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. Author manuscript; available in PMC: 2014 Dec 16.
Published in final edited form as: Dev Dyn. 2009 Apr;238(4):980–992. doi: 10.1002/dvdy.21912

Cloning and Spatiotemporal Expression of Zebrafish Neuronal Nicotinic Acetylcholine Receptor Alpha 6 and Alpha 4 Subunit RNAs

Kristin M Ackerman 1, Robin Nakkula 1, Jeffrey M Zirger 1, Christine E Beattie 1, R Thomas Boyd 1,*
PMCID: PMC4267763  NIHMSID: NIHMS646912  PMID: 19301390

Abstract

Acetylcholine plays an important role in regulation of nervous system development and function. We are developing zebrafish (Danio rerio) as a model system to study the role of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes in development and the effects of nicotine on the developing vertebrate nervous system. We previously characterized the expression of several zebrafish nAChR subunits. To further develop the zebrafish model, here we report a study on the molecular characterization of two additional nAChR subunit genes, designated chrna6 and chrna4. Both zebrafish nAChRs have a high degree of sequence identity to nAChRs expressed in a variety of mammalian species. Reverse transcription polymerase chain reaction was used to show that both nAChR subunit RNAs were expressed early in zebrafish development, with the chrna4 transcript present at 3 hours postfertilization (hpf) and the chrna6 RNA present at 10 hpf. In situ hybridization was used to localize chrna6 and chrna4 RNA expression in 24, 48, 72, and 96 hpf zebrafish. The chrna6 and chrna4 RNAs were each expressed in a unique pattern, which changed during development. At various ages, chrna6 was expressed in Rohon-Beard sensory neurons, trigeminal ganglion, retina, and the pineal gland. Most notably, chrna6 was expressed in catecholaminergic neurons in the midbrain, but was also present in noncatecholaminergic cells in both midbrain and hindbrain. The expression of chrna6 RNA in catecholaminergic cells supports the use of zebrafish as a valid model system to better understand the molecular basis of cholinergic regulation of dopaminergic signaling and the role of α6-containing nAChRs in Parkinson’s disease. The most notable chrna4 expression was in neural crest cells at 24 hpf and reticulospinal neurons in hindbrain at 48 hpf. chrna4 RNA exhibited a widespread and robust expression pattern in the midbrain in 72 hpf and 96 hpf zebrafish.

Keywords: cholinergic, RNA, development, RT-PCR, In situ hybridization, zebrafish

INTRODUCTION

Acetylcholine plays an important role in the regulation of nervous system development and function. Dysfunctions in cholinergic signaling have been shown to result in structural damage with subsequent effects on behavior (Hohmann et al., 1988, 1991; Bachman et al., 1994; Slotkin, 1999, 2004, 2008). In vertebrates, there are two main classes of cholinergic receptors: muscarinic and nicotinic acetylcholine receptors (nAChR). In a variety of models, cholinergic agents acting through nAChRs have been shown to exert effects on cell growth, neurite outgrowth, synaptogenesis, and apoptosis (Lipton and Kater, 1989; Chan and Quik, 1993; Pugh and Berg, 1994; Zheng et al., 1994; Hory-Lee and Frank, 1995; Schuller, 1995; Berger et al., 1998; Svoboda et al., 2002). nAChRs are ligand-gated ion channels that are classically responsible for rapid neurotransmission and expressed in many regions of the brain (neuronal nAChR), neuromuscular junction (muscle nAChR), and autonomic ganglia. Signaling through nAChRs also modulates the release of other neurotransmitters such as dopamine and glutamate (Gotti et al., 2006). nAChRs are allosteric membrane proteins that are assembled in complexes of five subunits, each monomer classified as either one of nine α or three β subunits (α2–10, β2–4; and Role, 1995; Gotti et al., 2006). Each nAChR subunit has four conserved transmembrane (TM) regions and a cytoplasmic loop of variable length and sequence. Receptor subtypes are designated as homo- or hetero-pentameric, depending on the subunit composition. Heteromeric receptors are assembled by both α and β subunits with the α4β2 receptor being the most prominent heteromer in the brain. Homomeric receptors are composed of only α subunits with the α7 receptor being the primary homomer. Distinct nAChR subtypes exist that can be stimulated by acetylcholine, nicotine, or by other cholinergic compounds. In recent years, it has become clear in adults that neuronal nAChRs are valid pharmacological targets in a variety of pathologies, including cognitive and attention deficits, Parkinson’s disease, Alzheimer’s disease, schizophrenia, epilepsy, Tourette’s syndrome, anxiety, and in pain management (Lloyd and Williams, 2000). The role of specific nAChRs during development remains unclear.

Disruption of cholinergic signaling mediated by nAChRs during fetal development can have serious consequences. It has been shown that smoking during pregnancy increases the risks of behavioral problems, cognitive impairment, learning disabilities, and attention deficit/hyperactivity disorders in offspring, in addition to causing several prenatal deaths (Sexton et al., 1990; Slotkin, 1992, 2004, 2008; Olds et al., 1994). This represents a major health concern in that approximately 25% of pregnant women in the United States smoke. Studies of fetal development in animal models showed specific effects of nicotine on fetal cell loss, brain damage, behavioral deficits, and synaptic abnormalities (Slotkin, 2004, 2008). We are developing the use of zebrafish (Danio rerio) as a model system to study the precise role of specific neuronal nAChR subtypes at a molecular genetic level during normal developmental processes and the effects of nicotine on the developing vertebrate nervous system. The major advantage of the zebrafish as a model system is the simultaneous application of embryological and advanced genetic techniques. These genetic and molecular studies can be done efficiently, as embryos can be obtained in large numbers, develop ex utero, are easy to maintain in relatively small facilities, and the embryos develop quickly. Moreover in zebrafish, gain of function and transient gene knockdown analysis can be efficiently performed by mRNA overexpression as well as antisense morpholino oligonucleotide gene knockdown, respectively (Nasevicius and Ekker, 2000). Furthermore, due to the transparency of the embryos, expression analysis by in situ hybridization can be easily performed to investigate genetic interactions. Thus, in zebrafish novel genes controlling important developmental processes can be rapidly identified and functionally analyzed.

To date, little work has been done to examine the effects of nicotine on zebrafish. Zebrafish have been used to study the effects of nicotine on motoneuron development (Svoboda et al., 2002) and have been recently used to assess the effects of nicotine on cognitive function (Levin and Chen, 2004; Levin et al., 2006). We previously characterized the expression of zebrafish nAChR α2, α7, and β3 subunits (Zirger et al., 2003) and, to further develop the zebrafish model, we have now cloned and characterized the expression pattern of the zebrafish nAChR subunits α6 (chrna6) and α4 (chrna4).

RESULTS AND DISCUSSION

Cloning and Analysis of Zebrafish chrna6 and chrna4 cDNAs

We used reverse transcription-polymerase chain reaction (RT-PCR) in combination with 5′ and 3′ rapid amplification of cDNA ends (RACE) to isolate full-length zebrafish cDNAs encoding presumptive zebrafish nAChR α6 and α4 subunits. The genes encoding the presumptive zebrafish cDNAs were designated chrna6 and chrna4 based on the level of DNA and protein sequence homologies to nAChRs from other species. The chrna6 cDNA (1,940 bp) encoded a 512 amino acid protein and included a 161-bp 5′ untranslated region and a 243-bp 3′ untranslated region. The 3′ end of the zebrafish cDNA did not have a consensus polyadenylation sequence and possibly does not represent the actual 3′ end of the native chrna6 RNA. However, a complete coding region was present and a consensus polyadenylation sequence (AATAAA) was present in the genomic sequence approximately 270 base pairs from the 3′ end of the chrna6 cDNA (R.T. Boyd, data not shown).

Genomic analysis using the zebrafish genomic assembly version 7 (www.sanger.ac.uk) indicates that there is one copy of the chrna6 on chromosome 1. The zebrafish chrna6 DNA and translated protein sequences were used for BLAST searches of Genbank. The chrna6 protein was most similar to a nAChR designated α3 from goldfish (Hieber et al., 1990), with a 95% identity at the protein level. Despite the homology to the goldfish nAChR α3 subunit, we have designated the zebrafish cDNA chrna6 because of several observations. First, we have cloned another zebrafish nAChR cDNA with closer sequence identity to nAChR α3 subunits from other species that we have designated chrna3 (Fig. 1C). In addition to the goldfish α3, the zebrafish chrna6 cDNA has a higher homology to the Fugu rubripes nAChR α6a subunit (Table 1; Fig. 1) than to any other nAChR subunits. In addition, and similar to other species, zebrafish chrna6 is closely linked to zebrafish chrnb3 (Zirger et al., 2003; www.sanger.ac.uk). This linkage of the zebrafish chrna6 and chrnb3 nAChR genes and the highest sequence identify to the Fugu α6a supports our conclusion that we have cloned a zebrafish nAChR α6 subunit orthologue.

Fig. 1.

Fig. 1

Fig. 1

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A,B: ClustalW2 Alignments (Larkin et. al., 2007) of chrna6 (A) and chrna4 (B) cDNAs to neuronal nicotinic acetylcholine receptors (nAChRs). The ClustalW2 alignment program was used on the EMBL-EBI site (http://www.ebi.ac.uk/Tools/clustalw2/). “*” identical residues in all sequences,“ : ” conserved substitutions, and “ . ” semiconserved substitutions. Zebrafish subunit transmembrane residues are in red and conserved cysteines are in green. C,D: Phylogenetic analysis of chrna6 (C) and chrna4 (D). Each tree search was conducted for 100 replicates in the RAXML Web server (Stamatakis et al., 2008) using tree building followed by branch swapping replicates under the JTT model of amino acid substitution. Bootstrapping was conducted for 100 pseudoreplicates.

TABLE 1.

DNA and Protein Identify (%DNA/%Protein) Using Pairwise Comparisons of chrna6 and chrna4 and Related Nicotinic Acetylcholine Receptor Subunit Genesa

Fugα6a Fugα6b Chickα3 Humα3 Ratα3 Chickα3 Mouα3
chrna6 72/81 61/72 64/67 65/67 66/67 63/66 63/64
chrna4 Chickα4
67/63		 Humα4
66/61		 Mouα4
67/60		 Ratα4
67/59		 Fugα4
65/63		 Zfα4
54/56
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a

Fug, Fugu; Hum, human; Mou, mouse; Zf, zebrafish; Mouse α6 (NM_021369); Fugu α6a (AY299463.1); Fugu α6b (AAP58379); Chick α3 (NM_204416.1); Human α 3(AY889804.1); Chick α6 (NM_205364.1); Rat α3 (NP_434692); Fugu α4 (AAP58377.1); Chick α4 (NP_990145); Mouse α 4(NP_056545.3); Human α4 (AAH96290); Rat α4 (NP_077330.1); Zf α 2(AY256908).

The zebrafish chrna6 protein sequence also had significant levels of protein homology (64–81% identity) with chick, human, mouse, rat, and Fugu nAChRs (Table 1; Fig. 1). The high sequence identity was also maintained at the DNA level. The levels of sequence similarity were comparable to the similarities between previously cloned zebrafish nAChRs and nAChRs from other species (Zirger et al., 2003). The homology to the other nAChRs was high throughout the sequence, especially in the four TM regions, including the comparison with Fugu α6 cDNA (Fig. 1A), but not in the cytoplasmic loop. The high degree of DNA sequence identity of the zebrafish chrna6 cDNA with the goldfish α3 subunit cDNA sequence was maintained throughout the cytoplasmic loop between transmembrane regions 3 and 4 (TM3-TM4), even though this sequence is known to be most variable between nAChR subunits (Fig. 1). Our analysis of chrna6 and the comparison to the goldfish α3 leads us to propose that the previously cloned goldfish α3 is actually an α6 nAChR orthologue. The chrna6 cDNA also had sequence features characteristic of all nAChR subunits including cysteines at amino acids 151 and 165 as well as vicinal cysteines at amino acids 215-216 typical of nAChR α subunits. The chrna6 sequence was submitted to GenBank (accession no. DQ822507).

The chrna4 cDNA (2,264 bp) encoded a 627 amino acid protein and included a 192-bp 5′ untranslated region and a 191-bp 3′ untranslated region. The 3′ end of the zebrafish chrna4 cDNA did not have a consensus polyadenylation sequence and possibly does not represent the actual 3′ end of the native chrna4 RNA. However, a complete coding region was present. Genomic analysis using the zebrafish genomic assembly version 7 (www.sanger.ac.uk) indicates that there are two tightly linked copies of chrna4 on chromosome 11. However, the coding regions of these copies are 99–100% identical and the sequences 2,000 bp upstream of the first exon are 96% identical, the differences being some small deletions or insertions. Due to the high sequence identity, it is possible that only one copy exists and that the duplicate is due to an error in the genomic assembly. The final resolution will be forthcoming upon completion of the zebrafish genomic sequencing. It should be noted that only one nAChR α4 subunit gene has been identified in another teleost, the pufferfish (Fugu rubripes; Jones et al., 2003).

The zebrafish chrna4 DNA and translated protein sequences were used for BLAST searches of GenBank. The chrna4 protein was most similar to the chick nAChR α4 subunit (63% identity). The zebrafish chrna4 protein sequence also had significant levels of protein sequence homology (56–63% identity) with chick, human, mouse, rat, and Fugu α4 nAChRs (Fig. 1; Table 1). High DNA sequence identity with other α4 nAChR subunits was also present. Interestingly, the percent of DNA identity was higher than the percent protein identity for many of the α4 subunits. The levels of sequence similarity were comparable to the similarities of previously cloned zebrafish nAChRs and had 56% identity to the previously cloned zebrafish chrna2 (Zirger et al., 2003). Sequence similarities with other nAChRs were maintained throughout the sequence with the exception of the cytoplasmic loop (Fig. 1B). The chrna4 cDNA also had sequence features characteristic of all nAChR subunits, including cysteines at amino acids 155 and 169 as well as vicinal cysteines at amino acids 219-220 typical of nAChR α subunits. The chrna4 sequence was also submitted to GenBank (accession no. DQ822508).

Zebrafish chrna6 RNA Expression

Time course

We used reverse transcription polymerase chain reaction (RT-PCR) with subunit-specific primers to determine the time course of chrna6 RNA expression during development (Fig. 2). Equal amounts of cDNA from each stage were used for the RT-PCR. β-actin transcripts were also co-amplified to allow semiquantitative comparisons and to ensure that the RNA was intact at all stages examined. The chrna6 RNA was first detected at 10 hpf, the onset of neurogenesis and somitogenesis. Low chrna6 RNA expression was apparent until 24 hpf with an increase until 60 hpf. The chrna6 RNA expression was maintained at 7 days (168 hpf). chrna6 RNA was not detected in embryos younger than 10 hpf including maternal RNA after 40 cycles of PCR (data not shown).

Fig. 2.

Fig. 2

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Expression of chrna6 and chrna4 RNA in zebrafish. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine when chrna6 and chrna4 nAChR subunit RNAs were expressed in embryos and larvae. Three micrograms of RNA from each age zebrafish (h, hpf; d, dpf) were reverse-transcribed and amplified using subunit-specific primers. The β-actin was also amplified for each age. As controls, PCR was performed using zebrafish RNA without reverse transcription (−r) or without cDNA (−d). The size of each PCR product was consistent with the size predicted by the cDNA sequences. The φx 174 HaeIII DNA (φχ) was used as a size marker.

Localization of chrna6 RNA expression by in situ hybridization

To reveal the temporal and spatial expression pattern of chrna6 during embryogenesis, we performed whole-mount RNA in situ hybridization at 24, 48, 72, and 96 hpf. We used krox20, also known as erg2, as a hindbrain marker of rhombomeres 3 and 5 (Oxtoby and Jowett, 1993) at 24 hpf. dlx2 was used as a marker at 24 and 48 hpf and is expressed in multiple domains, including telencephalon, diencephalon, the hypothalamus of embryonic forebrain, and in cranial neural crest cells at the pharyngeal arches (Akimento et al., 1994). A probe for tyrosine hydroxylase (TH; Holzschuh et al., 2001) was used to colocalize expression of α6 RNA with catecholaminergic neurons in 48, 72, and 96 hpf embryos.

In 24 hpf embryos, chrna6 RNA was present in a subset of Rohon-Beard neurons, as well as spinal cord neurons (Fig. 3A-D). Expression was also observed in ventral forebrain (Fig. 3B,D), specifically diencephalon (Fig. 3E) as labeled with dlx-2, trigeminal ganglion (Fig. 3C), pineal (Fig. 3B,D,E), and in the first hindbrain rhombomere (Fig. 3D,E) ventral to the cerebellum. In 48 hpf embryos, chrna6 was no longer observed in spinal neurons (data not shown), but was still expressed in trigeminal ganglion (Fig. 3F-I), pineal (Fig. 3F-J), and was now colocalized to the TH + locus coeruleus (Fig. 3H,I) and the TH + diencephalic catecholaminergic cluster (Holzschuh et al., 2001; Fig. 3H,J-N). A small amount of chrna6 expression was detected in tectum at 48 hpf (Fig. 3H). chrna6 RNA was also highly expressed in the 48 hpf retina (Fig. 3F-J), but was not detected in 24 hpf retina.

Fig. 3.

Fig. 3

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In situ hybridization analysis of chrna6 RNA expression in 24 hours postfertilization (hpf) and 48 hpf zebrafish embryos. The purple stain represents chrna6 neuronal nicotinic acetylcholine receptor (nAChR) subunit mRNA in all panels, and the orange labeling is either krox20 in rhombomeres 3 and 5, dlx-2 in telencephalon, diencephalon and pharyngeal arches, or tyrosine hydroxylase (TH) in catecholaminergic neurons. Use of these additional probes is indicated in each panel. All images are lateral views with the anterior to the left unless otherwise noted. Arrows point to specific brain regions and arrows with an asterisk denote co-labeling with TH. A: In 24 hpf embryos, the chrna6 transcript was seen in a subset of spinal neurons and a subset of Rohon-Beard sensory neurons in the trunk. B: 24 hpf, chrna6 expression was present in pineal, ventral forebrain, and Rohon-Beard sensory neurons. C: At 24 hpf, dorsal view, chrna6 expression in trigeminal ganglion and Rohon-Beard sensory neurons. D: At 24 hpf, chrna6 expression was seen in rhombomere 1 as well as forebrain, pineal, and spinal neurons. The Krox20 probe (orange) labeled rhombomeres 3 and 5. E: At 24 hpf, chrna6 expression was identified in diencephalon as shown by the colocalization with dlx2, a marker for telencephalon, diencephalon, and hypothalamus. Additionally, chrna6 is labeled in pineal and rhombomere 1. F: At 48 hpf, dorsal view, chrna6 transcript was heavily expressed in the retina with limited expression in midbrain and continued expression in pineal and trigeminal ganglion. G: At 48 hpf, dorsal view, expression in midbrain is not colocalized with dlx2 and is dorsal to the dlx2 expression domain (orange labeling) in telencephalon, diencephalon, and hypothalamus. Additionally, expression in retina and trigeminal ganglion was present. H: At 48 hpf, chrna6 was expressed in tectum, retina, pineal, and trigeminal ganglion. The diencephalic catecholaminergic cluster and locus coeruleus are detected by the TH probe (orange). As heavy retinal expression is evident in H that impedes with visualizing midbrain, I-N are 8–12-micron sagittal sections though the diencephalic regions marked by a line in H and L. I: At 48 hpf, chrna6 expression was clear in the pineal, retinal ganglion cells, trigeminal ganglion, and colocalization with TH in the locus coeruleus was evident. The insert is a magnification of locus coeruleus. J: At 48 hpf, indicates chrna6 expression in the most lateral regions of the diencephalic catecholaminergic cluster colocalizing with TH. Additionally, chrna6 expression in the pineal and retinal ganglion cells was present. K: At 48 hpf, higher magnification of K, colocalization of TH and chrna6 transcript in the lateral most region of the diencephalic catecholaminergic cluster in the midbrain. L: At 48 hpf, a midsagittal section through the diencephalic catecholaminergic cluster indicates chrna6 colocalization with TH. M,N: At 48 hpf, medial sagittal sections, indicate areas of colocalization with TH and areas solely expressing chrna6 RNA. dien, diencephalon; dcc, diencephalic catecholaminergic cluster; fb, forebrain; hypo, hypothalamus; lat, lateral; lc, locus coeruleus; 1, mandibular pharyngeal arch; mb, midbrain; obc, olfactory cluster in olfactory bulbs; pin, pineal; rgc, retinal ganglion cells; r1, rhombomere 1; rb, Rohon-Beard sensory neurons; sn, spinal neurons; tec, tectum; tg, trigeminal ganglion.

Zebrafish chrna6 continued to be expressed in trigeminal ganglion (Fig. 4A,D,E,F,H,J,L), retina (Fig. 4A,D,F,H,J), and pineal (Fig. 4A,B,F,H,I,J) at 72 and 96 hpf. chrna6 RNA was now expressed in tectum (Fig. 4A,B,H,I) in 72 and 96 hpf zebrafish, at robust levels with more widespread distribution than at 48 hpf. At 96 hpf, chrna6 expression was detected for the first time with a pattern consistent with cranial sensory neurons (Fig. 4L) in the hindbrain. At 72 hpf and 96 hpf, chrna6 RNA continued to be expressed in the diencephalic catecholaminergic cluster (Fig. 4A-H,J,K), but was also present in noncatecholaminergic cells in both midbrain and hindbrain (Fig. 4B-E,G,H,K).

Fig. 4.

Fig. 4

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In situ hybridization analysis of chrna6 RNA expression in 72 hours postfertilization (hpf) and 96 hpf zebrafish embryos. The purple stain represents chrna6 neuronal nicotinic acetylcholine receptor (nAChR) subunit mRNA in all panels, and the orange labeling is tyrosine hydroxylase (TH) mRNA. All images are lateral views with the anterior to the left unless otherwise noted. Arrows point to specific brain regions, arrowheads point to amacrine cells in the retina, and arrows with an asterisk denote co-labeling with TH. Line denotes diencephalic catecholaminergic cluster in A–G. A: At 72 hpf, chrna6 was now localized heavily to the eye and tectum, pineal, trigeminal ganglion, and colocalization with TH in the diencephalic catecholaminergic cluster and pretectal catecholaminergic cluster. B: At 72 hpf, midsagittal section, showed chrna6 expression in pineal, tectum, and colocalization with TH in a subset of cells in the diencephalic catecholaminergic cluster, and no expression in the olfactory bulb catecholaminergic cluster. C: At 72 hpf, midsagittal section, higher magnification of B, colocalization of chrna6 with TH in the diencephalic catecholaminergic cluster. D: At 72 hpf, sagittal section, chrna6 expression in amacrine cells of the retina, retinal ganglion cells, trigeminal ganglion, and colocalization with TH in a subset of cells in the diencephalic catecholaminergic cluster. E: At 72 hpf, sagittal section higher magnification of D, colocalization of chrna6 with TH in the diencephalic catecholaminergic cluster and expression in trigeminal ganglion. F: At 72 hpf, dorsal view of whole animal, showed chrna6 expression in retina, trigeminal ganglion, pineal, and colocalization with TH in the diencephalic catecholaminergic cluster and locus coeruleus. G: At 72 hpf, longitudinal section, showed chrna6 expression in a subset of TH+ cells in the diencephalic catecholaminergic cluster. H: At 96 hpf, expression was evident in retina, pineal, tectum, trigeminal ganglion, a subset of cells in the hindbrain consistent with cranial sensory neurons, and colocalization with TH in the diencephalic catecholaminergic cluster, locus coeruleus, and pretectal catecholaminergic cluster. I: At 96 hpf, longitudinal section, demonstrated colocalization of TH and chrna6 expression in the pretectal area, with pineal and tectum labeled. J: At 96 hpf, dorsal view whole-mount, chrna6 expression shown in retina, pineal, trigeminal ganglion, diencephalic catecholaminergic cluster, and locus coeruleus. K: At 96 hpf, dorsal view whole embryo, chrna6 colocalization with TH in the diencephalic catecholaminergic cluster. L: At 96 hpf, dorsal view whole embryo, chrna6 expression in hindbrain neurons consistent with the localization of cranial sensory neurons. ac, amacrine cells in the retina; dcc, diencephalic catecholaminergic cluster; hb, hindbrain nuclei; lc, locus coereuleus; obc, olfactory bulb catecholaminergic cluster; ptc, pretectal catecholaminergic cluster; pin, pineal; tec, tectum; tg, trigeminal ganglion.

The localization of chrna6 in catecholaminergic neurons advances the use of zebrafish as a model to study the role of nAChRs in dopaminergic transmission. chrna6 expression in several catecholaminergic regions of the zebrafish nervous system is consistent with studies in mammals where α6 nAChRs have been localized to locus coeruleus, mesostriatal dopaminergic neurons, substantia nigra, and VTA (Champtiaux et al., 2003; Gotti and Clementi, 2004). The α6 and β3 expression colocalize to retina and dopaminergic neurons (SN, VTA) in several species (Gotti and Clementi, 2004). This study in conjunction with our previous work (Zirger et al., 2003) indicates that chrna6 and chrnb3 RNA are also colocalized in zebrafish retinal ganglion. Zebrafish also express chrna6 RNA in trigeminal ganglion consistent with the expression pattern in adult rats. Zebrafish lack chrna6 expression in the olfactory bulb of 48, 72, and 96 hpf embryos, although the olfactory bulb catecholaminergic cluster (Holzschuh et al., 2001) was clearly detected by the TH probe in 72 and 96 hpf embryos. This is also consistent with rats in that only 17% of rats express α6 in adult olfactory bulbs (Liu et al., 1998; Keiger and Walker, 1999). Zebrafish chrna6 expression occurs in retina, consistent with α6 expression after embryonic day (E) 14 in chick retina. However, while chrna6 RNA was detected in developing optic tectum in zebrafish, α6 was not detected in chick optic tectum (Gotti and Clementi, 2004).

Zebrafish chrna4 RNA Expression

Time course

We used RT-PCR with subunit-specific primers to determine the time course of chrna4 RNA expression during development (Fig. 2). Equal amounts of cDNA from each stage were used for the RT-PCR. The β-actin transcripts were also co-amplified to allow semiquantitative comparisons and to ensure that the RNA was intact at all stages examined. The chrna4 RNA was first expressed at 3 hpf in blastula staged embryos and was transiently expressed at all stages (except 16 hpf) examined. 2.5 hpf is approximately when zygotic transcription begins during the mid-blastula transition (Kimmel et al., 1995). However, chrna4 expression was not present in maternal RNA (data not shown). The low expression of chrna4 RNA at 16 hpf was observed several times and the coamplification of zebrafish β-actin indicates that the cDNA derived from 16 hpf RNA was intact.

Localization of chrna4 RNA Expression by In Situ Hybridization

To reveal the temporal and spatial expression pattern of chrna4 during embryogenesis, we performed whole-mount RNA in situ hybridization at 24, 48, 72, and 96 hpf. Similar to the chrna6 in situ hybridization studies, the hindbrain marker krox20 and the forebrain marker dlx2 were used at 24 and 48 hpf. The zebrafish chrna4 expression pattern was distinct from that of the chrna6 nAChR subunit RNA. In 24 hpf embryos (Fig. 5A-E), chrna4 was detected in a subset of neurons in rhombomeres 4–7 (Fig. 5A-C) and expression possibly overlapped dlx2-expressing cells consistent with cranial neural crest cells migrating posterior to the pharyngeal mandibular arch and along the hyoid arch and brachial arch (Fig. 5D,E; Albertson et al., 2005). Limited expression was observed in forebrain and midbrain structures. At 48 hpf, significant bilateral expression was seen in both midbrain and hindbrain (Fig. 5F,G) consistent with the nucleus of the medial longitudinal fascicle and reticulospinal neurons (Metcalfe et al., 1986), with no expression detected in the spinal cord. At 72 hpf (Fig. 5H,I) and 96 hpf (Fig. 5J,K), chrna4 continues to be highly expressed in specific midbrain and hindbrain areas. No brain region-specific probes were used at these stages due to the obvious widespread expression in the midbrain.

Fig. 5.

Fig. 5

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Whole-mount in situ hybridization analysis of chrna4 RNA expression in 24–96 hours postfertilization (hpf) zebrafish embryos. The purple stain represents chrna4 neuronal nicotinic acetylcholine receptor (nAChR) subunit mRNA in all panels, and orange labeling denotes either krox20 RNA in rhombomeres 3 and 5 or dlx2 RNA in telencephalon, diencephalon, and the pharyngeal arches. All images are lateral views with the anterior to the left unless otherwise noted. Arrows point to specific brain regions and arrowheads point to spinal neurons. A: At 24 hpf, chrna4 expression was localized to a limited area of forebrain, a subset of cells along the yolk sac consistent with neural crest cells, and in a subset of hindbrain neurons with expression beginning in rhombomere 4. B: At 24 hpf, magnification of A, expression in forebrain, hindbrain neurons, and in dlx2-expressing areas likely to be neural crest. C: At 24 hpf, dorsal view whole embryo, possible neural crest expression at the level of, but outside of the rhombomeres, with expression also evident in spinal neurons. D: At 24hpf, chrna4 likely localization in neural crest cells in the dlx2-expressing domain of the mandibular (1), hyoid (2), and brachial (3) pharyngeal arches. E: At 24 hpf, higher magnification of D, chrna4 expression was evident in the dlx2 expression domain of the hyoid (2) pharyngeal arch and brachial (3) arch. F: At 48 hpf, dorsal view, localization consistent with the nucleus of the medial longitudinal fascicle and reticulospinal neurons of the hindbrain. G: At 48 hpf, chrna4 expression was not present in the telencephalon or hypothalamus as evident by using dlx2 expression to localize these structures. There was midbrain and hindbrain expression consistent with the localization pattern of the nucleus of the medial longitudinal fascicle and reticulospinal neurons. H: At 72 hpf, dorsal view and I: At 72 hpf, chrna4 expression was extensive in midbrain and limited in hindbrain. J: At 96 hpf, dorsal view and K: At 96 hpf, chrna4 expression was extensive in midbrain and limited in hindbrain similar to the pattern that seen in 72 hpf embryos (I). dien, diencephalon; fb, forebrain; hb, hindbrain; hn, hindbrain neurons; mb, midbrain; mhb, midbrain hindbrain boundary; nc, neural crest; otic, otic vesicle; pharyngeal arches: (1) mandibular, (2) hyoid, and (3) brachial arch; nmlf, nucleus of the medial longitudinal fascicle; rs, reticulospinal neurons; rhombomeres 3 (r3) and 5 (r5), telencephalon (te).

The zebrafish chrna4 RNA expression patterns differ from that characterized in other species. It is possible that multiple chrna4 nAChR genes are expressed in zebrafish and that we cloned a variant with a more restricted pattern of expression. As noted above, the two apparent copies of chrna4 contain identical coding regions and thus our probe should detect RNA transcribed from either gene. We previously demonstrated the presence of high affinity epibatidine binding sites in 2 day embryos (Zirger et al., 2003) consistent with the presence of α4 containing receptors and chrna4 RNA in 48 hpf embryos. Interestingly, we detected no chrna4 RNA in the zebrafish retina, although α4 nAChRs are expressed as early as E7 in chick retina (Gotti and Clementi, 2004) and α4 RNA was present in E15 rat retina (Hoover and Goldman, 1992; Zoli et al., 1995). Zebrafish chrna4 RNA was not detected in the optic tectum, in contrast to chick optic tectum, which expresses α4β2 nAChRs (Gotti and Clementi, 2004). In addition, zebrafish chrna4 was also not highly observed in the forebrain, in contrast to chick forebrain, which contains α4α5β2 and α4β2 subtypes (Conroy and Berg, 1998). It is documented in other species that migrating neural crest cells synthesize acetylcholine as evident by the presence of acetylcholinesterase (AChE) and choline acetyltransferase (CAT) enzymes (Smith et al., 1979; Cochard and Coltey, 1983) and that nAChR proteins are indeed present, but a function has yet to be determined. To date, cholinergic neural crest cells have not been described in zebrafish. Unexpectedly, and in contrast to other systems in which only α3,α5,α7,β2, and β4 nAChR subunit mRNAs and proteins have been detected in neural crest cell populations (Howard et al., 1995), zebrafish α4 nAChR mRNA was detected in areas of dlx2 expressing cell populations along the pharangeal arches consistent with a cranial neural crest population.

SUMMARY

We have cloned two additional zebrafish nAChR subunit cDNAs, chrna6 and chrna4, which are highly homologous to those present in rat, mouse, human, chick, and other species. They are both expressed early in development, consistent with a possible role in signaling events early in the formation of the nervous system. This early expression was also seen with other zebrafish nAChR subunit RNAs as well (Zirger et al., 2003). chrna6 expression was present in catecholaminergic regions analogous to those in mammals. The α6-containing nAChRs are involved in signaling in mammalian nigrostriatal pathways and modulating dopamine release. Because α6-containing nAChRs in zebrafish appear to be in analogous locations to those in mammals that are involved in dopamine release, zebrafish should be explored as an easily accessible model to study the role of α6-containing nAChRs in dopamine release. In addition, knowledge of the expression of specific nAChRs in zebrafish may aid studies related to the mechanisms of cell death which occur in Parkinson’s disease and the identification of protective agents. Previous work with zebrafish showed that MPTP induces cell loss in zebrafish brain dopaminergic neurons (Lam et al., 2005; McKinley et al., 2005) as it does in other species. Identification of chrna6 expression in zebrafish catecholaminergic neurons may aid in understanding the role of α6-containing nAChRs in cell death or neuroprotection during Parkinson’s disease.

EXPERIMENTAL PROCEDURES

Zebrafish Embryos

Zebrafish AB* embryos were collected, raised at 28.5°C and staged as described by Kimmel et al. (1995). To facilitate visualization of RNA during in situ hybridization, 0.2 mM phenylthiourea (PTU) was added to the fish water at approximately 24 hpf to prevent pigment formation (Westerfield, 2000).

Frozen Sectioning

Frozen sectioning of zebrafish embryos was performed as outlined in Passini et al. (1997) with minor changes. Embryos were fixed overnight at 4°C in 4% PFA made in 0.1 M phosphate buffer at pH 7.4 with 5% sucrose. The next day embryos were quickly washed five times with 1 ml of 0.1 M phosphate buffer at pH 7.4 with 5% sucrose. The embryos were then incubated in a graded series of 0.1 M phosphate buffer to sucrose (5%, 15%, and 30% sucrose) washes for 45 min at 4°C. The embryos were then placed in fresh 30% sucrose/0.1 M phosphate buffer overnight at 4°C. The next day embryos were brought to room temperature and incubated in 1 part OTC to 2 parts 30% sucrose/0.1 M phosphate buffer for at least 30 min with 1.5 hr generating optimal results. The embryos were then transferred to Biopsy Cryomolds (Tissue-Tek) with fresh 30% sucrose/0.1 M phosphate buffer and stored at −80°C overnight and up to 3 months. Embryos were mounted with OTC, allowed to equilibrate to −25° C, and then sectioned at 8–12 microns.

Cloning of Zebrafish nAChR cDNAs

Zebrafish RNA was isolated from 96 hpf and 7 days postfertilization (dpf) embryos using Trizol (Invitrogen) and reverse transcribed using random primers with the Superscript III Pre-amplification System (Invitrogen). The Sanger database (www.sanger.ac.uk) was searched using α6 and α4 nAChR sequences from other species. The 5′ and 3′ RACE primers were designed to sequences present in the zebrafish genome with high homology to presumptive zebrafish α6 and α4 nAChRs. RACE was performed using the FirstChoice RLM-RACE kit (Ambion) to clone 5′ and 3′ cDNAs. Additional primers from the 5′ and 3′ ends of the RACE cDNAs were designed and RT-PCR used to isolate full-length zebrafish chrna6 and chrna4 cDNAs.

Sequence Analysis

All RT-PCR and RACE products were cloned into pCRII-TOPO or pCRBlunt II (Invitrogen) and sequenced at the Ohio State University Plant-Microbe Genomics Facility with an Applied Biosystems 3730 DNA Analyzer. Custom primers used for sequencing and cloning the zebrafish α6 and α4 cDNAs were purchased from Invitrogen. DNA sequences were used for BLAST searches against GenBank and Sanger databases (Altschul et al., 1990). Additional sequence analysis was done using GeneWorks (Intelligenetics) and ClustalW2 alignment program (Larkin et al., 2007). The tree search was conducted for 100 replicates in the RAXML web server (Stamatakis et al., 2008) using tree building followed by branch swapping replicates under the JTT model of amino acid substitution. Bootstrapping was conducted for 100 pseudoreplicates.

RT-PCR

RNA was isolated (Trizol Reagent, Invitrogen) from whole embryos at various stages from 3 hpf–7 dpf. cDNA was amplified with zebrafish nAChR subunit specific primers for α4 sense (5′-TAT GAT CAA AAC TGG ACG AAG-3′) and antisense (5′-CAA TCA GTG GGA TGA CCA AT-3′) and α6 sense (5′-TTT GTT TTG GAA AGG GAG ACG GT-3′) and antisense (5′-GAC AAG AAG GAA CAC AGT GAG AGA T-3′) for 32 and 30 cycles, respectively, with an annealing temperature of 55°C. All PCR products were resolved in 1% agarose (Invitrogen) with ethidium bromide. Two negative controls were included in the experiment to rule out contamination: a –RT reaction for the presence of genomic DNA in the RNA preparation and a –DNA to control for the presence of DNA contaminates in the PCR reagents. Zebrafish β-actin was used as an internal control to ensure that equal amounts of cDNA were used during the amplification.

Riboprobe Synthesis

The chrna6 and chrna4 full-length cDNAs were cloned into pCRII-TOPO or pCRBlunt II (Invitrogen) vector following the manufacturer’s suggested protocol and antisense and sense digoxigenin (DIG) -labeled RNA probes were synthesized (Roche DIG RNA Labeling Kit SP6/T7). In addition to using 0.2 M ethylenediaminetetraacetic acid (EDTA) to stop the reaction, we also precipitated the RNA with ammonium acetate and 100% ethanol. The RNA was run on an agarose formaldehyde gel. The dlx2, krox20 (erb2), and TH RNA probes (provided by Drs. Paul Henion and Christine Beattie at the Center for Molecular Neurobiology, The Ohio State University) were labeled with fluorescein using standard methods.

In Situ Hybridization

We used procedures outlined by Thisse et al. (1993) and Beattie et al. (1997) with some modifications. Thirty whole embryos were placed in 1.5-ml Eppendorf tubes and fixed in fresh 4% paraformaldehyde (PFA)/1× phosphate buffered saline (PBS) overnight if 72 hpf or 96 hpf, 2 days if 48 hpf, and 3 days if 24 hpf, making the PFA fresh each day. Embryos were then washed at room temperature twice with 100% methanol for 5 min and one time for 10 min. Embryos were stored in fresh 100% methanol at −20°C. Twenty-four to forty-eight hpf embryos were stored at −20°C for up to 6 months, but 72–96 hpf embryos were used within a couple days for optimal expression. On day 1, all washes were performed with 750 μl of solution for 5 min at room temperature unless noted otherwise. Embryos were taken through graded methanol/1× PBS (75%, 50%, 25% methanol), washed four times with PBS-Tween, and then treated with 1 ml of 10 μg/ml proteinase K in 1× PBS-Tween for 5–6 min if 24 hpf, 10–12 min for 48 hpf, 20 min for 72 hpf, and 30 min for 96 hpf. After permeabilization, embryos were post-fixed in fresh 4% PFA for 20 min, rinsed five times with PBS-Tween, prehybridized in hybridization buffer (heparin concentration at 100 mg/ml) for 2 hr and 15 min at 68° C, and finally hybridized with 200 ng of RNA probe overnight at 68°C for at least 15 hr. In addition, 30 embryos were not used to hybridize probe, but instead incubated in 2 μl of anti-DIG antibody in 1 ml of blocking buffer (PBS-Tween, 2% bovine serum albumin and 3% normal goat serum) overnight at 4°C. On day 2, the embryos hybridized with probe were then washed in a graded series of hybridization buffer/2× standard saline citrate (SSC; 75%, 50%, 25% hybridization buffer), 2× SSC for 15 min at 68°C, 0.2× SSC for 30 min at 68°C, followed by a grades series of .02× SSC/PBS-Tween (25%, 50%, 75% 0.2× PBS-Tween) for 5 min at room temperature. The embryos are then incubated in blocking buffer for 1.5 hr at room temperature. The anti-DIG anti-body/blocking buffer solution incubated the previous night is diluted 1:5,000 with blocking buffer. Embryos were incubated overnight at 4°C for at least 15 hr. On day 3, the embryos were washed with PBS-Tween six times for 15 min at room temperature and then washed in alkaline phosphate coloration buffer (AP) buffer three times for 5 min at room temperature. A total of 22.5 μl of 50 mg/ml NBT (nitroblue tetrazolium) and 17.5μl 50 mg/ml BCIP (5-bromo-4-chloro-3-indolyl phosphate; Promega) in 5 ml of AP buffer was used as a developing stain. The developing stain was changed every 6–8 hr for up to 3 days. For in situ hybridization with two probes, 150 ng of each probe was included at the hybridization step. After NBT/BCIP detection of the chrna6 or chrna4 signal, the embryos were immediately fixed in 4% PFA overnight. On day 4, all washes were at room temperature unless otherwise noted. The embryos were washed twice in 1× maleic acid buffer (MAB) with .01% Tween (MABT) for 30 min, the anti-DIG antibody was inactivated with a 10-min wash with 1× MABT and 10 mM EDTA at 68°C, dehydrated in 100% methanol for 10 min, and then rehydrated in a graded series of methanol/ 1× MABT washes (75%, 50%, 25% methanol). The embryos were then washed four times in 100% MABT. The blocking reaction was performed in a buffer containing 2% blocking reagent (BMB) powder (Roche), MAB, and 20% NGS for 2 hr. Anti-Fluorecein antibody was diluted 1:5,000 in BMB blocking buffer and embryos were incubated overnight at 4°C. The embryos were washed six times with MABT, 3 times with AP buffer, and then the alkaline phosphate substrate solution used was INT/BCIP. The 17.5 μl INT (55 mg/ml) and 17.5 μl BCIP (50 mg/ml) were added to 5 ml of AP buffer with 10% polyvinyl alcohol (PVA) to accelerate the reaction. The INT/BCIP solution was changed every 4–6 hr for up to 3 days. The nAChR chrna6 and chrna4 RNAs labeled with digoxigenin were detected by NBT/BCIP giving a dark purple stain and the second fluorescein-labeled brain-specific marker RNA was detected with anti-fluorescein antibody and developed in INT/BCIP which produced an orange stain.

ACKNOWLEDGMENTS

We thank Dr. Paul Henion along with the members of the Henion and Beattie labs at The Ohio State University and all of the people at the Zebrafish Facility. We also thank Drs. Marnie Halpern and Kiran Santhakumar (Carnegie Institution of Washington) for expert advice as well as Dr. Dan Janies, Department of Biomedical Informatics, The Ohio State University College of Medicine who helped with the sequence analysis. Research described in this article was supported in part by Philip Morris Incorporated.

ABBREVIATIONS

AChR

acetylcholine receptor

MAB

maleic acid buffer

bp

base pairs

PBS

phosphate buffered saline

dpf

days postfertilization

PTU

phenylthiourea

hpf

hr postfertilization

PFA

paraformaldahyde

nAChR

nicotinic acetylcholine receptors

TM

transmembrane

RT-PCR

reverse transcription polymerase chain reaction

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