Structure, Developmental Expression, and Physiological Regulation of ...

0013-7227/02/$15.00/0 Printed in U.S.A.

Endocrinology 143(7):2722–2731 Copyright © 2002 by The Endocrine Society

Structure, Developmental Expression, and Physiological Regulation of Zebrafish IGF Binding Protein-1 TRAVIS J. MAURES

AND

CUNMING DUAN

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 The biological activity and availability of IGFs are regulated by a group of secreted proteins that belong to the IGF-binding protein (IGFBP) gene family. Although six IGFBPs have been identified and studied in mammals, their nonmammalian orthologs remain poorly defined. In this study, we cloned and characterized the full-length zebrafish IGFBP-1. Sequence analysis indicated that its structure is homologous to mammalian IGFBP-1. Using in situ RNA hybridization and RTPCR, we discovered that IGFBP-1 mRNA was present in all early embryonic stages albeit at very low levels. IGFBP-1 mRNA was initially expressed in multiple embryonic tissues but became restricted to the liver shortly after hatching. In the adult stage, IGFBP-1 mRNA was found only in the liver at low levels. Prolonged food deprivation caused a significant increase in the hepatic IGFBP-1 mRNA levels, and refeeding

I

GFs, INCLUDING IGF-I and IGF-II, are major fetal growth regulators. The importance of IGFs in fetal growth is underscored by the significantly reduced fetal size and birth weights of the IGF-I and IGF-II knockout mice (1, 2). In humans, the fetus size and birth weight are positively correlated to the IGF-I concentration in cord serum (3) and a homozygous deletion of exon 4 and 5 of the human IGF-I gene results in severe intrauterine as well as postnatal growth retardation (4). The biological effects of IGFs are dependent upon their interactions with specific receptors on the cell surface. In addition to the two types of IGF receptors, six secreted, high-affinity IGF binding proteins (IGFBPs), designated IGFBP-1 to -6, have been isolated and characterized in humans and other mammals (see Refs. 5–7 for reviews). These IGFBPs are structurally distinct from the receptors, bind IGFs with an affinity equal to or greater than that of the receptors, and are differentially regulated. They can control the distribution of IGF between extracellular environments and cell surface-binding sites and may regulate IGF bioactivity by modulating its interaction with the receptor (5–7). IGFBP-1 is the first characterized member of this gene family and has been studied extensively in mammals. IGFBP-1 has been shown to inhibit IGF actions in numerous in vitro studies and several in vivo animal studies (see Ref. 8 for review). In all mammal species studied to date, the expression of IGFBP-1 gene is highly tissue specific, being predominantly expressed in the liver with modest levels in the kidney, decidua, and ovary (8). IGFBP-1 is the most

Abbreviations: EST, Expressed sequence tag; hpf, hours post fertilization; IGFBP, IGF-binding protein; RACE, rapid amplification of cDNA ends; RGD, Arg-Gly-Asp.

restored the IGFBP-1 mRNA to the basal levels. When adult fish or embryos were subjected to hypoxic conditions, the IGFBP-1 mRNA expression increased dramatically. Intriguingly, the hypoxia-induced IGFBP-1 expression operated in different embryonic tissues in a developmental-stage-dependent manner. In early embryos, hypoxia-stimulated IGFBP-1 mRNA expression in the pharyngeal arches, ventricle, atrium, and brain. After hatching, the hypoxia-induced IGFBP-1 expression became liver specific. These results not only provide new information about the structural conservation, developmental expression, and physiological regulation of the IGFBP-1 gene but also present the opportunity to elucidate the developmental role of IGFBP-1 using a unique vertebrate model organism. (Endocrinology 143: 2722–2731, 2002)

metabolically responsive member of the IGFBP family. In adult mammals, the levels of circulating IGFBP-1 and hepatic mRNA rise markedly in catabolic or stressful conditions such as fasting, malnutrition, and diabetes (8). It is believed that the elevated IGFBP-1 in turn regulates the bioavailability of IGFs and inhibits IGF actions. Recent studies suggest that IGFBP-1 may also play an important role in modulating fetal growth in response to altered nutritional and environmental conditions such as fetal hypoxia, which is a leading cause of human intrauterine growth restriction (8). There is a very strong inverse correlation between human fetal IGFBP-1 levels and fetal size (3). In human and animal models, circulating IGFBP-1 and hepatic IGFBP-1 mRNA expression are elevated in fetuses with long-term chronic hypoxia and intrauterine growth restriction (9 –13). Using HepG2 cells and primary human fetal hepatocytes, more recent studies have shown that the human IGFBP-1 gene is directly regulated by chronic hypoxia through the hypoxia-inducible factor-1 pathway (13, 14). Therefore, regulation of IGFBP-1 may be a physiological mechanism operating in postnatal and fetal stages to inhibit the IGFmediated somatic growth under catabolic and stressful conditions. Although the physiological regulation of IGFBP-1 expression and the endocrine role of IGFBP-1 are becoming clear, many questions remain unresolved. For instance, when and where does the IGFBP-1 gene begin to be expressed during embryogenesis? Is the IGFBP-1 gene expressed before the liver is developed? If so, in what embryonic tissues? When does the hypoxia-HIF-1 pathway begin to activate the IGFBP-1 gene during fetal development? Does hypoxia increase the IGFBP-1 levels only in the fetal liver or does it also activate IGFBP-1 gene expression in other fetal tissues? Because most, if not all, previous studies were carried out using

2722

Maures and Duan • IGFBP-1, Zebrafish Embryos, and Hypoxia

mammalian fetuses of advanced stages, we are ignorant about the IGFBP-1 expression patterns in early embryonic stages (15–17). This gap in our knowledge is in part because of the inaccessibility of the mammalian fetus enclosed in the uterus. The goal of this study was to determine the developmental and physiological regulation of IGFBP-1 expression using a unique vertebrate model organism, the zebrafish (Danio rerio). Unlike mammalian embryos, zebrafish embryos live in water freely and are easily accessible for various experimental manipulations. Their transparent and free-living bodies also make it easy to monitor the cellular processes during early development. Furthermore, recent advances in systematic compilation of information on genomic sequences and expressed sequence tags (ESTs) have made the cloning of zebrafish genes easier. In this study, we isolated and characterized the zebrafish full-length IGFBP-1 by EST database searching and molecular cloning. Using the free-living and transparent zebrafish embryos, the spatial and temporal expression pattern of the IGFBP-1 gene throughout embryogenesis was determined. The results indicated that IGFBP-1 mRNA was present in all developmental stages. IGFBP-1 mRNA expression was initially detected in multiple embryonic tissues but became restricted to the liver shortly after hatching. When adult fish or embryos were subjected to hypoxia or fasting conditions, the hepatic IGFBP-1 mRNA expression increased dramatically. Intriguingly, the hypoxiainduced IGFBP-1 expression operated in embryonic stages even before the liver is formed. Materials and Methods Materials and animals All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Oligonucleotides were synthesized by Invitrogen Life Technologies, Inc. (Carlsbad, CA). Restriction and other modifying enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). Wild-type zebrafish (D. rerio) were maintained on a 14-h light/10-h dark cycle at 28 C and fed twice daily.

Cloning and sequence analysis RNA isolated from adult zebrafish liver was used as template and an initial RT-PCR was performed to amplify an approximately 220-bp fragment with a set of primers (forward primer, 5⬘ TAC GCA AGA CAC TGG AGG AAC AGG 3⬘; reverse primer, 5⬘ CAG GAT GAC ACA CAC CAA CAC TTC 3⬘). These two primers were designed based on an EST sequence (GI no. fb0401.y1) deposited in the zebrafish EST database (http://zfin.org/index.html). The full-length sequence was obtained by 5⬘ rapid amplification of cDNA ends (RACE) and 3⬘ RACE using the Generacer kit (Invitrogen Life Technologies, Inc.) following the manufacturer’s instruction. The RACE products were first screened by nested PCR using two internal primers (forward primer, 5⬘ AGC CTA ACC ACA GCC AAA GCG AGA C 3⬘; reverse primer, 5⬘ ACC AAC ACT TCC CCC TCT GAC CAT C 3⬘). The positive PCR products were further analyzed by Southern blot analysis using the 220-bp IGFBP-1 cDNA fragment as probe. The positive RACE products were cloned into a pCR4 Blunt End Topo vector (Invitrogen), and automated DNA sequencing was performed at the University of Michigan DNA Sequencing Core Facility. DNA sequence analysis, phylogenetic analysis, and primer designing were done using the MacVector software (Oxford Mol. Ltd., Madison, WI). Sequences used for alignment other than reported here were extracted from the public databases from the National Center for Biotechnology Information using BLAST searches.

Endocrinology, July 2002, 143(7):2722–2731 2723

Developmental and physiological regulation of IGFBP-1 expression The embryos were obtained by natural crosses. Fertilized eggs were collected, staged, and raised in embryo rearing solution at 28.5 C following standard methods. To determine the effect of food intake on IGFBP-1 mRNA expression, adult zebrafish were fasted for 1, 2, or 3 wk after 1 wk of acclimation. After 2 wk of fasting, the remaining fish were divided into two groups. The first group continued 1 wk of food deprivation, and the other group was refed for 1 wk. A normal feeding control group was run in parallel throughout the experiment. Three to six fish were killed at each sampling time. To examine the effect of hypoxia in zebrafish embryos and adults, oxygen levels were decreased by bubbling nitrogen gas into the fish tanks, and measurements were made using a dissolved oxygen meter (YSI Model 58, Fisher Scientific, Pittsburgh, PA). At the beginning of the experiment, the dissolved oxygen content was 0.60 mg/liter in the hypoxic group, which was significantly lower than the normal ambient range (6.50 mg/liter). After fish or embryos were transferred to the tanks, the tanks were sealed and monitored every 2 h over the 24-h course of the experiment to assure oxygen levels remained reduced. Control fish or embryos were transferred to tanks in which the dissolved oxygen levels were maintained at approximately 6.50 mg/liter. Four fish were killed in each group.

Northern blot analysis Total RNA was isolated from zebrafish embryos and adult tissues using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) following the manufacturer’s instruction. RNA samples were size fractionated on a 1.2% agarose gel, blotted, and fixed onto a Hybond-N membrane (ICN Biomedicals, Irvine, CA). The membrane was hybridized with [32P]dCTP (ICN Biomedicals) labeled zebrafish IGFBP-1 cDNA. A 700-bp zebrafish ␤-actin cDNA was used to determine levels of ␤-actin mRNA. The prehybridization, hybridization, washing, and autoradiograph were performed as described previously (18). Densitometry was performed and analyzed using the Scion Image software as previously reported (18).

RT-PCR analysis Five micrograms total RNA from embryo or adult tissues were reverse transcribed into single-stranded DNA using random primer and SuperScript II (Life Technologies, Inc.). PCRs were carried out using one fifth of the cDNA reaction in a final volume of 50 ␮l containing 50 mm KCl, 10 mm Tris-HCl (pH 8.0), 2.5 mm MgSO4, 200 mm each dNTP, 2 U of Vent polymerase, and 0.2 ␮m specific primers. The amplification profile was optimized for a Mastercycler gradient thermocycler (Brinkmann Instruments, Inc., Westbury, NY) and was as follows: one cycle of 1 min at 94 C; 25 cycles of 1 min at 94 C, 45 sec at 59.4 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 ␮g/ml ethidium bromide, and photographed under UV light. Each PCR experiment included a positive control containing 10 ng cloned IGFBP-1 cDNA as well as a negative control without template. Each gel was then transferred to a nylon membrane and subjected to Southern blot analysis. We validated the RT-PCR/Southern blotting as a semiquantitative assay by adding different amounts of zebrafish cDNA as template and obtained a proportionate amount of amplified DNA product.

Whole-mount in situ hybridization analysis Whole-mount in situ hybridization using digoxigenin-labeled RNA riboprobe was carried out essentially as reported previously (19). The plasmid DNA of zebrafish IGFBP-1 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. Before use, the specificity of the riboprobes was verified by dot-blot analysis. The embryos were dechorionated manually and fixed overnight with 4% paraformaldehyde in PBS. They were treated with ⫺20 C methanol and rehydrated through a descending methanol series in PBS. Embryos older than 18 h post fertilization (hpf) were permealized by proteinase K treatment. They were then hybridized with the ribo-

2724

Endocrinology, July 2002, 143(7):2722–2731

probe at 65 C for 16 h, followed by incubation with an antidigoxigenin antibody conjugated with alkaline phosphatase and stained with substrate nitro blue terazolium and 5-bromo, 4-chloro, 3-indolil phosphate to produce insoluble precipitates. Photographs were taken with a dissecting microscope (SMZ-10A, Nikon, Melville, NY) and a Coolpix 995 digital camera (Nikon).

Statistical analysis Values are means ⫾ sem. Differences among groups were analyzed by one-way ANOVA followed by Fisher’s protected least significance difference test using Statview (Abacus Concept, Inc., Berkeley, CA).

Results Molecular cloning of the zebrafish IGFBP-1 cDNA

By searching the zebrafish EST database, an EST sequence (GI no. fb0401.y1) that displayed significant homologies to members of the human IGFBP gene family was identified. The predicted amino acid sequence was 161 amino acids in length, and it showed the highest amino acid sequence identity to that of human IGFBP-1 (39%). Sequence alignment analysis indicated that the partial sequence of the putative


zebrafish IGFBP-1 covered part of L-domain and C-domain. Based on this partial sequence, the full-length IGFBP-1 cDNA was obtained using 5⬘ and 3⬘ RACE. The full nucleotide and deduced amino acid sequence (GenBank accession no. AF434664) is shown in Fig. 1A. The 1031-bp cDNA contains an initiation codon ATG that is flanked by sequences resembling the Kozak consensus sequence. A poly-A tail-like sequence is found at position 1007–1031. The complete open reading frame of 786 bp encodes a protein of 262 amino acids with a putative signal peptide of 25 residues. The 237-residue mature zebrafish IGFBP-1 has a predicted molecular size of 25, 478 Da. When the sequence of the mature protein was compared with the six human IGFBPs, it shows 40% sequence identity and 54% similarity to that of human IGFBP-1. It is 39% identical and 48% similar with that of human IGFBP-4. The sequence identity with IGFBP-2, -3, -5, and -6 was between 35% and 26%. To clarify its relationship with mammalian IGFBP-1 and IGFBP-4, we further compared the sequence of this zebrafish IGFBP with those of other mammalian IGFBP-1 and IGFBP-4. The results showed that the

FIG. 1. A, Nucleotide and deduced amino acid sequence of the zebrafish IGFBP-1. The GenBank database accession no. is AF434664. B, Phylogenetic tree of the IGFBP gene family. Full-length IGFBPs were analyzed using the neighbor-joining method. Numbers on branches are percentage of times that the two clades branched as sisters (1000 runs). The results indicate that the zebrafish IGFBP-1 is most closely related to members of the IGFBP-1 subgroup.


zebrafish IGFBP displayed greater sequence identity to IGFBP-1 than IGFBP-4 in nonprimate mammals. Its sequence identities to bovine, rat, and mouse IGFBP-1 are 45%, 41%, and 41%, which are higher, compared with its identities to that of bovine, rat, and mouse IGFBP-4 (38%, 37%, and 37%, respectively). Further evidence that this zebrafish cDNA encodes an IGFBP-1 homolog but not an IGFBP-4 homolog came from the phylogenetic analysis of the known vertebrate IGFBPs. As shown in Fig. 1B, the zebrafish IGFBP-1 is grouped into the IGFBP-1 clade (phylogenetically related gene group) with very high bootstrap support value (i.e. very high probability). The alignment of the zebrafish IGFBP-1 primary sequence with that of other known, full-length vertebrate IGFBP-1s is shown in Fig. 2. Zebrafish IGFBP-1 contains 18 cysteine residues and their alignment is identical with those of mammalian IGFBPs. There are two highly conserved regions in the mature protein: the cysteine-rich N-terminal domain (residues 26 –114) and C-terminal domain (residues 180 –262). The central L-domain (residues 115–179) shares little sequence identity among different vertebrate IGFBP-1s (15– 20%). It is believed that this domain acts structurally as a hinge between the N- and C-terminal domains (6). The greatest sequence identity is observed in the N-domain, being 56% and 52% identical with that of bovine and human. With the exception of the last 15 residues, the C-terminal domain is also highly conserved with an overall sequence identity of 50% and 45% to the C-domain of bovine and human IGFBP-1, respectively. The homology would be greater if the last 15 residues were excluded. The Arg-Gly-Asp (RGD) sequence, present in the C-terminal domain of all mammalian IGFBP-1, is not present in the zebrafish. The corresponding sequence in fish IGFBP-1 is PAD. Spatial and temporal expression pattern of IGFBP-1 mRNA during zebrafish embryogenesis

Using the cloned cDNA as probe, we analyzed the spatial and temporal expression pattern of the IGFBP-1 gene during zebrafish development. Initial Northern blot analysis failed to detect IGFBP-1 mRNA (data not shown). We therefore examined the temporal expression profile of IGFBP-1 mRNA using the sensitive RT-PCR assay. As shown in Fig. 3A, IGFBP-1 transcript was detected

FIG. 2. Comparison of the zebrafish IGFBP-1 primary sequence with that of mouse, rat, bovine, and human. Sequence alignment was obtained with MacVector by the Clustal method. Gaps were introduced to maximize sequence homologies. The first amino acid residue of the predicted mature protein is indicated by *. Residues that are identical or conserved are shaded. The bottom line shows the consensus sequence.


throughout the entire embryogenesis, ranging from fertilized eggs (0 hpf) to hatched embryos (60 hpf). There was limited variation in the IGFBP-1 mRNA levels. We next mapped the spatial expression profile of IGFBP-1 using in situ hybridization in whole-mounted embryos. Embryos of several critical stages in zebrafish development were used, including: 1) the 16-cell cleavage period (1–2 hpf); 2) blastula stage (3– 4 hpf); 3) gastrula stage (9 hpf); 4) segmentation stage (22 hpf); 5) the hatching stage (48 hpf); and 6) hatched embryos of 72 hpf and 96 hpf. No strong IGFBP-1 mRNA signal was detected in embryos of cleavage, blastula, and gastrula stages (Fig. 3B, panels a, c, e, and g). The IGFBP-1 mRNA signal became detectable at 24 hpf. At this stage, the IGFBP-1 transcript was ubiquitously distributed in the anterior portion of the body (Fig. 3B, panels i and q). At 48 hpf, high levels of IGFBP-1 mRNA were observed in the differentiating cartilage cells in the newly formed mandibular cartilage, pharyngeal pouch, and the otic vesicles (Fig. 3B, panels k, m, r, and s). After 72 hpf, the IGFBP-1 mRNA signals began to decrease and became undetectable at 96 hpf (Fig. 3, panels m and o). No signal was found in control experiments performed in parallel on the same batch of embryos with the corresponding sense RNA riboprobe (Fig. 3B, panels b, d, f, h, j, l, n, and p). These data suggest that IGFBP-1 transcript is probably maternally deposited in the eggs and present in early embryos at very low levels. During the pharyngula and hatching periods, the zygotic IGFBP-1 gene is expressed transiently in differentiating skeletal tissues and other tissues (Fig. 3B, panels k, m, r, and s). Tissue distribution and nutritional regulation of IGFBP-1 mRNA expression in adult zebrafish

The expression of zebrafish IGFBP-1 mRNA in various adult tissues of normally fed and 2-wk fasted animals was analyzed by Northern blot analysis. As shown in Fig. 4A, a weak band at the size of approximately 1.1 kb was recognized in the liver of fed animals after a prolonged exposure time (12 d). No IGFBP-1 mRNA was detected in other tissues of the fed adult animals. Two-week food deprivation increased the hepatic IGFBP-1 mRNA levels by more than 10-fold (Fig. 4B). In addition, modest levels of transcript were also detected in the gut/kidney of the fasted fish. Because of

2726



FIG. 3. Temporal and spatial expression pattern of the IGFBP-1 gene during zebrafish embryogenesis. A, RT-PCR analysis of IGFBP-1 mRNA. Upper panel is IGFBP-1 mRNA and bottom panel shows ␤-actin mRNA. The developing stage was shown at the bottom as hours post fertilization (hpf). B, In situ hybridization analysis of whole mounted embryos. Embryos of the following developmental stages were analyzed: 1) the 16-cell cleavage period (0 –1 hpf, panels a and b); 2) the blastula period (1–2 hpf, panels c and d); 3) the gastrula period (8 hpf, panels e and f); 4) the 5-somite segmentation period (11 hpf, panels g and h); 5) the prim.5 straightening period (24 hpf, panels i, j, and q); 6) the long bud stage (48 hpf, panels k, l, r, and s); 7) the 72-hpf hatched embryos (panels m–n); and 8) the 96-hpf hatched embryos (panels o and p). Showing in panels a, c, e, g, i, k, m, o, q, r, and s are embryos probed with the antisense IGFBP-1 RNA riboprobe (labeled as AS in the upper left corner). Panel q shows a closer lateral review of a 24-hpf embryo. Note the ubiquitous presence of hybridization signals in the head region. Panels r and s are closer lateral and ventral views of a 48-hpf embryo. Note the high levels of hybridization signal in the developing mandibular cartilage, pharyngeal pouch, and otic vesicle (indicated by arrows). Panels b, d, f, h, j, l, n, and p are embryos probed with the sense IGFBP-1 RNA riboprobe (labeled as S in the upper left corner). No hybridization signal was found in these controls.

the small size, we were not able to separate kidney from the gut. No IGFBP-1 mRNA was detectable in the brain, eye, ovary, and gill even after 2 wk of fasting (Fig. 4B). To determine the time course of nutritionally regulated IGFBP-1 mRNA expression in zebrafish, adult zebrafish were deprived of food for up to 3 wk. As shown in Fig. 5, the levels of IGFBP-1 mRNA in normal, well-fed adult animals were very low. When the animals were fasted, the steady-state levels of IGFBP-1 mRNA increased significantly. Two-week fasting increased the whole-body IGFBP-1 mRNA level by 5.3-fold (P ⬍ 0.05). By the end of the third week, IGFBP-1 mRNA levels began to decrease but still remained 2-fold higher than the fed control group. Refeeding of the fasted fish for 1 wk reduced the IGFBP-1 mRNA levels to the basal level. These changes were specific because no significant change was seen in the levels of ␤-actin mRNA (Fig. 5B). These results suggest that IGFBP-1 mRNA is expressed in the adult

liver and to a lesser degree in the gut/kidney. The hepatic IGFBP-1 mRNA is significantly increased by prolonged starvation. The IGFBP-1 mRNA expression is regulated by hypoxia in zebrafish adults and embryos

To examine whether reducing environmental oxygen levels has any effect on IGFBP-1 mRNA expression in fish, adult zebrafish were transferred to tanks containing low dissolved oxygen level (0.60 ⫾ 0.3 mg/liter) for 24 h. Fish kept in the ambient and normal oxygen levels (6.5 ⫾ 0.3 mg/liter) were used as controls. Fish were fed normally in all groups. As shown in Fig. 6, when exposed to the hypoxic environment, the IGFBP-1 mRNA levels increased dramatically. After normalizing the IGFBP-1 mRNA levels with ␤-actin mRNA levels, the mean value of the hypoxic group was 280-fold greater



FIG. 4. Tissue distribution and nutritional regulation of IGFBP-1 mRNA expression in adult zebrafish. Steady-state levels of IGFBP-1 mRNA in various tissues of fed (A) and 2-wk fasted adult zebrafish (B). Lane 1, Brain; lane 2, eye; lane 3, liver; lane 4, ovary; lane 5, gut/kidney; and lane 6, gill.

than those of the normal oxygen group (P ⬍ 0.01). We next tested the effect of hypoxia on IGFBP-1 mRNA expression at different embryonic stages. First, 72-hpf embryos were transferred to tanks filled with embryo rearing solution with normal or low oxygen levels. After 24-h exposure, the embryos at the stage of 96 hpf were fixed and subjected to wholemount in situ hybridization analysis. At this stage, the liver is differentiated. The level of IGFBP-1 mRNA in the embryos raised under normal oxygen was barely detectable in the liver by in situ hybridization (Fig. 7, panels a and b). Under the hypoxic condition (10% of normal oxygen levels), there was a marked increase in the IGFBP-1 mRNA signals in the liver. No IGFBP-1 mRNA signal was detected in other tissues (Fig. 7, panels e and f). These data indicate that hypoxia stimulates IGFBP-1 expression only in the liver in animals older than 72 hpf. To determine whether and in which tissues hypoxia stimulates IGFBP-1 gene expression in early embryonic stages, 48-hpf embryos, which do not have a distinguishable liver, were exposed to hypoxic conditions for 24 h. As shown in Fig. 8, embryos exposed to the hypoxic solution had significantly stronger IGFBP-1 mRNA signals, compared with embryos raised in water containing normal oxygen levels (Fig. 8, panels a, b, e, f, g, and j). At this stage, the hypoxia-regulated IGFBP-1 transcripts were highly expressed in the pharyngeal arches, brain, atrium, and ventricle (Fig. 8, panels e and j). These results suggest that the expression of IGFBP-1 is increased under hypoxic conditions in adult as well as embryonic stages. Although the hypoxiaregulated IGFBP-1 expression is restricted to the liver in adult and late embryonic stages, this regulation occurs in many embryonic tissues before the appearance of liver. Discussion

To study the developmental role of IGFBP-1 using the unique zebrafish embryos, we cloned and characterized the full-length zebrafish IGFBP-1 cDNA and studied the developmental and physiological regulation of IGFBP-1 expression. Structural analysis indicated that zebrafish IGFBP-1 shares similar domain arrangement with its mammalian counterparts. It has a highly cysteine-rich N-terminal domain, a cysteine-rich C-terminal domain, and a central domain with no cysteine residues. This domain arrangement is

FIG. 5. Time-course effect of food deprivation on the steady-state IGFBP-1 mRNA levels in adult zebrafish. A, Representative autoradiography. Total RNA was isolated from zebrafish that were sampled at the beginning of the experiment (lanes 1, 5), after feeding for 1 wk (lane 2), 2 wk (lane 3), 3 wk (lane 4), or after fasting for 1 wk (lanes 6), 2 wks (lane 7), 3 wk (lane 8), or refed for a week after 2-wk fasting (lane 9). RNA was subjected to Northern blot analysis as described in Materials and Methods. Upper panel, Autoradiography of IGFBP-1 mRNA; lower panel, autoradiograph of ␤-actin mRNA. B, Densitometric analysis result. OD was measured and normalized by the ␤-actin mRNA signal. Filled bars represent the normal feeding group, open bars represent the fasting group, and the refeeding group is shown as a hatched bar. The values are mean ⫾ SEM (n ⫽ 3– 6). *, P ⬍ 0.05, compared with 0 wk control; #, P ⬍ 0.05, compared with the fed control group.

also conserved in zebrafish IGFBP-2 (20). Sequence alignment reveals that the N- and C-domains are highly conserved across species as well as among various members of the IGFBP family within a given species. Twelve of the 18 conserved cysteines are located in the N-domain and the remaining six in the C-terminal domain. The cysteine residues in the N-terminal domain are arranged into two conserved Zn-finger motifs (CX2CX7C). The central region, L-domain, separates the N-terminal domain from the C-terminal domain. The amino acid sequence in this region is highly variable among different IGFBPs in a given species, with shared similarity less than 15%. It is believed that this region acts structurally as a hinge between the N- and C-terminal domains (6). Sequence comparison performed in this study indicated that the L-domain is highly divergent among various vertebrate IGFBP-1. The high degree of conservation of N- and C-domains is probably not surprising. Recent studies have indicated that both N- and C-terminal domains of IGFBPs are required for IGF binding (5–7). This is also in good agreement with recent biochemical studies showing that fish IGFBPs bind human IGF-I and IGF-II with high affinity and specificity (20, 21). Therefore, IGF binding de-

2728


FIG. 6. Effect of hypoxia on the steady-state IGFBP-1 mRNA levels in adult zebrafish. Total RNA was isolated from individual adult zebrafish that were kept in low (lanes 1– 4) or normal oxygen level condition (lanes 5– 8) for 24 h. RNA was subjected to Northern blot analysis as described in Materials and Methods. Upper panel, Autoradiography of IGFBP-1 mRNA; middle panel, autoradiography of ␤-actin mRNA; and lower panel, densitometric analysis result. OD was measured and calculated and normalized by the ␤-actin mRNA signal. The values are mean ⫾ SEM (n ⫽ 4). *, P ⬍ 0.01.

terminants of IGFBP-1 may have been preserved during the several hundred million years of vertebrate evolution. Despite the overall structural similarity, however, fish IGFBP-1 has diverged significantly from its mammalian homologues. The overall sequence identities between the fulllength, mature zebrafish IGFBP-1 and those of mammalian IGFBP-1s are 40 – 45%. Most of these changes occur in the central L-domain. Another highly diverged region is the last 15 residues at the C terminus. In all the mammalian IGFBP-1 studied, there is an RGD motif residing within this region. This RGD motif in human IGFBP-1 has been shown to be involved in the integrin binding of IGFBP-1, and this interaction was reported to mediate the IGF-independent actions of IGFBP-1 on Chinese hamster ovary cell migration (22). IGFBP-1 has also been shown to bind ␣5␤1 integrin in human trophoblasts and to stimulate trophoblast migration (23). The sequence in the corresponding region in the zebrafish IGFBP-1 is PAD. To date, the only available sequences of nonmammalian IGFBP-1 are two partial sequences obtained in rainbow trout (Oncorhynchus mykiss) and the goby (Gillichthys mirabilis) (21, 24). The partial rainbow trout sequence IGFBP-1 is limited to the N terminus. The goby partial sequence, however, covers the entire C domain. As in the case of zebrafish IGFBP-1, goby IGFBP-1 does not contain an RGD motif in the C-terminal region. Therefore, the RGD motif of IGFBP-1 and its potential biological actions may have been a later acquisition during vertebrate evolution. It is worthy to note the close relationship of fish IGFBP-1 with mammalian IGFBP-4. Besides mammalian IGFBP-1, zebrafish IGFBP-1 is most closely related to mammalian IGFBP-4. In fact, zebrafish IGFBP-1 is 37–39% identical with those of mammalian IGFBP-4, which is lower than that of mammalian IGFBP-1 (40 – 45%) but significantly higher than the identities with mammalian


IGFBP-2, -3, -5, and -6 (26 –35%). In addition, phylogenetic analyses indicated that the IGFBP-1 and IGFBP-4 clades are sister clades, suggesting that these two IGFBPs may have evolved from a common ancestral IGFBP gene that had already diverged from other IGFBPs. In all mammal species studied to date, the expression of IGFBP-1 gene is highly tissue specific, being predominantly expressed in the liver. In addition, IGFBP-1 mRNA has also been detected in kidney, decidua, ovary, and cultured human osteoblast-like cells (7). The expression of the mammalian IGFBP-1 gene is strongly and acutely stimulated under catabolic conditions. Both insulin-dependent diabetes mellitus and starvation resulted in elevated IGFBP-1 levels in the circulation (25–29). This has been linked to the regulatory effects of insulin and glucocorticoids (30 –33). The tissue distribution and physiological regulation patterns of IGFBP-1 are preserved in zebrafish. In the adult zebrafish, the IGFBP-1 mRNA is expressed specifically in the liver. When subjected to prolonged food deprivation, the hepatic IGFBP-1 mRNA rises significantly. Refeeding restored the IGFBP-1 mRNA to the basal levels. Compared with rodents, a much longer time of starvation is required to see changes in fish. For instance, 1–3 d of starvation can cause a significant change in the hepatic IGFBP-I mRNA levels in rat and human (25–29). In zebrafish, however, 2 wk is needed to observe a significant change in the hepatic IGFBP-I mRNA levels, and 1 wk of refeeding is necessary to restore it. Likewise, it takes 2 wk of starvation in coho salmon and trout to see a change in hepatic IGF-I mRNA levels (34). The slower response in fish may be a reflection of the generally slower metabolism of the ectothermal fish. In addition, many fish species are metabolically well adapted to long periods of starvation during their life cycle (34). Studies in mammals have shown that the hepatic IGFBP-1 expression is also strongly induced by hypoxia in both fetal and postnatal stages (9 –14). It is believed that the elevated IGFBP-1 in the circulation in turn regulates the bioavailaility of IGFs. Likewise, hypoxia dramatically increased IGFBP-1 mRNA levels in zebrafish. These results suggest that the zebrafish IGFBP-1 is highly responsive to the availability of food and oxygen. In agreement with our finding, several groups have reported a dramatic increase in the circulating levels of a 29- to 30-kDa IGFBP (measured by Western ligand blot using labeled human IGF-I as tracer) in other bony fishes under starvation (35–39). Collectively, these findings indicate that up-regulation of the IGFBP-1 gene under prolonged starvation or hypoxia may be an evolutionarily conserved adaptive response to limitation in the availability of food and oxygen. Starvation and hypoxia lead to dramatic adaptive changes in metabolic organization, for instance the activation of the anaerobic ATP-generating pathway, glycolysis. The adaptive significance of the expression of the IGFBP-1 gene in response to food deprivation and hypoxia may be to divert important energy resources away from growth toward those metabolic processes essential for survival. Recent studies in goby and rainbow trout have shown that the levels of cortisol are elevated under hypoxic (rainbow trout) and fasting conditions (goby) (39, 40). As mentioned above, glucocorticoids have been shown to regulate hepatic IGFBP-1 mRNA expression in mammals (30 –33). It is therefore possible that



FIG. 7. Hypoxia stimulates the IGFBP-1 mRNA expression in the liver of advanced embryos. Hatched embryos of 72 hpf were transferred to water containing normal (panels a– d) or low oxygen level (panels e– h). After 24 h, the embryos of 96 hpf were fixed and subjected to in situ hybridization analysis as described in Material and Methods. Showing in panels a, b, e, and f are embryos probed with the antisense IGFBP-1 RNA riboprobe (labeled as AS in the upper right corner). Panels a and e are lateral views and panels b and f are dorsal views. Note the intense IGFBP-1 mRNA signals in the liver (indicated by the arrow) of the embryo that was exposed to hypoxia (panels e and f). No signal was found in embryos probed with the corresponding sense (S) IGFBP-1 RNA riboprobe (panels c, d, g, and h).

FIG. 8. Hypoxia stimulates the IGFBP-1 mRNA expression in multiple nonhepatic tissues in early embryonic stages. The 48-hpf zebrafish embryos were transferred to water with normal (panels a– e) or low oxygen level (panels f–j). After 24 h, the embryos of 72 hpf were fixed and subjected to in situ hybridization analysis as described in Materials and Methods. Showing in panels a, b, e, f, g, and j are embryos probed with the antisense IGFBP-1 RNA riboprobe (AS). Panels a and f are lateral views and panels b and g are dorsal views. Showing in panels e and j are front views. Note the high levels of IGFBP-1 mRNA signals in the brain, atrium, ventricle, and pharyngeal arch in animals that were exposed to hypoxic condition. No signal was found in control experiments using the corresponding sense (S) IGFBP-1 RNA riboprobe (panels c, d, h, and i).

the elevated IGFBP-1 mRNA expression observed under hypoxic and fasting conditions may be mediated by cortisol in the adult fish. Further studies are needed to elucidate the role of cortisol and other hormones in regulating IGFBP-1 expression in zebrafish embryos and adults. Taking advantage of the free-living and transparent zebrafish embryos, we mapped the spatial and temporal expression pattern of the IGFBP-1 gene throughout the entire embryogenesis and studied the effect of hypoxia at different embryonic stages. These analyses led to several novel findings. First, the IGFBP-1 mRNA is present in fertilized eggs, apparently deposited from the maternal source. Several

groups have shown that the IGF-I, IGF-II, and IGF-IR transcripts are detectable in the zygotes of rodents and teleost fish, including the zebrafish (19, 41– 44). It appears likely that not only the transcripts of IGF ligands and receptors, but IGFBP transcripts are also deposited in the vertebrate eggs. Second, we found that the zygotic IGFBP-1 gene is expressed transiently in many embryonic tissues before its expression becomes liver specific. IGFBP-1 mRNA was expressed at low levels in a ubiquitous fashion in early embryonic stages. At 48 hpf, high levels of IGFBP-1 mRNA were found in the mandibular cartilage, pharyngeal pouch, fin buds, and to a lesser degree in the brain. This observation is intriguing in

2730


light of the recent finding that human osteoblastic cells in culture, express IGFBP-1 mRNA and protein when exposed to glucocorticoid (45). Not only is IGFBP-1 mRNA expressed in the embryonic skeletal tissues, but its levels are highly regulated. For instance, the IGFBP-1 mRNA levels were significantly increased under hypoxic conditions. These data suggest that IGFBP-1 mRNA is expressed in several nonhepatic tissues in a highly developmentally and physiologically regulated fashion. Therefore, the locally produced IGFBP-1 may have a previously unrecognized paracrine/autocrine role in the differentiation and/or growth of the skeletal tissues. Another interesting finding made in this study is that the hypoxia-IGFBP-1 pathway is operational in many nonhepatic tissues during early development. Our data suggest that hypoxia-regulated IGFBP-1 expression begins in multiple nonhepatic tissues at an early stage of embryogenesis before formation of the liver. Shortly after hatching, the hypoxia-regulated IGFBP-1 expression becomes liver specific. Thus, the hypoxia-induced IGFBP-1 occurs in different tissues at different developmental stages. To our knowledge, this is the first demonstration that the hypoxia regulation of IGFBP-1 expression occurs in the skeletal, cardiovascular, and nervous tissues in early embryonic stages. Because oxygen is the terminal electron acceptor in oxidative phosphorylation, oxygen levels are closely monitored in all cells. If the cellular oxygen concentration fails to match the requirements of energy metabolism or other biochemical reactions dependent on oxygen, a tightly controlled regulatory pathway is used to respond to the hypoxic environment. A well-characterized mediator of the hypoxic response in mammalian cells is the transcription factor HIF-1 and its close relatives HIF-2/endothelial PER-ARNT-SIM and HIF-3. To date, approximately 36 hypoxia-regulated genes have been reported in mammals, and the majority of these genes appear to be under HIF-1␣ control (24). A recent gene profiling study in the goby indicated that a large number of the same genes are induced by hypoxia (24), suggesting a similar transcriptional control mechanism in fish. Searching of the zebrafish EST database reveals the presence of HIF-1. The physiologic relevance of the hypoxia-regulated IGFBP-1 gene expression in early embryos is not yet known. However, several precedents for the suggestion that in addition to its known endocrine function, IGFBP-1 can also act as an autocrine/paracrine growth regulator. For example, the localized production of IGFBP-1 in the mammalian decidua appears to play a crucial role in the implantation (8). Likewise, the production of IGFBP-1 in the uterine endometrium has been proposed to have a local regulatory role in endometrial cells (16, 17). In vitro, IGFBP-1 inhibits IGF binding to the endometrial membrane and inhibits the mitogenic effects of IGF-I in these cells (46, 47). Likewise, our recent functional analyses have shown that fish IGFBP-1 inhibits IGF-I-induced zebrafish embryonic cell proliferation in vitro (21). Therefore, it is likely that the regulated IGFBP-1 in the early embryogenic stages may have local actions in the embryonic skeletal and cardiovascular tissues in response to hypoxia. It will be necessary to determine the in vivo function(s) of IGFBP-1 using a morpholino-based, targeted gene knockdown approach (48) in the future.


In summary, the results of this study have shown that the structure, developmental expression, and physiological regulation of the IGFBP-1 gene is conserved in zebrafish. Exploring the unique zebrafish model, we show that IGFBP-1 gene is expressed transiently in many embryonic tissues during early embryonic stages before its expression becomes liver specific. We further demonstrate that the hypoxiainduced IGFBP expression operates in early embryos before the appearance of the liver. Intriguingly, the hypoxiainduced IGFBP occurs in different tissues at different developmental stages. These results not only provide new information about the conservation of the structure, developmental expression, and physiological regulation of the IGFBP-1 gene but also present the opportunity to elucidate the role of IGFBP-1 during early development using a unique vertebrate model system. Acknowledgments We thank Ms. Tricia L. Royer for her assistance with staging embryos and in situ hybridization analysis. Received December 6, 2001. Accepted March 21, 2002. Address all correspondence and requests for reprints to: Cunming Duan, Ph.D., Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048. Email: [email protected]. The sequence reported in this paper has been deposited in the GenBank database under accession no. AF434664. This work was supported in part by NSF Grant IBN 0110864 (to C.D.) and NIH Grant (NIH P60 DK20572) to Michigan Diabetes Training and Research Center.

References 1. Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiades A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (IGF-I) and type I IGF receptor (IGFIR). Cell 75:59 –72 2. Baker J, Liu J-P, Robertson EJ, Efstratiades A 1993 Role of insulin like factors in embryonic and postnatal growth. Cell 75:73– 82 3. Fant M, Salafia C, Baxter RC, Schwander J, Vogel C, Pezzullo J, Moya F 1993 Circulating levels of IGFs and IGF binding proteins in human cord serum— relationship to intrauterine growth. Regul Peptides 48:29 –39 4. Woods KA, Camacho-Hubner C, Savage MO, Clark AJL 1996 Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med 335:1363–1367 5. Rechler MM, Clemmons DR 1998 Regulatory actions of insulin-like growth factor-binding proteins. Trends Endocrinol Metab 9:176 –183 6. Hwa V, Oh Y, Rosenfeld RG 1999 The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr Rev 20:761–787 7. Baxter RC 2000 Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. Am J Physiol 278:E967–E976 8. Lee PDK, Giudice LC, Conover CA, Powell DR 1997 Insulin-like growth factor binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med 216:319 –357 9. Unterman T, Lascon R, Gotway MB, Oehler D, Gounis A, Simmons RA, Ogata ES 1990 Circulating levels of insulin-like growth factor binding protein-1 (IGFBP-1) and hepatic messenger RNA are increased in the small-forgestation-age (SGA) fetal rat. Endocrinology 127:2035–2037 10. Unterman T, Simmons RA, Glick RP, Ogata ES 1993 Circulating levels of insulin, insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins in the small-for-gestation-age (SGA) fetal rat. Endocrinology 132:327–336 11. McLellan KC, Hooper SB, Bocking AD, Delhanty PJD, Phillips ID, Hill DJ, Han VKM 1992 Prolonged hypoxia induced by the reduction of maternal uterine blood-expression in the ovine fetus. Endocrinology 131:1619 –1628 12. Tapaninen PJ, Bang P, Wilson K, Unterman T, Verman HJ, Rosenfeld RG 1994 Maternal hypoxia as a model for intrauterine growth-retardation— effects on insulin-like growth factors and their binding proteins. Pediatr Res 36: 152–158 13. Tazuke SI, Mazure NM, Sugawara J, Carland G, Faessen GH, Suen LF, Irwin JC, Powell DR, Giaccia AJ, Giudice LC 1998 Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia. Proc Natl Acad Sci USA 95:10188 –10193


14. Popovici RM, Lu M, Bhatia S, Faessen GH, Giaccia AJ, Giudice LC 2001 Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency. J Clin Endocrinol Metab 86:2653–2659 15. Han VKM, Matsell DG, Delhanty PJD, Hill DJ, Shimasaki S, Nygard K 1996 IGF-binding protein mRNAs in the human fetus: tissue and cellular distribution of developmental expression. Horm Res 45:160 –166 16. Han VKM, Carter AM 2000 Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals. Placenta 21:289 –305 17. Lee CI, Goldstein O, Han VKM, Tarantal AF 2001 IGF-II and IGF binding protein (IGFBP-1, ICFBP-3) gene expression in fetal rhesus monkey tissues during the second and third trimesters. Pediatr Res 49:379 –387 18. Duan C, Liimatta MB, Bottum OL 1999 Insulin-like growth factor (IGF)-I regulates IGF-binding protein-5 gene expression through the phosphatidylinositol 3-kinase, protein kinase B/Akt, and p70 S6 kinase signaling pathway. J Biol Chem 274:37147–37153 19. Maures T, Chan SJ, Xu B, Ding J, Sun H, Duan C 2002 Structural, biochemical, and expression analysis of two distinct insulin-like growth factor (IGF)-I receptors and their ligands in zebrafish. Endocrinology 143:1858 –1871 20. Duan C, Ding J, Li Q, Tsai W, Pozias K 1999 Insulin-like growth factor (IGF) binding protein-2 is a growth inhibitory protein highly conserved in zebrafish. Proc Natl Acad Sci USA 96:15274 –15279 21. Bauchat JR, Busby Jr W, Garmany A, Moore J, Swanson P, Lin M, Duan C 2001 Biochemical and functional analysis of a conserved insulin-like growth factor binding protein (IGFBP) isolated from rainbow trout hepatoma. J Endocrinol 170:619 – 628 22. Jones JI, Gockerman A, Busby WH, Wright G, Clemmons DR 1993 Insulinlike growth factor binding protein 1 stimulates cell migration and binds to the ␣5␤1 intergrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–10557 23. Irwin JC, Giudice LC 1998 Insulin-like growth factor binding protein-1 binds to placental cytotrophoblast ␣(5)␤(1) integrin and inhibits cytotrophoblast invasion into decidualized endometrial stromal cultures. Growth Horm IGF Res 8:21–31 24. Gracey A, Troll J, Somero G 2001 Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis. Proc Natl Acad Sci USA 98:1993–1998 25. Busby WH, Snyder DK, Clemmons DR 1988 Radioimmunoassay of a 26,000dalton plasma insulin-like growth factor-binding protein: control by nutritional variables. J Clin Endocrinol Metab 67:11225–11230 26. Brismar K, Gutniak M, Povoa G, Werner S, Hall K 1988 Insulin regulates the 35 kDa IGF binding protein in patients with diabetes mellitus. J Endocrinol Invest 11:599 – 602 27. Cotterill AM, Cowell CT, Baxter RC, McNeil D, Silinik M 1988 Regulation of the growth hormone-independent growth factor-binding protein in children. J Clin Endocrinol Metab 67:882– 887 28. Straus DS, Takemoto CD 1990 Effect of dietary protein deprivation on insulinlike growth factor (IGF)-I and -II, IGF binding protein-2, and serum albumin gene expression in rat. Endocrinology 127:1849 –1860 29. Donovan SM, Atilano LC, Hintz RL, Wilson DM, Rosenfeld RG 1991 Differential regulation of the insulin-like growth factors (IGF-I and -II) and IGF binding proteins during malnutrition in the neonatal rat. Endocrinology 129: 149 –157 30. Suikkari A-M, Koivisto VA, Rutanen EM, Yki-Jarvinen H, Karonen SL, Seppala M 1988 Insulin regulates the serum levels of low molecular weight insulin-like growth factor-binding protein. J Clin Endocrinol Metab 66:266 –272


31. Luo J, Reid RE, Murphy LJ 1990 Dexamethasone increases hepatic insulin-like growth factor binding protein-1 (IGFBP-1) mRNA and serum IGFBP-1 concentrations in the rat. Endocrinology 127:1456 –1462 32. Ooi GT, Tseng LY, Tran MQ, Rechler MM 1992 Insulin rapidly decreases insulin-like growth factor-binding protein-1 gene transcription in streptozotocin-diabetic rats. Mol Endocrinol 6:2219 –2228 33. Unterman TG, Jentel JJ, Oehler DT, Lacson RG, Hofert J 1993 Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat. Endocrinology 133:2531–2539 34. Duan C 1998 Nutritional and developmental regulation of insulin-like growth factors in fish. J Nutr 128:306S–314S 35. Kelley KM, Siharath K, Bern HA 1992 Identification of insulin-like growth factor-binding proteins in the circulation of four teleost fish species. J Exp Zool 263:220 –224 36. Siharath K, Kelley KM, Bern HA 1996 A low-molecular-weight (25-kDa) IGF-binding protein is increased with growth inhibition in the fasting striped bass, Morone saxatilis. Gen Comp Endocrinol 102:307–316 37. Shimizu M, Swanson P, Dickhoff WW 1999 Free and protein-bound insulinlike growth factor-I (IGF-I) and IGF-binding proteins in plasma of coho salmon, Oncorhynchus kisutch. Gen Comp Endocrinol 115:398 – 405 38. Park R, Shepherd BS, Nishioka RS, Grau GE, Bern HA 2000 Effects of homologous pituitary hormone treatment on serum insulin-like growth factorbinding proteins (IGFBPs) in hypophysectomized tilapia, Oreochromis mossambicus, with special reference to a novel 20 kDa IGFBP. Gen Comp Endocrinol 117:404 – 412 39. Kelley KM, Haigwood JT, Perez M, Galima MM 2001 Serum insulin-like growth factor binding proteins (IGFBPs) as markers for anabolic/catabolic condition in fishes. Comp Biochem Phys B 129:229 –236 40. Van Raaij MT, Pit DSS, Balm PHM, Steffens AB, van den Thillart GEEJM 1996 Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia. Horm Behav 30:85–92 41. Telford NA, Watson AJ, Schultz GA 1990 Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 26:90 –100 42. Scavo LM, Shuldiner AR, Serrano J, Dashner R, Roth J, DePablo F 1991 Genes for the insulin receptor and the insulin-like growth factor I receptor are expressed in the chicken embryo blastoderm and throughout organogenesis. Biochem Biophys Res Commun 176:1393–1401 43. Funkeinstein B, Shemer R, Cohen I 1996 Nucleotide sequence of the promoter region of Sparus aurata insulin-like growth factor I gene and expression of IGF-I in eggs and embryos. Mol Mar Biol Biotechnol 5:43–51 44. Green MW, Chen TT 1997 Temporal expression pattern of insulin-like growth factor mRNA during embryonic development in a teleost, rainbow trout (Oncorynchus mykiss). Mol Mar Biol Biotechnol 6:144 –151 45. Conover CA, Lee PDK, Riggs BL, Powell DR 1996 Insulin-like growth factor binding protein-1 expression in cultured human bone cells: regulation by glucocorticoid and insulin. Endocrinology 137:3295–3301 46. Rutanen EM, Pekonen F, Makinen T 1988 Soluble 34 K binding protein inhibits the binding of insulin-like growth factor I to its receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action. J Clin Endocrinol Metab 66:173–180 47. Frost RA, Mazella J, Tseng L 1993 Insulin-like growth factor binding protein-1 inhibits the mitogenic effect of insulin-like growth factors and progestins in human endometrial stomal cells. Biol Reprod 49:104 –111 48. Nasevlclus A, Ekker SC 2000 Effective targeted gene “knockdown” in zebrafish. Nat Genet 26:216 –220