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Neuroligins (Nlgns) are transmembrane cell adhesion molecules that bind with high specificity and affinity to β-neurexins (Nrxns) (Ichtchenko et al., 1995, 1996; Scheiffele et al., 2000). Cell localization studies suggest that, in general, Nlgns are localized in dendrites at synapses, i.e., at the postsynaptic density (PSD), whereas Nrxns are principally found at presynaptic terminals of axons (Dean et al., 2003; Rosales et al., 2005). These molecules have been hypothesized to mediate synaptogenesis and/or synaptic maturation (Sudhof, 2008). This interpretation is based on the ability of Nlgns expressed in non-neuronal cells to induce the formation of presynaptic terminals in contacting axons (Scheiffele et al., 2000) and the ability of Nrxns to induce the formation of postsynaptic specializations within dendrites of cultured neurons (Nam and Chen, 2005; Barrow et al., 2009).
In humans, 5 NLGN genes have been identified: NLGN1, 2, 3, 4X, and 4Y (Bolliger et al., 2001). Both NLGN3 and NLGN4X are located on the X chromosome, while NLGN4Y is located on the male sex chromosome Y. Mice, on the other hand, possess only 4 Nlgn genes, with murine Nlgn4 being located on an autosome and demonstrating significant sequence divergence from the human proteins (Bolliger et al., 2008). In addition, Nlgn genes have been found in all vertebrates examined to date and are also present in invertebrates. Five NLGN genes have been found in honey bees (Biswas et al., 2008) and 1 NLGN gene exists in the Caenorhabditis elegans genome (GenBank: NM_077882). It is possible that the increase in complexity of the Nlgn gene family has permitted the proteins to mediate the formation of different types of synapses and to potentially mediate synaptic specificity. While Nlgn1 is mostly localized to glutamatergic synapses (Song et al., 1999; Chih et al., 2005), Nlgn2 is concentrated at GABAergic synapses (Varoqueaux et al., 2004; Chih et al., 2005; Levinson et al., 2005). Corroborating a possible role of Nlgn genes during the development of the nervous system, Nlgn expression patterns are mostly confined to the nervous system and their expression levels increase during nervous system development in both honey bees and mice (Varoqueaux et al., 2006; Biswas et al., 2008).
The Nlgn family of genes has recently come under scrutiny because mutations in NLGN3 and NLGN4 genes have been found in patients with familial autism, Asperger syndrome, and X-linked mental retardation (Jamain et al., 2003; Laumonnier et al., 2004; Yan et al., 2005). The majority of these mutations are located in the extracellular acetylcholine-esterase (AChE)-like domain, abrogating the interaction with Nrxns and causing the protein to be retained in the endoplasmic reticulum (Chih et al., 2004). In addition to the AChE-like domain, Nlgns possess intracellular interaction domains that are also important for function. The C-terminal PDZ (postsynaptic density 95/Discs large/Zona occludens1) binding motif can interact with several PDZ domain–containing proteins, most notably PSD-95 (Irie et al., 1997; Meyer et al., 2004). This small motif is necessary for PSD-95 recruitment to synapses and for the cotransport of Nlgn1 with NMDA-type glutamate receptors (NMDARs) (Barrow et al., 2009). In addition, a distinct intracellular motif can mediate an interaction with the synaptic scaffolding protein S-SCAM through its WW domain (Iida et al., 2004).
Ablation of individual Nlgn genes in the mouse have no overt effect on neural development or behavior; however, combined deletion of Nlgn genes 1, 2, and 3 results in neurotransmission deficits of inhibitory synapses in the hindbrain of mouse embryos, leading to respiratory difficulties (Varoqueaux et al., 2006). Knock-down studies in the amygdala of rats show a dependence of LTP and fear conditioning on Nlgn1 expression (Kim et al., 2008). Furthermore, close examination of inhibitory synapses in the retinas of Nlgn2 knockout mice reveals a decrease in the recruitment of postsynaptic GABA (A) receptors (Patrizi et al., 2008). These results suggest that Nlgn genes may not play a pivotal role in initial synapse formation, but may be critical for synaptic maturation. Alternatively, the synaptogenic function of Nlgn proteins may be redundant between individual Nlgn family members and other synaptic cell adhesion molecule families such as SynCAMs (Biederer et al., 2002) and netrin-G-ligands (Kim et al., 2006).
To shed more light on the role of Nlgns in the development of the nervous system, we have characterized the nlgn genes in zebrafish. Zebrafish have a relatively simple nervous system in which it is possible to detect expression patterns at the level of identifiable neurons. Furthermore, the teleost genome duplication has resulted in the duplication of roughly 50% of genes (Postlethwait et al., 2000). This duplication has permitted the partitioning of gene function or expression patterns (Cresko et al., 2003). Indeed, we have found that zebrafish possess 7 nlgn genes, 3 of which represent duplicates of mammalian Nlgn genes 2, 3, and 4. In contrast, nlgn1 is present as a single copy. They are expressed throughout the nervous system during development and in the adult, with increasing expression levels during the course of nervous system development. Here we describe highly divergent expression patterns within the nervous system between the 7 nlgn genes during development and in the adult brain. Our data suggest that these expression patterns may provide the specificity necessary to dissect the roles of these genes during neural development in vivo.
RESULTS AND DISCUSSION
Isolation and Characterization of the Zebrafish nlgn Genes
There are five distinct NLGN gene family members in humans: NLGN1, 2, 3, 4X, and 4Y. In contrast, mice only possess four, with Nlgn4 possessing great sequence divergence from other members of the Nlgn family (Bolliger et al., 2008). We have identified seven Nlgn orthologs in zebrafish by searching the zebrafish genome database and NCBI database using mammalian Nlgns as templates. Using reverse transcriptase PCR (RT-PCR), we have fully cloned and sequenced all seven zebrafish nlgn open reading frames, indicating all seven nlgn genes are expressed in zebrafish.
Alignment of the Nlgn amino acid sequences with those of human and mouse Nlgns showed that the primary structure of Nlgn proteins is largely conserved (see Supp. Fig. S1, which is available online). This alignment revealed that for each human NLGN protein, except NLGN1, there are two zebrafish proteins that show a high degree of similarity. Because the ancestor of teleosts underwent genome duplication after branching from the tetrapod lineage (Postlethwait et al., 2000), we hypothesized these genes are duplicates. Consistent with this hypothesis, the amino acid identity as a percentage between the different sets of duplicates and their human orthologs is greater than that of the other Nlgns to each other (Table 1). Phylogenetic analysis confirms that the duplicates segregate into distinct clades with their mammalian orthologs (Biswas et al., 2008; Bolliger et al., 2008). We have named these co-orthologs nlgn2a, nlgn2b, nlgn3a, nlgn3b, nlgn4a, and nlgn4b.
Table 1. Percent Amino Acid Identity
Amino acid conservation of Nlgn proteins. Percentage amino acid identity for pairwise alignments of the seven zebrafish Nlgn proteins (Danio rerio) and the five human NLGN proteins (Homo sapiens). Shaded boxes highlight comparisons between presumed paralogs within the zebrafish genome (left) and comparisons between human and zebrafish orthologs (right).
Interestingly, zebrafish Nlgn4a and 4b are more closely related, in terms of amino acid sequence identity, to the human orthologs NLGN4X and 4Y (between 78 and 82% identity, Table 1), than mouse Nlgn4 is to the human proteins (around 60% identity; Bolliger et al., 2008). It will be interesting to further analyze the commonalities between the zebrafish Nlgn4 and the human NLGN4 duplicates, as only higher primates (Homo sapiens and Pan troglodytes) and teleost fish (Danio rerio and Tetraodon nigroviridis) possess duplicated nlgn4 genes. BLAST searches of rhesus macaque (Macacca mulatta), dog (Canis lupus familiaris), rat (Rattus norvegicus), short-tailed opossum (Monodelphis domestica), and chicken (Gallus gallus) revealed only single copies of Nlgn4 (see also Bolliger et al., 2008).
We detected only one nlgn gene similar to human NLGN1 suggesting that this gene is a single ortholog and that its duplicate was likely lost during evolution. Furthermore, stickleback (Gasterosteus aculeatus) and puffer fish (Tetraodon nigroviridis) also possess a single nlgn1 gene whereas the other nlgns are duplicated, as in zebrafish (Bolliger et al., 2008), lending weight to the observation that only a single ortholog exists in zebrafish.
We performed an analysis of conserved synteny between zebrafish and human genomes to further test the hypothesis of orthology and to determine if nlgn2a, nlgn2b, nlgn3a, nlgn3b, nlgn4a and nlgn4b genes are duplicates resulting from a genome duplication (Postlethwait et al., 2000; Jaillon et al., 2004; Postlethwait, 2007). This was performed using synteny analysis software (Catchen et al., 2009) based on the seventh zebrafish genome assembly (Zv7). Due to errors that still may be present in this version, our analysis may be imperfect. Our results suggest that zebrafish nlgn1 is the single ortholog of human NLGN1, as several genes surrounding NLGN1 on human chromosome 3 (Hsa3) have preserved co-localization on zebrafish chromosome 11(Dre11) in the regions around nlgn1 (Fig. 1A). Three of the six genes upstream of nlgn1 (ect2, b3gnt5, zgc:110312) possess duplicates (on Dre2), whereas downstream genes of nlgn1 (rfc4, zgc92715, mfn1) also returned to singleton status after the genome duplication.
The regions flanking NLGN2 on Hsa17 display conserved synteny with regions flanking zebrafish nlgn2a and nlgn2b on Dre10 and 7, respectively (Fig. 1B). For the most part, genes on Dre10 display the same order as those on Hsa17 and three genes are in the same order on Dre7, suggesting minimal chromosomal rearrangement in this region. Similarly, we also see conserved synteny between NLGN3 on Hsa3 and nlgn3a on Dre14 and nlgn3b on Dre5 (Fig. 1C), with the flanking genes ASB12 and ZDHHC15 possessing orthologs on both zebrafish chromosomes.
We detected no conserved syntenies between HsaY and zebrafish nlgn4 paralogs on Dre9 and Dre1. This is consistent with the loss of many genes from HsaY, which occurred after the generation of the sex chromosomes by duplication of a single autosomal ancestor (Graves, 1995). We did find conserved synteny between HsaX and the zebrafish nlgn4 duplicates. In addition to NLGN4X, we found one other gene, GPM6B, which is a close neighbor to the nlgn4 genes and has orthologs on both Dre9 and Dre1. We found four genes surrounding nlgn4b that appear to be single orthologs and three genes surrounding nlgn4a that appear to be single orthologs (Fig. 1D). This represents a reduced amount of conserved synteny, as compared to the other nlgn genes. This may be due to the locations of NLGN4X and nlgn4a near the end of HsaX and Dre9, respectively, as chromosomal rearrangements, such as fissions, are more likely near chromosome ends (Bailey and Murnane, 2006). We conclude that all seven identified nlgn genes are indeed orthologs of four of the human NLGN genes.
Temporal Expression of nlgn Genes
We conducted RT-PCR to detect the presence of the nlgn genes during development and in the adult brain (Fig. 2). All seven nlgns were expressed at or before 24 hr postfertilization (hpf), with expression remaining present or increasing as development progressed. In addition, all seven genes were strongly expressed in the adult brain. We found that nlgn1 and nlgn4a were expressed at the 16-cell stage (1.5 hpf, Fig. 2A,F), before midblastula transition, indicative of maternal mRNA expression. This expression was then lost until it was faintly detected at 24 hpf. Expression of nlgn2a was first detectable at 16 hpf and nlgn2b expression was first detectable at the 3-somite stage (11 hpf, Fig. 2B,C). We found that nlgn3a was also expressed maternally. However, the low level made detection difficult (1.5 hpf, Fig. 2D). This expression was then lost and was again detected at 16 hpf. Expression of nlgn3b was first detected at 90% epiboly (9 hpf, Fig. 2E). This gene remained expressed at an intermediate level, and was then more strongly expressed at 48 hpf. nlgn4b was first detected at 3-somites (11 hpf, Fig. 2G) and its expression then appeared to increase through development. We conclude that all nlgn genes are expressed during the development of zebrafish and that they show distinct temporal expression profiles.
Expression Patterns in the Developing Brain
To gain insight into possible anatomical specificity of expression patterns for the seven nlgn genes, we assayed the temporal and spatial distribution of their mRNAs by in situ hybridization (ISH) in whole-mount and sectioned zebrafish in the embryonic and adult zebrafish nervous system. We are confident that the probes used were specific and did not cross-react, as their expression patterns are highly specific and sense probes did not reveal any staining.
Our RT-PCR analysis suggested that, during early development, zebrafish embryos showed either no or very weak expression of most nlgn genes until after 16 hpf (Fig. 2). Since we expected nlgns to be expressed in the developing nervous system, we decided to compare nlgn gene expression patterns at pharyngula stage (24–42 hpf) and hatching period (48–72 hpf) (Kimmel et al., 1995). At 24 hpf, the sensory-motor reflexive circuits are becoming functional (Drapeau et al., 2002), suggesting ongoing synapse formation in the spinal cord (Pietri et al., 2009). At 48 hpf, sensory organs are becoming more mature. Olfactory placodes reveal beating cilia and hair cells have differentiated in the sensory maculae of the otic vesicle and in neuromasts (Kimmel et al., 1995). In the eye at 30 hpf, the first post-mitotic neurons have differentiated (Hu and Easter, 1999), and by 48 hpf retinal axons reach the optic tectum (Burrill and Easter, 1995). We conclude our descriptive study by discussing the adult nlgn expression in the adult zebrafish brain and comparing the expression pattern to adult mouse brain Nlgn gene expression.
At both 24 and 48 hpf, the vast majority of expression for all nlgns was localized to the nervous system of zebrafish. Interestingly, close examination of the mRNA expression revealed very specific expression patterns, down to individual cells, particularly in the developing hindbrain and spinal cord (see below).
At 24 hpf, all seven nlgn genes were expressed in restricted regions in the developing forebrain (telencephalon [t] and diencephalon [d]) (Fig. 3A). We found weak and rather broad expression of nlgn1 in the telencephalon, diencephalon, and the eyes (Fig. 3A,A'). In contrast, all other nlgn genes presented more robust and specific expression patterns. We found nlgn2a (Fig. 3C,C') and nlgn2b (Fig. 3E,E') mRNA transcripts expressed in the dorsal-rostral and the ventral-rostral regions of the telencephalon, whereas nlgn3a (Fig.3G,G') and nlgn3b mRNA transcripts (Fig. 3I,I') were located more diffusely within the telencephalon. The expression of nlgn4a (Fig. 3K,K') was restricted to a dorsal and caudal aspect of the telencephalon, likely the olfactory bulbs (ob in Fig. 3K′). Interestingly, we found nlgn4b expression (Fig. 3M,M') in a ventro-caudal region of the telencephalon, presumably the dorso-rostral cluster (drc in Fig. 3M). In addition, nlgn4b was expressed in the dorsal-most cell layers of the telencephalon (arrowheads in Fig. 3M,M′). In general, all nlgn genes were expressed in the diencephalon in slightly different expression patterns. All nlgn genes except nlgn1 were expressed in the ventro-rostral cluster (vrc; Fig. 3C).
In the region of basal mesencephalon, we found clusters of cells expressing nlgn2a, nlgn2b, nlgn3a, nlgn3b, and nlgn4a, suggesting expression of these nlgn genes in the ventro-caudal cluster (vcc in Fig. 3E). Interestingly, nlgn2b was highly expressed in a small number of cells in the vcc (arrow in Fig. 3E'). In general, we found faint and diffuse signal of all seven nlgn genes in the dorsal midbrain, suggesting only weak or no expression in this region. While all seven nlgn genes were expressed in the cranial ganglia (cg in Fig. 3C′), the expression of nlgn2a (Fig. 3C') was significantly stronger compared to all other nlgn genes. As the expression of nlgn genes in the hindbrain was very restricted and selective at 24 hpf, we describe it in more detail in Figure 5.
At 48 hpf, nlgn1 expression in the forebrain (Fig. 3B,B′) appeared weaker compared to the other nlgn genes. Within the telencephalon, including the olfactory bulbs, nlgn2 and 3 genes had similar expression patterns, whereas the nlgn4 genes displayed more unique expression patterns. Also, nlgn4b showed a unique staining pattern in the diencephalon, likely in the posterior tuberculum, hypothalamus, and hypophysis (arrows in Fig. 3N). Within the diencephalon, all other nlgn genes were expressed in a similar and at least partially overlapping pattern, namely in the preoptic area, hypophysis, thalamus, and hypothalamus. Furthermore, all nlgn genes shared expression in the preoptic tectum, tegmentum, cerebellar plate, medulla oblongata, and patches of otic sensory neurons in the hindbrain.
While expression for all nlgn genes was detected in the developing eyes, the expression levels and distributions differed. nlgn1 demonstrated stronger expression in the ganglion cell layer at 48 hpf (Fig. 4A3); nlgn4b was expressed the strongest in the peripheral region of the retina (Fig. 4G2,G3). In contrast, expression for nlgn2a, 2b, 3a, 3b, and 4a was distributed evenly across all layers of retina at this age, albeit with different expression levels (Fig. 4B3–F3). nlgn2a, 2b, and 3b were expressed in the olfactory placode (op in Fig. 4B1,C1, and not shown), while expression was not detected for nlgn1, 3a, 4a, and 4b. At 48 hpf, the optic tectum (ot in Fig. 4F3) was clearly visible as the most dorsal and caudal part of the mesencephalon. Expression was weak in the ot for nlgn1, 2b, 3a and 4a, but strong in the tegmentum. This lack of expression was most clearly visible for nlgn4a (Fig. 4F3). Although broadly expressed, nlgn2a showed stronger expression in the tegmentum. nlgn3b appeared uniformly expressed throughout the midbrain. nlgn1, 2a, 2b, and 3b were expressed throughout the hypothalamus (ht in Fig. 4A3; Fig. 4B3,C3,E3), while nlgn4a was not expressed in this brain region (Fig. 4F3). In contrast, nlgn3a was almost exclusively expressed at its ventral portion (Fig. 4D3). At this level of the 48-hpf brain, this region corresponds to the ventral hypothalamus and the anterior-most portion of the hypophysis. nlgn2a also showed expression in the hypophysis (Fig. 4B3), whereas nlgn4b appeared to be exclusively localized to the hypophysis (hy in Fig. 4G3) and not to the hypothalamus. More posteriorly, expression was detected for all nlgn genes in the myelencephalon (Fig. 4A4-G4), with very distinct expression in ventral regions of this structure for nlgn2b and 3a (Fig. 4C4,D4) at the level of the otic vesicle (ov in Fig. 4A4). In the region of the hindbrain adjacent to the pectoral fin buds (pfb in Fig. 4A5), nlgns 1, 3b, and 4b displayed a rather uniform distribution. In contrast, nlgn3a was localized ventrally, while nlgn2a, 2b, and 4a were localized laterally. We conclude that all seven nlgn genes are expressed widely throughout the developing brain of zebrafish in highly specific but also partially overlapping patterns. Furthermore, we observed that the duplicate pairs (e.g., nlgn2a and 2b) show distinct expression patterns, suggesting subfunction partitioning between the duplicates.
Cell-Specific Expression in the Reticulospinal Neurons
Upon close examination of expression of the nlgn genes in the hindbrain of embryos at 24 hpf, we noticed very distinct and restricted expression patterns. It was possible to identify individual cells in particular rhombomeres (Fig. 5). For example, the Mauthner (M) cells are conspicuously large neurons located in rhombomere 4 (r4), just anterior to the otic vesicle (ov; Fig. 5A). The expression patterns detected for nlgn1 and nlgn4b suggest that they may be almost exclusively expressed in large cells in r4, perhaps Mauthner cells (arrows in Fig. 5A,G). In contrast, nlgn2a, 2b, 3a, and 3b were expressed symmetrically in multiple cells throughout r2–6 on either side of the midline (Fig. 5B–F). The large reticulospinal neurons located throughout this part of the hindbrain are involved in generating specific swim behaviors, such as the escape response (Gahtan et al., 2002). Thus, it is interesting to speculate that the nlgn genes are expressed in the reticulospinal neurons and may be aiding in the formation of specific connections necessary for sensory-motor control in zebrafish embryos and larvae.
Differential Expression in the Spinal Cord
Next we focused our attention on expression patterns of the nlgn genes in the developing trunk. nlgn1 was not detectable in the trunk (not shown). However, all other nlgn genes were expressed in the trunk, with the vast majority of ISH staining localizing exclusively to the spinal cord (sc in Fig. 6A) at 24 and 48 hpf. Interestingly, they showed highly varied patterns of expression that were also dynamic with development. nlgn2a was expressed in cells in the dorsal spinal cord, presumably Rohon-Beard sensory neurons (arrows in Fig. 6A) and dorsal interneurons at 24 hpf. This expression pattern transitioned to more ventrally located cells at 48 hpf (Fig. 6B). In contrast, nlgn2b, was expressed in ventral cells of the spinal cord, presumably motoneurons and interneurons, at both 24 and 48 hpf (Fig. 6C,D). Both nlgn2 genes were expressed with an anterior-to-posterior gradient at 24 hpf (Fig. 6A,C), suggesting that they are required during maturation of spinal cord development. nlgn3a and 3b were weakly expressed in spinal cord cells. nlgn3a was stronger expressed in clusters in the ventral spinal cord, likely motoneurons; no expression was detectable at 48 hpf (Fig. 6F). In addition, nlgn3a showed strong staining ventral to the somites at 24 hpf (arrow in Fig. 6E), consistent with expression in the mesoderm. This is interesting as NLGN3 was reported to be expressed outside of the nervous system in humans, but not in rodents (Philibert et al., 2000). While nlgn4a showed diffuse and weak expression at both 24 and 48 hpf (Fig. 6I,J), similarly to nlgn3b (Fig. 6G,H), nlgn4b showed a remarkably specific expression pattern in a limited number of interneurons at both time points (Fig. 6K,L). In conclusion, our analysis of nlgn gene expression in the trunks of developing zebrafish embryos demonstrated highly divergent expression patterns for the different nlgn genes, suggesting that the Nlgn proteins may participate in cell-type-specific functions during nervous system development.
Expression in the Adult Brain
In contrast to the specific expression patterns displayed by the nlgn genes in the developing nervous system, these genes were expressed in highly overlapping patterns in the adult brain of zebrafish (Fig. 7). In general, ISH staining was seen in most regions where neuronal cell bodies are to be expected, including the periventricular gray zone of the optic tectum (OT), periventricular pretectal nucleus, thalamus, parvocellular preoptic nucleus, hypothalamus, and the facial and vagal lobes of the hindbrain (see Fig. 7A′ for diagram of brain regions). However, we noticed that each nlgn gene was expressed in a distinct subregion of the telencephalon. For example, nlgn1 was expressed in the ventral nucleus of the ventral telencephalon (Vv in Fig. 7B), whereas nlgn2a was expressed in the dorsal nucleus of the ventral telencephalon (Vd) and in the medial zone of dorsal telencephalon (Dm, see Fig. 7C). nlgn2b showed a similar pattern of expression in the telencephalon as nlgn1, which contrasted sharply with nlgn3a, which was expressed exclusively in the Dm (Fig. 7E). nlgn3b expression was broad and uniform in the forebrain (Fig. 7F). nlgn4a and 4b were sparsely expressed in this forebrain structure, being mostly restricted to the Vd (Fig. 7G,H). In addition, we noticed that all but nlgn 4b were expressed in sparse cells in the dorsal layers of the optic tectum (OT, Fig. 7B′–H′).
In general, nlgn4a and 4b showed the most divergent expression patterns from all other nlgn genes. For example, nlgn4a (Fig. 7G) was not expressed in the torus longitudinalis (Tl, see Fig. 7C), whereas all other nlgn genes were expressed in this structure (expression of nlgn2a was reduced but present). nlgn4b expression was absent from the corpus mamillare (Cm in Fig. 7H), a brain nucleus that presented expression for all other nlgn genes. Expression in the cerebellum also demonstrated differential patterning for the nlgn4 genes. While most of the nlgn genes were expressed in the granule cell layer (GCL in Fig. 7E') of the corpus cerebelli (CCe, see Fig. 7H') and valvula cerebelli (VCe, see Fig.7G″), nlgn4a was only expressed in the Purkinje cell layer (PCL in Fig. 7G′) of the CCe, with almost no expression in the VCe. Furthermore, nlgn4b was expressed in the granule cell layer of the CCe, but not in the VCe (Fig. 7H′).
Our results suggest that, in general, the expression patterns of the nlgn genes in the adult zebrafish brain largely resemble the broad and overlapping expression patterns seen for Nlgn1, 2, and 3 in mice (http://mouse.brain-map.org), but do show some regional differences, especially for nlgn4a and 4b. To date, there is no information regarding the expression pattern of mouse Nlgn4, as this was only recently identified in the mouse genome due to unusually high variation in the mouse Nlgn4 sequence compared to other species (Bolliger et al., 2008). We conclude that the nlgn genes are expressed predominantly in the nervous system during zebrafish development and in the adult. Their dynamic and highly specific expression patterns suggest an important role in nervous system development.
Cloning of nlgn Genes
To clone the zebrafish nlgn genes, we searched the zebrafish genome assembly Zv7 from the Sanger Institute (http://www.ensembl.org/Danio_rerio) using the human NLGN1, NLGN2, NLGN3, and NLGN4X protein sequences (GenBank: NM_014932, NM_020795, NM_018977 and NM_020742, respectively). This search revealed seven zebrafish putative nlgn genes, with duplications at the nlgn2, nlgn3, and nlgn4 loci. The putative translated nlgn coding sequences contained signal peptide sequences and stop codons, indicating full-length open reading frames. PCR primers used in the cloning of the nlgn genes were designed 5′ and 3′ to these predicted coding sequences. RT-PCR was used to clone full-length zebrafish nlgn coding sequences from 6 days postfertilization cDNA and adult brain cDNA. The coding sequences for the zebrafish nlgn mRNAs have been deposited with NCBI under the following accession numbers: nlgn1, GQ892833; nlgn2a, GQ892834; nlgn2b, GQ892835; nlgn3a, GQ892836; nlgn3b, GQ892837; nlgn4a, GQ892838; nlgn4b, GQ892839.
Protein sequences deduced from the cloned sequences of the zebrafish nlgn genes were aligned using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). This alignment was used to predict the shared amino acid percent identity from the pairwise alignments of the seven Nlgn proteins.
RNA was isolated from whole embryos and adult brains using Trizol Reagent (Invitrogen, Carlsbad, CA). mRNA was isolated from total RNA and potential genomic DNA contamination using the Oligotex mRNA Midi Kit (Qiagen, Valencia, CA). First-Strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis System (Invitrogen). The cDNAs were screened for genomic contamination by performing a PCR reaction with a reverse primer specific to intron 1 of the nlgn1 gene (forward primer 5′-AACAACCAAACCGCCTGAGC-3′ and reverse primer 5′-GGATGGTGGGAAGGTTAGGACA-3′). Genomic DNA was used as a control. Concentrations of the cDNAs were determined and adjusted using primers specific to the tubulin-alpha gene (forward primer 5′-CTGTTGACTACGGAAAGAAGT-3′ and reverse primer 5′-TATGTGGACGCTCTATGTCTA-3′). cDNAs were amplified using forward primers designed to amplify a 3′ region of the nlgn gene low in nucleotide similarity and reverse primers in the 3′-UTR to ensure specificity. Primer sequences are listed in Supp. Table S1.
In Situ Hybridization
AB/Tübingen zebrafish embryos were raised at 28.5°C under standard procedures (Westerfield, 2000) and staged in hours post fertilization (hpf, Kimmel et al., 1995). To prevent pigment formation, embryos were treated with 0.003% 1-phenyl-2-thiourea (PTU) in embryo medium at 8 hpf. Following the manufacturer's protocol (Roche, Nutley, NJ), sense and antisense RNA probes were transcribed in vitro from linearized plasmids and tagged with digoxigenin. Purchased ESTs were used to synthesize the nlgn1 and nlgn3b probes (Open Biosystems catalog numbers EDR1052-5635463 and EDR442-98316200, respectively). Probes for nlgn2a, nlgn2b, nlgn3a, nlgn4a, and nlgn4b were synthesized from full-length cDNAs cloned in pCRII-TOPO (Invitrogen). Full-length probes were fragmented into 600–800-bp segments by incubating the probes in 2× carbonate buffer at 60°C for time = (Linitial–Lfinal)/(0.11 kb min−1 × Linitial-Lfinal) where L= length in kb. Fragmentation was stopped with 3M NaOAc and the probes were precipitated using standard protocol. In order to test the specificity of the full-length probes, we also synthesized probes in the 3′-UTR region; although this staining matched that of the fragmented probes, the signal was weaker. Thus, in this report we show whole-mount ISH stainings carried out with the full-length fragmented probes according to the procedures outlined in Thisse and Thisse (2008) with some minor modifications. ISH on frozen sections was performed as described in (Jensen et al., 2001) with some modifications. Sections were cut at 18 μm for 48-hpf embryos and 20 μm for adult brains. Embryos and sections were viewed with a Zeiss (Thornwood, NY) Axioplan2 microscope and photographed with a Zeiss AxioCam MRc5 camera. The descriptions of expression patterns are based on high-resolution close-up images and many additional sections that have not been reproduced due to limited space.
We thank J. Postlethwait (Univ. of Oregon) for suggestions regarding synteny analysis and the University of Oregon Zebrafish and Histology Facilites. This work was supported by grant R01NS065795 from the National Institute of Neurological Disorders to P. Washbourne and a NIH Developmental Biology Training Grant to C. Davey.