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Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system
Accepted Manuscript Title: Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system Author: Eunmi Kim Inyoung Jeong Suhyun Kim Hwan-Ki Kim Dong-won Lee Boa Kim Jae Young Seong Young-Ki Bae Jae-Ho Ryu Hae-Chul Park PII: DOI: Reference:
S0304-3940(16)30434-7 http://dx.doi.org/doi:10.1016/j.neulet.2016.06.025 NSL 32113
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
31-3-2016 14-5-2016 9-6-2016
Please cite this article as: Eunmi Kim, Inyoung Jeong, Suhyun Kim, HwanKi Kim, Dong-won Lee, Boa Kim, Jae Young Seong, Young-Ki Bae, Jae-Ho Ryu, Hae-Chul Park, Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.06.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system
Eunmi Kim, Inyoung Jeong, Suhyun Kim, Hwan-Ki Kim, Dong-won Lee, Boa Kim, Jae Young Seong1, Young-Ki Bae2, Jae-Ho Ryu3 and Hae-Chul Park
Graduate School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea; 1Graduate School of Medicine, Korea University, Seoul 136-705, Republic of Korea; 2Comparative Biomedical Research Branch, Research Institute, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea; 3Genomic
Design Bioengineering Company, Daejeon 306-230, Republic of Korea.
The number of text pages : 19 The number of figures : 4
Correspondence to : Hae-Chul Park, Graduate School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea. Telephone: +82-31-412-6712 Fax: +82-31-412-6718 Email: [email protected].
1
Highlights •
A novel galanin receptor 2b (galr2b) is expressed in the zebrafish nervous system.
•
The galr2b:egfp transgenic zebrafish was generated to visualize galr2b neurons.
•
Distribution of galr2b neurons and their interaction with galanin was analyzed in the zebrafish central nervous system.
ABSTRACT Galanin is a multifunctional neuropeptide that is implicated in the modulation of physiological processes, including nociception, cognition, feeding behavior, neuronal growth, and reproduction. The physiological effects of galanin are mediated through its interaction with three different G protein-coupled receptors, i.e., GALR1, GALR2, and GALR3. Unlike mammals, zebrafish have four different receptors for galanin, diversified from GALR1 (GAL1a and GALR1b) and GALR2 (GALR2a and GALR2b). Despite the importance of galanin in the central nervous system (CNS), no information has been reported regarding GalR2 in zebrafish CNS. In this study, we found that galr2a is expressed at low levels in restricted areas of the brain; however, galr2b was widely expressed in CNS including olfactory bulb, midbrain tegmentum, preoptic region, dorsal thalamus, posterior tuberculum, postoptic commissure, hindbrain, and spinal cord. To further analyze the distribution of GALR2b neurons and their interaction with GAL, we generated Tg(galr2b:egfp) zebrafish, which express enhanced green fluorescent protein (EGFP) under the control of a galr2b promoter. Investigation of the CNS of transgenic reporter zebrafish revealed that galr2b:EGFP+ neurons are distributed and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may have the potential to interact with other ligands instead of galanin in these regions. 2
Key words: neuropeptide, galanin, galanin receptor, central nervous system, zebrafish
1. Introduction
Galanin (GAL) is a multifunctional neuropeptide that has a widespread distribution in the peripheral and central nervous system (CNS). Galanin consists of 29–30 amino acid sequences and was first isolated from porcine intestine [23]. In mammals, galanin has been implicated in the modulation of several different physiological processes including nociception [4], cognition [21], feeding behavior [1], neuronal growth [11] and reproduction [20]. Galanin has also been shown to play a role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress, as well as in the pathogenesis of pituitary adenomas [25, 27]. In mammals, the physiological effects of galanin are mediated through its interaction with three galanin receptor subtypes GALR1, GALR2, and GALR3, which are G proteincoupled receptors (GPCRs). GALR1 expression is largely restricted to the nervous system including ventral hippocampus, thalamic nuclei, amygdala, brainstem nuclei, hypothalamus, posterior horns of the spinal cord, and dorsal root ganglia [2, 8]. Unlike GALR1, GALR2 is widely distributed in almost all tissues, including the brain, such as the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2, 3, 5, 6, 14]. GALR3 showed limited expression in CNS but is relatively abundant in peripheral tissues [12]. In zebrafish, immunohistochemical analysis using the anti-Galanin antibody—a marker for galanin neuropeptide—revealed that galanin-immunoreactive (galanin-IR) nerve cells are located in the preoptic region, hypothalamus, midbrain tegmentum, and rostral hindbrain [16]. In addition, nerve fibers of galanin-IR neurons have been detected primarily in the ventral region of the brain, including the subpallium, anterior commissure, postoptic commissure, and commissure of the posterior tuberculum [16]. A recent study has shown that galanin is the neuropeptide most frequently colocalized with histaminergic neurons and that galanin-IR fibers surround the 3
histaminergic neurons in the ventrocaudal hypothalamus of the adult zebrafish brain [22]. It plays a role in regulating blood glucose level, indicating that galanin function is evolutionarily conserved [15, 16]. BLAST survey and phylogenetic analysis have revealed that the zebrafish has three different receptors for galanin; GALR1a, GALR1b, and GALR2, while it lacks GALR3 [10]. Recent phylogeny and synteny analysis of the gene family have shown that zebrafish GALR2 is subdivided into GALR2a and GALR2b [7]. A recent study has shown that galr1a expression is mainly restricted to the brain and intestines, while galr1b mRNA was present in all tissues [9]. However, nothing has been reported about GALR2a and GALR2b in the zebrafish CNS. In this study, we describe the expression of galr2a and galr2b in zebrafish embryos using whole-mount in situ RNA hybridization. To further analyze the distribution
of
GALR2b
and
its
interactions
with
galanin,
we
generated
Tg(galr2b:egfp) zebrafish, which express EGFP under the control of a galr2b promoter. Investigation of CNS of transgenic reporter zebrafish revealed that galr2b:EGFP+ neurons are distributed, and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may interact with other ligands instead of galanin in these regions.
2. Materials and methods
2.1. Animals
Wild-type AB, Tg(galr2b:egfp) zebrafish of either sex were used for this study. Zebrafish embryos were staged according to hours post-fertilization (hpf), days postfertilization (dpf) and morphological charateristics.
2.2. Cloning of galr2a and galr2b, and in situ RNA hybridization
To clone galr2a and galr2b, we designed PCR primers using sequence from a GenBank (galr2a: XM_002664007.3, galr2b:XM_001339133.3) and amplified a 4
product from 2 dpf cDNA. PCR products were cloned by using pGEM-T easy vector (Promega). in situ RNA hybridization was performed as described previously [24].
2.3. DNA constructs and generation of transgenic zebrafish
To produce transgenic zebrafish, we first identified a BAC clone (CH73-52E11, BACPAC Resources Center/BPRC), which contained the genomic sequence of galr2b. To manipulate bacterial artificial chromosome (BAC) DNA, the zebrafish BAC clone for galr2b and BAC homologous recombination technique [18] was used. For the GFP construct, template was constructed by multisite Gateway system (Invitrogen) and consists of EGFP, a poly(A) signal, and a Kanamycin-resistant gene. To produce Tg(galr2b:EGFP) zebrafish, the modified galr2b BAC DNA was linearized and injected into fertilized one cell stage embryos. To screen for germ-line transmitted transgenic zebrafish, we mated injected founder fish with wild-type fish and screened the progeny for EGFP expression. The obtained transgenic founder fish were crossed with wild-type fish, and F1 transgenic fish [designated Tg(galr2b:egfp)] were raised to adulthood.
2.4. Immunohistochemistry
Embryos
were
anesthetized
by
200
mg/L
of
ethyl
3-aminobenzoate
methanesulfonate salt (MS222, Sigma) until movement ceased and fixed in 4% paraformaldehyde. Fixed embryos were embedded in 1.5% agar blocks containing 5% sucrose and equilibrated in a 30% sucrose solution. Frozen blocks were sliced into 10-µm sections using a cryostat microtome and mounted on glass slides. Sections were rinsed with PBS several times and then blocked in 2% bovine serum albumin with sheep serum. After blocking reaction, sections were treated with primary antibodies for 2 hours at the room temperature, washed out for 2 hours with PBS, than treated with secondary antibody for 2 hours at the room temperature. Images were
obtained
from
the
sections
of
12
Tg(galr2b:egfp)
embryos.
For
immunohistochemistry, we used following antibodies: mouse anti-HuC/D (1:200, Molecular Probes), rabbit anti-GAL (1:5000, Chemicon), rabbit anti-Pax2a (1:200, Covance), mouse anti-Hb9 (1:100, Developmental Studies Hybridoma Bank) and 5
chick anti-GFP (1:200, Abcam). Alexar Fluor 488- and 568-conjugated secondary antibodies were used for fluorescent detection of primary antibodies (1:1000, Invitrogen).
3. Results
galr2a and galr2b is specifically expressed in the zebrafish CNS In mammals, galr2 is widely expressed in the brain, including the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2, 3, 5, 6, 14]. To investigate the expression pattern of galr2a and galr2b in zebrafish, we first performed whole-mount in situ RNA hybridization with galr2a and galr2b. In contrast to the expression of zebrafish galr1a and galr1b, which are known to be expressed in the intestine and brain (galr1a), and all tissues (galr1b), respectively, [9], expression of galr2a and galr2b is restricted to CNS of zebrafish embryos. We found that galr2a was expressed at very low levels in a restricted area of the brain including the subpallium (Fig. 1A, B) and that this expression was not detectable with a sense probe for galr2a (Fig. 1C). Expression of galr2b was detectable in CNS including in the olfactory bulb (Fig. 1D, F, black arrowheads), midbrain tegmentum (Fig. 1E, black arrow) preoptic region (Fig. 1D, F, H, red arrowheads), dorsal thalamus (Fig. 1I, black arrowheads), posterior tuberculum (Fig. 1J, black arrowheads), postoptic commissure (Fig. 1I, red arrowhead), hindbrain (Fig. 1K) and the lateral margin of spinal cord (Fig. 1G, L). Semi-quantitative reverse transcription PCR (qRT-PCR) analysis revealed that galr2b expression was first detected at 24 hpf, and its expression was maintained in the brain and spinal cord through adulthood (data not shown). Restricted expression of galr2a and galr2b in the zebrafish CNS is very similar to mammalian galr2, indicating that zebrafish galr2a and galr2b are orthologs of mammalian galr2 [2, 3, 5, 6, 14]. In addition, widespread expression of zebrafish galr2b in the brain and spinal cord suggests that galr2b plays a role as a main receptor for galanin neuropeptides in CNS.
Tg(galr2b:egfp) zebrafish express EGFP in galr2b-expressing cells To investigate the identity and distribution of galr2b-expressing cells and their interaction with galanin in CNS of zebrafish embryos, we first established a 6
Tg(galr2b:egfp) transgenic zebrafish, which expresses EGFP under the control of a galr2b promoter. EGFP was specifically expressed in CNS of Tg(galr2b:egfp) transgenic fish including the brain and spinal cord (Fig. 2A-D), in a pattern similar to that of the endogenous galr2b gene. EGFP expression was observed in anterior brain regions including the thalamus (white arrowhead), midbrain tegmentum (white arrow), and olfactory bulb cells (red arrow; Fig. 2A, B). EGFP expression was also observed in the hindbrain and spinal cord (Fig. 2C, D). Tg(galr2b:egfp) embryos were further analyzed for EGFP expression by confocal microscopy to compare their expression with endogenous galr2b expression. Expression of both EGFP fluorescence and endogenous galr2b was observed in the brain of whole-mount embryos including olfactory bulb (red arrowheads), thalamus (white arrowheads), and midbrain tegmentum (white arrows; Fig. 2E, F). EGFP+ cells and galr2b expressing cells were also detected in the posterior tuberculum (red arrowheads) and hypothalamus (white arrowheads) of the brain section (Fig. 2G, H), and in the lateral margin of the spinal cord section (Fig. I, J). Altogether, these data show that Tg(galr2b:egfp) embryos express EGFP in CNS cells which express endogenous galr2b, indicating that Tg(galr2b:egfp) zebrafish can be used as a reporter transgenic fish to study distribution of the GALR2b cells and their interaction with galanin. Distribution of galr2b:EGFP+ cells and their interaction with galanin in the zebrafish CNS To investigate the identity of the galr2b:EGFP+ cells, we labeled brain and spinal cord sections of 3 dpf Tg(galr2b:egfp) embryos with anti-Hu antibody specific to postmitotic neurons. As shown in Fig. 3 and Fig. 4, all of the galr2b:EGFP+ cells were labeled by anti-Hu antibody in the brain (Fig. 3A-C) and spinal cord sections (Fig. 4A-C), indicating that galr2b:EGFP+ cells are postmitotic neurons in the zebrafish CNS. Next, we investigated the distribution of galr2b:EGFP+ neurons and their interactions with galanin-immunoreactive (galanin-IR) nerve cells and their fibers. Previous immunohistochemical analysis with anti-Galanin antibody have shown that that galanin-IR nerve cells are located in the preoptic region, hypothalamus, midbrain tegmentum, and rostral hindbrain, and nerve fibers of galanin-IR neurons have been mainly been detected in the ventral region of the brain including the subpallium, 7
anterior commissure, postoptic commissure, and commissure of the posterior tuberculum [16]. Therefore, we analyzed the distribution of galr2b:EGFP+ neurons and their interactions with galanin-IR nerve cells in these regions by labeling brain and spinal cord sections of Tg(galr2b:egfp) zebrafish with anti-Galanin antibody. In the horizontal sections of the brains of Tg(galr2b:EGFP) zebrafish, galr2b:EGFP+, neurons were distributed in the anterior commissure (Cant; Fig. 3D, arrowhead), postoptic commissure (Cpop) of the preoptic region (PO; Fig. 3E, arrowhead), and hypothalamus (Hyp; Fig 3F, arrowhead). In these regions, we observed the galr2b:EGFP+ neurons and their nerve fibers closely interacting with galanin-IR nerve fibers, indicating that galanin functions through the GALR2b. We next examined the distribution of galanin-IR and galr2b:EGFP+ neurons in the pituitary gland, and found that galr2b:EGFP+ neurons are intermingled with the galanin-IR cells in the pituitary gland (Fig 3G, arrowhead), indicating that like mammals, galanin-GALR2b signaling is involved in neuroendocrine systems of the HPA-axis in zebrafish. Interestingly, we do not observe galanin-IR cells or nerve fibers in some regions of the brain. Galanin-IR nerve fibers were detectable near the postoptic commissure (Cpop; Fig. 3H, arrowheads) but not in the vicinity of posterior tuberculum (PT) in the midbrain (Fig. 3H, arrows). Galanin-IR signal was detectable in the ventral hindbrain (Fig. 3I, arrowheads) but not in the dorsal hindbrain (Fig. 3I, arrows), olfactory bulb (OB; Fig. 3J, arrows), and midbrain tegmentum (Fig. 3K, arrows). The lack of galanin immunoreactivity we observed in the dorsal hindbrain is consistent with previous data showing that the descending nerve fibers, originating from neurons of the midbrain tegmentum and the anterior portion of the hindbrain, run in the ventral portion of the medulla oblongata and project to the ventral portion of the spinal cord [16]. These data suggest that galr2b:EGFP+ neurons interact with galanin-IR cells in some regions, but there may be other ligands that interact with galr2b:EGFP+ neurons in the regions where galanin-IR signals do not exist. In rat, galr2 mRNA has been shown to be detected in the dorsal horn and motor neurons of the ventral horn of the spinal cord [14]. To reveal the identity and distribution of galr2b:EGFP+ neurons and their interaction with galanin-IR nerve cells in the spinal cord, we labeled transverse sections of the spinal cord of Tg(galr2b:egfp) embryos with various cell type-specific markers (Fig. 4). At 3 dpf, all galr2b:EGFP+ cells are labeled by anti-Hu antibody, indicating that galr2b:EGFP+ cells are 8
postmitotic neurons (Fig. 4A-C). Interestingly, labeling of Tg(galr2b:egfp) embryos with anti-Hb9 antibody, which is a marker for motor neurons in zebrafish, revealed that dorsally located Hb9+ motor neurons are galr2b:EGFP+ neurons (Fig. 4D-F, arrows), but ventrally located Hb9+ motor neurons are galr2b:EGFP- cells (Fig. 4D-F, arrowheads), suggesting that galr2b:EGFP+ neurons are partly involved in the control of motor activity. Labeling of Tg(galr2b:egfp) embryos with anti-Pax2 antibody, which marks interneurons in the dorsal neural tube, revealed that a large population of galr2b:EGFP+ neurons located in the dorsal spinal cord are Pax2 + interneurons (Fig. 4G-I, arrows). These data indicate that galr2b:EGFP+ neurons consist of various neuronal cell types, including motor neurons and interneurons, throughout the dorsoventral axis of the spinal cord of zebrafish embryos. However, galanin-IR nerve fibers were bilaterally located only in the white matter of the ventral spinal cord of the zebrafish embryo (Fig. 4J-L, arrows). We did not detect any galanin-IR nerve fibers in the dorsal spinal cord, near Pax2-expressing interneurons. Together with the midbrain and hindbrain, in which galanin-IR nerves are mostly detectable in the ventral region, but galr2b:EGFP+ neurons are located throughout the dorsoventral axis (Fig. 3H, I), these data suggest that galr2b:EGFP+ neurons interact with galaninIR nerves in the ventral region of the neural tube, but dorsally located galr2b:EGFP+ neurons may interact with other ligands instead of galanin.
4. Discussion
In the present study, we describe the expression of galr2a and galr2b genes in the zebrafish CNS, and identify the distribution of galr2b:EGFP neurons and their interactions with galanin-IR nerve cells by analyzing CNS of Tg(galr2b:egfp) zebrafish. This is the first report examining galr2a and galr2b expression and neuronal connections between GALR2b and galanin-IR nerve cells in zebrafish. In zebrafish embryos, expression of galr2a and galr2b was restricted to CNS. We found that galr2a was expressed at very low levels in a restricted area of the brain; however, galr2b was widely expressed in the brain and spinal cord in a pattern similar to mammalian galr2, indicating that zebrafish galr2b is functional ortholog of mammalian galr2 (Fig. 1). Immunohistochemical analysis of CNS of Tg(galr2b:egfp) 9
zebrafish with anti-GAL antibody revealed that galr2b:EGFP+ neurons are distributed and interact with galanin-IR in the various regions of the brain and ventral spinal cord including the anterior commissure, postoptic commissure of the preoptic region, hypothalamus, and commissure of posterior tuberculum (Fig. 3). However, galaninIR nerve cells were not observed near galr2b:EGFP neurons in the olfactory bulb (Fig. 3J), posterior tuberculum of the midbrain (Fig. 3H), dorsal area of the hindbrain (Fig. 3I), midbrain tegmentum (Fig. 3K), and spinal cord (Fig. 4L). Although GALR2b is a receptor for galanin, these data suggest that GALR2b may interact with other ligands instead of galanin in these regions. A possible candidate that interacts with GALR2b is spexin (SPX). SPX is a recently identified novel secreted neuropeptide, which is composed of 14 amino acids and highly conserved in different vertebrates [13, 19, 26, 29]. Several previous studies have revealed that SPX is widely expressed in the brain, skin, respiratory system, digestive system, reproductive system, and endocrine system, indicating that SPX may have multiple functions [17, 28]. Recently, it was reported that SPX is a functional agonist for GALR2 and GALR3 in humans as well as GALR2a and GALR2b in zebrafish. In this report, SPX has been shown to activate GALR2a and GALR2b in zebrafish in vitro by a ligand-receptor interaction assay [7]. Interestingly, zebrafish SPX has been shown to have higher potencies toward GALR2b than galanin, while GALR2a responds to SPX and GAL with similar potency, suggesting that SPX is a functional agonist for GALR2b in zebrafish [7]. Therefore, we hypothesize that galr2b:EGFP neurons may interact with SPX in the regions where galanin-IR nerve cells do not exist. To probe this hypothesis, we need to investigate the distribution and interactions between GALR2b and SPX neurons in the zebrafish CNS. Since neuropeptides mediate signals through specific GPCRs, it is important to analyze neuronal circuits including the neuropeptide and its receptor to understand the regulation and function of neuropeptides. In this study, we generated Tg(galr2b:egfp) zebrafish which expresses EGFP under the control of a galr2b promoter. We also have shown that galr2b:EGFP is expressed in the endogenous galr2b-expressing
neurons
and
successfully analyzed
the
distribution
and
interactions of galr2b:EGFP and galanin neurons. Altogether, our data indicate that Tg(galr2b:egfp) zebrafish can be a valuable tool to study the functions of and 10
neuronal circuit formation between GALR2b and its interacting neuropeptides.
Acknowledgements
This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2011-0019233) and by the grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A121793).
11
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Figure legends
Figure 1. galr2a and galr2b expression revealed by in situ RNA hybridization. (A-C) RNA in situ hybridization with galr2a anti-sense (A, B) and sense probes (C). (A, C) Dorsal views of the brain (anterior to the left) and (B) transverse section of the brain ( dorsal at the top). Black arrowheads indicate subpallium. (D-G) Lateral (D, G) and dorsal (E, F) views of the brain with different focus and lateral view of the spinal cord (G) of 3 dpf embryos, anterior to the left. Diagram shows the plane of cryostat section in H, I, J, K, and L. Black arrows indicate midbrain tegmentum and black arrowheads indicate olfactory bulb. (H-L) Transverse sections of the brain (H-K) and spinal cord (L) of 3 dpf embryos, dorsal at the top. Red arrowheads indicate preoptic region (D, F, H) and postoptic commissure (I). Black arrowheads indicate dorsal thalamus (I) and posterior tuberculum (J). Abbreviations: nc, notochord. Scale bar: AC, 100 µm; D, 150 µm; E-I, 30 µm.
Figure 2. Tg(galr2b:egfp) zebrafish express EGFP in galr2b-expressing cells. (A-D) EGFP expression in 3 dpf Tg(galr2b:egfp) embryos. (A) Lateral view image of the whole embryo. (B-D) dorsal view images of the boxed areas in panel (A). Forebrain, midbrain (B), hindbrain (C) and spinal cord (D), anterior to the left. (E) Expression of galr2b revealed by in situ RNA hybridization at 3 dpf and (F) expression of EGFP in 3 dpf Tg(galr2b:egfp) embryos. Dorsal views, anterior to the left. Diagram shows the plane of cryostat section in G, H, I and j. (B, E, F) White arrows indicate midbrain tegmentum, white arrowheads indicate thalamus, and red arrowheads indicate olfactory bulb.
(G-J) Transverse sections of the brain (G, H) and spinal cord (I, J),
dorsal at the top. (G, I) galr2b expression revealed by in situ RNA hybridization and (H, J) EGFP expression in 3 dpf Tg(galr2b:egfp) embryos. (G, H) Red arrowheads indicate posterior tuberculum and white arrowheads indicate hypothalamus. Scale bar: A, 100 µm; B-D, 60 µm; E-F, 50 µm; G-J, 20 µm. Figure 3. Distribution of galr2b:EGFP+ cells and their interactions with galanin in the brain of zebrafish embryos. (A-G, J) Horizontal sections of the 3 dpf Tg(galr2b:egfp) embryos, Anterior to the left. (H, I, K) Transverse sections of the 3 dpf Tg(galr2b:egfp) embryos, dorsal to the top. (A-C) Labeling of the brain sections of 3 dpf 15
Tg(galr2b:egfp) embryos with anti-Hu antibody. (B, C) High magnification images of the boxed areas in panel (A). (D-K) Labeling of the brain sections of 3 dpf Tg(galr2b:egfp) embryos with anti-Galanin antibody. Cant, anterior commissure; PO, preoptic region; Cpop, postoptic commissure; Hyp, hypothalamus; Pit, pituitary gland; PT, posterior tuberculum; Rho, rhombencephalon; OB, olfactory bulb; Ctub, commissure of posterior tuberculum. Scale bar: A, 50 µm; B-K, 20 µm. Figure 4. Distribution of galr2b:EGFP+ cells and their interactions with galanin in the spinal cord of zebrafish embryos. All images are transverse sections of the spinal cord, dorsal at the top. Labeling of the spinal cord of 3 dpf Tg(galr2b:egfp) embryos with an anti-Hu antibody (A-C), anti-Hb9 antibody (D-F), anti-Pax2 antibody (G-I) and anti-Galanin antibody (J-L). (F) Arrows indicate GALR2b+ motor neurons and arrowheads indicate GALR2b- motor neurons. (I) Arrows indicate Pax2+, GALR2b+ interneurons and (L) arrow indicate galanin-IR nerve fibers. Scale bar: 20 µm.
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S0304-3940(16)30434-7 http://dx.doi.org/doi:10.1016/j.neulet.2016.06.025 NSL 32113
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
31-3-2016 14-5-2016 9-6-2016
Please cite this article as: Eunmi Kim, Inyoung Jeong, Suhyun Kim, HwanKi Kim, Dong-won Lee, Boa Kim, Jae Young Seong, Young-Ki Bae, Jae-Ho Ryu, Hae-Chul Park, Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.06.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system
Eunmi Kim, Inyoung Jeong, Suhyun Kim, Hwan-Ki Kim, Dong-won Lee, Boa Kim, Jae Young Seong1, Young-Ki Bae2, Jae-Ho Ryu3 and Hae-Chul Park
Graduate School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea; 1Graduate School of Medicine, Korea University, Seoul 136-705, Republic of Korea; 2Comparative Biomedical Research Branch, Research Institute, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea; 3Genomic
Design Bioengineering Company, Daejeon 306-230, Republic of Korea.
The number of text pages : 19 The number of figures : 4
Correspondence to : Hae-Chul Park, Graduate School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea. Telephone: +82-31-412-6712 Fax: +82-31-412-6718 Email: [email protected].
1
Highlights •
A novel galanin receptor 2b (galr2b) is expressed in the zebrafish nervous system.
•
The galr2b:egfp transgenic zebrafish was generated to visualize galr2b neurons.
•
Distribution of galr2b neurons and their interaction with galanin was analyzed in the zebrafish central nervous system.
ABSTRACT Galanin is a multifunctional neuropeptide that is implicated in the modulation of physiological processes, including nociception, cognition, feeding behavior, neuronal growth, and reproduction. The physiological effects of galanin are mediated through its interaction with three different G protein-coupled receptors, i.e., GALR1, GALR2, and GALR3. Unlike mammals, zebrafish have four different receptors for galanin, diversified from GALR1 (GAL1a and GALR1b) and GALR2 (GALR2a and GALR2b). Despite the importance of galanin in the central nervous system (CNS), no information has been reported regarding GalR2 in zebrafish CNS. In this study, we found that galr2a is expressed at low levels in restricted areas of the brain; however, galr2b was widely expressed in CNS including olfactory bulb, midbrain tegmentum, preoptic region, dorsal thalamus, posterior tuberculum, postoptic commissure, hindbrain, and spinal cord. To further analyze the distribution of GALR2b neurons and their interaction with GAL, we generated Tg(galr2b:egfp) zebrafish, which express enhanced green fluorescent protein (EGFP) under the control of a galr2b promoter. Investigation of the CNS of transgenic reporter zebrafish revealed that galr2b:EGFP+ neurons are distributed and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may have the potential to interact with other ligands instead of galanin in these regions. 2
Key words: neuropeptide, galanin, galanin receptor, central nervous system, zebrafish
1. Introduction
Galanin (GAL) is a multifunctional neuropeptide that has a widespread distribution in the peripheral and central nervous system (CNS). Galanin consists of 29–30 amino acid sequences and was first isolated from porcine intestine [23]. In mammals, galanin has been implicated in the modulation of several different physiological processes including nociception [4], cognition [21], feeding behavior [1], neuronal growth [11] and reproduction [20]. Galanin has also been shown to play a role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress, as well as in the pathogenesis of pituitary adenomas [25, 27]. In mammals, the physiological effects of galanin are mediated through its interaction with three galanin receptor subtypes GALR1, GALR2, and GALR3, which are G proteincoupled receptors (GPCRs). GALR1 expression is largely restricted to the nervous system including ventral hippocampus, thalamic nuclei, amygdala, brainstem nuclei, hypothalamus, posterior horns of the spinal cord, and dorsal root ganglia [2, 8]. Unlike GALR1, GALR2 is widely distributed in almost all tissues, including the brain, such as the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2, 3, 5, 6, 14]. GALR3 showed limited expression in CNS but is relatively abundant in peripheral tissues [12]. In zebrafish, immunohistochemical analysis using the anti-Galanin antibody—a marker for galanin neuropeptide—revealed that galanin-immunoreactive (galanin-IR) nerve cells are located in the preoptic region, hypothalamus, midbrain tegmentum, and rostral hindbrain [16]. In addition, nerve fibers of galanin-IR neurons have been detected primarily in the ventral region of the brain, including the subpallium, anterior commissure, postoptic commissure, and commissure of the posterior tuberculum [16]. A recent study has shown that galanin is the neuropeptide most frequently colocalized with histaminergic neurons and that galanin-IR fibers surround the 3
histaminergic neurons in the ventrocaudal hypothalamus of the adult zebrafish brain [22]. It plays a role in regulating blood glucose level, indicating that galanin function is evolutionarily conserved [15, 16]. BLAST survey and phylogenetic analysis have revealed that the zebrafish has three different receptors for galanin; GALR1a, GALR1b, and GALR2, while it lacks GALR3 [10]. Recent phylogeny and synteny analysis of the gene family have shown that zebrafish GALR2 is subdivided into GALR2a and GALR2b [7]. A recent study has shown that galr1a expression is mainly restricted to the brain and intestines, while galr1b mRNA was present in all tissues [9]. However, nothing has been reported about GALR2a and GALR2b in the zebrafish CNS. In this study, we describe the expression of galr2a and galr2b in zebrafish embryos using whole-mount in situ RNA hybridization. To further analyze the distribution
of
GALR2b
and
its
interactions
with
galanin,
we
generated
Tg(galr2b:egfp) zebrafish, which express EGFP under the control of a galr2b promoter. Investigation of CNS of transgenic reporter zebrafish revealed that galr2b:EGFP+ neurons are distributed, and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may interact with other ligands instead of galanin in these regions.
2. Materials and methods
2.1. Animals
Wild-type AB, Tg(galr2b:egfp) zebrafish of either sex were used for this study. Zebrafish embryos were staged according to hours post-fertilization (hpf), days postfertilization (dpf) and morphological charateristics.
2.2. Cloning of galr2a and galr2b, and in situ RNA hybridization
To clone galr2a and galr2b, we designed PCR primers using sequence from a GenBank (galr2a: XM_002664007.3, galr2b:XM_001339133.3) and amplified a 4
product from 2 dpf cDNA. PCR products were cloned by using pGEM-T easy vector (Promega). in situ RNA hybridization was performed as described previously [24].
2.3. DNA constructs and generation of transgenic zebrafish
To produce transgenic zebrafish, we first identified a BAC clone (CH73-52E11, BACPAC Resources Center/BPRC), which contained the genomic sequence of galr2b. To manipulate bacterial artificial chromosome (BAC) DNA, the zebrafish BAC clone for galr2b and BAC homologous recombination technique [18] was used. For the GFP construct, template was constructed by multisite Gateway system (Invitrogen) and consists of EGFP, a poly(A) signal, and a Kanamycin-resistant gene. To produce Tg(galr2b:EGFP) zebrafish, the modified galr2b BAC DNA was linearized and injected into fertilized one cell stage embryos. To screen for germ-line transmitted transgenic zebrafish, we mated injected founder fish with wild-type fish and screened the progeny for EGFP expression. The obtained transgenic founder fish were crossed with wild-type fish, and F1 transgenic fish [designated Tg(galr2b:egfp)] were raised to adulthood.
2.4. Immunohistochemistry
Embryos
were
anesthetized
by
200
mg/L
of
ethyl
3-aminobenzoate
methanesulfonate salt (MS222, Sigma) until movement ceased and fixed in 4% paraformaldehyde. Fixed embryos were embedded in 1.5% agar blocks containing 5% sucrose and equilibrated in a 30% sucrose solution. Frozen blocks were sliced into 10-µm sections using a cryostat microtome and mounted on glass slides. Sections were rinsed with PBS several times and then blocked in 2% bovine serum albumin with sheep serum. After blocking reaction, sections were treated with primary antibodies for 2 hours at the room temperature, washed out for 2 hours with PBS, than treated with secondary antibody for 2 hours at the room temperature. Images were
obtained
from
the
sections
of
12
Tg(galr2b:egfp)
embryos.
For
immunohistochemistry, we used following antibodies: mouse anti-HuC/D (1:200, Molecular Probes), rabbit anti-GAL (1:5000, Chemicon), rabbit anti-Pax2a (1:200, Covance), mouse anti-Hb9 (1:100, Developmental Studies Hybridoma Bank) and 5
chick anti-GFP (1:200, Abcam). Alexar Fluor 488- and 568-conjugated secondary antibodies were used for fluorescent detection of primary antibodies (1:1000, Invitrogen).
3. Results
galr2a and galr2b is specifically expressed in the zebrafish CNS In mammals, galr2 is widely expressed in the brain, including the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2, 3, 5, 6, 14]. To investigate the expression pattern of galr2a and galr2b in zebrafish, we first performed whole-mount in situ RNA hybridization with galr2a and galr2b. In contrast to the expression of zebrafish galr1a and galr1b, which are known to be expressed in the intestine and brain (galr1a), and all tissues (galr1b), respectively, [9], expression of galr2a and galr2b is restricted to CNS of zebrafish embryos. We found that galr2a was expressed at very low levels in a restricted area of the brain including the subpallium (Fig. 1A, B) and that this expression was not detectable with a sense probe for galr2a (Fig. 1C). Expression of galr2b was detectable in CNS including in the olfactory bulb (Fig. 1D, F, black arrowheads), midbrain tegmentum (Fig. 1E, black arrow) preoptic region (Fig. 1D, F, H, red arrowheads), dorsal thalamus (Fig. 1I, black arrowheads), posterior tuberculum (Fig. 1J, black arrowheads), postoptic commissure (Fig. 1I, red arrowhead), hindbrain (Fig. 1K) and the lateral margin of spinal cord (Fig. 1G, L). Semi-quantitative reverse transcription PCR (qRT-PCR) analysis revealed that galr2b expression was first detected at 24 hpf, and its expression was maintained in the brain and spinal cord through adulthood (data not shown). Restricted expression of galr2a and galr2b in the zebrafish CNS is very similar to mammalian galr2, indicating that zebrafish galr2a and galr2b are orthologs of mammalian galr2 [2, 3, 5, 6, 14]. In addition, widespread expression of zebrafish galr2b in the brain and spinal cord suggests that galr2b plays a role as a main receptor for galanin neuropeptides in CNS.
Tg(galr2b:egfp) zebrafish express EGFP in galr2b-expressing cells To investigate the identity and distribution of galr2b-expressing cells and their interaction with galanin in CNS of zebrafish embryos, we first established a 6
Tg(galr2b:egfp) transgenic zebrafish, which expresses EGFP under the control of a galr2b promoter. EGFP was specifically expressed in CNS of Tg(galr2b:egfp) transgenic fish including the brain and spinal cord (Fig. 2A-D), in a pattern similar to that of the endogenous galr2b gene. EGFP expression was observed in anterior brain regions including the thalamus (white arrowhead), midbrain tegmentum (white arrow), and olfactory bulb cells (red arrow; Fig. 2A, B). EGFP expression was also observed in the hindbrain and spinal cord (Fig. 2C, D). Tg(galr2b:egfp) embryos were further analyzed for EGFP expression by confocal microscopy to compare their expression with endogenous galr2b expression. Expression of both EGFP fluorescence and endogenous galr2b was observed in the brain of whole-mount embryos including olfactory bulb (red arrowheads), thalamus (white arrowheads), and midbrain tegmentum (white arrows; Fig. 2E, F). EGFP+ cells and galr2b expressing cells were also detected in the posterior tuberculum (red arrowheads) and hypothalamus (white arrowheads) of the brain section (Fig. 2G, H), and in the lateral margin of the spinal cord section (Fig. I, J). Altogether, these data show that Tg(galr2b:egfp) embryos express EGFP in CNS cells which express endogenous galr2b, indicating that Tg(galr2b:egfp) zebrafish can be used as a reporter transgenic fish to study distribution of the GALR2b cells and their interaction with galanin. Distribution of galr2b:EGFP+ cells and their interaction with galanin in the zebrafish CNS To investigate the identity of the galr2b:EGFP+ cells, we labeled brain and spinal cord sections of 3 dpf Tg(galr2b:egfp) embryos with anti-Hu antibody specific to postmitotic neurons. As shown in Fig. 3 and Fig. 4, all of the galr2b:EGFP+ cells were labeled by anti-Hu antibody in the brain (Fig. 3A-C) and spinal cord sections (Fig. 4A-C), indicating that galr2b:EGFP+ cells are postmitotic neurons in the zebrafish CNS. Next, we investigated the distribution of galr2b:EGFP+ neurons and their interactions with galanin-immunoreactive (galanin-IR) nerve cells and their fibers. Previous immunohistochemical analysis with anti-Galanin antibody have shown that that galanin-IR nerve cells are located in the preoptic region, hypothalamus, midbrain tegmentum, and rostral hindbrain, and nerve fibers of galanin-IR neurons have been mainly been detected in the ventral region of the brain including the subpallium, 7
anterior commissure, postoptic commissure, and commissure of the posterior tuberculum [16]. Therefore, we analyzed the distribution of galr2b:EGFP+ neurons and their interactions with galanin-IR nerve cells in these regions by labeling brain and spinal cord sections of Tg(galr2b:egfp) zebrafish with anti-Galanin antibody. In the horizontal sections of the brains of Tg(galr2b:EGFP) zebrafish, galr2b:EGFP+, neurons were distributed in the anterior commissure (Cant; Fig. 3D, arrowhead), postoptic commissure (Cpop) of the preoptic region (PO; Fig. 3E, arrowhead), and hypothalamus (Hyp; Fig 3F, arrowhead). In these regions, we observed the galr2b:EGFP+ neurons and their nerve fibers closely interacting with galanin-IR nerve fibers, indicating that galanin functions through the GALR2b. We next examined the distribution of galanin-IR and galr2b:EGFP+ neurons in the pituitary gland, and found that galr2b:EGFP+ neurons are intermingled with the galanin-IR cells in the pituitary gland (Fig 3G, arrowhead), indicating that like mammals, galanin-GALR2b signaling is involved in neuroendocrine systems of the HPA-axis in zebrafish. Interestingly, we do not observe galanin-IR cells or nerve fibers in some regions of the brain. Galanin-IR nerve fibers were detectable near the postoptic commissure (Cpop; Fig. 3H, arrowheads) but not in the vicinity of posterior tuberculum (PT) in the midbrain (Fig. 3H, arrows). Galanin-IR signal was detectable in the ventral hindbrain (Fig. 3I, arrowheads) but not in the dorsal hindbrain (Fig. 3I, arrows), olfactory bulb (OB; Fig. 3J, arrows), and midbrain tegmentum (Fig. 3K, arrows). The lack of galanin immunoreactivity we observed in the dorsal hindbrain is consistent with previous data showing that the descending nerve fibers, originating from neurons of the midbrain tegmentum and the anterior portion of the hindbrain, run in the ventral portion of the medulla oblongata and project to the ventral portion of the spinal cord [16]. These data suggest that galr2b:EGFP+ neurons interact with galanin-IR cells in some regions, but there may be other ligands that interact with galr2b:EGFP+ neurons in the regions where galanin-IR signals do not exist. In rat, galr2 mRNA has been shown to be detected in the dorsal horn and motor neurons of the ventral horn of the spinal cord [14]. To reveal the identity and distribution of galr2b:EGFP+ neurons and their interaction with galanin-IR nerve cells in the spinal cord, we labeled transverse sections of the spinal cord of Tg(galr2b:egfp) embryos with various cell type-specific markers (Fig. 4). At 3 dpf, all galr2b:EGFP+ cells are labeled by anti-Hu antibody, indicating that galr2b:EGFP+ cells are 8
postmitotic neurons (Fig. 4A-C). Interestingly, labeling of Tg(galr2b:egfp) embryos with anti-Hb9 antibody, which is a marker for motor neurons in zebrafish, revealed that dorsally located Hb9+ motor neurons are galr2b:EGFP+ neurons (Fig. 4D-F, arrows), but ventrally located Hb9+ motor neurons are galr2b:EGFP- cells (Fig. 4D-F, arrowheads), suggesting that galr2b:EGFP+ neurons are partly involved in the control of motor activity. Labeling of Tg(galr2b:egfp) embryos with anti-Pax2 antibody, which marks interneurons in the dorsal neural tube, revealed that a large population of galr2b:EGFP+ neurons located in the dorsal spinal cord are Pax2 + interneurons (Fig. 4G-I, arrows). These data indicate that galr2b:EGFP+ neurons consist of various neuronal cell types, including motor neurons and interneurons, throughout the dorsoventral axis of the spinal cord of zebrafish embryos. However, galanin-IR nerve fibers were bilaterally located only in the white matter of the ventral spinal cord of the zebrafish embryo (Fig. 4J-L, arrows). We did not detect any galanin-IR nerve fibers in the dorsal spinal cord, near Pax2-expressing interneurons. Together with the midbrain and hindbrain, in which galanin-IR nerves are mostly detectable in the ventral region, but galr2b:EGFP+ neurons are located throughout the dorsoventral axis (Fig. 3H, I), these data suggest that galr2b:EGFP+ neurons interact with galaninIR nerves in the ventral region of the neural tube, but dorsally located galr2b:EGFP+ neurons may interact with other ligands instead of galanin.
4. Discussion
In the present study, we describe the expression of galr2a and galr2b genes in the zebrafish CNS, and identify the distribution of galr2b:EGFP neurons and their interactions with galanin-IR nerve cells by analyzing CNS of Tg(galr2b:egfp) zebrafish. This is the first report examining galr2a and galr2b expression and neuronal connections between GALR2b and galanin-IR nerve cells in zebrafish. In zebrafish embryos, expression of galr2a and galr2b was restricted to CNS. We found that galr2a was expressed at very low levels in a restricted area of the brain; however, galr2b was widely expressed in the brain and spinal cord in a pattern similar to mammalian galr2, indicating that zebrafish galr2b is functional ortholog of mammalian galr2 (Fig. 1). Immunohistochemical analysis of CNS of Tg(galr2b:egfp) 9
zebrafish with anti-GAL antibody revealed that galr2b:EGFP+ neurons are distributed and interact with galanin-IR in the various regions of the brain and ventral spinal cord including the anterior commissure, postoptic commissure of the preoptic region, hypothalamus, and commissure of posterior tuberculum (Fig. 3). However, galaninIR nerve cells were not observed near galr2b:EGFP neurons in the olfactory bulb (Fig. 3J), posterior tuberculum of the midbrain (Fig. 3H), dorsal area of the hindbrain (Fig. 3I), midbrain tegmentum (Fig. 3K), and spinal cord (Fig. 4L). Although GALR2b is a receptor for galanin, these data suggest that GALR2b may interact with other ligands instead of galanin in these regions. A possible candidate that interacts with GALR2b is spexin (SPX). SPX is a recently identified novel secreted neuropeptide, which is composed of 14 amino acids and highly conserved in different vertebrates [13, 19, 26, 29]. Several previous studies have revealed that SPX is widely expressed in the brain, skin, respiratory system, digestive system, reproductive system, and endocrine system, indicating that SPX may have multiple functions [17, 28]. Recently, it was reported that SPX is a functional agonist for GALR2 and GALR3 in humans as well as GALR2a and GALR2b in zebrafish. In this report, SPX has been shown to activate GALR2a and GALR2b in zebrafish in vitro by a ligand-receptor interaction assay [7]. Interestingly, zebrafish SPX has been shown to have higher potencies toward GALR2b than galanin, while GALR2a responds to SPX and GAL with similar potency, suggesting that SPX is a functional agonist for GALR2b in zebrafish [7]. Therefore, we hypothesize that galr2b:EGFP neurons may interact with SPX in the regions where galanin-IR nerve cells do not exist. To probe this hypothesis, we need to investigate the distribution and interactions between GALR2b and SPX neurons in the zebrafish CNS. Since neuropeptides mediate signals through specific GPCRs, it is important to analyze neuronal circuits including the neuropeptide and its receptor to understand the regulation and function of neuropeptides. In this study, we generated Tg(galr2b:egfp) zebrafish which expresses EGFP under the control of a galr2b promoter. We also have shown that galr2b:EGFP is expressed in the endogenous galr2b-expressing
neurons
and
successfully analyzed
the
distribution
and
interactions of galr2b:EGFP and galanin neurons. Altogether, our data indicate that Tg(galr2b:egfp) zebrafish can be a valuable tool to study the functions of and 10
neuronal circuit formation between GALR2b and its interacting neuropeptides.
Acknowledgements
This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2011-0019233) and by the grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A121793).
11
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Figure legends
Figure 1. galr2a and galr2b expression revealed by in situ RNA hybridization. (A-C) RNA in situ hybridization with galr2a anti-sense (A, B) and sense probes (C). (A, C) Dorsal views of the brain (anterior to the left) and (B) transverse section of the brain ( dorsal at the top). Black arrowheads indicate subpallium. (D-G) Lateral (D, G) and dorsal (E, F) views of the brain with different focus and lateral view of the spinal cord (G) of 3 dpf embryos, anterior to the left. Diagram shows the plane of cryostat section in H, I, J, K, and L. Black arrows indicate midbrain tegmentum and black arrowheads indicate olfactory bulb. (H-L) Transverse sections of the brain (H-K) and spinal cord (L) of 3 dpf embryos, dorsal at the top. Red arrowheads indicate preoptic region (D, F, H) and postoptic commissure (I). Black arrowheads indicate dorsal thalamus (I) and posterior tuberculum (J). Abbreviations: nc, notochord. Scale bar: AC, 100 µm; D, 150 µm; E-I, 30 µm.
Figure 2. Tg(galr2b:egfp) zebrafish express EGFP in galr2b-expressing cells. (A-D) EGFP expression in 3 dpf Tg(galr2b:egfp) embryos. (A) Lateral view image of the whole embryo. (B-D) dorsal view images of the boxed areas in panel (A). Forebrain, midbrain (B), hindbrain (C) and spinal cord (D), anterior to the left. (E) Expression of galr2b revealed by in situ RNA hybridization at 3 dpf and (F) expression of EGFP in 3 dpf Tg(galr2b:egfp) embryos. Dorsal views, anterior to the left. Diagram shows the plane of cryostat section in G, H, I and j. (B, E, F) White arrows indicate midbrain tegmentum, white arrowheads indicate thalamus, and red arrowheads indicate olfactory bulb.
(G-J) Transverse sections of the brain (G, H) and spinal cord (I, J),
dorsal at the top. (G, I) galr2b expression revealed by in situ RNA hybridization and (H, J) EGFP expression in 3 dpf Tg(galr2b:egfp) embryos. (G, H) Red arrowheads indicate posterior tuberculum and white arrowheads indicate hypothalamus. Scale bar: A, 100 µm; B-D, 60 µm; E-F, 50 µm; G-J, 20 µm. Figure 3. Distribution of galr2b:EGFP+ cells and their interactions with galanin in the brain of zebrafish embryos. (A-G, J) Horizontal sections of the 3 dpf Tg(galr2b:egfp) embryos, Anterior to the left. (H, I, K) Transverse sections of the 3 dpf Tg(galr2b:egfp) embryos, dorsal to the top. (A-C) Labeling of the brain sections of 3 dpf 15
Tg(galr2b:egfp) embryos with anti-Hu antibody. (B, C) High magnification images of the boxed areas in panel (A). (D-K) Labeling of the brain sections of 3 dpf Tg(galr2b:egfp) embryos with anti-Galanin antibody. Cant, anterior commissure; PO, preoptic region; Cpop, postoptic commissure; Hyp, hypothalamus; Pit, pituitary gland; PT, posterior tuberculum; Rho, rhombencephalon; OB, olfactory bulb; Ctub, commissure of posterior tuberculum. Scale bar: A, 50 µm; B-K, 20 µm. Figure 4. Distribution of galr2b:EGFP+ cells and their interactions with galanin in the spinal cord of zebrafish embryos. All images are transverse sections of the spinal cord, dorsal at the top. Labeling of the spinal cord of 3 dpf Tg(galr2b:egfp) embryos with an anti-Hu antibody (A-C), anti-Hb9 antibody (D-F), anti-Pax2 antibody (G-I) and anti-Galanin antibody (J-L). (F) Arrows indicate GALR2b+ motor neurons and arrowheads indicate GALR2b- motor neurons. (I) Arrows indicate Pax2+, GALR2b+ interneurons and (L) arrow indicate galanin-IR nerve fibers. Scale bar: 20 µm.
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