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Expression pattern of two zebrafish genes, cxcr4a and cxcr4b
Mechanisms of Development 109 (2001) 347–354 www.elsevier.com/locate/modo
Gene expression pattern
Expression pattern of two zebrafish genes, cxcr4a and cxcr4b Shang Wei Chong a, Alexander Emelyanov a, Zhiyuan Gong b, Vladimir Korzh a,b,* a
Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore
b
Received 25 June 2001; received in revised form 13 August 2001; accepted 13 August 2001
Abstract We cloned and mapped two novel zebrafish genes, cxcr4a and cxcr4b, which are closely related to mammalian CXCR4. Expression analysis by reverse transcription-polymerase chain reaction and in situ hybridization demonstrated that these two genes are expressed in most cell lineages known to express Cxcr4 in mammals. These genes are co-expressed in lateral mesoderm and posterior midbrain. The transcripts of cxcr4a were detected in interneurons and endoderm, whereas cxcr4b was specifically expressed in sensory neurons, motoneurons and cerebellum. In the lateral mesoderm, cxcr4b transcripts appeared earlier than those of cxcr4a. Thus, the function of mammalian CXCR4 could be split between the two zebrafish genes. These genes probably derived from the genome duplication event, which occurred during the evolution of teleosts. Similar pairs of Cxcr4 may exist in other species, where genome duplication has occurred. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: G-protein coupled receptors; Chemokine; Lateral mesoderm; Genome duplication; Primary neurons; Islet-1
1. Results and discussion CXCR4 is a predominant chemokine receptor detected in vertebrate embryos. Murine CXCR4 has a dynamic expression pattern during organogenesis, particularly in the developing neuronal, cardiac, vascular, hematopoietic and craniofacial systems, suggesting a role for chemokine signaling in multiple embryogenic events (McGrath et al., 1999). In zebrafish, the developmental roles of mammalian CXCR4 appear to be split between two related genes, cxcr4a and cxcr4b. Initially, a clone containing a partial cDNA of the 3 0 terminal of cxcr4b was identified by the expressed sequence tag (EST) approach (Gong et al., 1997). The full size cDNA of this gene was obtained using 5 0 rapid amplification of cDNA ends (RACE)-PCR. The clone containing cxcr4a was found during the search for the full size cxcr4b cDNA. Following sequencing, BLAST analysis indicated a close similarity of deduced zebrafish proteins with the piscine (carp and trout) Cxcr4s (Fig. 1A,B). The putative proteins are less related to the mammalian CXCR4s, however, the level of conservation is relatively high (Fig. 1A), suggesting that the zebrafish
* Corresponding author. Tel.: 165-872-7418; fax: 165-872-7007. E-mail address: [email protected] (V. Korzh).
Cxcr4s may perform a function similar to that of the mammalian CXCR4. The two zebrafish cxcr4s were mapped using the T51 radiation hybrid panel (zebrafish–hamster hybrid cell lines; Research Genetics, USA; Kwok et al., 1998; Geisler et al., 1999). cxcr4a maps to LG6 and cxcr4b to LG9. Detailed information about the primers used is available from the authors upon request. The mapping data suggests that the chromosomal regions containing the two cxcr4s could be evolutionarily related (Fig. 1C,D). These regions of LG6 and LG9 contain three consecutive duplicated genes when compared with human chromosome 2 (Fig. 1D). To our knowledge, there are no mutants mapped to either location of the cxcr4 genes. The transcripts of the two genes were detected immediately after fertilization, suggesting the presence of maternally-derived mRNA of both genes. Transcription of cxcr4a strongly increased after the mid-blastula transition (MBT). In contrast, the transcription of cxcr4b only mildly increased after the MBT (Fig. 2). Later, the increase of transcription at the early neurula stage was observed by in situ hybridization (see below). Whole-mount in situ hybridization and immunohistochemistry were performed as reported (Oxtoby and Jowett, 1993; Dheen et al., 1999). Results of the analyses are presented in Figs. 3–5. In general, the combination of
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00520-2
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Fig. 1. (a,b)
expression patterns of the two zebrafish cxcr4 genes is similar to that of the single mammalian Cxcr4 gene. To simplify this presentation, we compared the expression domains of the two zebrafish genes with that of murine Cxcr4 (McGrath et al., 1999; Table 1). In most cell lineages, the two zebrafish genes have mutually exclusive patterns of expression. For example, cxcr4a is expressed broadly in the neural plate (Fig. 3I,K) and endo-
derm (Fig. 3A–D), and later on in the endothelium (Table 1; and not shown), while cxcr4b is expressed in sensory neurons and kidneys (Figs. 4E and 5D,F). However, in some regions, zebrafish cxcr4s are co-expressed. In the lateral mesoderm, the murine Cxcr4 is expressed only before the formation of somites (McGrath et al., 1999). cxcr4b is expressed at a low level in most, if not all, cells of the lateral mesoderm in the tail bud region (Fig.
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Fig. 1. A comparative analysis of the two zebrafish CXCR4s. (A) Predicted amino acid sequences of the two zebrafish Cxcr4 genes aligned with those of carp, mouse and human. The GenBank accession numbers for zebrafish sequences are – for cxcr4a – AY057095 and for cxcr4b – AY057094 (B) Dendrogram comparison of similarity within a group of vertebrate CXCR4s, where the two zebrafish proteins form a group with CXCR4s of other teleosts. Since all teleosts have probably undergone a genome duplication, it would be reasonable to expect that genomes of other teleosts within this group (trout and carp) must contain at least one more cxcr4 gene. (C) The two zebrafish cxcr4s mapped to LG6 (cxcr4a) and LG9 (cxcr4b) using the radiation hybrid panel T51 (Kwok et al., 1998; Geisler et al., 1999). (D) Based on the results of genetic mapping, we suggest conservation of syntenies among chromosomes containing zebrafish cxcr4 genes. The loci syntenic on individual chromosomes are arranged in columns with apparent orthologues in the mouse (chromosome 1 and chromosome 2) and human (chromosome 2).
4C,F) and increases prior to somite formation (Fig. 4C,D). After the formation of somites, expression is transiently maintained in the relatively undifferentiated anterior part of somites (Fig. 4C). In contrast, the expression of cxcr4a starts more anteriorly in the adaxial cells (Fig. 3G).
Following somite formation, the expression of cxcr4a appears in paraxial cells. Similar to cxcr4b, the expression of cxcr4a is maintained in the anterior part of somites (Fig. 3I). Thus, the expression of cxcr4b in the lateral mesoderm starts early, while that of cxcr4a is delayed and more
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Table 1 Expression of murine and zebrafish Cxcr4 a Tissue
Mice CXCR4 b
Cxcr4a
Cxcr4b
Extraembryonic mesoderm Endoderm Mesoderm
1 1 1 (posterior to the last somite)
NA 1 1 (late adaxial cells, somites)
Sensory neurons Interneurons Motoneurons Cerebellum Midbrain–hindbrain boundary Hypothalamus Telencephalon Eye field Neural crest Anterior axial mesoderm Endothelium (blood vessels)
drg1 NA 1 1 1 NA NA NA 1 1 1
– 1 – – 1 – – 1 1 1 1
NA – 1 (early adaxial cells, somitic primordia, somites) rb cells1 – 1 1 1 1 1 – – – –
a b
NA, not available; drg, dorsal root ganglion; rb, Rohon–Beard; (–), not observed. The expression data on murine Cxcr4 is adapted from McGrath et al. (1999).
restricted, suggesting different developmental roles of these genes in derivatives of the lateral mesoderm. These observations provide evidence to support the idea that after duplication, gene pairs have partitioned ancestral functions between the two copies (Force et al., 1999). The two zebrafish cxcr4 genes split not only tissue-specific, but also developmental, stage-specific regulatory elements. Thus, it seems that during evolution, cxcr4a maintained most of the functions associated with the murine CXCR4. Also, the time course of its expression is more reminiscent of that of mammalian CXCR4. In contrast, cxcr4b acquired some novel early functions. It is expressed
in structures associated with the escaping reaction, such as the primary sensory cells (RB cells), motoneurons and early muscles (Fig. 5E–G). Therefore, cxcr4b could play a role in the evolutionary adaptation mechanism that was developed to accelerate the embryonic development of zebrafish (reviewed in Kimmel and Westerfield, 1990; Korzh, 1994).
Acknowledgements The Mab 3A10 developed by Dr T. Jessell and Dr J.
Fig. 2. Reverse transcription (RT)-PCR detects the continuous presence of transcripts of the two cxcr4s during early development. cxcr4b mRNA is present at a low level during the first 6 h of development. As shown by whole-mount in situ hybridization, its level starts to increase at the early neurula stage (see Fig. 4). cxcr4a transcripts are present at a low level during blastula stage and zygotic transcription is strongly activated at 3.3 hpf. RT-negative control was carried out at the same time and no band was detected (not shown).
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Fig. 3. Embryonic expression of cxcr4a. The expression pattern of cxcr4a includes: (A–D), the early endoderm (en); (G,H), the primordium of Kupffer’s vesicle (pkv); and (E,F), the forebrain (fb). (G) The expression appeared relatively late in adaxial cells (ac). (D) is a sagittal section of the embryo shown in (C). In (F), the white broken lines define a section shown in (E). In (G), the white line defines a section shown in (H). During somitogenesis, cxcr4a is expressed in somites (sm) immediately after their separation from the unsegmented lateral mesodermal plate (black dotted line) and in the tail bud (I). In addition, ventral interneurons (in) express this gene (J). In somites, expression of cxcr4a is confined to the sclerotome (sc) (J). In the tail bud (K), cxcr4a expression is found mainly in the neural plate (np) and endoderm (en). am, axial mesoderm; d, dorsal; es, embryonic shield; hgg, hatching gland; n, notochord; rb, Rohon–Beard sensory cells; v, ventral. Black arrowheads in (D) indicate endodermal cells. The broken black line represents the border of segmented and unsegmented lateral mesoderm. Anterior in all figures is oriented to the left unless otherwise indicated. Broken white lines represent the section level. Scale bar in (A–C), 250 mm; remainder, 50 mm.
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Fig. 4. Expression of cxcr4b during early zebrafish development. Expression of cxcr4b has been detected by whole-mount in situ hybridization mainly in deep cells starting from the blastula stage (A). Later on during early neurulation, the level of expression increases in specific regions of the CNS, including the posterior midbrain, the set of sensory neurons (trigeminal ganglia, dorsal sensory cells, epiphysis and telencephalon) (B,C), anterior neural ridge, ventral diencephalon and ventral midbrain (B). In addition, expression is detected in the posterior lateral mesoderm (C,F), where the level of expression increases just before formation of somites. White dashed lines in (C) indicate the level of cross-sections in reference to (D–F). The broken black line shows the border of segmented and unsegmented lateral mesoderm. The complementary mode of expression of the two genes is most obvious in the tail bud. Here, cxcr4a transcripts are in the neural plate and endodermal precursors, while cxcr4b transcripts are in mesodermal cells lateral to the notochord bud (modified from Figs. 3K and 4F). This results in the bulb of the notochord being surrounded by cells specifically expressing one of the two genes (see schematic in (G)). ac, adaxial cells; anr, anterior neural ridge; ep, epiphysis; kv, Kupffer’s vesicle; k, kidney; m, motoneuron; n, notochord; rb, Rohon–Beard cells; sm, somite; tb, tail bud; tg, trigeminal ganglion; tgm, tegmentum; tc, telencephalon; vd, ventral diencephalon. Bar, 50 mm ((A), 250 mm).
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Fig. 5. Expression of cxcr4b during late zebrafish development. To confirm the identity of cxcr4b-positive cells in various areas of the CNS, we performed double staining using whole-mount in situ hybridization with the cxcr4b probe in combination with other neuronal markers as indicated on the figure. cxcr4b is expressed in the nucleus of the posterior commissure and locus coeruleus (A), as well as brachiomotor neurons of the hindbrain (B,C). The small box inserted in (B) shows an enlarged view of brachiomotor neurons. A cross-section level through r4 is shown in (G). Note the co-expression of cxcr4b and Islet-1 in the posterior part of the epiphysis (D). The lack of expression of cxcr4b in the anterior epiphysis (arrow), where only Islet-1 is expressed, implies that cells in the epiphysis undergo differentiation according to the same antero-posterior (A–P) time course as the rest of the CNS. (E,F) cxcr4b co-localizes with Islet-1 in trigeminal ganglion (tg) and Rohon–Beard cells (rb). In the eye (H–J), expression starts in the ventral patch and later continues in retinal ganglial cells. (K,L) The expression of cxcr4b is detected in the anterior (aht) and posterior hypothalamus (pht). (M) In the telencephalon (tc), the most intense expression was detected in the olfactory placode (op). In the dorsal diencephalon, expression occurs in the epiphysis (ep) and nuclei of posterior commissure (npc). bmn, brachiomotor neurons; ep, epiphysis; lc, locus coeroleus; ma, Mauthner neuron; op, olfactory placode; mhb, midbrain–hindbrain boundary; nmlf, nucleus of medial longitudinal fascicle; npc, nuclei of posterior commissure; pllo, primordium lateral line organ; r, rhombomeres; rgc, retinal ganglial cells; tc, telencephalon; vp, ventral patch; PTU, embryos treated with 1-phenyl-2-thiourea. Bar, 50 mm.
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Dodd was obtained from The Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. The authors are obliged to Dr R. Geisler and Dr G.-J. Rauch for help in mapping genes on the T51 panel and Dr Michael Richardson and Ms Lee Thean Chu for careful reading of this paper. This work is supported by a research grant from the National Science and Technology Board of Singapore to V.K. References Dheen, S.T., Sleptsova-Friedrich, I., Xu, Y., Clark, M., Gong, Z., Lehrach, H., Korzh, V., 1999. Zebrafish tbx-c functions during formation of midline structures. Development 126, 2703–2713. Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.-L., Postlethwait, J., 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151, 1531–1545. Geisler, R., Rauch, G., Baier, H., van Bebber, F., Brobeta, L., Dekens, M., Finger, K., Fricke, C., Gates, M., Geiger, H., Geiger-Rudolph, S.,
Gilmour, D., Glaser, S., Gnugge, L., Habeck, H., Hingst, K., Holley, S., Keenan, J., Kirn, A., Knaut, H., Lashkari, D., Maderspacher, F., Martyn, U., Neuhauss, S., Neumann, C., Nicolson, T., Pelegri, F., Ray, R., Rick, J.M., Roehl, H., Roeser, T., Schauerte, H.E., Schier, A.F., Schonberger, U., Schonthaler, H., Schulte-Merker, S., Seydler, C., Talbot, W., Weiler, C., Nusslein-Volhard, C., Haffter, P., 1999. A radiation hybrid map of the zebrafish genome. Nat. Genet. 23, 86–89. Gong, Z., Yan, T., Liao, J., Lee, S., He, J., Hew, C., 1997. Rapid identification and isolation of zebrafish cDNA clones. Gene 201, 87–98. Kimmel, C., Westerfield, M., 1990. Primary neurons of the zebrafish. In: Edelman, G., Gall, W., Gowan, W. (Eds.). Signals and Sense, Wiley Liss, New York, pp. 561–588. Korzh, V., 1994. Genetic control of early neuronal development in vertebrates. Curr. Opin. Neurobiol. 4, 21–28. Kwok, C., Korn, R., Davis, M., Burt, D., Critcher, R., McCarthy, L., Paw, B., Zon, L., Goodfellow, P., Schmitt, K., 1998. Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates. Nucleic Acids Res. 26, 3562–3566. McGrath, K., Koniski, A., Maltbi, K., McGann, J., Palis, J., 1999. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213, 442–456. Oxtoby, E., Jowett, T., 1993. Cloning of the zebrafish krox-20 gene and its expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095.
Gene expression pattern
Expression pattern of two zebrafish genes, cxcr4a and cxcr4b Shang Wei Chong a, Alexander Emelyanov a, Zhiyuan Gong b, Vladimir Korzh a,b,* a
Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore
b
Received 25 June 2001; received in revised form 13 August 2001; accepted 13 August 2001
Abstract We cloned and mapped two novel zebrafish genes, cxcr4a and cxcr4b, which are closely related to mammalian CXCR4. Expression analysis by reverse transcription-polymerase chain reaction and in situ hybridization demonstrated that these two genes are expressed in most cell lineages known to express Cxcr4 in mammals. These genes are co-expressed in lateral mesoderm and posterior midbrain. The transcripts of cxcr4a were detected in interneurons and endoderm, whereas cxcr4b was specifically expressed in sensory neurons, motoneurons and cerebellum. In the lateral mesoderm, cxcr4b transcripts appeared earlier than those of cxcr4a. Thus, the function of mammalian CXCR4 could be split between the two zebrafish genes. These genes probably derived from the genome duplication event, which occurred during the evolution of teleosts. Similar pairs of Cxcr4 may exist in other species, where genome duplication has occurred. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: G-protein coupled receptors; Chemokine; Lateral mesoderm; Genome duplication; Primary neurons; Islet-1
1. Results and discussion CXCR4 is a predominant chemokine receptor detected in vertebrate embryos. Murine CXCR4 has a dynamic expression pattern during organogenesis, particularly in the developing neuronal, cardiac, vascular, hematopoietic and craniofacial systems, suggesting a role for chemokine signaling in multiple embryogenic events (McGrath et al., 1999). In zebrafish, the developmental roles of mammalian CXCR4 appear to be split between two related genes, cxcr4a and cxcr4b. Initially, a clone containing a partial cDNA of the 3 0 terminal of cxcr4b was identified by the expressed sequence tag (EST) approach (Gong et al., 1997). The full size cDNA of this gene was obtained using 5 0 rapid amplification of cDNA ends (RACE)-PCR. The clone containing cxcr4a was found during the search for the full size cxcr4b cDNA. Following sequencing, BLAST analysis indicated a close similarity of deduced zebrafish proteins with the piscine (carp and trout) Cxcr4s (Fig. 1A,B). The putative proteins are less related to the mammalian CXCR4s, however, the level of conservation is relatively high (Fig. 1A), suggesting that the zebrafish
* Corresponding author. Tel.: 165-872-7418; fax: 165-872-7007. E-mail address: [email protected] (V. Korzh).
Cxcr4s may perform a function similar to that of the mammalian CXCR4. The two zebrafish cxcr4s were mapped using the T51 radiation hybrid panel (zebrafish–hamster hybrid cell lines; Research Genetics, USA; Kwok et al., 1998; Geisler et al., 1999). cxcr4a maps to LG6 and cxcr4b to LG9. Detailed information about the primers used is available from the authors upon request. The mapping data suggests that the chromosomal regions containing the two cxcr4s could be evolutionarily related (Fig. 1C,D). These regions of LG6 and LG9 contain three consecutive duplicated genes when compared with human chromosome 2 (Fig. 1D). To our knowledge, there are no mutants mapped to either location of the cxcr4 genes. The transcripts of the two genes were detected immediately after fertilization, suggesting the presence of maternally-derived mRNA of both genes. Transcription of cxcr4a strongly increased after the mid-blastula transition (MBT). In contrast, the transcription of cxcr4b only mildly increased after the MBT (Fig. 2). Later, the increase of transcription at the early neurula stage was observed by in situ hybridization (see below). Whole-mount in situ hybridization and immunohistochemistry were performed as reported (Oxtoby and Jowett, 1993; Dheen et al., 1999). Results of the analyses are presented in Figs. 3–5. In general, the combination of
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00520-2
348
S. Wei Chong et al. / Mechanisms of Development 109 (2001) 347–354
Fig. 1. (a,b)
expression patterns of the two zebrafish cxcr4 genes is similar to that of the single mammalian Cxcr4 gene. To simplify this presentation, we compared the expression domains of the two zebrafish genes with that of murine Cxcr4 (McGrath et al., 1999; Table 1). In most cell lineages, the two zebrafish genes have mutually exclusive patterns of expression. For example, cxcr4a is expressed broadly in the neural plate (Fig. 3I,K) and endo-
derm (Fig. 3A–D), and later on in the endothelium (Table 1; and not shown), while cxcr4b is expressed in sensory neurons and kidneys (Figs. 4E and 5D,F). However, in some regions, zebrafish cxcr4s are co-expressed. In the lateral mesoderm, the murine Cxcr4 is expressed only before the formation of somites (McGrath et al., 1999). cxcr4b is expressed at a low level in most, if not all, cells of the lateral mesoderm in the tail bud region (Fig.
S. Wei Chong et al. / Mechanisms of Development 109 (2001) 347–354
349
Fig. 1. A comparative analysis of the two zebrafish CXCR4s. (A) Predicted amino acid sequences of the two zebrafish Cxcr4 genes aligned with those of carp, mouse and human. The GenBank accession numbers for zebrafish sequences are – for cxcr4a – AY057095 and for cxcr4b – AY057094 (B) Dendrogram comparison of similarity within a group of vertebrate CXCR4s, where the two zebrafish proteins form a group with CXCR4s of other teleosts. Since all teleosts have probably undergone a genome duplication, it would be reasonable to expect that genomes of other teleosts within this group (trout and carp) must contain at least one more cxcr4 gene. (C) The two zebrafish cxcr4s mapped to LG6 (cxcr4a) and LG9 (cxcr4b) using the radiation hybrid panel T51 (Kwok et al., 1998; Geisler et al., 1999). (D) Based on the results of genetic mapping, we suggest conservation of syntenies among chromosomes containing zebrafish cxcr4 genes. The loci syntenic on individual chromosomes are arranged in columns with apparent orthologues in the mouse (chromosome 1 and chromosome 2) and human (chromosome 2).
4C,F) and increases prior to somite formation (Fig. 4C,D). After the formation of somites, expression is transiently maintained in the relatively undifferentiated anterior part of somites (Fig. 4C). In contrast, the expression of cxcr4a starts more anteriorly in the adaxial cells (Fig. 3G).
Following somite formation, the expression of cxcr4a appears in paraxial cells. Similar to cxcr4b, the expression of cxcr4a is maintained in the anterior part of somites (Fig. 3I). Thus, the expression of cxcr4b in the lateral mesoderm starts early, while that of cxcr4a is delayed and more
350
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Table 1 Expression of murine and zebrafish Cxcr4 a Tissue
Mice CXCR4 b
Cxcr4a
Cxcr4b
Extraembryonic mesoderm Endoderm Mesoderm
1 1 1 (posterior to the last somite)
NA 1 1 (late adaxial cells, somites)
Sensory neurons Interneurons Motoneurons Cerebellum Midbrain–hindbrain boundary Hypothalamus Telencephalon Eye field Neural crest Anterior axial mesoderm Endothelium (blood vessels)
drg1 NA 1 1 1 NA NA NA 1 1 1
– 1 – – 1 – – 1 1 1 1
NA – 1 (early adaxial cells, somitic primordia, somites) rb cells1 – 1 1 1 1 1 – – – –
a b
NA, not available; drg, dorsal root ganglion; rb, Rohon–Beard; (–), not observed. The expression data on murine Cxcr4 is adapted from McGrath et al. (1999).
restricted, suggesting different developmental roles of these genes in derivatives of the lateral mesoderm. These observations provide evidence to support the idea that after duplication, gene pairs have partitioned ancestral functions between the two copies (Force et al., 1999). The two zebrafish cxcr4 genes split not only tissue-specific, but also developmental, stage-specific regulatory elements. Thus, it seems that during evolution, cxcr4a maintained most of the functions associated with the murine CXCR4. Also, the time course of its expression is more reminiscent of that of mammalian CXCR4. In contrast, cxcr4b acquired some novel early functions. It is expressed
in structures associated with the escaping reaction, such as the primary sensory cells (RB cells), motoneurons and early muscles (Fig. 5E–G). Therefore, cxcr4b could play a role in the evolutionary adaptation mechanism that was developed to accelerate the embryonic development of zebrafish (reviewed in Kimmel and Westerfield, 1990; Korzh, 1994).
Acknowledgements The Mab 3A10 developed by Dr T. Jessell and Dr J.
Fig. 2. Reverse transcription (RT)-PCR detects the continuous presence of transcripts of the two cxcr4s during early development. cxcr4b mRNA is present at a low level during the first 6 h of development. As shown by whole-mount in situ hybridization, its level starts to increase at the early neurula stage (see Fig. 4). cxcr4a transcripts are present at a low level during blastula stage and zygotic transcription is strongly activated at 3.3 hpf. RT-negative control was carried out at the same time and no band was detected (not shown).
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Fig. 3. Embryonic expression of cxcr4a. The expression pattern of cxcr4a includes: (A–D), the early endoderm (en); (G,H), the primordium of Kupffer’s vesicle (pkv); and (E,F), the forebrain (fb). (G) The expression appeared relatively late in adaxial cells (ac). (D) is a sagittal section of the embryo shown in (C). In (F), the white broken lines define a section shown in (E). In (G), the white line defines a section shown in (H). During somitogenesis, cxcr4a is expressed in somites (sm) immediately after their separation from the unsegmented lateral mesodermal plate (black dotted line) and in the tail bud (I). In addition, ventral interneurons (in) express this gene (J). In somites, expression of cxcr4a is confined to the sclerotome (sc) (J). In the tail bud (K), cxcr4a expression is found mainly in the neural plate (np) and endoderm (en). am, axial mesoderm; d, dorsal; es, embryonic shield; hgg, hatching gland; n, notochord; rb, Rohon–Beard sensory cells; v, ventral. Black arrowheads in (D) indicate endodermal cells. The broken black line represents the border of segmented and unsegmented lateral mesoderm. Anterior in all figures is oriented to the left unless otherwise indicated. Broken white lines represent the section level. Scale bar in (A–C), 250 mm; remainder, 50 mm.
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Fig. 4. Expression of cxcr4b during early zebrafish development. Expression of cxcr4b has been detected by whole-mount in situ hybridization mainly in deep cells starting from the blastula stage (A). Later on during early neurulation, the level of expression increases in specific regions of the CNS, including the posterior midbrain, the set of sensory neurons (trigeminal ganglia, dorsal sensory cells, epiphysis and telencephalon) (B,C), anterior neural ridge, ventral diencephalon and ventral midbrain (B). In addition, expression is detected in the posterior lateral mesoderm (C,F), where the level of expression increases just before formation of somites. White dashed lines in (C) indicate the level of cross-sections in reference to (D–F). The broken black line shows the border of segmented and unsegmented lateral mesoderm. The complementary mode of expression of the two genes is most obvious in the tail bud. Here, cxcr4a transcripts are in the neural plate and endodermal precursors, while cxcr4b transcripts are in mesodermal cells lateral to the notochord bud (modified from Figs. 3K and 4F). This results in the bulb of the notochord being surrounded by cells specifically expressing one of the two genes (see schematic in (G)). ac, adaxial cells; anr, anterior neural ridge; ep, epiphysis; kv, Kupffer’s vesicle; k, kidney; m, motoneuron; n, notochord; rb, Rohon–Beard cells; sm, somite; tb, tail bud; tg, trigeminal ganglion; tgm, tegmentum; tc, telencephalon; vd, ventral diencephalon. Bar, 50 mm ((A), 250 mm).
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Fig. 5. Expression of cxcr4b during late zebrafish development. To confirm the identity of cxcr4b-positive cells in various areas of the CNS, we performed double staining using whole-mount in situ hybridization with the cxcr4b probe in combination with other neuronal markers as indicated on the figure. cxcr4b is expressed in the nucleus of the posterior commissure and locus coeruleus (A), as well as brachiomotor neurons of the hindbrain (B,C). The small box inserted in (B) shows an enlarged view of brachiomotor neurons. A cross-section level through r4 is shown in (G). Note the co-expression of cxcr4b and Islet-1 in the posterior part of the epiphysis (D). The lack of expression of cxcr4b in the anterior epiphysis (arrow), where only Islet-1 is expressed, implies that cells in the epiphysis undergo differentiation according to the same antero-posterior (A–P) time course as the rest of the CNS. (E,F) cxcr4b co-localizes with Islet-1 in trigeminal ganglion (tg) and Rohon–Beard cells (rb). In the eye (H–J), expression starts in the ventral patch and later continues in retinal ganglial cells. (K,L) The expression of cxcr4b is detected in the anterior (aht) and posterior hypothalamus (pht). (M) In the telencephalon (tc), the most intense expression was detected in the olfactory placode (op). In the dorsal diencephalon, expression occurs in the epiphysis (ep) and nuclei of posterior commissure (npc). bmn, brachiomotor neurons; ep, epiphysis; lc, locus coeroleus; ma, Mauthner neuron; op, olfactory placode; mhb, midbrain–hindbrain boundary; nmlf, nucleus of medial longitudinal fascicle; npc, nuclei of posterior commissure; pllo, primordium lateral line organ; r, rhombomeres; rgc, retinal ganglial cells; tc, telencephalon; vp, ventral patch; PTU, embryos treated with 1-phenyl-2-thiourea. Bar, 50 mm.
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