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Laboratory of Reproductive Biology (K.O., F.S., E.L.L., Y.N.), National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan; Department of Marine Biosciences (G.Y., Y.T.), Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan; Department of Biological Sciences (K.N.), Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan; and Department of Aquatic Bioscience (K.A.), Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
Address all correspondence and requests for reprints to: Yoshitaka Nagahama, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. E-mail: nagahama{at}nibb.ac.jp.
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Abstract |
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Introduction |
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It is generally accepted that, unlike most central nervous system neurons, forebrain GnRH neurons, including the preoptic and terminal nerve ganglion populations, originate peripherally in the olfactory placode, from which they migrate to their final destinations (3). However, recent studies have questioned whether the olfactory placode is the exclusive source of forebrain GnRH neurons. For instance, a few populations of GnRH neurons of nonolfactory origin have been described in the mouse brain (4). Moreover, the embryological origin of forebrain GnRH neurons is controversial in nonmammalian taxa, with some data suggesting that the olfactory region is the source of the hypophysiotropic GnRH neurons in the preoptic area in nonmammalian vertebrates, as has been described in mammals (5), whereas others have reported that they arise within the diencephalic zone (6, 7) or anterior telencephalon (8).
When GnRH neurons fail to develop normally, reproductive development suffers, resulting in delayed, or the absence of, pubertal maturation (9). X-linked Kallmann syndrome (X-KS) is a human disorder characterized by the absence of gonadal function secondary to a deficiency in gonadotropin secretion (10). It is known to be caused by a mutation in the KAL1 gene on the X chromosome, which encodes what appears to be an extracellular matrix protein (11, 12). In the only human fetal brain with X-KS that has been studied, GnRH neurons were found to be inappropriately clustered in the olfactory compartment and absent from the forebrain, suggesting that the syndrome is caused by the inability of GnRH neurons to migrate from the olfactory region to the forebrain and that, by extension, KAL1 regulates this process (13). The foregoing notwithstanding, the pathogenesis of X-KS is still largely unknown.
One of the greatest difficulties in exploring developmental aspects of GnRH neuronal maturation and migration, as well as the pathogenesis of human disorders in GnRH deficiency, including X-KS, has been the lack of an animal model. Mammals have highly scattered and scarce populations of forebrain GnRH neurons that express Gnrh1. Because rodents curiously seem to lack a KAL1 ortholog in their genome, it has not been possible to generate a mouse/rat genetic model of X-KS (14). In recent years, a small freshwater fish, i.e. the medaka Oryzias latipes, has emerged as an important vertebrate model for developmental and reproductive studies (15, 16, 17). The medaka has three paralogous GnRH genes, gnrh1, gnrh2, and gnrh3, and its forebrain neuronal populations express either gnrh1 or gnrh3 (18). Its preoptic population that regulates gonadotropin secretion expresses gnrh1, and its terminal nerve ganglion that regulates reproductive behavior expresses gnrh3, the latter of which has been lost in mammals but whose function may be compensated for by Gnrh1 (19). Thus, the medaka can be used to examine the independent function of these two GnRH neuronal populations. What makes this model particularly useful is the fact that embryos can be created by external fertilization, and because they exhibit optical clarity throughout embryogenesis, living cells labeled with the green fluorescent protein (GFP) gene can be directly monitored (20, 21). This technique is clearly useful for the investigation of GnRH neuronal development. To date, although transgenic mice, rats, and zebrafish that expressed GFP in their GnRH neurons have been generated and used to study the electrophysiology of GnRH neurons (22, 23, 24, 25), there have been no reports of the use of such animals for the study of GnRH neuronal development in vivo.
In the present study, we report the establishment of transgenic medaka that express GFP under the control of the gnrh1 and gnrh3 promoters; these animals were used to track the development of GnRH neurons from embryogenesis to adulthood. We also identified two medaka homologs of KAL1, designated kal1.1 and kal1.2, assessed their chromosomal location and developmental expression, and showed that the X-KS phenotype could be mimicked by antisense knockdown of kal1.1. Our analyses showed that these transgenic medaka represent a valuable model system for studying GnRH neuronal development as well GnRH deficiencies, including X-KS.
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Materials and Methods |
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Generation of constructs
The medaka gnrh1 and gnrh3 loci were isolated by screening a bacterial artificial chromosome library, as previously described (19). A fragment containing the 5'-flanking region (
6.0 kb) along with exon 1 of gnrh1 was PCR amplified from a bacterial artificial chromosome clone screened, as was a fragment of the 5'-flanking region (5.8 kb) and exon 1 of gnrh3. In each gene, exon 1 coded a 5'-untranslated region, and the initiation methionine codon was present at the 5' end of exon 2. Each fragment was fused with the GFP-coding sequence followed by the polyadenylation signal of the simian virus 40 (SV40) t antigen gene, which was excised from phrGFP-Nuc (Stratagene, La Jolla, CA), and subcloned into the cloning vector pcR-XL-TOPO (Invitrogen, Groningen, The Netherlands) (Fig. 1
). These constructs were purified, linearized with XhoI, and dissolved in 10 mM Tris-HCl (pH 8.0) containing 0.1 mM EDTA.
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In situ hybridization
Digoxigenin-labeled RNA probes of gnrh1, gnrh3, kal1.1, and kal1.2 sense and antisense strands were synthesized by in vitro transcription using digoxigenin-labeling mix (Roche, Grenzach-Wyhlen, Germany) according to the manufacturer’s instructions. Whole-mount and section in situ hybridization experiments were carried out with standard protocols using embryos obtained before hatching and 3-month-old fish, respectively. Section in situ hybridization was carried out on transversely cut (8 µm), paraffin-embedded brain sections. After whole-mount in situ hybridization, the embryos were paraffin embedded and transversely sectioned (8 µm). In no case was staining observed with the control sense probes (data not shown).
cDNA cloning
Double-strand cDNA was synthesized from the medaka brain using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA) (26). Fragments encoding two medaka homologs of KAL1, designated kal1.1 and kal1.2, were amplified using degenerated PCR primers. The PCR products were analyzed on agarose gels and ligated into the pGEM-Teasy vector (Promega, Madison, WI). The plasmid DNA was purified and sequenced using an ABI Prism 3100 sequencer (Applied Biosciences, Branchburg, NJ). After determination of the partial cDNA sequences, gene-specific PCR primers were designed and rapid amplification of cDNA ends was carried out to isolate the full-length cDNAs using the Marathon kit according to the manufacturer’s instructions. Electrophoresis, subcloning, and sequencing were performed as described above. The deduced amino acid sequences of kal1.1 and kal1.2 in the medaka and their counterparts in other species were aligned using CLUSTAL W (27) using the default setting. A phylogenetic tree was generated with PHYLIP software (28) using the neighbor-joining method (29).
Linkage analysis
Several parts of kal1.1 and kal1.2 from two inbred strains of the medaka, HNI and AA2, were sequenced, and insertion/deletion polymorphisms between the two strains were detected. The oligonucleotide primer pairs, K1-F (5'-GGTGGTTTATTATGAGAATGTACAGATAGTG-3')/K1-R (5'-CGGTACTCATATACAGTCAGCGGACTA-3') and K2-F (5'-ACTATGGATGGAGCAGTTATAACACTGA-3')/K2-R (5'-GGCTTCAAATCAATGCAATAAACTCTTATTTCA-3'), were designed to amplify the polymorphism-containing sequences in kal1.1 and kal1.2, respectively. PCR amplification of the HNI and AA2 genomic DNAs with K1-F/K1-R yielded DNA fragments of 129 and 101 bp, respectively. Amplification of the HNI and AA2 genomic DNAs with K2-F/K2-R produced fragments of 71 and 86 bp, respectively. The chromosomal location of these two genes was determined using reference-typing DNA panels derived from 39 offspring of a backcross between an HNI/AA2 male F1 and an AA2 female parental line and 52 offspring of a backcross between an HNI/AA2 female F1 and an AA2 male parental line (30, 31). Genotypes were analyzed by amplification of the polymorphic DNA regions followed by agarose gel electrophoresis.
Gene knockdown
Antisense peptide nucleic acid (PNA) (32, 33, 34) was designed to overlap the translational start sites of kal1.1 (5'-AAACATCGTCCCGCTGCT-3') and kal1.2 (5'-CGGAGCACTGACATCGCT-3') (gripNA; Active Motif, Carlsbad, CA). PNA was solubilized in PBS (pH 7.5) at a final concentration of 1.0–2.0 mM and injected into the cytoplasm of one-cell-stage embryos of gnrh1-GFP and gnrh3-GFP transgenic and wild-type medaka. A control experiment was carried out using PNA targeted against GFP (5'-TGCTTGCTCACCATGGTT-3'). Injection of the GFP PNA silenced GFP expression for at least 8 d and did not result in the formation of any abnormal phenotypes during that period (data not shown). Images were acquired as described above. At least 20 embryos were treated for each PNA.
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Results |
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Characterization of the gnrh1-GFP transgenic medaka
In the gnrh1-GFP transgenic medaka, GFP expression was first identified bilaterally, as early as 2 dpf, in the neuronal clusters at the junction between the olfactory compartment and olfactory bulb and in the dorsal telencephalon (Fig. 2A). The olfactory neuronal population migrated caudally to the forebrain and passed through the ventral telencephalon to reach the ventral preoptic area by 4 dpf (Fig. 2, B and C). During this process, GFP-expressing neurons continued to appear in the olfactory region and migrate through to the ventral preoptic area (Fig. 2C). By around 20 dpf, the ventral preoptic neurons extended projections to the anterior pituitary (Fig. 2E). In 3-month-old fish, GFP-expressing neurons were seen in the ventral preoptic area (Fig. 2F). The GFP-expressing neurons that originated in the dorsal telencephalon, on the other hand, migrated in ventral and caudal directions to reach the dorsal region of the preoptic area by 4 dpf (Fig. 2, B–D).
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). These neurons advanced caudally, taking a more medial migratory route than that of the olfactory-derived neurons, and reached the medial ventral telencephalic region by 10 dpf (Fig. 2D). GFP fluorescence persisted in this neuronal population through the adult stage (Fig. 2, E and F). Finally, nonmigratory GFP-expressing neuronal populations were seen in the ventral hypothalamus and lateral myelencephalon (Fig. 2, C and D).
Unfortunately, endogenous gnrh1 mRNA expression during embryogenesis could not be detected by whole-mount in situ hybridization. We therefore carried out section in situ hybridization using adult brains, within which we were able to detect endogenous gnrh1 mRNA expression in the medial ventral telencephalon (Fig. 3A), ventral preoptic area (Fig. 3B), dorsal preoptic area (Fig. 3B), and ventral hypothalamus (Fig. 3C). These results confirmed that the GFP fluorescence observed in these sites actually reflected the endogenous expression of gnrh1.
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) and in the blastodisc of one-cell-stage fertilized embryos (Fig. 2H) in the gnrh3-GFP transgenic medaka, suggesting that it reflected maternal expression. To verify this, homozygous gnrh3-GFP transgenic males and females were mated with wild-type fish. All embryos produced by the gnrh3-GFP transgenic females were green (Fig. 2, I–K), whereas no embryos produced by the gnrh3-GFP males exhibited fluorescence (Fig. 2, L–N), supporting the notion that GFP expression was maternally inherited.
A single neuronal population that zygotically expressed GFP was detected in the forebrain. This population was encountered at the junction site between the olfactory compartment and olfactory bulb as early as 2 dpf, after which it coursed caudally into the forebrain (Fig. 2, P and Q), where they reached as far as the terminal nerve ganglion; a subset of these neurons advanced further to reach the preoptic area/anterior mesencephalon by 4 dpf (Fig. 2R). These neurons were distributed throughout their migratory route at this stage and converged on the terminal nerve ganglion and preoptic area/anterior mesencephalon by 5 dpf (Fig. 2S). No axonal projections to the pituitary were detected in preoptic GFP-expressing neurons (Fig. 2U). In 3-month-old fish, strong GFP expression was observed in the terminal nerve ganglion (Fig. 2, V–X). GFP signal was also seen in the preoptic area and anterior mesencephalon (Fig. 2, V and X).
Zygotic GFP expression was also detected in neurons, along with their central axons, in the trigeminal ganglion on 2 dpf and thereafter (Fig. 2, O–U). Careful observation revealed that their peripheral axons also fluoresced (data not shown). After 4 dpf, GFP fluorescence appeared in the retina and optic nerve (Fig. 2, T and U) whereas after 5 dpf, GFP-expressing neurons were demonstrable in sympathetic ganglion (Fig. 2, S and T). Each somite contained one set of clustered GFP-expressing neurons.
Using whole-mount in situ hybridization, gnrh3 mRNA expression was detected in the olfactory-forebrain area and trigeminal ganglion of medaka embryos (Fig. 3, D–K), supporting the notion that the GFP fluorescence seen in these sites of the gnrh3-GFP transgenic medaka reflects endogenous gnrh3 expression. We also carried out brain section in situ hybridization in 3-month-old fish and detected gnrh3 mRNA expression in the terminal nerve ganglion (Fig. 3L), ventral telencephalon (Fig. 3M), preoptic area (Fig. 3N), and anterior mesencephalon (Fig. 3, O and P), thus providing additional confirmation of endogenous gnrh3 mRNA expression in these regions.
Presence of two KAL1 homologs in the medaka
To determine whether the gnrh1-GFP and gnrh3-GFP transgenic medaka can be used as an in vivo vertebrate model for X-KS, we identified medaka KAL1 homologs, assessed their chromosomal location and developmental expression, and examined their effects on GnRH neuronal development.
Two medaka homologs of KAL1, designated kal1.1 and kal1.2, were identified by cDNA cloning. The medaka kal1.1 and kal1.2 sequences appear in the DDBJ/EMBL/GenBank database under accession numbers AB191203 and AB191204, respectively. Analysis of the deduced amino acid sequences revealed that the medaka kal1.1 has 87, 82, and 72% similarity to the human KAL1 and zebrafish kal1.1 and kal1.2, respectively, at the amino acid level. The medaka kal1.2 exhibited 64, 63, and 74% similarity to the human KAL1 and zebrafish kal1.1 and kal1.2, respectively. A phylogenetic tree was generated by the neighbor-joining method, using the Caenorhabditis elegans KAL1 homolog as the outroot (Fig. 4). The tree showed that kal1.1 and kal1.2 seen in the medaka and zebrafish arose from a gene duplication predating the divergence of teleosts and tetrapods and that the medaka kal1.1 is orthologous to the human KAL1, whereas the medaka kal1.2 is its paralogous form. High bootstrap values of more than 90% for each node supported this contention.
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Prominent kal1.1 mRNA expression was detected from 3 dpf onward in the olfactory bulb, medial region of the telencephalon, branchial arches, cerebellum, and pectoral fin using whole-mount in situ hybridization (Fig. 5A). From 4 dpf onward, kal1.1 mRNA was also detected in the dorsal telencephalon and retina and in the vicinity of the pharynx (Fig. 5, B–E). On the other hand, prominent kal1.2 mRNA expression was seen around the otic vesicle and in the medial aspect of the two telencephalic lobes on and beyond 3 dpf (Fig. 5, F–J).
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Injection of kal1.1 PNA into gnrh1-GFP medaka embryos led to the accumulation of GFP-expressing neurons in the olfactory region and dorsal telencephalon and inhibition of their migration into the preoptic area in approximately 73% of treated embryos (Fig. 6A). Similarly, injection of kal1.1 PNA into gnrh3-GFP embryos resulted in the suppression of migration of GFP-expressing neurons from the olfactory region into the forebrain in approximately 83% of embryos (Fig. 6B). The distribution of GFP-expressing neurons in other populations was not affected. Interestingly, injection of kal1.1 PNA resulted in the enlargement of the body cavity in approximately 93% of treated gnrh1-GFP, gnrh3-GFP, and wild-type medaka embryos (Fig. 6C). On the other hand, injection of kal1.2 PNA did not affect phenotype, i.e. it blocked neither gnrh1 nor gnrh3 neuronal migration, and had no effect on the size of the body cavity (Fig. 6, D–F).
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Discussion |
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Our studies using the gnrh1-GFP transgenic medaka, which included in situ hybridization assessment of endogenous gnrh1 expression, led to the identification of the following four independent gnrh1 neuronal populations with differing embryological origins and developmental behaviors: 1) an olfactory-derived ventral preoptic population, 2) a dorsal preoptic population that migrates from the dorsal telencephalon, 3) a medial ventral telencephalic population that migrates from the anterior telencephalic area, and 4) a nonmigratory ventral hypothalamic population. The developmental sequence of the olfactory-derived ventral preoptic neuronal population appeared to be identical to that of the authentic septohypothalamic Gnrh1 neuronal population previously described in mammals (35). Consistent with our findings, the medial ventral telencephalic and ventral hypothalamic GnRH populations have recently been described in other species (5, 8, 36, 37). In our study, GFP-expressing neurons were also observed in the lateral myelencephalon of the gnrh1-GFP medaka; in situ hybridization, however, failed to detect endogenous gnrh1 mRNA in this region. The lateral myelencephalon has not, to our knowledge, been identified as a site of GnRH expression in any vertebrates. Additional experiments will need to be carried out to examine whether the GFP-expressing neurons in this region are indeed gnrh1 neurons.
Although it is generally accepted that in mammals, the hypophysiotropic Gnrh1 neuronal population in the preoptic area originates in the olfactory compartment (3), this has been a matter of controversy in nonmammalian vertebrates (6, 7, 8). Our analysis of the gnrh1-GFP transgenic medaka provides direct evidence that the ventral preoptic gnrh1 neurons in these animals specifically arise from the olfactory region, supporting the notion that, contrary to popularly held views, they have an evolutionarily conserved embryological origin. A hypophysiotropic role for these ventral preoptic gnrh1 neurons was supported by our direct observation of their projection to the anterior pituitary. In addition to the prominent ventral preoptic area, gnrh1 neurons were found in the medial ventral telencephalon, dorsal preoptic area, and ventral hypothalamus of the adult medaka brain. In contrast to the ventral preoptic population, however, cells in these latter regions originated within the forebrain and did not appear to send projections into the pituitary; their function is still unclear.
Recent studies have shown that the olfactory region is also the source of gnrh3 neurons in the terminal nerve ganglion (8, 24). Our data derived from gnrh3-GFP transgenic medaka, which included in situ hybridization analysis, indeed did show that gnrh3 neurons in the terminal nerve ganglion originated in the olfactory region. Our analysis also revealed that olfactory gnrh3 neurons migrate further and reach the preoptic area and anterior mesencephalon. These cells seem to share a common embryological origin and migratory pathway with olfactory-derived gnrh1 neurons. In contrast to the ventral preoptic population of gnrh1 neurons, however, gnrh3 neurons did not appear to project to the pituitary. This latter finding is in contrast to some previous studies in other teleosts that reported the presence of a few gnrh3-associated peptide-immunoreactive fibers in the pituitary (38, 39). Additional studies are needed to elucidate the anatomical origin of these fibers. Unexpectedly, we also found as yet unreported gnrh3 expression in the trigeminal ganglion, suggesting that GnRH plays a role in the somatosensory innervation of the cranial region by acting as a neurotransmitter or neuromodulator. Indeed, several other neuropeptides have been identified in the trigeminal ganglion, including somatostatin, neuropeptide Y, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, and galanin, which have also been suggested to act as neurotransmitters or neuromodulators (40). Additionally, GFP signal was identified in the retina, optic nerve, and sympathetic ganglia in gnrh3-GFP transgenic medaka. Unfortunately, however, we were unable to detect endogenous gnrh3 mRNA expression in these sites by in situ hybridization. In light of the fact that the expression of the GnRH gene or its product has been detected in the eye (41) and sympathetic ganglion (42) in other vertebrates, it seems likely that these sites indeed express gnrh3.
Unexpectedly, we also found that gnrh3 was maternally expressed in medaka oocytes and embryos. In support of this finding, gnrh3 transcript was detected in embryos as early as the day of fertilization by PCR analysis (unpublished data). These data suggest that gnrh3 may play a previously unsuspected role in embryogenesis. Alternatively, given the recent evidence that oocytes express gonadotropins (43), a GnRH-gonadotropin autocrine pathway may exist in oocytes that regulates their maturation.
Two KAL1 homologs, designated kal1.1 and kal1.2, were identified in the medaka, consistent with a previous description in zebrafish (44). Phylogenetic analysis indicated that a gene duplication that gave rise to kal1.1 and kal1.2 likely occurred before the divergence of teleosts and tetrapods and that kal1.1 is the medaka ortholog of human KAL1, whereas kal1.2 is the paralogous form. Linkage analysis revealed that the medaka kal1.1 and kal1.2 are located on autosomes LG21 and LG4, respectively, whereas KAL1 was assigned to Xp22.3 (12). Two medaka expression sequence tags that have been mapped around the kal1.1 locus on LG21, i.e. OLe1415a and AU169918, have their human orthologs on Xp22.3 and Xp22.2, respectively (31). This syntenic conservation lends credence to the idea that kal1.1 is the medaka ortholog of KAL1.
Knockdown of kal1.1 resulted in the inappropriate accumulation of gnrh1 and gnrh3 neurons in the olfactory compartment and loss of their ability to migrate into the forebrain. This result is in good agreement with that reported in a fetus with X-KS, the latter of which exhibited clusters of GnRH neurons in the olfactory compartment but not forebrain (13). Thus, knockdown of kal1.1 appears to mimic the X-KS phenotype. Our observations demonstrate that kal1.1 is crucial for GnRH neuronal migration from the olfactory region to the forebrain, but not in their neurogenesis within the olfactory compartment. These data were supported by the demonstration of kal1.1 expression in the olfactory bulb and telencephalon, but not in the peripheral olfactory region. Similarly, strong kal1 expression was reported in the mitral cells of the olfactory bulb, but not in the olfactory epithelium or surrounding nasal mesenchyme in chicken embryos (45, 46, 47). The evolutionarily conserved expression pattern of kal1 suggests that the GnRH neuronal anomaly in X-KS results from a central, but not peripheral, target cell defect. Importantly, we also found that the migration of the dorsal telencephalic gnrh1 neurons to the dorsal preoptic area was also suppressed by the kal1.1 knockdown; this may yet be found to hold true in X-KS patients. Interestingly, knockdown of kal1.1 resulted in body cavity enlargement. Some patients with X-KS were reported to exhibit congenital abnormalities that included unilateral renal agenesis (9). The congenital abnormalities seen in X-KS patients may result from the enlargement of the body cavity during embryogenesis. In contrast, knockdown of kal1.2 did not result in any specific alterations in phenotype, although kal1.2 expression was detectable in the cranial region during embryogenesis. These results suggest that kal1.1, but not kal1.2, plays a critical role in forebrain GnRH neuronal migration and that the medaka kal1.1 is functionally equivalent to the human KAL1. Thus, our data suggest that X-KS can be phenocopied by antisense knockdown of kal1.1 and can be directly monitored in the transgenic medaka. Although a few studies on pathogenesis of this syndrome have been conducted in C. elegans and in vitro (48, 49), there have been no descriptions of in vivo genetic models in vertebrates until now. Additional analysis using the medaka system should promote our understanding of the pathophysiology of this syndrome.
In conclusion, we established transgenic medaka lines in which gnrh1- and gnrh3-expressing neurons were tagged with GFP. These fish allowed for the in vivo imaging of multiple populations of GnRH neurons comprising the central network that regulates reproductive competence. We also demonstrated that these animal lines, in conjunction with loss-of-function analysis of the KAL1 ortholog, can be used as an in vivo vertebrate model of X-KS.
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Acknowledgments |
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Footnotes |
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First Published Online November 17, 2005
Abbreviations: dpf, Days post fertilization; GFP, green fluorescent protein; LG, linkage group; PNA, peptide nucleic acid; SV40, simian virus 40; X-KS, X-linked Kallmann syndrome.
Received April 20, 2005.
Accepted for publication November 8, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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