kanoko Yanagi, Hiroaki Suzuki, Takaaki Mori, Rie
Ohga, Kiyohito Taimatsu
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Laboratory for Developmental Biology, Center for Medical Education and Sciences,
Yamanashi, 409-3989, Japan
* Corresponding author
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Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo,
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E-mail: akawahara@yamanashi.ac.jp (A. Kawahara)
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Abstract
The cocaine- and amphetamine-regulated transcript (CART) genes are involved in the
neural regulation of energy homeostasis; however, their developmental expressions and
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functions are not fully understood in vertebrates. We have identified a novel zebrafish
cart-like gene that encodes a protein of 105 amino acids possessing sequence similarity
to zebrafish and mammalian CART proteins. RT-PCR analysis revealed that the
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cart-like transcripts were maternally supplied and gradually decreased during the
cleavage, blastula and gastrula stages; then, transcripts subsequently reaccumulated at
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the segmentation, pharyngula and hatching stages. Based on a whole-mount in situ
hybridization analysis using an antisense cart-like RNA probe, we found that the
cart-like transcript was predominantly expressed in both the Rohon-Beard neurons and
trigeminal ganglia, suggesting the involvement of the cart-like gene in zebrafish neural
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development.
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Keywords: cart-like, cart1, cart2, Rohon-Beard neuron, trigeminal ganglia, zebrafish
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1. Introduction
CART (cocaine- and amphetamine-regulated transcript) mRNA was identified as a
specific mRNA upregulated by acute administration of cocaine or amphetamine (Kuhar
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et al., 1999). The CART gene is highly conserved among vertebrates, and the
posttranslational processing of the CART protein generates a biologically active
neuropeptide. The CART peptide possesses six cysteine residues in the carboxy
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terminus that are strictly conserved among vertebrates (Kehoe et al., 2007), suggesting
that a similar tertiary conformation is formed by disulfide bounds.
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The expression of CART mRNA has been detected in the hypothalamus and implicated
in the control of feeding behavior. Interestingly, injection of the CART peptide
intracerebroventricularly into rats causes an inhibition of feeding (Kristensen et al.,
1998). In contrast, injection of a CART antibody stimulates feeding in rats (Lambert et
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al., 1998). The expression level of the CART transcript is decreased in some
hypothalamic areas in leptin-deficient mice. Injection of leptin into these
leptin-deficient mice results in an accumulation of CART mRNA in the lateral
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hypothalamus (Kristensen et al., 1998). Thus, these findings suggest that CART peptide
is involved in the control of feeding behavior.
a
whole-mount
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During
in
situ
hybridization
(WISH)-based
screening
of
uncharacterized zebrafish genes containing short reading frames, we identified a novel
gene, the cocaine- and amphetamine-regulated transcript-like (cart-like) gene. We
found that the cart-like gene has sequence similarity to zebrafish cart genes and exhibits
unique expression profiles during zebrafish neurogenesis as described below.
2. Results
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2.1 A novel cart-like gene in zebrafish
The zebrafish cart-like gene encodes 105 amino acids and possesses amino acid
sequence similarity to zebrafish Cart proteins (Fig. 1A) (Akash et al., 2014). Zebrafish
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Cart1 and Cart2, compared to zebrafish Cart-like, are highly homologous to medaka
CART, frog CART, chick CART and mammalian CART protein (Fig. 1A and B)
(Mckinzie, 1995; Kuhar et al., 1999; Roubos et al., 2008; Murashita et al., 2011; Gai et
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al., 2015; Nishio et al., 2018). The cysteine residues presumably involved in disulfide
bonding are completely conserved between the Cart-like and CART proteins (Rogge et
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al., 2008), whereas potential proteolytic cleavage sites (KR or KK) processed by
prohormone convertases in CART proteins are not perfectly matched in the Cart-like
protein (Fig. 1A). A phylogenetic tree analysis indicates that Cart-like is diverged from
mammalian CART proteins (Fig. 1B), suggesting that the cart-like gene encodes a novel
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Cart family protein. CART transcripts in mammals are detected in the hypothalamus
(Kristensen et al., 1998), whereas the zebrafish cart3 gene is expressed in the
hypothalamus at 24 hours post-fertilization (hpf) (Nishio et al., 2018), suggesting the
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importance of CART genes in neuroendocrine functions. Therefore, we examined the
developmental expression pattern of this novel cart-like gene during zebrafish early
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embryogenesis.
2.2 Temporal expression of the cart-like transcript during zebrafish embryogenesis
Total RNA was isolated from embryos at different developmental stages and the
expression of cart-like was examined by reverse-transcriptase PCR (Fig. 2). The
cart-like transcript was weakly detected in 2-cell stage embryos, and it gradually
decreased in the cleavage, blastula and gastrula stages (Fig. 2A). cart-like gene
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expression was detected in the late segmentation and pharyngula and hatching stages,
while the β-actin transcript level gradually increased during zebrafish embryogenesis.
The temporal expression pattern of cart-like was also confirmed by real-time PCR
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analysis (Fig. 2B), suggesting a possible role for cart-like gene regulation during
zebrafish embryogenesis.
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2.3 Developmental expression of the cart-like gene in zebrafish neurogenesis
We performed WISH with embryos at different developmental stages to examine the
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distribution of the cart-like transcript during zebrafish embryogenesis. Consistent with
the results of the reverse-transcriptase PCR and real-time PCR (Fig. 2), the cart-like
transcript was detected at the 2-cell stage (Fig. 3A), and the expression of cart-like in
the blastomeres was weak or marginal at the dome, shield, bud, 5-somite and 10-somite
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stages (Fig. 3B-E). cart-like gene expression was initially induced in a small population
of neural tissues (Fig. 3F). At the 15-somite stage, the number of cells and intensity of
cart-like expression in prospective Rohon-Beard (RB) neurons were increased (Fig. 3G:
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arrowheads), and cart-like expression was detected in prospective trigeminal ganglia
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(Fig. 3G: asterisk). The expression of cart-like in prospective RB neurons and
trigeminal ganglia was maintained at the 20-somite, 24 hpf, 48 hpf and 72 hpf stages
(Fig. 3H-K). Uemura et al. have reported that zCREST2 (the zebrafish conserved
regulatory element of the islet-1 gene 2) enhancer is sufficient to drive GFP expression
in the RB neurons, trigeminal ganglia and notochord (Uemura et al., 2005). In fact, GFP
expression was specifically detected in the RB neurons, trigeminal ganglia and
notochord of Tg(zCREST2-hsp70:GFP)rw011 embryos (Fig. 4A and B). WISH analysis
using the antisense cart-like probe showed that cart-like expression was largely merged
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with GFP expression in the RB neurons and trigeminal ganglia, but not in the notochord.
Furthermore, cart-like gene expression at the RB neurons in the neural tube was
observed in trunk transverse sections of embryos stained with an antisense cart-like
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probe (Fig. 5A), while cart-like expression in the trigeminal ganglia was confirmed in
head horizontal sections (Fig. 5B). Thus, the cart-like gene is predominantly expressed
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in the RB neurons and trigeminal ganglia during zebrafish embryogenesis.
3. Discussion
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We have demonstrated the molecular structure of a novel cart-like gene that has
sequence similarity to zebrafish cart and mammalian CART genes (Fig. 1A and B). Six
cysteine residues of the Cart-like protein are conserved in zebrafish Cart and
mammalian CART proteins, but the potential proteolytic cleavage sites (KR or KK) of
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Crat-like only partially match those of CART proteins. Phylogenetic tree analysis also
suggests that Cart-like is diverged from mammalian CART proteins, suggesting that
zebrafish Cart-like is a unique member of the CART family of proteins.
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Both reverse-transcriptase PCR and real-time PCR demonstrated that cart-like mRNA
is maternally supplied (Fig. 2A and B). The cart-like transcript gradually decreased
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during the blastula and gastrula stages and was detected in the late segmentation,
pharyngula and hatching stages. WISH analysis revealed that the cart-like is
predominantly expressed in the RB neurons and trigeminal ganglia (Figs. 3-5). These
expression results are quite unique, because this expression pattern has not been
observed for any CART genes in mammals or fish (Kristensen et al., 1998; Subhedar et
al., 2011; Akash et al., 2014). In fact, zebrafish cart1 is detected in the diencephalon at
72 hpf, while cart2 is expressed in the hindbrain at 36 hpf and in the medulla oblongata
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at 48 hpf (Nishio et al., 2018). The expression of cart3 is detected in the hypothalamus
and hindbrain at 36 hpf. The cart-like expression domains do not overlap the expression
domains of these cart genes; therefore, it is important to clarify the contribution of
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cart-like gene to the formation of RB neurons and trigeminal ganglia. In mammals, the
CART transcript is expressed in the hypothalamus, which regulates various metabolic
processes, including energy homeostasis (Koylu et al., 1997). Interestingly,
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intracerebroventricular injections of the CART protein in rodents decreases food intake,
whereas central administration of a CART antibody stimulates feeding (Kristensen et al.,
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1998; Lambert et al., 1998), demonstrating a function for CART proteins in energy
homeostasis. Therefore, the developmental expression profiles of the cart-like transcript
will contribute to a better understanding of the physiological functions of the cart-like
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gene in zebrafish neurogenesis.
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4. Experimental procedures
4.1 Ethics statement
All animal experiments were performed in accordance with the animal protocol
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approved by the Institutional Animal Care and Use Committee (IACUC) of the
University of Yamanashi. The IACUC of the University of Yamanashi approved this
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study (Approval Identification Number: A25-28).
4.2 Isolation of zebrafish cart-like
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The cart-like gene was isolated from a zebrafish cDNA library from 1 day
post-fertilization (1 dpf) by PCR amplification using the following specific primers;
cart-like-BamHI-F,
5’-CGGGATCCACCATGACGAGCTCTGAGATGC-3’
and
cart-like-XbaI-R, 5’-GCTCTAGATCAGATGCATTTTAAGAGAA-3’. The amplified
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cart-like gene fragment was digested by BamHI and XbaI, and inserted into the
BamHI-XbaI-cleaved pCS2P vector. We deposited the cart-like gene in the DNA Data
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Bank of Japan (DDBJ); the accession number of cart-like is LC378415.
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4.3 Phylogenetic analysis
Protein sequence alignments were performed using ClustalW. A phylogenetic tree was
constructed based on the amino acid sequences with the neighbor-joining method and
500 bootstrap replicates using MEGA7 (Kumar et al., 2016) for tree building and
drawing.
4.4 RNA extraction, reverse-transcriptase PCR and real-time PCR
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Total RNA was isolated from embryos at different developmental stages using TRIzol
reagent (Thermo Fisher Scientific), and cDNA was synthesized using the SuperScript
IV First-Strand kit (Thermo Fisher Scientific). Reverse-transcriptase PCR was
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performed under the following conditions: 94 °C for 3 min and 33 cycles of 94 °C for
30 sec, 55 °C for 30 sec and 72 °C for 30 sec for cart-like; and 94 °C for 3 min and 25
cycles of 94 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec for β-actin. To amplify
cart-like
gene,
the
following
primers
5’-ACTCTTCACCGTCCTCTGC-3’
were
used:
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the
and
cart-like-F,
cart-like-R,
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5’-CAGTTTGATCCTCTGCCACA-3’. β-actin was amplified as the control using the
following primers: β-actin-F: 5’-CTGGCCTCCCTGTCCACCTT-3’ and β-actin-R:
5’-ACAGGTTGGCCCCACCAAAT-3’. The PCR products were resolved on a 1%
agarose gel with 0.5 µg/ml of ethidium bromide. For real-time PCR, primers
5’-AAGGCCAACAGGGAAAAGATG-3’,
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(β-actin-RT-F2:
β-actin-RT-R2:
5’-CAGCCTGGATGGCAACGTA-3’, cart-like-RT-F:
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5’-TGTCCCCGCAGGAAACA-3’, cart-like-RT-R:
5’-TGATATCTCTCCAAAAGTGCTGTCA-3’) and TaqMan probes (β-actin-FAM:
cart-like-FAM:
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Fam-ACACAGATCATGTTCGAGAC-MGB,
Fam-AATCAGCTGGTTGAGGCT-MGB) were purchased from Thermo Fisher
Scientific. Real-time PCR using StepOnePlus (Thermo Fisher Scientific) was performed
under the following conditions: 95 °C for 20 sec and 40 cycles of 95 °C for 1 sec and
60 °C for 20 sec for cart-like and β-actin. TaqMan assays of triplicate samples were
performed and analyzed by StepOne software. The data of β-actin were used for
normalizing.
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4.5 Whole-mount in situ hybridization (WISH)
WISH using zebrafish embryos was performed as described previously (Hanaoka et al.,
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2006; Kawahara et al., 2009). The digoxigenin (DIG)-labeled antisense cart-like probe
was synthesized using T7 RNA polymerase from the BamHI-cleaved pCS2P-cart-like
plasmid using an RNA labeling kit (Roche). Zebrafish embryos hybridized with the
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DIG-labeled RNA probe were incubated with alkaline phosphatase-conjugated anti-DIG
antibody. To visualize the RNA probe recognized by the anti-DIG antibody, the embryos
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were subsequently incubated with BM Purple (Roche) as the substrate. The color
reaction was terminated by washing the embryos with PBS containing Tween 20 (0.1%),
and the embryos were fixed in 4% paraformaldehyde.
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4.6 Transgenic zebrafish line
Tg(zCREST2-hsp70:GFP)rw011 was obtained from the National BioResource Project in
Japan. GFP expression was observed in the RB neurons, trigeminal ganglia and
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notochord (Uemura et al., 2005).
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4.7 Histological analysis
Embryos were dehydrated in 80% ethanol and embedded using the Technovit kit
(Kulzer). Embedded embryos were sectioned on a Leica RM2125 microtome at 4 µm
and mounted on slides. Embryos were stained with neutral red after sectioning.
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Acknowledgements
We would like to thank A. Nagase for maintaining the zebrafish. We also would like to
thank the National BioResource Project for providing Tg(zCREST2-hsp70:GFP)rw011.
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This work was supported in part by grants from the Ministry of Education, Science,
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Technology, Sports and Culture of Japan and by the Takeda Science Foundation (A.K.).
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Figure legend
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Fig. 1 Zebrafish cart-like gene encodes a novel Cart protein. (A) Multiple amino acid
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sequence alignment of Cart proteins. Amino acid sequences were aligned based on the
ClustalW program and conserved amino acids are highlighted in black. Black bars (KR
or KK) and asterisks are potential proteolytic cleavage sites processed by prohormone
convertases and conserved cysteine residues presumably involved in disulfide bonding,
respectively. (B) Phylogenetic tree of Cart proteins. The evolutionary distances were
computed using the p-distance method and are in units representing the number of
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amino acid differences per site. Evolutionary analyses were conducted in MEGA7
(Kumar et al., 2016). The accession numbers of the amino acid sequences used are as
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follows: human CART (CAG47012), mouse CART (NP_001074962), rat CART
(NP_058806), chick CART (AGG54990), Xenopus laevis CART (NP_001087565),
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medaka CART ch4 (BAJ39822), medaka CART ch3 (BAJ39821), zebrafish Cart1
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(ADB12484), zebrafish Cart2 (ADB12486), zebrafish Cart3 (GU057836) and zebrafish
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Cart-like (LC378415).
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Fig. 2 Temporal expression of cart-like transcripts during zebrafish embryogenesis. (A)
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Zebrafish cart-like and β-actin genes at the indicated stages were amplified by
reverse-transcriptase PCR. The upper panel exhibits the results of cart-like, whereas the
bottom panel represents the results of the β-actin control. Similar results were obtained
in three independent experiments. (B) Real-time PCR represents the temporal
expression of the cart-like gene relative to that of the β-actin gene. Relative
quantification maximum and relative quantification minimum are indicated as bars. 5S,
5-somite stage; 10S, 10-somite stage; 15S, 15-somite stage; 20S, 20-somite stage.
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Fig. 3 Spatiotemporal expression of the cart-like gene during zebrafish embryogenesis.
(A) 1-cell stage. (B) Dome stage. (C) Shield stage. (D) Bud stage. (E) 5-somite stage.
(F) 10-somite stage. (G) 15-somite stage. (H) 20-somite stage. (I) 24 hpf stage. (J) 48
hpf stage. (K) 72 hpf stage. All pictures show the lateral view. (D-K) Anterior to the left,
dorsal up. Arrowheads and asterisks represent prospective Rohon-Beard (RB) neurons
and trigeminal ganglia, respectively. Scale bars, 100 µm.
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Fig. 4 Rohon-Beard (RB) neurons and trigeminal ganglia expressing the cart-like gene.
(A, B) GFP expression at 24 hpf was detected in the RB neurons (arrowheads),
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trigeminal ganglia (asterisk) and notochord (arrows) of Tg(zCREST2-hsp70:GFP)rw011.
After recording GFP expression, a WISH analysis of individual embryos was performed
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using the DIG-labeled antisense cart-like probe. (C, D) The cart-like gene was detected
in the RB neurons and trigeminal ganglia, but not in the notochord. Scale bars, 100 µm.
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Fig. 5 Cross sections of neutral-red-stained embryos. (A) The trunk transverse section of
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the embryos stained with the antisense cart-like probe (arrowheads). The neural tube is
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indicated by a dotted line, whereas an asterisk indicates the position of the notochord.
(B) Head horizontal section of the embryos stained with the antisense cart-like probe
(arrows). Scale bars, 50 µm. Crosses and red arrow indicate the positions of the lenses
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and hindbrain ventricle, respectively.
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