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Duplicate Zebrafish pth Genes Are Expressed along the Lateral Line and in the Central Nervous System during Embryogenesis

Ludwig Institute for Cancer Research (B.M.H., J.E.L., N.E.H., J.K.H., G.J.L.), The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; and St. Vincent’s Institute of Medical Research (J.A.D.), Fitzroy, Victoria 3065, Australia

Address all correspondence and requests for reprints to: Dr. Graham J. Lieschke, P.O. Box 2008, The Royal Melbourne Hospital, Victoria 3050, Australia. E-mail: graham.lieschke{at}ludwig.edu.au.

	Abstract

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PTH plays a critical role in calcium metabolism in tetrapods. The primary site of PTH expression is the parathyroid glands, although it is also detected in the thymus and hypothalamus. Fish lack anatomically distinct parathyroid glands, and the first animals to evolve parathyroid glands were the amphibians. However, fish do have PTH family ligands and receptors, which are functionally similar to their mammalian counterparts. We report the expression patterns of duplicate zebrafish pth genes during embryogenesis. Both zebrafish pth1 and pth2 transcripts are expressed along the lateral line before the migration of the lateral line primordium and later in development Pth protein is detected in lateral line neuromasts by immunohistochemistry. pth1 Transcripts are also detected in the central nervous system in the ventral neural tube. These temporally and anatomically restricted expression patterns imply a novel role for PTH family hormones during embryonic development of the zebrafish and allow for the genetic dissection of PTH function in this model organism.

	Introduction

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THE MAMMALIAN PARATHYROID glands regulate serum calcium levels through the release of PTH into the bloodstream in response to low serum calcium. Through the endocrine activation of cell surface PTH receptors stimulating intracellular cAMP and/or inositol 1,4,5-triphosphate/calcium, PTH stimulates the molecular pathways that ultimately regulate bone turnover, renal calcium, and phosphate handling and, through the generation of 1,25 dihydroxyvitamin D₃, the intestinal absorption of calcium. When calcium levels are restored, the release of PTH from the parathyroid glands is inhibited. The first animals to have evolved parathyroid glands are the amphibians, and the absence of anatomically distinct parathyroids in fish is one of the major differences between marine and terrestrial vertebrate endocrinology (1, 2, 3).

Recent studies in the teleost Danio rerio (zebrafish) have uncovered a surprising level of conservation in Pth receptor activity with higher vertebrates. The zebrafish Pth receptors Pth1r, Pth2r, and Pth3r have been previously described (4, 5). Recently duplicate pth-like ligands from both zebrafish and Takifugu rubripes (fugu) have been identified (6, 7). Both zebrafish Pth1 and Pth2 are capable of activating the zebrafish Pth receptors (although with varying efficiencies), uncovering significant conservation in the biochemical activity of these ligands despite their evolutionary duplication and sequence divergence (6, 7). However, this biochemical study did not describe the anatomical expression patterns of the pth genes and the extent of physiological conservation of the role of Pth in zebrafish remains unclear.

We independently cloned these duplicate pth genes and now report their expression patterns during zebrafish embryogenesis. Our goal was to gain a further understanding of the potential roles of these ligands in the development of a representative teleost and attempt to determine whether a fish parathyroid gland equivalent may exist.

	Materials and Methods

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Zebrafish
Wild-type zebrafish stocks were obtained from a local pet shop and held in the Ludwig Institute for Cancer Research Aquarium facility using standard husbandry techniques.

Embryos for developmental studies were incubated at 28 C on a plate warmer. Embryos for in situ hybridization and whole-mount immunohistochemistry were incubated in 0.003% 1-phenyl-2-thiourea before fixation to inhibit pigmentation. All protocols were approved by the Ludwig Institute for Cancer Research Animal Ethics Committee.

Microinjection
Capped mRNA for microinjection was produced by using the mMessage Machine kit (Ambion, Austin, TX). The final resuspension of mRNA was in water, and the concentration was determined spectrophotometrically. mRNA was microinjected into one to four cell embryos at a concentration of 100 ng/µl. Integrity of mRNA was checked by visualization after denaturing formaldehyde agarose gel electrophoresis.

Cloning of duplicate pth genes
The primers 5'-CCCGCAACCTGCAATATTAC-3' and 5'-AAGCTTCTCTCTTCAGAAAC-3' were used to PCR amplify the pth1 cDNA from 48 h post fertilization (hpf) cDNA. The PCR product was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and then subcloned into pBluescript II KS (Stratagene, La Jolla, CA) as an EcoRV, BamHI fragment.

The primers 5'-GCGCGAATTCATGCTTATTATTGTGCTGTGG-3' and 5'-GCGCCTCGAGACATAAGTAAATCAGTGGAC-3' were used to amplify a partial cDNA clone of pth2 from 48 hpf cDNA. Using EcoRI and XhoI recognition sites introduced into the primer sequences, the PCR product was directionally cloned into pCS2+ (8).

For mRNA synthesis, the coding sequences of only pth1 and pth2 were amplified using a 1:7 ratio of Pfu polymerase (Promega, Madison, WI) to Taq polymerase using the following primers: pth1, 5'-GGCCGAATTCATGGTTTCCATCAACGGG-3' and 5'-GGCCCTCGAGTCACGATGGGTTCATGAG-3', and pth2, 5'-GGCCGAATTCATGTTACTCATACGTTGTTT-3' and 5'-GCGCCTCGAGTCAGTAAAAGCTCCAGGTTG-3'. The products were subcloned into pCS2+ (8), linearized with NotI and transcribed with SP6 polymerase.

Whole-mount in situ hybridization
Whole-mount in situ hybridization analyses were performed as previously described (9). pth1 and pth2 riboprobes were produced using the following restriction enzymes and RNA polymerases: pth1 antisense, BamHI and T3, sense, EcoRV and T7; and pth2 antisense, EcoRI and T7, sense, XhoI and SP6.

Antibodies
The N terminus region of the fugu Pth1 peptide (1–14) was synthesized using a 433A peptide synthesizer (Applied Biosystems, Foster City, CA). The completed peptides were purified, verified, and analyzed (7). The peptides were conjugated with keyhole limpet hemocyanin and mixed with Freund’s adjuvant and used to immunize New Zealand white rabbits with sc injections at five sites. Peptide (100 µg) was administered at each immunization. Antibody content and specificity was assessed every 14 d.

Antibody specificity was tested in an immunoblot with 0, 10, 25, and 50 µg fugu Pth1 (1–34) and four negative controls were included (7). These were synthetic peptides of human PTH (1–14), human PTH (1–34), fugu Pthrp (1–34), and human PTHrP (1–34). The fugu Pth1 antisera did cross-react with 10, 25, and 50 µg fugu Pth1 (1–34) but not with any of the other PTH or PTHrPs.

The fugu Pth1 (1–14) antisera were used in immunohistochemical staining of an extensive panel of fish, rat, and human tissues. A peroxidase-antiperoxidase method (10, 11) localized antigen in tissues from T. rubripes and Oncorhynchus mykiss (data not shown). Panels of positive and negative controls were included in each assay. There was no demonstration of fugu Pth1 in any of the human or rat tissues even at very high (x500) antibody concentrations.

Western blotting
Embryonic lysates were prepared from identical numbers of age-matched embryos by dounce homogenization in hypotonic lysis buffer [5 mM Tris/HCl (pH 7.5), 2.5 mM KCl, 1 mM MgCl₂, 0.5 mM dithiothreitol] with Complete protease inhibitor (Boehringer, Ingelheim, Germany) followed by centrifugation at 1000 x g to remove cellular debris. Lysates were separated on precast 4–12% BIS-Tris gradient gels, using MES SDS running buffer (Novex, Invitrogen) and transferred to a nitrocellulose membrane (Osmonics, Medos, Melbourne, Australia) using standard techniques. Membranes were blocked in 5% skim milk and incubated in antibody in 1% BSA (overnight for primary, 2 h for secondary), signal was detected using SuperSignal West Pico chemoluminescent substrate (Pierce, Rockford, IL), and development was performed in a developer (Kodak, Rochester, NY) with standard x-ray film (Fuji, Tokyo, Japan).

Whole-mount immunohistochemistry
Embryos were prepared by fixation in 4% paraformaldehyde in PBS with 0.1% Tween (PBST) for 2 h and then were washed in 0.3% Triton X-100/PBST, blocked in 2% BSA in 0.3% Triton X-100/PBST, and incubated in primary and secondary antibodies overnight in 0.2% BSA in 0.3% Triton X-100/PBST. Antibody staining was detected using the liquid DAB substrate-chromogen system (Dako, Carpinteria, CA) to the manufacturer’s specifications. The rabbit antifugu Pth1 primary antibody was used at a concentration of 0.6 µg/ml, and nonimmune controls were included at the same concentration.

Calcein staining of bone in zebrafish larvae
Staining for calcified bone in developing zebrafish larvae was performed essentially as described (12).

	Results

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Cloning of pth1 and pth2
A 673-bp expressed sequence tag sequence (GenBank accession no. BM776837) from an adult male zebrafish cDNA library was identified using an NCBI BLAST search of current zebrafish expressed sequence tag sequences and corresponded to the pth1 cDNA now reported (accession number AY275669) (6). We cloned a full 618-bp fragment containing the pre-pro-Pth protein coding sequence and 38 bp of 5' and 271 bp of 3' untranslated region by PCR from 48 hpf embryonic cDNA (Fig. 1A).

View larger version (51K): [in this window] [in a new window]   FIG. 1. In situ hybridization analysis of the expression of pth1 and pth2 in zebrafish embryogenesis. A, Depiction of the pth1 and pth2 cDNA structures as described by Gensure et al. (19 ) and comparison with riboprobes used here. Riboprobe clones are depicted above as empty boxes. Untranslated regions are shown in black, translated regions are shown in white and gray, where gray indicates the functional 1–34 region of the processed peptide product. Numbers indicate clone lengths in base pairs, italicized numbers indicate corresponding amino acids encoded, and numbers in brackets indicate residues in the published sequences at which riboprobe clones begin and end. Both riboprobe clones span the sequence coding for the functional peptide but share just 52.2% sequence similarity, making cross-hybridization of riboprobes with both mRNA species highly unlikely. The 395-bp pth2 clone contains an alternate 3' untranslated region sequence to that of the published cDNA, containing an 8-bp extension of the untranslated region (indicated by *). B–G, Expression of pth1 in the lateral line and central nervous system during embryogenesis. B, Lateral view displaying the expression of pth1 in cells along the lateral line at 24 hpf by in situ hybridization (arrowheads). C, Dorsal view showing the lateral distribution of pth1-expressing cells at 24 hpf (arrowhead indicates the lateral pth1-expressing cells). D, Lateral view at 48 hpf of pth1 expression in the ventral neural tube immediately dorsal to the notochord (the position of the notochord is indicated by the bracket; arrowheads indicate pth1 expression). E, Dorsal view corresponding to (D) of pth1 expression along the ventral neural tube immediately adjacent to the notochord (the position of the notochord is indicated by the bracket; arrowhead indicates pth1 expression). F and G, pth1 expression at 4 dpf in the central nervous system. Lateral view (F) and a section (G) through a 4-dpf embryo (section orientation indicated by hashed line in F) showing pth1 expression (arrowhead) in a distinct bundle of cells no longer adjacent to the notochord. Scale bar, 20 µm. nt, Neural tube; g, gut; n, notochord. Inset in G is an expanded image of pth1 expression, indicating that only four to five axial cells express pth1. Scale bar, 2.5 µm. H and I, Expression of pth2 along the lateral line. H, Lateral view of pth2 expression (arrowheads) along the lateral line at 24 hpf. I, Dorsal view displaying the lateral distribution of pth2-expressing cells (arrowheads) at 24 hpf.

Expression of pth1 and pth2 during zebrafish embryogenesis
We examined pth1 expression throughout development using in situ hybridization. An antisense riboprobe transcribed from the full-length pth1 clone produced the pth1 expression pattern described, whereas a sense riboprobe transcribed from the same template produced no staining.

pth1 expression was first detected in a population of cells dispersed along the lateral line in the posterior of the animal at 24 hpf (Fig. 1, B and C). Although they could not be lateral line neuromasts because they were present before the migration of the lateral line primordium, these cells ran bilaterally either side of the midline at 24 hpf. pth1 transcript expression in these cells disappeared by 48 hpf, although at this time point, transient expression in a discrete zone of cells immediately dorsal to the notochord was observed in the ventral neural tube (Fig. 1, D and E). At 4 d post fertilization (dpf), this restricted transient expression was consistently observed in the midline of the neural tube (Fig. 1, F and G). This central nervous system expression at 48 hpf appears similar to that of sox10 and olig2 dorsal to the notochord (13, 14). sox10 and olig2 are the earliest known markers of the oligodendrocyte lineage, identifying the cells as putative oligodendrocyte precursors or similarly located cells (13, 14).

The expression of pth2 in embryogenesis was also analyzed by in situ hybridization. The cloned partial cDNA was used as a template for antisense riboprobe synthesis and sense controls were negative. pth2 transcripts were detected in two populations of cells running bilaterally either side of the midline of the animal; the anatomical distribution of these cells was apparently identical with those expressing pth1, although expression was somewhat variably attributable to slight scatter in developmental age within any given clutch of embryos (Fig. 1, H and I). No later expression of pth2 was observed, indicating that there was no cross-reactivity of the pth2 riboprobe with pth1 transcripts, particularly because no central nervous system staining was observed.

Spatiotemporal detection of Pth peptides during zebrafish embryogenesis
An antibody to fugu Pth1 was raised to the 1–14 primary Pth1 peptide from fugu (7). Zebrafish Pth peptides are more closely related to fugu Pth1 at the amino acid level than any other PTH peptides described.

We used this antibody for whole-mount immunohistochemistry to examine the distribution of total Pth protein in staged zebrafish embryos. At 3 and 4 dpf, we detected signal in lateral line neuromasts and anterior neuromasts (Fig. 2). We also detected signal in small cells dispersed in the anterior of the animal and the developing jaw (Fig. 2, green arrows). Normal rabbit serum IgG and secondary antibody only controls confirmed the specificity of this expression pattern (Fig. 2, K–O). Interestingly, the staining of fugu Pth1 immunohistochemistry in the developing jaw was coincident with the earliest calcification of this region of the jaw as stained with calcein (Fig. 2, E–H), perhaps suggesting a role in bone development.

View larger version (59K): [in this window] [in a new window]   FIG. 2. Analysis of Pth protein distribution in zebrafish embryogenesis. A–D, Anatomical analysis of Pth immunostaining in anterior neuromasts and the developing jaw by whole-mount immunohistochemistry during zebrafish embryogenesis. A and B. Lateral views of 3 (A) and 4 dpf (B) embryos showing anterior neuromasts (black arrowheads) and the developing jaw (green arrowheads) stained positive using anti-fugu Pth1 antiserum. C and D, Ventral views of 3 (C) and 4 dpf (D) embryos showing anterior neuromasts (black arrowheads) and dispersed weakly staining cells; green arrowheads indicate staining in the developing jaw. E–H, Light and fluorescence images of calcein staining at 4 dpf indicate that the earliest calcification of the developing jaw is coincident with early Pth protein immunohistochemistry staining. E, Lateral light microscope image of the developing jaw. F, Fluorescent image of calcein staining (arrowhead) showing the earliest calcification of developing bone. G, Ventral light microscope image of the developing jaw, H, Fluorescent image of calcein staining (arrowhead) showing the earliest calcification of developing bone. I and J, Lateral views of embryos with Pth immunostaining in the lateral line neuromasts at 3 and 4 dpf, respectively (arrowheads indicate lateral line neuromasts). Inset (J), A high-power image of Pth-positive cells displaying the typical morphology of lateral line neuromasts. Scale bar, 5 µm. K–O, Antibody and method specificity controls including normal rabbit serum and secondary antibody only control reactions. K and L, Representative lateral views at 3 dpf of normal rabbit serum controls displaying the specificity of staining with the antifugu Pth1 primary antibody. M and N, Representative lateral views at 4 dpf of normal rabbit serum controls displaying the specificity of staining with the antifugu Pth1 primary antibody. O, Representative lateral view at 3 dpf of a secondary antibody only control-stained embryo. P, Peptide alignment of the 1–14 fugu Pth1 immunizing peptide with the corresponding zebrafish (1–14) Pth1 and (1–14) Pth2 sequences and Western blot analysis of embryonic lysates with antifugu Pth1 polyclonal antibody. Three species of the predicted sizes were identified, and all three were increased in intensity in lysates prepared from pth1 mRNA-injected embryos lysed at 12 hpf and pth2 mRNA-injected embryos lysed at 24 hpf in comparison with lysates from uninjected controls. PpPth, Pre-pro-Pth; pPth, pro-Pth; size bars on the right of Western panels indicate approximately 6 and 3 kDa. Peptide alignment was generated with the ClustalX algorithm using default settings.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We detected pth1 and pth2 mRNA at 24 hpf in cells along a track marking the position of the future lateral line, a sensory system specific to fish. This expression is similar to that of sdf1, a chemokine that lays a trail down the lateral line along which the primordium will migrate (15). Pth protein was subsequently detected in the lateral line neuromasts and anterior neuromasts (3 and 4 dpf), sometime after the migration of the lateral line primordium and development of the neuromasts. The localization of Pth in neuromasts was somewhat consistent with the distribution of mRNA for both pth genes along the lateral line, although spatially and temporally coincident mRNA localization and protein localization was not observed. In fact, transient pth mRNA expression along the lateral line was observed only during d 1 of development and was absent when anterior and lateral line neuromast Pth protein expression was observed from 3 dpf. This is perhaps indicative of low-level transcription during d 2 and 3 of development, which cannot be detected by in situ hybridization. Consistent with this hypothesis, both pth1 and pth2 cDNAs were amplified from 48 hpf cDNA, a time point when in situ hybridization does not detect any pth2 signal.

In mammals and fish, PTH ligands act through two PTH receptors (PTHRs), PTHR1 and PTHR2. These receptors are activated by not only PTH but also two additional families of PTH-related molecules, namely PTHrP (16) and tuberoinfundibular peptide 39 (TIP39) (17). The presence of several conserved residues between these family members suggests that a common mechanism may mediate ligand-receptor interactions. In all cases, receptor engagement results in an accumulation of cAMP. TIP39 has been shown to be particularly potent at stimulating cAMP accumulation in cells expressing PTHR2, and it has been suggested that TIP39 may be the primary ligand for PTHR2 (18). Of particular relevance here is a recent study that points to a significant role for the TIP39-PTHR2 system in the developing zebrafish central nervous system (19). Zebrafish TIP39 is expressed in two discrete patches rostral and dorsal to the hypothalamus at 48 hpf (as well as in developing cardiac tissue), whereas at this stage, zebrafish PTHR2 is widely expressed throughout the developing central nervous system (19). Our demonstration here of the expression of zebrafish pth ligands in the ventral neural tube and the position along which the cells of the lateral line sensory organ migrate and develop presents further evidence for a role in neural development of a signaling axis involving pth, pth-related peptides, and their receptors that is worthy of further investigation. Perhaps understanding the role of zebrafish pth genes in zebrafish neural development may lead to insights into potential but poorly understood neurological roles of mammalian PTH peptides, such as are implied by two independent studies demonstrating PTH expression in the rodent hypothalamus (20, 21).

The PTH-secreting parathyroid glands derive from the endoderm of pharyngeal pouch three in the mouse, which expresses the regulator of parathyroid development Gcm2 (21, 22). In this study, no expression of Pth was detected with in situ hybridization or immunohistochemistry in any cells likely to correspond to a pharyngeal-derived parathyroid gland equivalent or in the thymus. This lack of conserved expression may confirm the long-held belief that fish lack parathyroid glands, underpinning a divergence in the anatomy of the endocrine regulation of calcium in marine vertebrates. Consistent with this hypothesis, we have recently shown that the zebrafish ortholog of Gcm2 is required for the development of gills and also does not identify any likely parathyroid gland equivalent (23).

However, it remains possible that zebrafish Pth arising from the sites we describe plays an endocrine role in calcium metabolism or that calcium-regulating Pth comes from an as-yet-unidentified source. Supporting this, Pth immunohistochemistry also detected staining in the developing jaw, which labeled the first region of the jaw cartilage to become calcified, perhaps indicative of a role in early bone development. Distinguishing between the possibilities presented here will require an in-depth functional analysis based on the expression patterns we have described.

	Acknowledgments

 
We thank Sony Varma and Andrew Trotter for expert technical assistance.

	Footnotes

 
This work was supported by Australian Research Council, Discovery Grant DP0346823, National Health and Medical Research Council Grant 234708. B.M.H. was the recipient of an Australian Postgraduate Award scholarship. G.J.L. was the recipient of a Wellcome Senior Research Fellowship in Medical Sciences in Australia

First Published Online November 11, 2004

Abbreviations: dpf, Days post fertilization; hpf, hours post fertilization; PBST, PBS with 0.1% Tween; PTHR, PTH receptor; TIP39, tuberoinfundibular peptide 39.

Received March 8, 2004.

Accepted for publication November 4, 2004.

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