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Prolactin Receptor Signaling Mediates the Osmotic Response of Embryonic Zebrafish Lactotrophs

Departments of Medicine (N.-A.L., Q.L., K.W., S.M.) and Molecular, Cell and Developmental Biology (Z.Y., S.L.), Cedars-Sinai Research Institute, University of California Los Angeles, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail: Melmed{at}CSMC.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.

	INTRODUCTION

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IN MAMMALS, the hormone prolactin (PRL), secreted by the pituitary lactotroph cell, regulates salt and water balance by increasing intestinal water and salt absorption and reducing renal Na⁺ and K⁺ excretion through modulating Na⁺-K⁺ ATPase activity (1). Although the pathophysiological relevance remains elusive, mammalian PRL is likely involved in fetal osmoregulation during early gestation (2). In fish, PRL is the fresh water-adapting hormone, where prevention of ion loss to the external hypo-osmotic environment is crucial for survival and homeostasis. PRL increases ion retention (primarily Na⁺ and Cl^–) and decreases water uptake via osmoregulatory surfaces including the gill, skin, kidney, intestine, and urinary bladder (3, 4). Consistent with the role of PRL in salt and water balance control, teleost pituitary lactotroph activity is inversely related to serum osmolarity and corresponding environmental salinity (5, 6, 7). However, little is known about molecular mechanisms underlying lactotroph osmotic response.

Mature pituitary lactotrophs are mostly regulated by tonic hypothalamic dopamine (DA) inhibition via the Drd2 dopamine receptor subtype (8). PRL, in turn, regulates its own secretion by stimulating hypothalamic dopaminergic neurons, up-regulating tyrosine-hydroxylase (TH) expression and activity, the rate-limiting enzyme in dopamine synthesis, and exerting neurotrophic effects on tuberoinfundibular neuron differentiation and maintenance (8, 9, 10, 11, 12). In addition, PRL inhibits lactotrophs via intrinsic, dopamine-independent regulatory pathways in an autocrine and/or paracrine manner (13). During mammalian gestation, the PRL receptor (PRLR) is expressed in multiple cell types derived from distinct embryonic primordia, implying the important roles of PRL in embryonic organ development and function (14). However, the role of PRL signaling on lactotroph development and function, particularly in association with osmotic homeostasis, is not known.

We use zebrafish, a fresh-water teleost, as a model organism to investigate functional regulation of early pituitary ontogeny. Molecular mechanisms underlying adenohypophyseal induction, patterning, specification, proliferation, and functional regulation are highly conserved between zebrafish and mouse (15, 16, 17, 18, 19, 20, 21). In teleosts, lactotrophs and corticotrophs represent two distinct pituitary lineages involved in osmo-adaptation to fresh water and sea water, respectively (3). Here, we generated stable double transgenic zebrafish coexpressing red fluorescent protein (RFP) directed by Prl regulatory elements and green fluorescent protein (GFP) by the pro-opiomelanocortin (Pomc) promoter. Transparent zebrafish embryos expressing fluorescent markers specifically targeted to pituitary lactotroph and corticotroph lineages allowed us to follow the dynamic process of pituitary ontogeny in live animals at different environment salinities. Our results show that PRL autoinhibition dominantly regulates embryonic lactotrophs and is permissive for osmotic signaling to the pituitary gland.

	RESULTS

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Generation and Characterization of Germline Double Transgenic Zebrafish, POMC-GFP/PRL-RFP
During early development, six pituitary cell lineages arise within the adenohypophysis, which, in the case of zebrafish, is composed of rostral pars distalis, proximal pars distalis and pars intermedia. Corticotroph and lactotroph lineages are situated in the rostral pars distalis, the most anterior aspect of the gland (18). To monitor dynamic pituitary ontogeny, we generated germline double transgenic zebrafish, POMC-GFP/PRL-RFP, by introducing a bacterial artificial chromosome (BAC) construct containing the zebrafish Prl regulatory element fused with sequence encoding the monomeric red fluorescent protein gene, RFP, into a previously established germline transgenic zebrafish expressing Pomc promoter-driven GFP specifically targeted to pituitary corticotrophs (19).

Time-lapse confocal microscopy analysis on live POMC-GFP/PRL-RFP embryos show that the RFP signal was apparent at 18 h post fertilization (hpf) within the anterior neural ridge (Fig. 1, A–F). Onset of lactotroph differentiation was asymmetric and followed by that of POMC-GFP-positive cells on the contra lateral side approximately 4 h later, whereas lactotrophs continued to migrate from bilateral to more medial regions of the anterior neural ridge, before further internalizing into the head. This dynamic process was recorded from live transgenic embryos by time-lapse confocal microscopy and is depicted in supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. PRL-RFP and POMC-GFP transgene expression patterns faithfully recapitulated endogenous pituitary Prl and Pomc mRNA expression in wild-type zebrafish embryos as shown by in situ hybridization (Fig. 1, G and H). By 3–4 d post fertilization (dpf), PRL-RFP expressing cells localized to the most anterior pituitary domain, the rostral pars distalis. In contrast, POMC-GFP-positive cells organized within both the rostral pars distalis and the posterior pars intermedia (Fig. 1I). These results are consistent with a previous report that zebrafish lactotrophs are the initial pituitary lineage to differentiate, whereas the pituitary primordium is still organized within the anterior neural placode, indicating the primary function of PRL as an osmoregulator in freshwater teleosts, critical for adaptation and survival especially in young embryos (18). Furthermore, the early spatial pattern of lactotroph induction is clearly distinct from that of the corticotroph, although they both differentiate bilaterally within the anterior neural ridge.

View larger version (142K): [in this window] [in a new window]   Fig. 1. Embryonic Pituitary Lactotroph and Corticotroph Ontogeny in Germline POMC-GFP/PRL-RFP Transgenic Zebrafish A–F, In vivo time-lapse imaging of PRL-RFP and POMC-GFP expressing-cell ontogeny in zebrafish pituitary anlage visualized from 18–26 hpf. The first PRL-RFP expressing cell (arrow) appears on the right lateral side of the anterior neural ridge at 18-somite stage, 18 hpf (A), which is then followed by bilateral lactotroph differentiation and medial migration (B–C; B: 19.5 hpf, C: 20 hpf). Four hours, POMC-GFP-positive cells appear on the left side of anterior neural ridge and continue to differentiate thereafter (D–F: D, 22 hpf, E: 23 hpf, F: 26 hpf). Using whole-mount in situ hybridization, endogenous pituitary Pomc (G) and Prl (H) expression were shown to be similar to those of the POMC-GFP (green) and PRL-RFP (red) transgenes (I, confocal microscopy image, x20) in 72 hpf embryos. Arrowheads, Pomc-positive cells corresponding to endorphin-generating cells in the hypothalamus. A–F: Frontal view; G-I: ventral view. Left, anterior; right, posterior. The eyes are indicated (e) as reference points. ANR, Anterior neural ridge.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Environment Salinity Effects on Embryonic Pituitary PRL-RFP Signals A, Immediately after fertilization, live transgenic embryos were divided into culture medium with salinity of 500, 1000, or 2000 eV (electron volt), equivalent to 0.5x, 1x, and 2x routine medium (0.025%, 0.05%, or 0.1% salt concentration). At 48 and 72 hpf, RFP signal intensity and area size were analyzed using fluorescent microscopy and Openlab software. The inverse association of PRL-RFP signals with medium salinity is apparent, and is not blocked by sulpride. PRL-RFP is presented as mean ± SEM of intensity x area, adjusted so that the value of the group without sulpride and in 500 eV salinity is 1. Inset, Comparison of lactotroph numbers in embryos cultured in 0.5x or 2x routine medium at 30 hpf. The y-axis shows the number of RFP-positive cells per embryo. A total of 116 embryos were used in the experiment. Similar results were obtained in two independent experiments. B, Live transgenic embryos were continuously treated with bromocriptine starting from the 20-somite stage, in the presence or absence of 200 µM sulpride at 1000 eV salinity. At 48 hpf, PRL-RFP signal intensity and area size were analyzed using fluorescent microscopy and Openlab software. PRL-RFP signals were dose-dependently suppressed by bromocriptine, and were reversed by sulpride. PRL-RFP is presented as mean ± SEM of intensity x area. A total of 140 embryos were used in the experiment. Similar results were obtained in three independent experiments.

To further examine the specificity of dopaminergic signaling on embryonic lactotrophs, we treated live PRL-RFP/POMC-GFP transgenic embryos with bromocriptine, a DA receptor agonist, starting at the 20-somite stage. By 48 hpf, dose-dependent bromocriptine suppression of pituitary PRL-RFP signals was reversed by sulpride (Fig. 2B). No significant effects of bromocriptine or sulpride on POMC-GFP signals were observed using the same doses (data not shown). These results indicate that, although dopaminergic and osmotic effects on embryonic lactotrophs both begin during early ontogeny, they function independently of each other.

Drd2c Is the Major Pituitary D2 Receptor Subtype during Early Ontogeny
To further validate the pharmacological observations of dopaminergic effects on embryonic lactotrophs, we examined pituitary Drd2 expression at an early developmental stage. Zebrafish Drd2 are encoded by three separate genes, Drd2a, Drd2b, and Drd2c, representing three subtypes sharing 66–71% amino acid homology with that of the human Drd2 (22). Our RNA whole-mount in situ hybridization analysis of 48 hpf embryos showed Drd2a, Drd2b, and Drd2c expressions in specific tegmental, diencephalon, and hindbrain regions (Fig. 3, A–H), consistent with a previous report (22). Pituitary Drd2c expression is low but clearly present (Fig. 3, D and H). Although Drd2b is very weakly expressed, no pituitary Drd2a is detected (Fig. 3, B, C, F, and G).

View larger version (54K): [in this window] [in a new window]   Fig. 3. Pituitary Dopamine D2 Receptor Subtypes A–H, mRNA whole-mount in situ hybridization using antisense mRNA of PRL (A and E), Drd2a (B and F), Drd2b (C and G) and Drd2c (D and H) as probes. Pituitary expression of Drd2c, although weak, is clearly detectable at 48 hpf, whereas Drd2b expression is only faintly detected and no pituitary Drd2a expression was detected. A–D, Dorsal views; E–H, lateral views. Left, Anterior; right, posterior; pit, pituitary; di, diencephalon; hi, hindbrain; hy, hypothalamus; rh, rhombomere; tg, tegmentum. I, MO-injected embryos are viable at 48 hpf but show cardiac congestion, a small brain and overall growth retardation. J, One- to two-cell stage homozygous PRL-RFP/POMC-GFP double transgenic embryos were injected with Drd2a, Drd2b, and Drd2c MO or vehicle (mock injection) and then cultured in routine medium. At 48 hpf, PRL-RFP signal intensity and area size were analyzed using fluorescent microscopy and Openlab software. Transgenic embryos injected with Drd2c MO exhibit markedly increased PRL-RFP signals presented as mean ± SEM of intensity x area, adjusted so that the value of the mock injection group is 1. Statistical analyses were performed by two-tailed Student’s t test. *, P < 0.003 compared with mock injection group; n = 8–10 embryos for each injected group.

Molecular and Genetic Characterization of the Zebrafish Prlr Gene
Mining the zebrafish genome assembly database generated by the zebrafish genome sequencing project (http://www.ensembl.org/Danio-rerio) led to identification of two genes on chromosome 5 and 10 (Ensembl Gene ID: ENSDARG00000028219 and ENSDARG00000016570) with sequence similarity to the mammalian Prlr gene. Further basic local alignment and search tool analysis revealed the zebrafish PRLR cDNA sequence, Prlr, with a predicted open reading frame encoding 602 amino acid residues (GenBank accession no. AY375318), and a corresponding genomic contig sequence (zK264M23.01125 and zK264M23.00015) containing 8 exons, 7 introns, and 5', 3' noncoding segments, which shares more than 97% sequence identity with ENSDARG00000016570 on chromosome 10. The putative polypeptide sequence shares 38% amino acid identity and 70% similarity with that of human PRLR (GenBank accession no. BC059392). The two pairs of disulfide-linked cysteines in the N-terminal subdomain and the WS motif in the C-terminal subdomain of the cytokine receptor homology region, both critical for PRLR function, are appropriately conserved in zebrafish Prlr (Fig. 4A). ENSDARG00000028219 on chromosome 5 represents a predicted transcript encoding 441 amino acid residues corresponding to the N-terminal sequence of Prlr. Phylogenetic analysis indicates that Prlr on chromosome 10 is the bona fide zebrafish ortholog of mammalian Prlr (Fig. 4B).

View larger version (56K): [in this window] [in a new window]   Fig. 4. Embryonic Zebrafish Prlr Expression and Morpholino Knockdown A, Conservation of the two pairs of disulfide-linked cysteines and the WS motif (marked by red letters) in human and zebrafish Prlr proteins. Solid lines and double dots represent identical and similar amino acids, respectively. B, Phylogenetic analysis of zebrafish, bovine, human, mouse, chick, and tilapia Prlr protein. The analysis was performed using the Jotun Hein method with DNA Star MegAlign software. C–E, Prlr mRNA whole-mount in situ analysis at 24 hpf. C, Lateral view; left, anterior; right, posterior. D, Frontal view; head to the top. E, Dorsal view; left, anterior; right, posterior. pa, pancreas; pit, pituitary; arrowhead: pro-nephrotic ducts. F, One- to two-cell homozygous PRL-RFP/POMC-GFP double transgenic embryos were injected with Prlr MO or a control MO with 5 bp mismatch (control) then cultured in medium with 500 or 2000 electron volt (eV) salinity, in the presence or absence of 400 µM sulpride. At 48 hpf, PRL-RFP signal intensity and area size were analyzed using fluorescent microscopy and Openlab software. Transgenic embryos injected with Prlr MO exhibited markedly increased PRL-RFP signals, and no longer respond to altered medium salinity or sulpride. PRL-RFP signal is presented as mean ± SEM of intensity x area, adjusted so that the value of controls cultured in 2000 eV without sulpride is 1. Statistical analyses were performed by two-tailed Student’s t test. **, P < 0.0001; *, P < 0.0005 compared with each respective control group; n = 8–10 embryos for each injected group. Similar results were obtained from two independent experiments. G, Expression of pituitary Prl in Prlr morphants at 48 hpf. One- to two-cell homozygous PRL-RFP/POMC-GFP double transgenic embryos were injected with Prlr or 5 bp mismatch MOs then cultured in medium with 500 or 2000 eV salinity, in the presence or absence of 400 µM sulpride. At 48 hpf, embryos were collected for whole-mount in situ hybridization analysis of Prl expression. Sulpride+, Sulpride present in culture medium; sulpride–, no sulpride present in culture medium; MO+, injected with antisense morpholino; MO–, injected with 5 bp mismatch morpholino. Prl expression in embryonic pituitary is increased in Prlr MO injected embryos, however, without further response to altered medium salinity or sulpride. Prlr morphants also show smaller head and eyes compared with control embryos.

Osmotic Regulation of Embryonic Lactotrophs Is Blocked by Prlr Morpholino Knockdown
To examine effects of PRLR signaling on osmotic responsiveness of embryonic lactotrophs, we blocked Prlr synthesis in PRL-RFP/POMC-GFP double transgenic embryos using zebrafish Prlr MO, which was then immediately followed by culturing the embryos in different salinities equivalent to 0.5x or 2x routine medium. One-cell embryos were injected with Prlr MO, or a control MO that contains 5 bp in mismatch sequence leading to inefficient binding to the target mRNA. At 48 hpf, Prlr morphants (Fig. 4F, closed bars) showed more than 2- to 8-fold enhanced PRL-RFP signals (Fig. 4F, open bars), indicating that endogenous PRL inhibits embryonic lactotrophs during early ontogeny.

However, PRL-RFP signals did not differ between Prlr morphants cultured with either 0.5x or 2x routine medium (Fig. 4F, closed bars), indicating a diminished lactotroph response to environmental salinity changes. This pattern of PRL-RFP signals was confirmed by RNA whole-mount in situ hybridization detecting endogenous pituitary Prl expression (Fig. 4G), which was also semiquantified by measuring signal intensity and area size (data not shown). Therefore, Prlr signaling is required for the embryonic lactotroph response to physiologic ranges of environment salinity changes.

Dopamine-Independent PRL Autoinhibition Regulates Embryonic Lactotrophs
At 48 hpf, Prlr morphants also exhibit attenuated endogenous dopaminergic inhibition of lactotrophs (Fig. 4, F and G), consistent with previous findings in mice suggesting that PRL exerts stimulating and trophic effects on hypothalamic dopaminergic neurons. On the other hand, interrupting the DA receptor did not block endogenous PRL autoinhibition mediated via the Prlr (Fig. 4, F and G). Furthermore, dopamine-independent PRL inhibition exerts greater control of embryonic lactotrophs than dopaminergic inhibition, as indicated by the magnitude of enhanced PRL-RFP signals of Prlr and Drd2c morphants, respectively (Figs. 3J and Fig. 4F).

To extend our investigations of PRL autoinhibition on embryonic lactotrophs, we injected synthetic PRL mRNA into one-cell embryos and then cultured them in routine medium with or without sulpride. By 48 hpf, hypothalamic TH mRNA levels were significantly increased in PRL mRNA-injected embryos compared with mock-injected embryos (Fig. 5A), consistent with PRL function known to stimulate TH gene expression via positive feedback (25). Sulpride treatment increased PRL-RFP signals almost 2-fold, which was suppressed by approximately 50% with exogenous PRL mRNA injection (Fig. 5B). Because interrupting the dopaminergic receptor does not abrogate lactotroph inhibition induced by exogenous PRL, PRL appears to regulate embryonic lactotrophs through dopamine-independent pathway(s). No significant effect of PRL mRNA injection on POMC-GFP signals was observed (Fig. 5B), indicating that PRL autoinhibition is specific for the lactotroph lineage, although its effect on pituitary lineages other than corticotrophs is still unknown.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Effect of PRL mRNA Injection on Embryonic Dopaminergic Neurons and Pituitary Lactotrophs A, One- to two-cell stage zebrafish embryos were injected with PRL mRNA (a) or vehicle (b). Tyrosin hydroxylase mRNA whole-mount in situ hybridization was performed at 48 hpf. Ventral view: left, anterior; right, posterior. Hy, Hypothalamus; AAN, arch-associated neurons. B, 1–2 cell PRL-RFP/POMC-GFP double transgenic embryos were injected with zebrafish PRL mRNA or vehicle then cultured in the presence or absence of 400 µM sulpride, which was added to the medium at 20 hpf. By 48 hpf, PRL-RFP and POMC-GFP signals were monitored using confocal microscopy. Fluorescent signal intensity and area size were analyzed using fluorescent microscopy and Openlab software. PRL mRNA injection resulted in PRL-RFP repression, which is not altered by sulpride treatment. POMC-GFP is not affected by PRL mRNA or sulpride. Insets, Individual confocal pituitary images; top panel, PRL-RFP; bottom panel, POMC-GFP; S+, with sulpride; PRL+, with PRL. PRL-RFP and POMC-GFP are presented as mean ± SEM of intensity x area, adjusted so that the value of controls is 1. Statistical analyses were performed by two-tailed Student’s t test. n = 8 embryos for each injected group. Similar results were obtained from three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Osmotic cell adaptation involves responding to extracellular osmotic deviations through cell volume counterregulation, which is mediated by multiple intracellular signaling pathways including nonselective stretch-activated cation channels (26, 27). Osmotic alterations of cell volume stimulate gene expression, and modify cell differentiation and proliferation (26). Mature lactotrophs respond to extracellular hypotonicity through opening of stretch-activated ion channels resulting from increased cell volume, which triggers extracellular Ca²⁺ influx and PRL release (26, 28, 29, 30). Embryonic lactotroph proliferation and/or PRL expression may be enhanced by extracellular hypotonic osmolarity through similar mechanism(s) of cell volume regulation. Furthermore, our results indicate that extracellular osmolarity does not influence commitment or induction of the lactotroph lineage because temporal and spatial patterns of PRL-RFP signals are not altered by environment salinity changes.

In mammals, classic osmotic sensory organs are located in the blood-brain barrier-deficient lamina terminalis, the anterior wall of the third ventricle adjacent to the anterior hypothalamus. There have been no previous reports indicating that the pituitary is a directly osmoreceptive organ. However, the anterior pituitary is also devoid of blood-brain barrier constraints, and in vitro data suggest direct lactotroph response to osmotic stimulations by triggering PRL gene expression, synthesis and secretion (4). Lactotrophs may therefore directly detect extracellular tonicity via cell-specific osmotic sensor(s), although more indirect mechanisms cannot be excluded. The observed osmo-regulatory effect on embryonic lactotrophs is apparently lineage specific because the corticotroph lineage, known to be involved in teleost sea-water adaptation (3), is not affected by similar ranges of osmolarity changes, indicating a unique osmo-sensing mechanism for lactotrophs.

Lactotrophs perceive and respond to local PRL in an autocrine/paracrine manner by inhibiting autosecretion, which is diminished in physiological and pathological conditions such as lactation or prolactinoma (13). Our observations indicate additional functions of PRL autocrine/paracrine effects in controlling developmental lactotroph osmotic responses. Osmotic feedback on lactotrophs mediated by PRL signaling via its pituitary receptor appears to be a component for early osmo-adaptation and homeostasis, in addition to the peripheral effects of PRL on osmoregulation.

Importantly, dopamine-independent PRL autoinhibition exerts a more robust effect on embryonic lactotrophs than tonic dopaminergic signaling. This appears reversed in adult lactotrophs, where dopamine serves almost exclusively as the primary control. Dopamine-independent PRL signaling may be partially attributed to the intrinsic regulatory mechanisms involving factors such as vasoactive intestinal peptide, galanin, TGF-ß, epidermal growth factor, and nerve growth factor, which, especially the latter growth factors, stimulate differentiation and proliferation during early development (13). The dependency of embryonic lactotroph osmotic responses on intrinsic PRL autoregulation indicates the importance of central-peripheral interactions for growth, development, and hormonal homeostasis.

	MATERIALS AND METHODS

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Generation of Modified BAC PRL-RFP Reporter Construct and Germline PRL-RFP/POMC-GFP Double Transgenic Zebrafish
The zebrafish genome database from Sanger Institute website (http://www.ensembl.org/Danio_rerio) was searched using basic local alignment and search tool with zebrafish PRL cDNA sequence. Zebrafish prolactin gene, Prl, was identified in super contig ctg9476. A pair of primers corresponding to Prl 5' noncoding sequences, PRL1: 5'-AAC GCT GGA CCT TAG CGA AGA CT-3'and PRL2: 5'-TAC TAG AAC AGG TTT CTT TAT TTT CTT GCG TG-3', were used to select zebrafish BAC clones covered by the contig from CHORI-211 library. A genomic clone, zC224H17, was identified. zC224H17 is located within Zv4_scaffold252 that contains seven exons of the Prl gene with approximately 195 kb 5' upstream sequence from the ATG initiation codon. Clone zC224H17 was modified by homologous recombination as described previously (31). In brief, PCR amplification of zC224H17 was performed using primers PRL1 and PRL2, and the 351-bp product, prl351, was linked with a vector carrying the monomeric red fluorescent protein gene, RFP, to provide a homologous arm (32). The RFP was then introduced into zC224H17 by homologous recombination. The modified zC224H17 clone was verified by PCR using primer P1 (5'-CTC AGT TGG TCA TGG CCA TTT CT-3'), 76 bp upstream of prl351 in zC224H17, and primer P2 (5'-AAG TTC ATC AGC CCC TAC AGC C-3'), 250 bp downstream from the ATG initiation codon of RFP, and further confirmed by restriction enzyme digestion. Modified BAC DNA was linearized by restriction digestion with NotI followed by dialysis, and resuspended at a final concentration of 100 µg/ml. Fertilized embryos from POMC-GFP germline transgenic zebrafish were microinjected with linearized PRL-RFP BAC DNA at the one-cell stage as described (19, 33). Injected founder fish were mated to wild-type fish, and their progeny observed for GFP and RFP expression under a Zeiss fluorescent microscope (Carl Zeiss, Jena, Germany). Founder fish that produced GFP- and RFP-positive eggs were considered transgenic and bred to generate F1 heterozygotes and F2 homozygotes.

Whole-Mount RNA in Situ Hybridization
A 1.5-kb zebrafish Prlr PCR product was subcloned into the pCR4-TOPO vector, which was subsequently linearized by NotI and transcribed with T3 polymerase to generate Prlr antisense mRNA. Zebrafish PRL and Pit-1 antisense mRNA were generated from cDNA constructs provided by Dr. M. Hammerschmidt (Max-Planck Institute for Immunology, Freiburg, Germany) using T3, T7, or Sp6 polymerase after linearization with NotI, KpnI, and EcoRV, respectively. Zebrafish Drd2 antisense probes were synthesized from Drd2a, Drd2b, and Drd2c cDNA constructs, pBS-D2a, pBS-D2b, pBS-D2c (kindly provided by Dr. R. Levenson, Pennsylvania State College of Medicine, Hershey, PA), which were linearized by XbaI or BamHI, and transcribed with T3 polymerase using a DIG/Genius 4 RNA Labeling kit (Roche, Germany). RNA in situ hybridizations of zebrafish whole-mount embryos were performed as described (34).

Morpholino Antisense and mRNA Microinjection
The Prlr morpholino antisense and control morpholino (5-bp mismatch) have the following respective sequences: ZFPRLR: 5'-TCA GCA CAG CGG CGG AAA TCC TCA T-3'; ZFPRLR/5mis: 5'-TCA cCA Cac Cgc Cgc AAA TCC TgA T-3'. The five mismatched base pairs in the control morpholino are shown in lowercase. The dopamine receptor morpholino antisense sequences are as follows: Drd2a, 5'-AGG CAT ACG CTG TGA AGA CTT CCA T-3'; Drd2b, 5'-CAC GTT CAG GAC AGG CATGAT GAA G-3'; Drd2c, 5'-GGA TAC TCC GTG AGG AAA TCC ATG A-3'. The complementary sequence of the putative ATG start site is underlined in each morpholino sequence. For injection, the morpholinos were diluted in 1x Danieu’s buffer to a concentration of 1 mM. Each one- to two-cell stage embryo was injected with 2 nl of morpholino. For mRNA injection, the capped PRL mRNA was synthesized from linearized plasmid using an RNA transcription kit (Ambion, Austin, TX), quantitated by spectrophotometer and adjusted to a concentration of 1ng/nl in diethylpyrocarbonate water.

Cell Count
Embryonic cells were mechanically dissociated at 30 hpf as previously described (34). RFP-positive cells were counted using a Zeiss Axioplan2 microscope.

Fluorescent Microscopy and Confocal Microscopy
Transgenic embryos were examined at various developmental stages under a fluorescein isothiocyanate filter on a Zeiss microscope (Zeiss Axioplan-2). Live embryo images were generated with an Axiocam video system (Zeiss). Fluorescence of POMC-GFP and PRL-RFP-positive cells was measured using Openlab software (Improvision, Lexington, MA). Confocal microscopy studies were performed on transgenic zebrafish embryos using a Leica TCS SP confocal microscope (Leica Microsystems, Mannheim, Germany). Pictures represent an area of 500 µm x 500 µm imaged with a 20x/0.7 numerical aperture HC PlanApo lens. GFP was detected at a spectral range from 507–550 nm and RFP from 585 to 690 nm.

Maintenance of Zebrafish and in Vivo Drug Treatment
Zebrafish embryos were maintained as described (34). Embryo staging was carried out according to Kimmel (35). Bromocriptine (Sigma, St. Louis, MO) was dissolved in methanol, sulpride dissolved in 0.01 M acetic acid, and each diluted in fish medium immediately before adding to live embryos at the stages indicated. Controls were cultured in fish medium containing the same final concentration of solvent.

	ACKNOWLEDGMENTS

 
We thank Ms. Hong Jiang for technical assistance and Dr. Martha Cruz-Soto for helpful discussions.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grants KO8 DK 064806 (to N.L.), CA75979 (to S.M.), RR13227 (to S.L.), and the Doris Factor Molecular Endocrinology Laboratory.

The authors have nothing to declare.

First Published Online December 8, 2005

Abbreviations: BAC, Bacterial artificial chromosome; DA, dopamine; Drd2, D2 dopamine receptor; dpf, days post fertilization; GFP, green fluorescent protein; hpf, hours post fertilization; MO, morpholino oligonucleotide; RFP, red fluorescent protein; PRL, prolactin; PRLR, prolactin receptor; POMC, pro-opiomelanocortin; PRLR, PRL receptor; MO, morpholino; TH, tyrosine-hydroxylase.

Received for publication October 7, 2005. Accepted for publication November 28, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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