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Endocrinology Vol. 143, No. 5 1858-1871
Copyright © 2002 by The Endocrine Society

RECEPTORS

Structural, Biochemical, and Expression Analysis of Two Distinct Insulin-Like Growth Factor I Receptors and Their Ligands in Zebrafish*

Travis Maures, Shu Jin Chan, Bin Xu, Hanshi Sun, Jun Ding and Cunming Duan

Department of Molecular, Cellular and Developmental Biology, University of Michigan (T.M., H.S., J.D., C.D.), Ann Arbor, Michigan 48109; and Howard Hughes Medical Institute, University of Chicago (S.J.C., B.X.), Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Cunming Duan, Ph.D. Department of Molecular, Cellular and Developmental Biology University of Michigan, Natural Science Building, Ann Arbor, Michigan 48109-1048. E-mail: .


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have cloned and characterized cDNAs encoding the zebrafish IGF ligands and receptors. Sequence comparison showed that the primary structures of zebrafish IGF-I, IGF-II, and IGF-I receptors (IGF-IRs) have been highly conserved in vertebrates. In contrast to the presence of a single IGF-IR gene in mammals, two distinct IGF-IR genes, termed igf-1ra and igf-1rb, were found in zebrafish. Structural and phylogenetic analyses indicated that both genes are orthologous to the human igf-1r gene. Immunoprecipitation studies with specific antibodies showed that both IGF-IR genes are expressed and both receptors bind to IGFs and des(1–3)IGF-I, but not to insulin. The spatio-temporal expression patterns of the two IGF-IRs and their ligands were determined using a combination of RT-PCR, whole mount in situ hybridization, and immunocytochemistry. Transcripts for both IGF-I and -II mRNAs were found throughout embryogenesis in a ubiquitous manner. In adult tissues, IGF-I mRNA was more abundant in liver and testis, and its level was increased after GH treatment, whereas IGF-II mRNA was not regulated by GH. IGF-IRa and IGF-IRb mRNAs and proteins were expressed in overlapping spatial domains, but exhibited distinct temporal expression patterns. In particular, the relative level of IGF-IRa mRNA was low during early embryogenesis and increased in the hatched larva, whereas the situation was reversed for IGF-IRb mRNA. In adult zebrafish, the overall tissue distribution patterns of the two IGF-IRs were similar, but there were differences in their cellular localization and relative abundance in defined cells/regions. The differential expression pattern of IGF-IRa and IGF-IRb suggest that they may play distinct roles in regulating the growth and development of zebrafish.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW well established that the IGF signals, mediated through the IGF-I receptor (IGF-IR) and modulated by IGF-binding proteins (IGFBPs), are essential for normal organismal growth and development (1, 2). Although the global importance of the IGF signaling system in growth regulation has become clear, the specific role of each individual member of the IGF signaling system in determining embryonic growth and development and how they interact with each other in vivo to regulate cell proliferation, differentiation, apoptosis, and migration in a developing vertebrate embryo are not well defined. Research on the developmental roles of the IGF signaling system have relied heavily on rodent models and knockout mice with null mutations in these genes (3, 4). However, attempts to elucidate the cellular actions of IGF, IGF-R, and, in particular, IGFBP actions, have not been easy in part due to the inaccessibility of the mammalian fetus enclosed in the uterus. Unlike mammalian embryos, fish embryos and larva live in water freely and are easily accessible for various experimental manipulations, which make them well suited for discerning the developmental roles of IGFs, IGF receptors, and IGFBPs. In the past decade, there has been a rapid accumulation of knowledge of IGF biology in teleost fish. The structures of IGF-I and IGF-II have been determined in a number of teleost species (see Refs. 5, 6, 7, 8 for reviews). One full-length and several partial sequences of IGF-IR have been reported for turbot, coho salmon, and rainbow trout (9, 10, 11, 12). Several fish IGFBP cDNAs or proteins have been cloned and characterized (13, 14, 15), and major intracellular signaling pathways of IGF actions have been elucidated (16, 17). The accumulated evidence to date suggests that the major components of the IGF signaling system in teleosts are structurally and functionally similar to those in mammals. However, there are several key differences. First, unlike mammals, most teleosts do not reach a static adult size, but continue to grow indeterminantly well beyond puberty (18). Given its central importance in growth regulation, it seems likely that the IGF signaling system may play an important role in this growth pattern. Second, during mammalian fetal development, the mannose-6-phosphate/IGF-II receptor has an important function as a biological sink to prevent tissue overgrowth stimulated by IGF-II (19). Comparative studies indicate that the mannose-6-phosphate receptors of nonmammalian species do not possess the capacity to bind IGF with high affinity (20, 21, 22). A third difference between mammals and teleosts is the recent finding that fish may contain multiple copies of IGF-IR and the insulin receptor (IR) (9, 12, 23). Therefore, study of IGF biology in fish may yield novel insights on their functions in growth control and on the evolution of growth regulatory mechanisms in vertebrates.

Among the numerous teleost species, zebrafish (Danio rerio) has recently become one of the standard model organisms in the study of vertebrate development, joining Xenopus, chick, and mouse. The free-living, fast-developing, and transparent zebrafish embryos make them well suited for investigating the mechanisms by which IGFs, IGF-IR, and IGFBPs act to regulate cell proliferation, differentiation, and apoptosis during early development. The availability of numerous genetic mutants provides an additional immense advantage of using the zebrafish model system. Despite the many advantages, little is known about the zebrafish IGF signaling system. To study IGF biology using this unique model organism, we have cloned and characterized cDNAs encoding for IGF-I, IGF-II, and two nonalleleic IGF-IRs from zebrafish and examined their temporal and spatial expression profiles in developing embryos, larvae, and adult zebrafish. Our results revealed that the structures of zebrafish IGF-I, IGF-II, and IGF-IRs are highly similar to their mammalian counterparts. Unlike mammalian model animals, however, we found that zebrafish possess two structurally distinct IGF-IR genes. The two zebrafish IGF-IR mRNAs and proteins display different temporal expression patterns, suggesting that they play distinct roles in regulating the growth and development of zebrafish.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All chemicals were molecular biology or reagent grade and were purchased from Sigma (St. Louis, MO) or VWR Scientific Products Inc. (Chicago, IL) unless noted otherwise. Human IGF-I and IGF-II were purchased from GroPep Pty. Ltd. (Adelaide, Australia), and [125I]IGF-I was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Disuccinimidyl suberate (DSS) was obtained from Pierce Chemical Co. (Rockford, IL). FBS was purchased from Life Technologies, Inc. (Grand Island, NY).

Fish
Wild-type zebrafish (Danio rerio) were maintained on a 14-h light, 10-h dark photoperiod cycle at 28 C and fed twice daily. The embryos were obtained by natural crosses. Fertilized eggs were collected, staged, and raised in embryo rearing solution at 28.5 C following standard procedures (24) that were approved by the University of Michigan University committee on use and care of animals. The ages of the embryos are given as hours post fertilization (hpf). Frozen zebrafish larva, purchased from Scientific Hatcheries (Huntington Beach, CA), were also used for some studies. To determine the effect of GH on IGF mRNA levels, purified seabass GH or saline was ip injected into adult zebrafish at a dose of 1 &mgr;g/g BW, and the fish were killed 15 h after the injection (13).

RNA isolation
Total RNA was isolated from zebrafish embryos and adult tissues using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) following the manufacturer’s instruction or using guanidinium thiocyanate as reported previously (9). The concentration of RNA was determined by measurement of absorbance at 260 nm. RNA integrity was examined by electrophoresis in 1% agarose gels.

Cloning zebrafish IGF-IR and IGF cDNAs
For cloning zebrafish IGF-IR cDNAs, RNA isolated from zebrafish larva (~60 hpf) was used as template, and an initial RT-PCR was performed with degenerate primers used to amplify an approximately 440-bp fragment of the tyrosine kinase domain that is highly conserved in all members of the IR family (9). Two distinct sequences were obtained that shared high sequence identity with human IGF-IR, and these were expanded to obtain full coding sequences using the combination of anchored PCR, rapid amplification of 5'-cDNA ends (5'RACE), and 3'RACE. Anchored PCR was performed using gene-specific primers and the following degenerate primers (with aa coding sequence shown in parentheses): 5'-CGNCTNGAPAAQTGQACNGT (sense, RLENCTV), 5'-AAPCCNTGGACNCAPTAQGC (sense, KPWTQYA), 5'-CATNAGQTCPAANAGCATPTC (cDNA, DMLFELM), and CCPTTCATPTGNGCPTANGG (cDNA, PYAHMNG). 5'RACE and 3'RACE were performed as described by Frohman et al. (25). The zebrafish IGF-I and -II cDNAs were obtained by RT-PCR using primers designed based on conserved sequences found in other teleosts. After amplification, the PCR products were cloned into the pCR 2.1 vector, and automated DNA sequencing was performed with ABI PRISM 377 (PE Applied Biosystems, Foster City, CA) using the Big Dye Terminator cycle sequencing kit (Perkin-Elmer Corp., Norwalk, CT). DNA sequences were analyzed using MacVector software (Oxford Molecular Ltd., Madison, WI) and/or DNASTAR (Madison, WI). Sequences used for alignment other than those reported here were extracted from the public databases from the National Center for Biotechnology Information using BLAST searches.

Peptide synthesis and antibody production
Two peptides, pepZ7 = DTEELDMEVENVPLDP and pepZ8 = PTSAANGPSMVLRGPFEEGQ (corresponding to unique sequences in the divergent C-terminal domains of IGF-IRa and IGF-IRb), were synthesized and purified at Howard Hughes Medical Institute, University of California (San Francisco, CA). The peptides were conjugated to keyhole limpet hemocyanin and used to generate polyclonal rabbit antiserum, anti-IGF-IRa (Z7), and anti-IGF-IRb (Z8) by Covance Research, Inc. (Richmond, CA).

Biochemical analysis of zebrafish IGF-IRs
Frozen zebrafish larva were homogenized in 10 vol buffer I [25 mM HEPES (pH 7.5), 4 mM EDTA, 4 mM EGTA, 2 mM phenylmethylsulfonylfluoride, and 50 &mgr;g/ml leupeptin] and centrifuged at 20,000 x g for 30 min. The crude membrane pellet was resuspended in fresh buffer I, and 0.1 vol 20% Triton X-100 was slowly added with stirring. After 1 h, the homogenate was centrifuged at 150,000 x g for 90 min, and the clear supernatant, containing solubilized IGF-IRs, was collected and stored at -70 until use. [125I]IGF-I binding was assayed by precipitation with polyethylene glycol (PEG). Zebrafish lysate was incubated in 200 &mgr;l binding buffer [30 mM HEPES (pH 7.5), 500 &mgr;g/ml BSA, and 500 &mgr;g/ml bacitracin] containing 12 pM [125I]IGF-I overnight at 4 C. After the addition of 0.5 ml 0.2% &ggr;-globulin (Sigma) and 0.5 ml 25% (wt/vol) PEG8000 (Sigma), the sample was microfuged for 10 min, and the precipitate was counted in a &ggr;-counter. For immunoprecipitation, 1 &mgr;l rabbit antiserum was added to [125I]IGF-I-bound zebrafish lysate and incubated at 4 C for 2 h, followed by the addition of 30 &mgr;l protein A-Sepharose (Amersham Pharmacia Biotech) to precipitate the immunocomplex. The immunocomplex was collected by centrifugation, rinsed with 1 ml 30 mM HEPES (pH 7.5) and 150 mM NaCl, and counted in a &ggr;-counter. For affinity cross-linking, zebrafish larva lysate was incubated with 1 x 105 cpm [125I]IGF-I in 100 &mgr;l binding buffer overnight at 4 C and cross-linked by adding 2 &mgr;l 10 mM DSS freshly dissolved in dimethylsulfoxide. After incubation on ice for 15 min, the reaction was terminated by adding 2 &mgr;l 1 M Tris-HCl (pH 7.4), and the samples were analyzed by SDS-PAGE. The gel was fixed, dried, and exposed to x-ray film for autoradiography.

Whole mount in situ hybridization
Whole mount in situ hybridization using digoxigenin (DIG)-labeled RNA riboprobe was carried out essentially as reported by Thisse et al. (26). The plasmid DNA of zebrafish IGF-I, IGF-II, IGF-IRa, or IGF-IRb was linearized by restriction enzyme digestion, followed by in vitro transcription reactions with either T7 or T3 RNA polymerase to generate the antisense or sense RNA riboprobes. Embryos were dechorionated manually and were fixed overnight with 4% paraformaldehyde in PBS. They were treated with cold methanol and dehydrated through a descending methanol series in PBS. Embryos older than 18 hpf were permeabilized by protease K treatment. Embryos were hybridized with the appropriate riboprobe at 65 C, followed by incubation with an anti-DIG antibody conjugated with alkaline phosphatase and stained with substrate nitro blue tetrazolium and 5-bromo-4-chloro-3-indolil phosphate to produce purple insoluble precipitates. Photographs were taken with a Nikon EC600 fluorescence microscope (Melville, NY).

RT-PCR assay
To examine the relative expression levels of IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNA, RT-PCR assays were performed using the primers shown in Table 1Go. Five micrograms of total RNA from embryo, larvae, or adult tissues were reverse transcribed into single-stranded DNA using random primer and SuperScript II (Life Technologies, Inc.). PCR reactions were carried out using one fifth of the cDNA reaction in a final volume of 50 &mgr;l containing 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgSO4, 200 mM of each dNTP, 2 U Vent polymerase (New England Biolabs, Inc., Beverly, MA), and 0.2 &mgr;M specific primers. The amplification profile was optimized for a MJ Research, Inc. Thermocycler (Cambridge, MA) and was as follows: one cycle of 1 min at 94 C; 29 cycles of 1 min at 94 C, 1 min at 50-58 C, and 1 min at 72 C; followed by a final extension of 10 min at 72 C. The RT-PCR products were separated by 1.0% agarose gel electrophoresis, stained with 0.5 &mgr;g/ml ethidium bromide, and photographed under UV light. Each PCR experiment included a positive control containing 10 ng cloned cDNA as well as a negative control with no template. In control experiments, we verified the specificity of each primer set by isolating the amplified fragment and performing DNA sequence analysis. We also validated the RT-PCR as a semiquantitative assay in several experiments by adding different amounts of zebrafish cDNA as template and obtained a proportionate amount of amplified DNA product (data not shown).


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  		Table 1. Sequences of primers and predicted PCR product sizes

 
Immunocytochemistry
Both fixed zebrafish embryos and larvae or tissue sections were used for immunohistochemical analysis of the two IGF-IR proteins. The embryos/tissues were fixed in 4% paraformaldehyde in PBS. Selected embryos/tissues were further embedded and stored at -80 after cryoprotecting in various sucrose solutions. Sections were cut at 10-&mgr;m thickness and mounted onto glass slides. After being permeated in 1% Triton X-100 in PBS and treated with PBS (pH 7.4) containing 3% normal goat serum to reduce nonspecific binding, the embryos, larvae, or tissue sections were incubated with the primary antiserum (diluted in 3% normal goat serum/1% Triton X-100/PBS at 1:100) at 4 C for overnight. After washing four times in 1x PBS, the primary antibody was detected using Cy3-conjugated donkey antirabbit IgG at 1:500 dilution (The Jackson Laboratory, Bar Harbor, ME) for 1 h at room temperature in the dark. They were again washed four times in 1x PBS. Microphotographs were taken with a Nikon EC600 fluorescence microscope with a TRIC filter. The specificity of the immunocytochemical reaction was verified using the following controls: 1) omission of the primary antibody, 2) replacement of the primary antibody with nonimmune serum, and 3) preabsorption of the primary antibody with the IGF-IR peptides.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of the IGF receptors and ligands from zebrafish
To clone IGF-IR cDNAs from zebrafish, we first performed RT-PCR with zebrafish larva RNA using degenerate primers to amplify a 440-bp DNA representing part of the receptor tyrosine kinase domain. These primers were designed to encode highly conserved amino acid (aa) sequences found in all three members of the insulin receptor (IR) family, including the IR, IGF-IR, and insulin receptor-related receptor (IRR), and have been used previously to clone four distinct partial IR cDNAs and two partial IGF-IR cDNAs from salmon cartilage (9). The predicted 440-bp PCR fragment was isolated and cloned into a plasmid vector. Fifty clones were analyzed by DNA sequencing and/or restriction endonuclease mapping. Six distinct sequences were obtained. Based on homology to known mammalian genes, these cDNAs were identified as follows: two IGF-IR cDNAs, zebrafish IGF-IRa, and IGF-IRb (GenBank accession no. AF400269 and AF400270); two IR cDNAs, zebrafish IRa and IRb (GenBank accession no. AF400271 and AF400272); one zebrafish homologue of c-ros (GenBank accession no. AF400273); and one zebrafish homolog of cell adhesion protein (GenBank accession no. AF400274; Fig. 1Go). Interestingly, no cDNA clone with significant homology to the mammalian IRR was obtained. Based on the partial sequences, the complete sequences of zebrafish IGF-IRa and zebrafish IGF-IRb were obtained using a combination of anchored RT-PCR, 5'RACE, and 3'RACE and have been deposited in GenBank (accession no. AF400275 and AF400276). The deduced primary sequences of zebrafish IGF-IRa and IGF-IRb precursors contain 1405 and 1380 aa, respectively. Pairwise comparisons revealed that zebrafish IGF-IRa and IGF-IRb share an overall sequence identity with human IGF-IR of 63.2% and 59.6%, respectively. Their sequence identity to that of human IR or IRR is significantly lower, 50.9% and 49.0% of human IR,and 50.7% and 48.7% of human IRR, respectively. Phylogenetic analyses group the two IGF-IRs in the vertebrate IGF-IR clade, whereas the two IRs are grouped in the IR clade (Fig. 1BGo). Interestingly, zebrafish IGF-IRa and IGF-IRb are only 69.8% identical to each other in primary sequence. The divergent residues are distributed throughout the entire molecules (Fig. 2BGo). Judging from these divergences, IGF-IRa and IGF-IRb cannot be isoforms derived from translational initiation at two in-phase ATG codons or alternative splicing of the same gene. These two cDNAs are very likely derived from two distinct genes.



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  		Figure 1. A, Partial sequences of six distinct tyrosine kinase domains identified in zebrafish. Zebrafish larva RNA was amplified by RT-PCR as described in Materials and Methods. Six distinct cDNA clones were obtained, and their deduced aa sequences were aligned by the Clustal method. Gaps were introduced to maximize sequence homologies. Residues that are identical or conserved are shaded. The GenBank database accession no. is AF400269 for IGF-IRa, AF400270 for IGF-IRb, AF400271 for IRa, AF400272 for IRb, AF400273 for c-ros, and AF400274 for the zebrafish cell adhesion protein. B, Phylogenetic tree of the IR/IGF-IR/IRR gene family. The full-length aa sequences of various IR, IGF-IR, and IRR were extracted from GenBank and analyzed using the UPGMA Bootstrap method. The GenBank database accession no. is GI13786530 for C. elegans IR; GI1079095 for Drosophila IR; GI6648596 for silkworm insulin-like peptide receptor; GI7511794 for mosquito IR; GI2497557 for Amphioxus insulin-like peptide receptor; GI3129622 for Turbot IGF-IR; GI3037089 for Xenopus IGF-IR; GI8393621 and GI2117841 for rat IR and IGF-IR; GI6754360, GI12644455, and GI675362 for mouse IR, IGF-IR, and IRR; and GI4557884, 2144422, and 12644000 for human IR, IGF-IR, and IRR, respectively. Numbers on branches are the percentage of times that the two clades branched as sisters (with 1000 runs). The results indicate that the two IGF-IRs are grouped in the IGF-IR clade, whereas the two zebrafish IRs IRs are grouped in the IR clade with high Bootstrap support.

 



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  		Figure 2. A, Comparison of the zebrafish IGF-IRa and IGF-IRb precursor sequences with that of the human IGF-IR. The aa sequences were deduced from the cloned full-length cDNAs of zebrafish IGF-IRa (GenBank accession no. AF400275) and IGF-IRb cDNA (AF400276). The first aa residue of the predicted mature protein is indicated by an asterisk. Sequence alignment was obtained by the Clustal method. Gaps were introduced to maximize sequence homologies. Residues that are identical or conserved are shaded. B, Schematic diagram comparing the domain structures of zebrafish IGF-IRa and IGF-IRb. In addition to the leader sequence, &agr;-subunit, and &bgr;-subunit, the cysteine-rich region, the tetrabasic proteolytic cleavage site (RKRR IGF-IRa, and RRRR for IGF-IRb), the tyrosine kinase domain, the potential ATP-binding site, and the IRS-1-binding site are indicated. The extra 15 aa found in IGF-IRa is indicated by an open box. The percentage of sequence identity for each domain was calculated and is presented in the middle column.

 
As shown in Fig. 2GoGo, both of the IGF-IRs can be divided into an N-terminal domain, a cysteine-rich domain, a tetrabasic proteolytic cleavage site (RKRR at positions 734–737 for IGF-IRa and RRRR at positions 727–730 for IGF-IRb), a transmembrane domain with juxtamembrane motifs, a tyrosine kinase domain, and a carboxyl-terminal domain. Among these domains, the tyrosine kinase domain is the most highly conserved. Moreover, both tyrosine kinase domains contain structural features with known functional significance, including a potential ATP-binding site (GXGXXXG21XK, positions 1004–1033 and 994-1021 for IGF-IRa and IGF-Ib, respectively), a triple tyrosine cluster that constitutes the major autophosphorylation site for mammalian IGF-IR (YXXXYY, positions 1159–1164 and 1148–1153). A consensus insulin receptor substrate-1 (IRS-1)-binding motif (NPEY), which is known to be critical for IGF-IR signaling, is located at positions 975–978 and 965–968 in IGF-IRa and IGF-IRb, respectively. The cysteine-rich regions, which are known to be important for ligand binding specificity, contain 26 conserved cysteine residues in a stretch of 196 aa extending from positions 156–352 and 151–348 in IGF-IRa and IGF-Ib, respectively. With the exception of the last 24 aa at the very C terminus, the cytoplasmic regions of these IGF-IRs are highly divergent. In addition, an extra 15-aa sequence (APSSQGNTGIQSQNP) is present in this part of the IGF-IRa (Fig. 2GoGo).

In addition to the receptors, cDNAs encoding the complete coding sequences for zebrafish prepro-IGF-I and IGF-II have been cloned. The predicted mature zebrafish IGF-I is 70 aa. It is 96–97% identical to that of carp and goldfish IGF-I (27, 28) and is identical to the recently published zebrafish IGF-I Ea-2 sequence (29). The zebrafish IGF-II sequence is identical to a sequence deposited in GenBank (AF194333) and is highly similar to other vertebrate IGF-II, with the highest sequence identity being 77% to trout IGF-II (30).

Biochemical and functional characterization of the IGF-IRa and IGF-IRb protein
To determine whether the two distinct IGF-IR mRNAs are translated into two functional receptor proteins in zebrafish, tissue lysate was prepared from zebrafish larva and assayed for IGF-I binding. As shown in Fig. 3AGo, zebrafish larva lysate specifically bound to the added 125I-labeled IGF-I, and the radioactive tracer was completely displaced by adding 106 M IGF-I, IGF-II, or des(1–3)-IGF-I, but not by insulin. Because des(1–3)-IGF-I has been shown to have impaired affinity for IGFBPs in mammals and in zebrafish (13, 31), the competition data indicated that [125I]IGF-I was bound to IGF-I receptors rather than IGFBPs in the tissues lysate. To further characterize the IGF-IRs, the [125I]IGF-I-bound IGF-IR was immunoprecipitated with anti-IGF-IRa or anti-IGF-IRb. These two antibodies against each receptor were generated using synthetic peptides corresponding to unique sequences in their divergent C-terminal domains, i.e. pepZ7 = DTEELDMEVENMENVPLDP for IGF-IRa and pepZ8 = PTSAANGPSMVLRGPFEEGQ for IGF-IRb. Anti-IGF-IRa and anti-IGF-IRb specifically brought down 42% and 19% of the total bound [125I]IGF-I, respectively (Fig. 3BGo). We were unable to achieve 100% immunoprecipitation because about half of the bound [125I]IGF-I was rapidly dissociated from the receptor during the buffer wash step in the assay (data not shown). The addition of excess unlabeled IGF-I reduced the immunoprecipitable binding to the background level, whereas insulin had no effect (Fig. 3BGo). When both anti-IGF-IRa and anti-IGF-IRb antisera were added together, 52% of the bound [125I]IGF-I activity was immunoprecipitated, indicating that the IGF-IRs immunoprecipitable by either anti-IGF-IRa or anti-IGF-IRb antiserum are partially additive. Based on these results we concluded that zebrafish IGF-IRa and IGF-IRb can account for most of the IGF-binding activity present in zebrafish larvae.



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  		Figure 3. The two IGF-IR cDNAs encode structurally distinct, specific IGF-I receptor proteins. A, Specific [125I]IGF-I binding to IGF-IRs in zebrafish larval tissues. Increasing amount of lysates were incubated with [125I]IGF-I in the absence ({circ}) and presence of 106 M unlabeled IGF-II ({bullet}), des(1–3)-IGF-I ({bullet}), or insulin (X). The bound [125I]IGF-I was precipitated with PEG. B, [125I]IGF-I binding to IGF-IRa and IGF-IRb. Zebrafish larval lysates were incubated with [125I]IGF-I in the absence or presence of 106 M unlabeled IGF-I or insulin and immunoprecipitated with anti-IGF-IRa (Z7) and anti-IGF-IRb (Z8), respectively. C, Affinity cross-linking of [125I]IGF-I to zebrafish IGF-IRs. Zebrafish larval lysates were incubated with [125I]IGF-I, cross-linked by adding DSS, and immunoprecipitated with preimmune serum (lane 2), anti-IGF-IRA (lanes 3), or anti-IGF-IRb (lane 4) antisera. Lane 1 is the total specific binding. The samples were analyzed by SDS-PAGE under reducing conditions.

 
The two IGF-IRs were further characterized by affinity cross- linking studies. Under nonreducing condition, [125I]IGF-I bound to proteins with an estimated molecular mass of 400 kDa, which may represent a disulfide-linked heterotetrameric receptor. After the samples were reduced with &bgr;-mercaptoethanol, however, [125I]IGF-I was found in a broad band of approximately 120–130 kDa. This band was specifically immunoprecipitable with antiserum anti-IGF-IRa or anti-IGF-IRb and is consistent in size with the glycosylated receptor &agr;-subunit (Fig. 3CGo). Consistent with data shown in Fig. 3BGo, the intensity of the band precipitated by antiserum anti-IGF-IRa was about 3 times greater than that of anti-IGF-IRb antiserum. Taken together, these results indicated that the two IGF-IR cDNAs encode for two structurally distinct, functional IGF-I receptors. Both of these receptors are expressed and functional in zebrafish larvae, with the IGF-IRa being the predominant form.

Spatial expression patterns of IGF ligand and receptor mRNAs and proteins during early development
Using the available cloned cDNAs as probes, we next analyzed the spatial expression profiles of IGF ligands and receptors. For each molecule, sense and antisense DIG- labeled riboprobes were synthesized and used to perform in situ hybridization at several critical stages in zebrafish development: 1) the 8- to 16-cell cleavage period (1.5 hpf); 2) the 26-somite stage (22 hpf), which corresponds to undergoing somitogenesis; 3) the hatching stage (48 hpf), which marks the end of embryogenesis; and 4) hatched larvae (72 hpf). Before use, the specificity of these riboprobes was verified by dot-blot analysis (data not shown). In all developmental stages, positive signal for IGF-I mRNAs was detected in a ubiquitous fashion throughout the body (Fig. 4AGo). Likewise, the ubiquitous presence of IGF-II transcripts was observed in the embryos (Fig. 4BGo, a, c, d, f, and h). After hatching, the expression of IGF-II mRNA was higher in the anterior portion of the body, most obviously in the eyes, brain, and other nervous tissues (Fig. 4BGo, j and l). Figure 6Go, A and B, shows the spatial distribution of zebrafish IGF-IRa and IGF-IRb mRNAs. At 8- to 16-cell stages, IGF-IRa and IGF-IRb mRNAs were easily detected in all cells (Fig. 5Go, A and B, a). Spatially both mRNAs were ubiquitously expressed throughout embryogenesis. However, some localized variations in the intensity of hybridization signals were observed. For example, strong hybridization signals for both receptors were found in several fast-growing areas, most notably in areas where the fin buds developed (Fig. 5Go, A and B, g, i, k, and m). No signal was found in control experiments performed in parallel on the same batch of embryos with the corresponding sense RNA riboprobes (Fig. 5Go, A and B, c, f, h, j, l, and n), demonstrating the specificity of the hybridization signal.



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  		Figure 4. Whole mount in situ hybridization analysis of IGF-I mRNA (A) and IGF-II mRNA (B) in zebrafish embryos and larvae. Embryos from four developmental stages were used: 1) the 8- to 16-cell cleavage period (1.5 hpf; a and b), 2) the 20- to 26-somite stage (18–22 hpf; c–e), 3) the hatching stage (48 hpf; f–i), and 4) hatched larvae (72 hpf; j–m). a, d, h, and l are lateral views. Dorsal views are shown in c, f, and j. No signal was found in control experiments with the corresponding sense RNA riboprobe (, b, e, g, i, k, and m).

 


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  		Figure 6. Immunocytochemical localization of zebrafish IGF-IRa and IGF-IRb proteins in zebrafish embryos and larvae. A and B, Whole mounted embryos and larvae were immunostained for IGF-IRa (A) or IGF-IRb (B). Embryos from seven developmental stages were used: 1) the dour-cell cleavage stage (1 hpf; a), 2) the 16-cell cleavage stage (1.5 hpf; b), 3) the sphere blastula stage (4 hpf; c), 4) the 20-somite segmentation stage (19 hpf; d), 5) the prim-15 pharyngula stage (30 hpf; e), 6) the hatching stage (48 hpf; f), and 7) the hatched larvae (72 hpf; g). No signal was found in 24 or 48 hpf embryos (h) when the primary antibody was omitted. C and D, Immunostaining of the serial cross-sections of larval fish (72 hpf) for IGF-IRa (C) and IGF-Rb (D). The fish were sectioned serially from head to tail and subjected to immunostaining as described in Materials and Methods. IGF-IRa and IGF-IRb immunoreactivity was detected throughout the body (magnification, x40). The micrographs are presented from the anterior to posterior order from top to bottom (a–f) with the dorsal side of the fish facing down. Similar results were obtained with two other individuals of the same stage. No signal was found after the primary antibodies were preabsorbed with the IGF-IR peptides (g).

 


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  		Figure 5. Whole mount in situ hybridization analysis of zebrafish IGF-IRa mRNA (A) and IGF-IRb mRNA (B) in zebrafish embryos and larvae. Embryos from four developmental stages were used: 1) the 8- to 16-cell cleavage period (1.5 hpf; a–c), 2) the 26-somite stage (22 hpf; d–f), 3) the hatching stage (48 hpf; g–j), and 4) hatched larvae (72 hpf; k–n). Dorsal views are shown in a, d, i, and m. Lateral views are shown in b, e, g, and k. No signal was found in control experiments using the corresponding sense RNA riboprobe (c, f, h, j, l, and n).

 
We next examined the expression of the two distinct IGF-IR proteins during zebrafish embryogenesis by immunostaining of whole mounted zebrafish embryos and larvae. As shown in Fig. 6Go, A and B, the spatial expression patterns of IGF-IR proteins resembled those of mRNAs. At 8- to 16-cell stages, IGF-IRa and IGF-IRb immunoactivity was detected on the surface of all cells (Fig. 6Go, A and B, a and b). Both receptors were ubiquitously expressed throughout embryogenesis (Fig. 6Go, A and B), although some localized variations in the intensity of signals were observed. Strong signals for both receptors were found in the fast-growing areas such as the budding fins in 48 hpf embryos and 72 hpf larvae (Fig. 6Go, A and B, f and g). To further determine whether the cellular localization of these two zebrafish IGF-IR proteins is different, serial cross-sections of zebrafish larvae (72 hpf) were subjected to immunocytochemical staining. Consistent with the whole mount results, immunoreactive IGF-IRa and IGF-IRb were detected in all cell types in zebrafish larvae (Fig. 6Go, C and D). The signals were specific, because omission of the primary antibody completely abolished the immunostaining signal (h in Fig. 6Go, A and B), and absorption of either antisera with the respective antigen resulted in a nearly complete loss of immunostaining signal (g in Fig. 6Go, C and D). This spatial overlapping expression pattern appeared to be limited in early development. In the adult stage, the two receptors displayed differential cellular localization in defined tissues. For instance, although both immunoreactive IGF-IRa and IGF-IRb were detected in the brain, the overall staining for IGF-IRa was stronger (data not shown).

Temporal expression patterns of IGF ligand and receptor mRNAs during early development
We next examined the temporal expression profiles of IGF ligand and receptor mRNAs during zebrafish embryogenesis by RT-PCR assays. Based on the cloned cDNA sequences, specific oligonucleotide primers were designed to amplify single bands of 500, 593, 748, and 488 bp, representing zebrafish IGF-I, IGF-II, IGF-IRa, and IGF-IRb cDNA (Table 1Go). Moreover, the RT-PCR assay was semiquantitative, in that the intensity of the amplified cDNA band obtained varied proportionately to the amount of template added (data not shown). As shown in Fig. 7Go, both IGF-I and -II transcripts were detected throughout embryogenesis and larva stages, including unfertilized eggs (1-cell, 0 hpf), embryos of cleavage period (2–32 cells, 1–2 hpf), blastula period (3–4 hpf), gastrula period (5–12 hpf), segmentation period (14–22 hpf), pharyngula period (30–40 hpf), and posthatching larvae (60 hpf). The levels of IGF-I and -II mRNA were relatively unchanged in all stages examined. However, the two IGF-I receptor genes exhibited different temporal expression patterns. The IGF-IRa mRNA levels were relatively low in early embryos, including fertilized eggs, and embryos of cleavage and blastula periods, but then gradually increased, and higher levels were seen at 35–40 and 60 hpf. In contrast, higher levels of IGF-IRb mRNA were found in zygotes and embryos of the cleavage and blastula periods. Its levels decreased at 5–8 hpf and remained at a relatively lower level through 60 hpf. These results indicate that IGF-I, IGF-II, and IGF-IR transcripts are expressed throughout zebrafish embryogenesis with distinct temporal patterns.



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  		Figure 7. Expression of IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNAs in zebrafish eggs, embryos, and larvae. cDNA was synthesized, and 30 cycles of PCR were performed. Amplified PCR products were analyzed by electrophoresis. -, Negative control with no DNA template. Similar results were obtained with two other experiments.

 
Tissue distribution and hormonal regulation of IGF ligand and receptor mRNAs in adult zebrafish
The expression of zebrafish IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNA in various adult tissues was analyzed by RT-PCR. As shown in Fig. 8AGo, IGF-I mRNA was detected in all tissues studied, with the highest levels in the liver and testes. High levels of IGF-II mRNA were found in brain, eyes, muscle, liver, testes, and intestine. The levels of IGF-II mRNA in the ovary were relatively low. As observed with embryos and larvae, the two IGF-IR mRNAs showed largely overlapping tissue distribution patterns in adult zebrafish, with the exception of gill and skeletal muscle, where IGF-IRb mRNA levels were considerably higher. Both mRNAs were expressed in all tissues examined, with the highest levels detected in ovary, testes, and brain and relatively high levels detected in the eye and intestine. Of the tissues examined, liver had the lowest levels of IGF-IRa and IGF-IRb mRNAs. To analyze the effect of GH treatment on IGF-I, IGF-II, and IGF-IR mRNA expression, adult zebrafish were injected with GH at a dose of 0.5 &mgr;g/g BW and were killed 15 h after the injection. GH treatment resulted in increased IGF-I mRNA levels. No marked difference was observed with IGF-II mRNA or either of the IGF-IR mRNA (Fig. 8BGo). These results suggest that the expression of IGF-I, but not IGF-II or IGF-IR mRNAs, is regulated by GH in adult zebrafish.



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  		Figure 8. Tissue distribution and hormonal control of IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNA expression in adult zebrafish. A, Tissue distribution of IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNAs in adult fish. cDNA was synthesized from 5 &mgr;g total RNA prepared from brain (b), eye (e), skeletal muscle (m), liver (l), ovary (o), testis (t), intestine (i), and gill (g). Thirty cycles of PCR were performed. +, Positive control with cloned DNA; -, negative control with no DNA template. B, GH treatment increases IGF-I, but not IGF-II or IGF-IR, mRNA levels in adult zebrafish. Total RNA was isolated from adult fish 15 h after they were injected with GH or saline (control) and subjected to RT-PCR analysis as described above. -, Negative control with no DNA template.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we determined the full-length structures of zebrafish IGF ligand and two IGF-IRs and analyzed their spatio-temporal expression and hormonal regulation. Sequence comparisons show that the primary structures of zebrafish IGF ligands and receptors are very similar to those of other vertebrates. The mature zebrafish IGF-I is 97% and 81% to that of carp and human. The mature zebrafish IGF-II is 77% identical to trout IGF-II and 71% identical to human IGF-II. The structure of the zebrafish IGF-IRs is highly similar to that of human IGF-IR. Not only is the overall sequence identity between zebrafish IGF-IRs and their human counterpart greater than 60%, most of the structural motifs that are known to be critical of IGF-IR functions, such as the ligand-binding region, tyrosine kinase domain, ATP-binding site, autophosphorylation site, and IRS-I docking site, are conserved in the two zebrafish IGF-IRs. This high degree of structural conservation of the IGF ligands and receptors is in good agreement with the results of recent functional studies showing that fish, chicken, and human IGFs are equally effective in stimulating zebrafish cell proliferation (17). Unlike the situation in mammals, however, two igf-1r genes are present in zebrafish, and they each encode a structurally distinct and functional IGF receptor. Phylogenetic analyses of both protein and DNA sequences by neighbor-joining and maximum likelihood methods group igf-1ra and igf-1rb in the vertebrate IGF-IR clade, distinct from the IR clade or the IRR clade. Furthermore, our biochemical analysis showed that the two IGF-IR proteins bound to IGF-I, IGF-II, and des(1–3)-IGF-I, but not to insulin, confirming that both proteins are functional IGF-IRs. As the primary sequences between the two IGF-IRs are significantly divergent (~30% residues are different from each other) and because these aa changes are distributed throughout the entire molecule, we concluded that these two cDNAs are derived from two distinct genes. The relatively high divergence suggests that a gene duplication event of the IGF-IR locus occurred early during teleost evolution. This conclusion is consistent with the theory that zebrafish and other fishes in the killifish lineage may have experienced an extra gene duplication during evolution based on studies of the Hox gene family in zebrafish and Fugu (32). Indeed, there appeared to be many examples of duplicated genes in zebrafish similar to that of igf-1r, such as msx and sox9 genes (33, 34). A recent study has characterized one zebrafish IGF-I gene (the IGF-Ia gene) and indicated that zebrafish may have more than one IGF-I gene, but the structure of the other zebrafish IGF-I gene has yet to be determined (29). To date, there is no report pertaining to the IGF-II gene(s) in zebrafish.

The finding that two igf-1r genes are present in zebrafish and that they each encode a functional IGF receptor is interesting when considering the evolutionary history of the IR/IGF-IR gene family. In the Caenorhabditis elegans and Drosophila genomes, there is only a single gene of this receptor gene family (35, 36). A single receptor equally related to human IR and IGF-IR has been identified in Amphioxus (37). The Amphioxus receptor may represent a reminiscence of the ancestral gene that duplicated to give rise to the IR and IGF-IR genes. All vertebrates studied to date possess at least two distinct receptors: an IR and an IGF-IR. Full-length or partial IR and IGF-IR cDNA sequences are known for mammals, birds, amphibians, and several bony fish (3, 9, 10, 11, 12, 23). In humans and rodents there is a third receptor in this gene family, the IRR. In this study we have cloned and characterized two full-length cDNAs encoding for two distinct IGF-IRs. In addition, two cDNAs encoding two structurally distinct IRs were obtained. Interestingly, no cDNA with significant homology to the human IRR was found in zebrafish despite the fact that our primers were designed based on sequences conserved in all three members of the IR/IGF-IR/IRR family, and that we were able to pull out the more remotely related members of the receptor tyrosine family, such as c-ros and cell adhesion protein, from zebrafish using these primers. These results suggest that IRR may have been a later invention during vertebrate evolution, possibly after the emergence of teleost fish.

The finding that both of the duplicated IGF-IR genes are preserved and functional in zebrafish also raised an important question regarding their functional relationship. Gene duplication followed by mutation and selection are thought to be critical events that drive biological diversity. Permanent preservation of both duplicates requires that each contributes in some distinct capacity to the viability of the organisms. Changes in gene expression, function of the protein products, or both may contribute to the endowment of differential properties upon duplication. In this study we explored the possible difference in the spatio-temporal expression patterns of the zebrafish igf-1ra and igf-1rb genes. Our expression analysis results suggest that although the overall spatial patterns in embryos, larvae, and adult zebrafish are similar, there are differences in their relative abundance in defined tissues/organs. For instance, IGF-IRa is more abundant in the cerebellum and pituitary gland in the adult brain. Also, although the levels of IGF-IRa mRNA and proteins gradually increased during zebrafish embryogenesis, those of IGF-IRb were relatively high in zygotes and early embryos, but decreased in later stages of embryogenesis and in larvae. Thus, although the two IGF-IRs are expressed in largely overlapping spatial domains during early development, they exhibit distinct temporal expression patterns and therefore may each make distinct contributions to the regulation of growth and development. As mentioned earlier, most teleosts do not reach a static adult size, but continue to grow indeterminantly after puberty (18). Given the central importance of IGF signaling in growth regulation, it is possible that the duplicated IGF-IR genes may attribute to the adult stage growth. It will be necessary to determine the in vivo function(s) of the two IGF-IR genes using a morpholino-based, targeted gene knockdown approach (38) in the future.

In addition to the differential expression patterns, it is also possible the function of the protein products of the gene duplicates may diverge. There are precedents for two or multiple receptor isoforms of a growth factor exerting different functions. The two mammalian platelet-derived growth factor (PDGF) receptors (PDGFR): PDGF&agr;R and PDGF&bgr;R, for example, are believed to have resulted from a gene duplication event predated the divergence of cyclostomes (nonjawed vertebrates) and gnathostomes (jawed vertebrates) (39). In addition to the difference in spatio-temporal expression patterns, these two receptors display very different ligand binding properties: whereas PDGF&agr;R binds and is activated by all forms of PDGF (AA, BB, and AB), PDGF&bgr;R is activated exclusively by PDGF-BB (40, 41). To this end, it is noteworthy that the cytoplasmic regions of the two IGF-IRs are very different. In particular, there is an extra 15-aa sequence in the IGF-IRa, but not in the IGF-IRb. It is conceivable that these diverged sequences may allow for different signaling capacities of the two receptors. As it is generally believed that the mannose-6-phosphate receptor in nonmammalian vertebrate does not possess IGF-binding ability, and because a recent biochemical study suggested the presence of a specific IGF-II receptor-like protein in rainbow trout embryos (42), it is also possible that these two IGF-IRs may have distinct ligand binding preferences. Although our affinity cross-linking and immunoprecipitation data have shown that both zebrafish IGF-IRs bind IGFs, but not insulin, this technique does not allow for a more quantitative comparison. Future studies are needed to determine whether the two zebrafish IGF-IRs possess similar or different ligand-binding and signaling properties.

Using the sensitive RT-PCR technique, several groups have shown that the IGF-I, IGF-II, and IGF-IR transcripts are detectable in the zygotes and embryos of rodents and other teleost fish (43, 44, 45, 46, 47, 48). The spatial expression profiles of these molecules, however, have not been studied. More importantly, whether and when these IGF and IGF-IR transcripts are translated into peptides and begin to function remain unknown. Taking advantage of the transparent zebrafish embryos, we have mapped the spatial expression profiles of the IGF ligands and receptors by whole mount in situ hybridization and immunocytochemistry. Our results reveal that IGF-I, IGF-II, IGF-IRa, and IGF-IRb mRNAs and proteins are ubiquitously expressed in embryos and larvae. Therefore, IGF-I, IGF-II, and IGF-IR are present in most, if not all, cells throughout the entire early life stages. In addition to the ubiquitous expression patterns, higher levels of IGF-IR mRNAs and proteins are observed in several fast-growing areas, most notably in the developing fin buds, suggesting that localized increases in the IGF-IR levels may be related to the elevated growth in these tissues. In agreement with this finding, our previous in vitro study showed that IGF-I and IGF-II, but not insulin, are potent mitogens for these cells (17). IGFs stimulate zebrafish embryonic cell proliferation by activating the ras-raf-MAPK and the PI3 kinase pathway. In mice, IGF or IGF-IR knockout results in a miniature fetus (1, 2). In Drosophila, genetic manipulation of either the insulin/IGF receptor, Chico (a fly homologue of human IRS), PI3K, PTEN (a lipid phosphatase that antagonizes PI3K, Akt, or p70S6 kinase resulted in alterations in cell, organ, and organism sizes (49, 50, 51, 52). Therefore, the IGF signaling system may act as a major regulator that determines the size of animals as diverse as mice, fish, and flies.

In adult zebrafish, IGF-I and IGF-II mRNAs are found in a wide variety of tissues, with the highest levels in the liver and testes. High levels of IGF-II mRNA were found all the tissues examined with the exception of the ovary. This tissue distribution pattern in zebrafish is consistent with what has been reported in other teleost fish. In juvenile salmon, for instance, IGF-I mRNA is detectable in virtually all tissues studied, with the highest levels observed in the liver (6). There is a large amount of evidence indicating that the GH-IGF-I axis operates in teleost fish, as in mammals and birds. In coho salmon, rainbow trout, and seabream, GH treatment significantly increased IGF-I mRNA levels in liver (6). The IGF-I mRNA level in adult zebrafish was elevated after GH treatment, suggesting that GH regulates the level of IGF-I expression in zebrafish as in other vertebrates. It was reported that GH treatment significantly increases IGF-II mRNA in the liver and pyloric ceca of rainbow trout (53). In the seabream, however, GH treatment does not increase tissue IGF-II mRNA levels (54). In the zebrafish, we found that GH injection did not significantly change whole body IGF-II mRNA levels.

In summary, the results of this study strongly support the concept that the IGF signaling system is highly conserved in vertebrates. Our results revealed that the structures of zebrafish IGF-I, IGF-II, and IGF-IRs are highly similar to their mammalian counterparts. Unlike mammalian model animals, however, zebrafish possess two structurally distinct IGF-IRs. The two zebrafish IGF-IR mRNAs and proteins display different temporal expression patterns, and there are differences in their cellular localization and relative abundance in defined cells/regions, suggesting that they may make distinct contributions in regulating zebrafish development and growth. Further studies in zebrafish, focusing on determining whether the two zebrafish IGF-IRs possess similar or different ligand-binding and signaling properties, will be needed to elucidate the physiological functions of the two IGF-IRs.


   Acknowledgments
 
S. J. Chan and B. Xu thank Dr. Donald F. Steiner and the Steiner laboratory for providing intellectual and material support.


   Footnotes
 
This work was supported by NSF Grant IBN 9728911 and IBN 0110864 (to C.D.) and Howard Hughes Medical Institute, NIH Grant DK-13914 (to Dr. D. F. Steiner).

Abbreviations: aa, Amino acid; DIG, digoxigenin; DSS, disuccinimidyl suberate; hpf, hours post fertilization; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; IR, insulin receptor; IRR, insulin receptor-related receptor; IRS-1, insulin receptor substrate-1; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PEG, polyethylene glycol; 5'RACE, rapid amplification of 5'-cDNA ends.

1 The sequence data reported in this paper have been submitted to the GenBank database under accession numbers AF400269, AF400270, AF400271, AF400272, AF400273, AF400274, AF400275, and AF400276. Back

Received November 13, 2001.

Accepted for publication January 8, 2002.


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 Discussion
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