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Expression pattern of neuronal nitric oxide synthase in embryonic zebrafish
Gene Expression Patterns 3 (2003) 463–466 www.elsevier.com/locate/modgep
Expression pattern of neuronal nitric oxide synthase in embryonic zebrafish Kar Lai Poona, Michael Richardsona, Chen Sok Lama, Hoon Eng Khoob, Vladimir Korzha,c,* a Institute of Molecular and Cell Biology, 1 Research Link, Singapore, Singapore Department of Biochemistry, National University of Singapore, Singapore, Singapore c Department of Biological Sciences, National University of Singapore, Singapore, Singapore b
Received 15 January 2003; received in revised form 10 March 2003; accepted 13 March 2003
Abstract Nitric oxide synthase catalyzes the production of nitric oxide, a multifunctional signaling molecule that affects diverse aspects of animal physiology such as cell proliferation, differentiation, neurotransmission and apoptosis. Here, we report the cloning and expression pattern of the zebrafish nnos. This gene was mapped to zebrafish linkage group 5. The spatial and temporal expression pattern of nnos in embryonic zebrafish was analyzed by whole mount in situ hybridization. nnos is widely expressed in the embryonic nervous system. Expression of zebrafish nnos appeared at 16 hours post-fertilization in the hypothalamus and by 3 days post-fertilization was present in discrete locations in the central nervous system as well as the enteric nervous system. Some nnos-positive cells were mapped to specific locations in the central nervous system using tyrosine hydroxylase as a specific marker indicating that nnos transcripts were present in the olfactory bulb, anterior diencephalon, posterior hypothalamus and anterior hindbrain. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Hypothalamus; Tyrosine hydroxylase; Axonogenesis; Catecholaminergic neurons; Enteric neurons
1. Results and discussion Nitric oxide (NO) and the enzyme, which controls production of NO in the nervous system, neuronal nitric oxide synthase (nNOS) have been implicated in multiple aspects of neuronal development and activity (reviewed in Christopherson and Bredt, 1997). During development these factors have been linked to the control of the transition of neural precursor cells from proliferation to differentiation (Kuzin et al., 1996, 2000; Peunova et al., 2001) and formation of synaptic connections (Verge et al., 1992; Herdegen et al., 1993; Roskams et al., 1994; Siedel and Bicker, 2000; Wu et al., 2000, 2001). Since a complete range of activities of NO and nitric oxide synthase (NOS) in vertebrates is far from being understood, a comparatively simpler organization of the central nervous system (CNS) was attractive enough to initiate studies in the zebrafish aiming to analyze the role of * Corresponding author. Institute of Molecular and Cell Biology, 1 Research Link, 117604 Singapore. Tel: þ 65-68727418; fax: þ 6567791117. E-mail address: [email protected] (V. Korzh).
NO and NOS during formation of the CNS. Expression of nNOS in zebrafish has been studied in the retina by immunological techniques (Shin et al., 2000), which showed that nNOS is expressed in the ganglial cell layer (GCL), the inner (IP) and outer plexiform (OP) layers and the photoreceptor layer (PR). Further, a partial clone of nnos was obtained and its expression in the adult zebrafish characterized demonstrating distribution of its transcripts in the distinct central nuclei and proliferation zones (Holmqvist et al., 2000). Here we extended this analysis by characterizing a complete cDNA encoding zebrafish nNOS, mapping this gene and describing its expression pattern in detail during embryonic development of the zebrafish. The EST database contained information only about short stretches of sequence homologous to NOS-encoding genes of other species. Thus, we employed 50 - and 30 -RACE PCR to generate the complete cDNA of the zebrafish nnos. This led to the isolation of a 4552 bp zebrafish nnos cDNA clone, including a complete coding region of 4293 bp and a 30 untranslated region of 247 bp (GenBank accession no. AY211528). The putative zebrafish nNos polypeptide is
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00063-2
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1432 amino acid long. Protein alignment demonstrated that the zebrafish nNos is 86% homologous to fugu nNos, 82% homologous to human nNOS and 83% homologous to murine nNos (not shown). The zebrafish nnos was mapped using the T51 radiationhybrid panel (zebrafish-hamster hybrid cell lines; Research Genetics, USA; Kwok et al., 1998; Geisler et al., 1999) to LG5 between z26471 and pescadillo (not shown). Detailed information about the primers used for mapping is available from authors upon request. The mapping data suggested that the organization of the chromosomal regions containing nNOS encoding genes have been preserved during evolution of vertebrates (not shown). Expression of nnos was first detected at 16 hours postfertilization (hpf) in the ventral forebrain (not shown), which coincided with the beginning of primary axonogenesis (Chitnis and Kuwada, 1990; Wilson et al., 1990; Ross et al., 1992) and is several hours after the beginning of neural differentiation (Korzh et al., 1993; Blader et al., 1997; Korzh et al., 1998). Since the expression pattern of nnos was similar to that of tyrosine hydroxylase (TH), we decided to compare the expression of these two markers using a combination of WISH and immunostaining. The distribution of TH-positive cells was recently characterized in the embryonic zebrafish in detail (Holzchuh et al., 2001; Kaslin and Panula, 2001; Lam, 2002; Lam et al., 2003; Rink and Wullimann, 2002). The expression domains of nnos were much wider compared to expression of TH and were located adjacent to TH-expressing cells. After 16 hpf, expression of nnos in the ventral forebrain increased and by
24 hpf became very intense (Fig. 1A). Expression was detected in the anterio-ventral diencephalon, where the ventro-rostral cluster of Islet1-positive cells has been described (Korzh et al., 1993), which is one of the first neuronal clusters to initiate axonogenesis. It establishes the ventral tract of post-optic commissure (TPOC), which more posteriorly contributes into the medial-longitudinal fascicle (MLF) (Ross et al., 1992; Kimmel, 1993). This domain of nnos expression appeared earlier than expression of TH and was located anteriorly to the first cluster of TH-positive neurons in the ventral diencephalon (Holzchuh et al., 2001). Expression of TH indicated that these cells had initiated production of catecholamines used in the CNS as neurotransmitters (reviewed in Reiner, 1994). Thus, nnos-positive cells were adjacent to cells that had entered a program of differentiation resulting in the formation of catecholaminergic neurons. In addition, expression domains with low levels of nnos transcripts were detected in the ventral telencephalon and tegmentum (Fig. 1A). Expression of nnos in these clusters increased at 36 hpf (Fig. 1C). By 48 hpf (Fig. 1D) nnos expression domains were found in the subventricular zone of the telencephalon, the dorsal hypothalamus, midbrain and hindbrain, including the locus coeruleus (LC), which projects into the lateral longitudinal fascicle (LLF; Ma, 1994; Lam et al., 2003). In all these positions nnos-positive cells were found in close proximity to TH-positive cells (Fig. 1D). The mutant mindbomb (mib2/2 ) shows an increase in the number of neurons (Hong et al., 2002). In this mutant, nnos expression increased dramatically in the ventral
Fig. 1. Expression of nnos in zebrafish embryos as detected by WISH. The distribution of nnos mRNA (blue) was compared to that of TH (brown). (A) Twentyfour hpf embryo showing strong nnos expression anterior to the diencephalic cluster of TH-positive cells in the hypothalamus and weaker expression in the ventral telencephalon and tegmentum. The inset shows a dorsal view of the hypothalamus. (B) mindbomb (mib) mutant at 24 hpf shows ectopic expression of nnos in the dorsal telencephalon and the up-regulation and expansion of expression domains in the diencephalon. (C) 36 hpf embryo showing that the nnospositive cells in the diencephalon maintain an anterior position compared to TH-expressing cells. The inset shows a dorsal view of the hypothalamus at higher magnification. (D) Forty-eight hpf embryo indicating nnos transcripts in the telencephalon, midbrain and the LC in the anterior hindbrain. The inset shows a higher magnification lateral view of the LC. Abbreviations: d, diencephalon; ht, hypothalamus; lc, locus coeruleus; ot, optic tectum; poa, pre-optic area; t, telencephalon; tg, tegmentum. Scale bar—50 mm.
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telencephalon, hypothalamus and tegmentum suggesting that during this developmental stage nnos-positive cells probably represent neurons (Fig. 1B). Intense expression of nnos was also detected in the dorsal telencephalon. At 72 hpf, expression of nnos became more complicated in the brain (Fig. 2A) and also expanded into the trunk (Fig. 2B, I) where nnos-positive cells were found in the ventral neural tube and intestine. To resolve the expression pattern at this stage, we analyzed it on cross sections. Expression of nnos was found in all major regions of the brain. In several regions expression of this gene was
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associated with expression of TH, including the olfactory bulb (Fig. 2C), pre-optic area (Fig. 2E), areas of diencephalon (Fig. 2G), LC in the anterior hindbrain (Fig. 2H) and the inner nuclear layer of the neuroretina (Fig. 2J). In some regions within the telencephalon, midbrain, spinal cord, intestine and GCL of the retina (Fig. 2D, F, I, J) no TH-positive cells were detected in association with expression domains of nnos. The expression of nnos in cell types other than those associated with differentiation of TH-positive cells indicated that other classes of neurons had started to express nnos.
Fig. 2. Expression of nnos in zebrafish embryos during hatching. (A) Brain, (B) trunk. Arrowhead in A and B indicates the spinal cord. Arrows in B show cells of the enteric nervous system. The anterior–posterior position of cross sections through the brain (A) and trunk (B) of a 72 hpf embryo is indicated. Crosssection analysis shows discrete clusters of nnos-positive cells in the telencephalon (C), sub-pallium of telencephalon (D), pre-optic area (E) and posterior hypothalamus (F,G) and intense nnos expression immediately below the ventricular layer in the anterior hindbrain (H). In the trunk (I) nnos transcripts present in secondary motor neurons (arrowhead) above the floorplate (red arrow) and in two bilateral rows of cells corresponding to myenteric neurons in the gut or adjacent tissues (arrows in B,I). Cross section through the eye (J) shows intense nnos expression in the amacrine cells present in the inner nuclear layer of the retina with less intense nnos expression in the GCL. (J– M) nnos-positive cells intermingle with and are also adjacent to TH-positive cells in the inner nuclear layer of the retina (J), and in several regions of the brain–olfactory bulb (C,K), pre-optic area (E,L) and the posterior tuberculum (G,M). Some cells show the presence of both nnos and TH (arrowhead in K –M). Yolk and eyes were removed from the embryo for clearer observation in lateral view (A). Anterior is to the left and dorsal is to the top in all pictures. All cross sections are shown with dorsal to the top. All insets were marked with a letter corresponding to a frame in a bottom row. Abbreviations: d, diencephalon; dp, dorsal pallium; fp, floor plate; g, gut; gcl, ganglion cell layer; hb, hindbrain; hbl, habenula; ht, hypothalamus; inl, inner nuclear layer of retina; lc, locus coeruleus; n, notochord; nsc, nucleus suprachiasmatic; ob, olfactory bulb; on, optic nerve; op, olfactory placode; ot, optic tectum; poa, pre-optic area; poc, post-optic commissure; pt, posterior tuberculum; s, somite; sc, spinal cord; sp, sub-pallium; t, telencephalon; tg, tegmentum; vt, ventral thalamus. Scale bar—50 mm.
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This latter case is represented by expression in the spinal cord (Fig. 2B, I) in cells above the floor plate. These cells may represent secondary motor neurons. Thus, expression of nnos at 72 hpf is characterized by its appearance in many classes of neurons, including myenteric neurons outside of the CNS in the gut (Fig. 2B, I).
Acknowledgements We are obliged to Drs N. Peunova and G. Enikolopov for fruitful discussion during the early phase of this project. Dr Yun-Jin Jiang provided us with mindbomb mutants. VK’s lab is supported by a research grant from the Agency for Science, Technology and Research (A-STAR) of Singapore.
References Blader, P., Fischer, N., Gradwohl, G., Guillemot, F., Stra¨hle, U., 1997. The activity of Neurogenin1 is controlled by local cues in the zebrafish embryo. Development 124, 4557–4569. Chitnis, A.B., Kuwada, J.Y., 1990. Axonogenesis in the brain of zebrafish embryos. J. Neurosci. 10, 1892–1905. Christopherson, K., Bredt, D., 1997. Nitric oxide in exitable tissues: physiological roles and disease. J. Clin. Invest. 100, 2424–2429. Geisler, R., Rauch, G.J., Baier, H., van Bebber, F., Brobeta, L., Dekens, M.P., Finger, K., Fricke, C., Gates, M.A., 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., Scho¨nberger, U., Scho¨nthaler, H.-B., Schulte-Merker, S., Seydler, C., Talbot, W.S., Weiler, C., Nu¨sslein-Volhard, C., Haffter, P., 1999. A radiation hybrid map of the zebrafish genome. Nat. Genet. 23, 86–89. Herdegen, T., Brecht, S., Mayer, B., Leah, J., Kummer, W., Bravo, R., Zimmermann, M., 1993. Long lasting expression of JUN and KROX transcription factors and nitric oxide synthase in intrinsic neurons of the rat brain following axotomy. J. Neurosci. 13, 4130–4145. Holmqvist, B., Ellingsen, B., Alm, P., Forsell, J., Oyan, A.M., Goksoyr, A., Fjose, A., Seo, H.C., 2000. Identification and distribution of nitric oxide synthase in the brain of adult zebrafish. Neurosci. Lett. 292, 119–122. Holzchuh, J., Ryu, S., Aberger, F., Driever, W., 2001. Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech. Dev. 101, 237 –243. Hong, S.K., Kim, C.-H., Yoo, K.-W., Kim, H.S., Kudoh, T., Dawid, I.B., Huh, T.L., 2002. Isolation and expression of a novel neuron-specific onecut homeobox gene in zebrafish. Mech. Dev. 112, 199 –202. Kaslin, J., Panula, P., 2001. Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J. Comp. Neurol. 440, 342–377. Kimmel, C., 1993. Patterning the brain of the zebrafish embryo. Ann. Rev. Neurosci. 16, 707 –732. Korzh, V., Edlund, T., Thor, S., 1993. Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation. Development 118, 417 –425.
Korzh, V., Sleptsova, I., Liao, J., He, J.Y., Gong, Z., 1998. Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn. 213, 92–104. Kuzin, B., Roberts, J., Peunova, N., Enikolopov, G., 1996. Nitric oxide regulates cell proliferation during Drosophila development. Cell 87, 639 –649. Kuzin, B., Regulski, M., Stasiv, Y., Scheinker, V., Tully, T., Enikolopov, G., 2000. Nitric oxide interacts with the retinoblastoma pathway to control eye development in Drosophila. Curr. Biol. 10, 459–462. Kwok, C., Korn, R.M., Davis, M.E., Burt, D.W., Critcher, R., McCarthy, L., Paw, B.H., Zon, L.I., Goodfellow, P.N., Schmitt, K., 1998. Characterization of whole genome radiation hybrid mapping resources for nonmammalian vertebrates. Nucleic Acids Res 26, 3562–3566. Lam, C.S., 2002. Embryonic neuroanatomy and the localization of the tyrosine hydroxylase immunoreactive neurons in the zebrafish, Danio rerio. MSc thesis, Department of Biological Sciences, National University of Singapore. Lam, C.S., Sleptsova-Friedrich, I., Munro, A.D., Korzh, V., 2003. Developmental analysis of signaling pathways involved in induction of catecholaminergic neurons in the locus coeruleus. Mol. Cell Neurosci. 22, 501–515. Ma, P.M., 1994. Catecholaminergic systems in the zebrafish. II. Projection pathways and pattern of termination of the locus coeruleus. J. Comp. Neurol. 344, 256–269. Peunova, N., Scheinker, V., Cline, H., Enikolopov, G., 2001. Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain. J. Neurosci. 21, 8809–8818. Reiner, A., 1994. The study of catecholaminergic perikarya and fibres in the nervous system: methodological considerations and technical limitations. In: Smeets, W.J.A.J., Reiner, A. (Eds.), Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates, Cambridge University Press, Cambridge, pp. 1– 6. Rink, E., Wullimann, M.F., 2002. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Brain Res. Dev. Brain Res. 137, 89–100. Roskams, A., Bredt, D., Dawson, T., Ronnett, G., 1994. Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons. Neuron 13, 289–299. Ross, L.S., Parrett, T., Easter, S.S. Jr, 1992. Axonogenesis and morphogenesis in the embryonic zebrafish brain. J. Neurosci. 12, 467 –482. Siedel, C., Bicker, G., 2000. Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons. Development 127, 4541–4549. Shin, D.H., Lim, H.S., Cho, S.K., Lee, H.Y., Lee, H.W., Lee, K.H., Chung, Y.H., Cho, S.S., Ik Cha, C., Hwang, D.H., 2000. Immunocytochemical localization of neuronal and inducible nitric oxide synthase in the retina of zebrafish, Brachydanio rerio. Neurosci. Lett. 292, 220– 222. Verge, V., Xu, Z., Xu, X.J., Weisenfeld-Hallin, Z., Ho¨kfelt, T., 1992. Marked increase in nitric oxide synthase mRNA in the rat dorsal root ganglia after peripheral axotomy: in situ hybridization and functional studies. Proc. Natl. Acad. Sci. USA 89, 11617–11621. Wilson, S.W., Ross, L.S., Parrett, T., Easter, S.S. Jr, 1990. The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio. Development 108, 121 –145. Wu, H., Cork, R., Huang, P., Shuman, D., Mize, R., 2000. Refinement of the ipsilateral retinocollicular projection is disrupted in double endothelial and neuronal nitric oxide synthase gene knockout mice. Dev. Brain Res. 120, 105–111. Wu, H., Selski, D., El-Fakahany, E., McLoon, S., 2001. The role of nitric oxide in development of topographic precision in the retinotectal projection of chick. J Neurosci 21, 4318–4325.
Expression pattern of neuronal nitric oxide synthase in embryonic zebrafish Kar Lai Poona, Michael Richardsona, Chen Sok Lama, Hoon Eng Khoob, Vladimir Korzha,c,* a Institute of Molecular and Cell Biology, 1 Research Link, Singapore, Singapore Department of Biochemistry, National University of Singapore, Singapore, Singapore c Department of Biological Sciences, National University of Singapore, Singapore, Singapore b
Received 15 January 2003; received in revised form 10 March 2003; accepted 13 March 2003
Abstract Nitric oxide synthase catalyzes the production of nitric oxide, a multifunctional signaling molecule that affects diverse aspects of animal physiology such as cell proliferation, differentiation, neurotransmission and apoptosis. Here, we report the cloning and expression pattern of the zebrafish nnos. This gene was mapped to zebrafish linkage group 5. The spatial and temporal expression pattern of nnos in embryonic zebrafish was analyzed by whole mount in situ hybridization. nnos is widely expressed in the embryonic nervous system. Expression of zebrafish nnos appeared at 16 hours post-fertilization in the hypothalamus and by 3 days post-fertilization was present in discrete locations in the central nervous system as well as the enteric nervous system. Some nnos-positive cells were mapped to specific locations in the central nervous system using tyrosine hydroxylase as a specific marker indicating that nnos transcripts were present in the olfactory bulb, anterior diencephalon, posterior hypothalamus and anterior hindbrain. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Hypothalamus; Tyrosine hydroxylase; Axonogenesis; Catecholaminergic neurons; Enteric neurons
1. Results and discussion Nitric oxide (NO) and the enzyme, which controls production of NO in the nervous system, neuronal nitric oxide synthase (nNOS) have been implicated in multiple aspects of neuronal development and activity (reviewed in Christopherson and Bredt, 1997). During development these factors have been linked to the control of the transition of neural precursor cells from proliferation to differentiation (Kuzin et al., 1996, 2000; Peunova et al., 2001) and formation of synaptic connections (Verge et al., 1992; Herdegen et al., 1993; Roskams et al., 1994; Siedel and Bicker, 2000; Wu et al., 2000, 2001). Since a complete range of activities of NO and nitric oxide synthase (NOS) in vertebrates is far from being understood, a comparatively simpler organization of the central nervous system (CNS) was attractive enough to initiate studies in the zebrafish aiming to analyze the role of * Corresponding author. Institute of Molecular and Cell Biology, 1 Research Link, 117604 Singapore. Tel: þ 65-68727418; fax: þ 6567791117. E-mail address: [email protected] (V. Korzh).
NO and NOS during formation of the CNS. Expression of nNOS in zebrafish has been studied in the retina by immunological techniques (Shin et al., 2000), which showed that nNOS is expressed in the ganglial cell layer (GCL), the inner (IP) and outer plexiform (OP) layers and the photoreceptor layer (PR). Further, a partial clone of nnos was obtained and its expression in the adult zebrafish characterized demonstrating distribution of its transcripts in the distinct central nuclei and proliferation zones (Holmqvist et al., 2000). Here we extended this analysis by characterizing a complete cDNA encoding zebrafish nNOS, mapping this gene and describing its expression pattern in detail during embryonic development of the zebrafish. The EST database contained information only about short stretches of sequence homologous to NOS-encoding genes of other species. Thus, we employed 50 - and 30 -RACE PCR to generate the complete cDNA of the zebrafish nnos. This led to the isolation of a 4552 bp zebrafish nnos cDNA clone, including a complete coding region of 4293 bp and a 30 untranslated region of 247 bp (GenBank accession no. AY211528). The putative zebrafish nNos polypeptide is
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00063-2
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1432 amino acid long. Protein alignment demonstrated that the zebrafish nNos is 86% homologous to fugu nNos, 82% homologous to human nNOS and 83% homologous to murine nNos (not shown). The zebrafish nnos was mapped using the T51 radiationhybrid panel (zebrafish-hamster hybrid cell lines; Research Genetics, USA; Kwok et al., 1998; Geisler et al., 1999) to LG5 between z26471 and pescadillo (not shown). Detailed information about the primers used for mapping is available from authors upon request. The mapping data suggested that the organization of the chromosomal regions containing nNOS encoding genes have been preserved during evolution of vertebrates (not shown). Expression of nnos was first detected at 16 hours postfertilization (hpf) in the ventral forebrain (not shown), which coincided with the beginning of primary axonogenesis (Chitnis and Kuwada, 1990; Wilson et al., 1990; Ross et al., 1992) and is several hours after the beginning of neural differentiation (Korzh et al., 1993; Blader et al., 1997; Korzh et al., 1998). Since the expression pattern of nnos was similar to that of tyrosine hydroxylase (TH), we decided to compare the expression of these two markers using a combination of WISH and immunostaining. The distribution of TH-positive cells was recently characterized in the embryonic zebrafish in detail (Holzchuh et al., 2001; Kaslin and Panula, 2001; Lam, 2002; Lam et al., 2003; Rink and Wullimann, 2002). The expression domains of nnos were much wider compared to expression of TH and were located adjacent to TH-expressing cells. After 16 hpf, expression of nnos in the ventral forebrain increased and by
24 hpf became very intense (Fig. 1A). Expression was detected in the anterio-ventral diencephalon, where the ventro-rostral cluster of Islet1-positive cells has been described (Korzh et al., 1993), which is one of the first neuronal clusters to initiate axonogenesis. It establishes the ventral tract of post-optic commissure (TPOC), which more posteriorly contributes into the medial-longitudinal fascicle (MLF) (Ross et al., 1992; Kimmel, 1993). This domain of nnos expression appeared earlier than expression of TH and was located anteriorly to the first cluster of TH-positive neurons in the ventral diencephalon (Holzchuh et al., 2001). Expression of TH indicated that these cells had initiated production of catecholamines used in the CNS as neurotransmitters (reviewed in Reiner, 1994). Thus, nnos-positive cells were adjacent to cells that had entered a program of differentiation resulting in the formation of catecholaminergic neurons. In addition, expression domains with low levels of nnos transcripts were detected in the ventral telencephalon and tegmentum (Fig. 1A). Expression of nnos in these clusters increased at 36 hpf (Fig. 1C). By 48 hpf (Fig. 1D) nnos expression domains were found in the subventricular zone of the telencephalon, the dorsal hypothalamus, midbrain and hindbrain, including the locus coeruleus (LC), which projects into the lateral longitudinal fascicle (LLF; Ma, 1994; Lam et al., 2003). In all these positions nnos-positive cells were found in close proximity to TH-positive cells (Fig. 1D). The mutant mindbomb (mib2/2 ) shows an increase in the number of neurons (Hong et al., 2002). In this mutant, nnos expression increased dramatically in the ventral
Fig. 1. Expression of nnos in zebrafish embryos as detected by WISH. The distribution of nnos mRNA (blue) was compared to that of TH (brown). (A) Twentyfour hpf embryo showing strong nnos expression anterior to the diencephalic cluster of TH-positive cells in the hypothalamus and weaker expression in the ventral telencephalon and tegmentum. The inset shows a dorsal view of the hypothalamus. (B) mindbomb (mib) mutant at 24 hpf shows ectopic expression of nnos in the dorsal telencephalon and the up-regulation and expansion of expression domains in the diencephalon. (C) 36 hpf embryo showing that the nnospositive cells in the diencephalon maintain an anterior position compared to TH-expressing cells. The inset shows a dorsal view of the hypothalamus at higher magnification. (D) Forty-eight hpf embryo indicating nnos transcripts in the telencephalon, midbrain and the LC in the anterior hindbrain. The inset shows a higher magnification lateral view of the LC. Abbreviations: d, diencephalon; ht, hypothalamus; lc, locus coeruleus; ot, optic tectum; poa, pre-optic area; t, telencephalon; tg, tegmentum. Scale bar—50 mm.
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telencephalon, hypothalamus and tegmentum suggesting that during this developmental stage nnos-positive cells probably represent neurons (Fig. 1B). Intense expression of nnos was also detected in the dorsal telencephalon. At 72 hpf, expression of nnos became more complicated in the brain (Fig. 2A) and also expanded into the trunk (Fig. 2B, I) where nnos-positive cells were found in the ventral neural tube and intestine. To resolve the expression pattern at this stage, we analyzed it on cross sections. Expression of nnos was found in all major regions of the brain. In several regions expression of this gene was
465
associated with expression of TH, including the olfactory bulb (Fig. 2C), pre-optic area (Fig. 2E), areas of diencephalon (Fig. 2G), LC in the anterior hindbrain (Fig. 2H) and the inner nuclear layer of the neuroretina (Fig. 2J). In some regions within the telencephalon, midbrain, spinal cord, intestine and GCL of the retina (Fig. 2D, F, I, J) no TH-positive cells were detected in association with expression domains of nnos. The expression of nnos in cell types other than those associated with differentiation of TH-positive cells indicated that other classes of neurons had started to express nnos.
Fig. 2. Expression of nnos in zebrafish embryos during hatching. (A) Brain, (B) trunk. Arrowhead in A and B indicates the spinal cord. Arrows in B show cells of the enteric nervous system. The anterior–posterior position of cross sections through the brain (A) and trunk (B) of a 72 hpf embryo is indicated. Crosssection analysis shows discrete clusters of nnos-positive cells in the telencephalon (C), sub-pallium of telencephalon (D), pre-optic area (E) and posterior hypothalamus (F,G) and intense nnos expression immediately below the ventricular layer in the anterior hindbrain (H). In the trunk (I) nnos transcripts present in secondary motor neurons (arrowhead) above the floorplate (red arrow) and in two bilateral rows of cells corresponding to myenteric neurons in the gut or adjacent tissues (arrows in B,I). Cross section through the eye (J) shows intense nnos expression in the amacrine cells present in the inner nuclear layer of the retina with less intense nnos expression in the GCL. (J– M) nnos-positive cells intermingle with and are also adjacent to TH-positive cells in the inner nuclear layer of the retina (J), and in several regions of the brain–olfactory bulb (C,K), pre-optic area (E,L) and the posterior tuberculum (G,M). Some cells show the presence of both nnos and TH (arrowhead in K –M). Yolk and eyes were removed from the embryo for clearer observation in lateral view (A). Anterior is to the left and dorsal is to the top in all pictures. All cross sections are shown with dorsal to the top. All insets were marked with a letter corresponding to a frame in a bottom row. Abbreviations: d, diencephalon; dp, dorsal pallium; fp, floor plate; g, gut; gcl, ganglion cell layer; hb, hindbrain; hbl, habenula; ht, hypothalamus; inl, inner nuclear layer of retina; lc, locus coeruleus; n, notochord; nsc, nucleus suprachiasmatic; ob, olfactory bulb; on, optic nerve; op, olfactory placode; ot, optic tectum; poa, pre-optic area; poc, post-optic commissure; pt, posterior tuberculum; s, somite; sc, spinal cord; sp, sub-pallium; t, telencephalon; tg, tegmentum; vt, ventral thalamus. Scale bar—50 mm.
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This latter case is represented by expression in the spinal cord (Fig. 2B, I) in cells above the floor plate. These cells may represent secondary motor neurons. Thus, expression of nnos at 72 hpf is characterized by its appearance in many classes of neurons, including myenteric neurons outside of the CNS in the gut (Fig. 2B, I).
Acknowledgements We are obliged to Drs N. Peunova and G. Enikolopov for fruitful discussion during the early phase of this project. Dr Yun-Jin Jiang provided us with mindbomb mutants. VK’s lab is supported by a research grant from the Agency for Science, Technology and Research (A-STAR) of Singapore.
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