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Published as doi: 10.1096/fj.05-3882fje.
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(The FASEB Journal. 2006;20:1230-1232.)
© 2006 FASEB

Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors

Peter J. Schlueter1, Tricia Royer1, Mohamed H. Farah1, Benjamin Laser1, Shu Jin Chan, Donald F. Steiner and Cunming Duan1

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109; and Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637

1Correspondence: Department of MCDB, University of Michigan, Kraus Natural Science Bldg., Ann Arbor, MI 48109, USA. E-mail: cduan{at}umich.edu

ABSTRACT

Insulin-like growth factor (IGF) 1 receptor (IGF1R)-mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb. In this study, we tested the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event at the ig f1r locus and that this has led to their functional divergence. The genomic structures of zebrafish ig f1ra and ig f1rb were determined and their loci mapped. While zebrafish ig f1ra has 21 exons and is located on linkage group (LG) 18, zebrafish ig f1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IG F1R gene. Using a MO-based loss-of-function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish ig f1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.—Schlueter, P. J., Royer, T., Mohamed, H. F., Laser, B., Chan, S. J., Steiner, D. F., Duan, C. Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors.


Key Words: IGF signaling • growth • developmental timing • retina • inner ear • heart • muscle

THE TYPE-1 IGF receptor (IGF1R), which binds to and is activated by IGF1 or IGF2, plays key roles in human growth, development, and physiology (1 2 3) . The human IGF1R is a heterotetrameric transmembrane protein, consisting of two {alpha} and two ß subunits, linked by disulfide bonds (4) . Ligand binding induces IGF1R autophosphorylation at tyrosine residues, activating multiple downstream signal transduction cascades, including the mitogen-activated protein kinase (MAPK) pathway and phosphoinositide-3-kinase (PI3K)-Akt pathway (1 , 2) . Patients with point mutations in the IGF1R gene exhibited severe intrauterine growth restriction and poor postnatal growth (5 , 6) . Likewise, loss-of-function mutations in the human IGF1 gene resulted in severe fetal and postnatal growth failure, and in mental retardation and sensorineural deafness (7 8 9) .

The central role of IGF1R-mediated signaling in growth or size control has been demonstrated by mouse genetic studies. Homozygous Ig f1r null mutant mice exhibited severe but proportional growth retardation (45% of the wild type littermates) and neonatal lethality (10 , 11) . Although Ig f1r knockout mice exhibited decreased numbers of specific neurons and reduced myelination, there was no evidence of major organ loss or patterning abnormalities (10 , 11) . Therefore, the IGF1R-mediated signaling is believed to be a key growth regulator in development (12 , 13) . Recent studies in Xenopus suggested that IGF1R-mediated signaling is important for anterior neural induction (14 15 16) ; similarly, a study using zebrafish reported that overexpression of a similar dominant negative IGF1R construct caused defects in head and central nervous system development (17) .

Recently we have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb, and that they are expressed in overlapping spatial domains throughout embryogenesis (18) . Like the human IGF1R, both zebrafish Igf1ra and Igf1rb are heterotetrameric transmembrane proteins, consisting of two {alpha} and two ß subunits, and they bind to IGFs but not to insulin. The relationship between the two zebrafish ig f1r genes, however, is not clear and their functions are unknown. In this study, we tested the hypothesis that ig f1ra and ig f1rb originated from a gene duplication event, which led to their functional divergence. Our results suggest that zebrafish ig f1ra and ig f1rb are encoded by distinct genetic loci believed to evolve from a common ancestral gene, and there is strong syntenic correspondence between zebrafish ig f1ra and ig f1rb and the human IGF1R gene. Employing a MO-based targeted gene knockdown approach, we provide evidence that ig f1ra and ig f1rb play largely overlapping, but not identical roles in zebrafish development.

MATERIALS AND METHODS

Materials
All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. RNase-free DNase was purchased from Promega (Madison, WI). Restriction endonucleases were purchased from New England BioLabs (Beverly, MA). PCR primers were synthesized by Invitrogen Life Technologies, Inc. (Carlsbad, CA). Akt antibodies were purchased from Cell Signaling (Beverly, MA), the anti-Tubulin antibody (Ab) was purchased from Sigma (St. Louis, MO), and the SV2 and Zn5 monoclonal antibodies were purchased from the Developmental Studies Hybridoma Bank (University of Iowa). The LN54 radiation hybrid panel was kindly provided by Dr. M. Ekker, and the F59 Ab by Dr. F. Stockdale. Plasmid DNAs for making various riboprobes were generously provided by Drs. R. Kollmar (claudin-a), S. Lyons (nkx2.5), P. Raymond (rx1, rx2) and S.J. Du (myoD, myogenin).

Experimental animals
Adult wild-type (WT) zebrafish (Danio rerio) were maintained at 28 C on a 14h:10h (light: dark) cycle, and fed twice daily. Embryos were generated from natural crosses. Fertilized eggs were raised in embryo medium at 28.5°C and staged according to Kimmel et al. (19) . All experiments were conducted in accordance with guidelines approved by the University Committee on the Use and Care of Animals, University of Michigan.

Determining the structure and physical mapping of zebrafish igf1ra and igf1rb
The zebrafish ig f1ra and ig f1rb genomic structures were determined by searching zebrafish genome (http://www.ensembl.org/Multi/blastview?species=Danio rerio) and PCR. Physical mapping was carried out using the LN54 radiation hybrid panel (20) . Primers used for mapping Ig f1ra amplified the last 197 bp of exon 21 and the first 142bp of the 3' UTR (forward 5'-CAGGCCTGGCTCTGGATAAGCACTCAG-3' and reverse 5'-TGCCCAAACCGTCCTCCGTCATTCCAA-3'). Primers used for mapping Ig f1rb amplified a 222 bp portion of exon 22 (forward 5'-GATGCGTCGGATGTGTGTCAAGCCACT-3' and reverse 5'- CAGTCAGTGATCCTGTCTGGCGGAAAT-3').

Morpholino knockdown
MOs were designed according to criteria provided by the commercial supplier (Gene Tools, LLC; Corvallis, OR). Two antisense MOs were designed against distinct sequences in the 5' UTR of each zebrafish ig f1r. The two ig f1ra targeting MOs are: ig f1ra MO 1, 5'-TCGCTGTTCCAGATCTCATTCCTAA-3'; and ig f1ra MO 2, 5'-TGAAATTGCAGAAAAACGCGAGGCT -3'. The two ig f1rb targeting MOs are: ig f1rb MO 1, 5'-TGTTTGCTAGACCTCATTCCTGTAC-3'; and ig f1rb MO 2, 5'AGAAATTAGGGAGAGACACCTCAAC-3'. In addition, we used two gene-specific mis-sense control MOs (ig f1ra Control MO, 5'-TCGgTGTagCAGATCTCtTTCgTAA-3'; and ig f1rb Control MO, 5'- TGaaTGCaAGAtCTCATaCCTcTAC-3'; small letters indicate nucleotide substitutions).

Construction of the igf1ra- and igf1rb-GFP reporter plasmids
A 650 bp cDNA fragment corresponding to a portion of the 5' UTR of the zebrafish ig f1ra gene (including the MO targeting regions) was generated by RT-PCR (forward primer, 5'-TTTTTGTTGGAGGAGAAGCCG-3'; reverse primer, 5'-TTACCATGGTCAACTTGGGGA-3'). The amplified DNA fragment was digested with EcoRI and PstI and was then subcloned into the pEGFP-N1 plasmid (Clontech; Palo Alto, CA). Similarly, an 880 bp DNA fragment corresponding to a portion of the 5' UTR of the zebrafish ig f1rb gene (including the MO target regions) was generated by RT-PCR (forward primer, 5'-ATCTCGAGAGAACCGCGCTGCTGAGGT-3'; reverse primer: 5'-TAGGATCCTTGCGGAAGACCTCCTGAT-3'). The amplified DNA fragment was digested with BamHI and XhoI andesubcloned into the pEGFP-N1 plasmid. The orientation and accuracy of sequences were verified by DNA sequencing.

Microinjection
MOs and/or plasmid DNA were injected into 1–2 cell stage embryos as reported (21) . In pilot experiments, control and gene-specific MOs were injected at a range of doses. A nominal concentration of 2.5 ng for each of the two ig f1ra MOs (5 ng total MO injected per embryo) and 4 ng for each of the two ig f1rb MOs (8 ng total per embryo) yielded consistent and reproducible phenotypes. These doses were used for all experimental analysis, except for analysis of spontaneous muscle contractility and motoneuron quantification where the same amount of ifg1ra MOs and ig f1rb MOs were injected per embryo (5 ng total MO per embryo). For the GFP reporter assays, 100 pg of plasmid and 2 ng of the indicated MO were injected per embryo.

Western blot
Twenty five embryos from each treatment group were dechorionated, deyolked, and homogenized in 100 µl of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, pH 7.5) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 100 mM PMSF, and 0.1 M sodium orthovanadate. The homogenates were briefly centrifuged to pellet cellular debris and the supernatant was retained. Protein levels of each sample were quantified using a protein assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein were analyzed by SDS-PAGE and Western blot as described previously (22) . The total Akt Ab and the phospho-Akt Ab (Ser-473 and Thr308) were both used at a 1:1000 dilution.

Whole mount in situ hybridization and antibody staining
Whole mount in situ hybridization using digoxigenin (DIG)-labeled RNA riboprobes and Ab staining were carried out as reported previously (18) . Ventricle tissue and skeletal muscle fibers were labeled by F59 staining at 1:10 dilution, hair cells in the otic vesicles were labeled using an anti-Tubulin Ab (1:1000 dilution), and motoneuron axons were labeled by SV2 staining (1:2000 dilution). Microphotographs were taken with a Nikon EC600 fluorescence microscope or acquired by laser scanning confocal microscopy (Model LSM 510, Carl Zeiss, Germany).

Immunocytochemistry
Cryosections were prepared following Hu and Easter et al. (23) . The sections (10 µm) were collected and air-dried at room temperature for 2 h before immunocytochemistry or storage at -20 C. After washing, sections were incubated in PBS at 37 C for 15 min to remove excess gelatin. Nonspecific binding was blocked by incubation in 5% goat serum/0.5% Triton/PBS for 1 h at room temperature. Muscle tissue staining was performed with a 1:20 dilution of F59. The retinal ganglion cell (RGC) layer was immunostained using a 1:500 dilution of Zn5. Primary antibodies were visualized with a 1:500 dilution of a Cy3-conjugated goat antimouse secondary Ab. Nuclei were counterstained with 50 nM sytox (Molecular Probes, Eugene, OR). Images were acquired as described above.

Body size, heart rate, and motoneuron measurements
Body length, somite number, and heart rates were measured as reported (21) . The caudal primary motoneurons (CaP) located above the yolk sac extension were quantified in 48 hpf embryos after staining with SV2 followed by color detection using a Vectastain avidin-biotin complex detection kit (Vector Labs; Burlingame, CA). Motoneurons were scored as present regardless of length or organization.

Statistics
Quantitative data are presented as means ± SE. (SEM). Differences among groups were statistically compared using one-way ANOVA followed with PLSD or Student’s t test. Statistical significance was accepted when p < 0.05.

RESULTS

Genomic structure and chromosomal locations of zebrafish igf1ra and igf1rb
The genomic structures of zebrafish ig f1ra and ig f1rb were determined by searching the zebrafish genome database and PCR. Zebrafish ig f1ra spans 151.5 kb in the genome. Like the human IGF1R, zebrafish ig f1ra has 21 exons. Zebrafish ig f1rb spans 156.5 kb in the genome and has an extra exon (#22) in its 3' end (Fig. 1 A, Table 1 ). Comparisons of exon lengths of the two zebrafish ig f1r genes with the human IGF1R gene indicated a high degree of cross-species conservation (Table 1) . We physically mapped ig f1ra and ig f1rb using the LN54 radiation hybrid mapping panel (20) . While ig f1rb mapped to linkage group (LG 7) as previously reported (24) , ig f1ra mapped to zebrafish LG 18 (Fig. 1B ). Further analysis suggested that ig f1rb and six other zebrafish genes (fb50h01, cox5a, herc1, mfap1, p24b and gro1) located on LG 7 have orthologs on human chromosome 15 (Table 2 ) (20 , 25 26 27) . Similarly, ig f1ra and four other zebrafish genes on LG 18 (arl, fa08a06, mef2a, and sema) also have orthologs on human chromosome 15 (Table 2) (20 , 25 26 27) . This result supports existing data that human chromosome 15 shares conserved synteny with zebrafish LGs 7 and 18 (27 , 28) and suggests a common ancestry for the human and zebrafish IGF1Rs.


Figure 1
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  		Figure 1. The genomic structure and physical mapping of zebrafish ig f1ra and ig f1rb. A) A schematic diagram comparing the structures of the zebrafish ig f1ra and ig f1rb genes. The horizontal lines represent introns, and the vertical black rectangles represent exons. B) Zebrafish ig f1ra is located on linkage group 18, 2.53 centiRay from marker fb09d12, and ig f1rb is located on linkage group 7, 4.71 centiRay from marker fc09c10. 1 centiRay = 148 kb.


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  		Table 1. Structural conservation between the human IGF1R and the duplicate zebrafish igf1rsa


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  		Table 2. Comparisons of genes mapped on zebrafish LGs 7 and 18 with those on human chromosome 15

Targeted knockdown of zebrafish igf1ra and igf1rb and inhibition of Igf signaling
To determine the functions of ig f1ra and ig f1rb, we ablated each gene product using two independent antisense MOs for each gene. Two gene-specific mis-sense MOs were used as controls. The efficacy and specificity of these MOs in knocking down their respective target gene product were determined using ig f1ra-GFP and ig f1rb-GFP, two reporter genes generated by fusing a portion of the 5'UTR of each receptor (containing the MO targeting sequences) upstream of GFP. Injection of either the ig f1ra-GFP or ig f1rb-GFP plasmids into zebrafish embryos resulted in mosaic GFP expression (Fig 2 A). As shown in Fig. 2A and 2B , coinjection of the ig f1ra-GFP plasmid with either ig f1ra MO significantly reduced the number of GFP-expressing embryos from 81.40 ± 3.76% in embryos injected with control MOs (n=149), to 1.67 ± 0.58% in embryos injected with ig f1ra MO 1 (n=143) and 1.33 ± 0.58% in embryos injected with ig f1ra MO 2 (n=159). In contrast, coinjection of ig f1rb MO 1 + 2 had no such effect (85.93±1.99%, n=172). Similarly, coinjection of the ig f1rb-GFP plasmid with either ig f1rb MO significantly reduced the number of GFP-expressing embryos from 86.0 ± 3.13% in embryos injected with control MOs (n=121), to 3.3 ± 0.47% in embryos injected with ig f1rb MO 1 (n=94) and 4.5 ± 0.57% in embryos injected with ig f1rb MO 2 (n=111). Coinjection of ig f1ra MO 1 + 2 had no such effect (78.7±2.72%, n=94). To show that the endogenous Igf1r-mediated signaling was indeed impaired, we analyzed the levels of Akt phosphorylation. Akt is a major downstream effector of the IGF1R in mammals and in zebrafish (29) . Western blot analysis indicated a marked reduction in the levels of phosphorylated Akt in the ig f1ra and ig f1rb MO-injected groups compared to controls. There was a further decrease in the group of embryos injected with a combination of ig f1ra and ig f1rb MOs. Together, these data indicate that the ig f1r MOs efficiently and specifically target their respective gene product and disrupt Igf1r-mediated signaling.


Figure 2
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  		Figure 2. Specific knockdown of ig f1ra and ig f1rb. A) Fluorescence microscopy images of zebrafish embryos injected with either ig f1ra-GFP or ig f1rb-GFP reporter plasmids, with or without ig f1ra and ig f1rb MOs. Lateral views are shown with anterior to the left and dorsal up. Each GFP plasmid was injected with or without the indicated MO. The yolk sac is autofluorescent. Scale bar = 250 µm. B) Quantification of (A). Data are presented as means ± SEM Results were obtained from three independent microinjection experiments, each with at least 30 embryos per group. *, P < 0.0001 compared to the control MO-injected group. C) Knockdown of ig f1ra and/or ig f1rb reduces phosphorylated Akt levels. Embryos injected with either ig f1ra MOs, ig f1rb MOs, or ig f1ra MO and ig f1rb MOs were subjected to Western blot analysis using antibodies for total and phospho-Akt. Similar results were obtained in two other experiments.

Igf1ra and Igf1rb are both required for proper embryonic growth, development, and survival
Embryos injected with control MOs were indistinguishable from wild type embryos (Fig. 3 A). In contrast, injection of either ig f1ra MO 1 or ig f1ra MO 2 resulted in embryos that were smaller and developmentally delayed (Fig. 3A ). Likewise, injecting either ig f1rb MO 1 or ig f1rb MO 2 caused similar growth and developmental retardation (Fig. 3A ). Because injecting multiple targeting MOs is known to exert maximal effects (30 , 31) , we injected both MOs for each receptor. Indeed, these embryos exhibited more severe phenotypes (Fig. 3A ) and were used for subsequent analysis. All ig f1ra and ig f1rb MO-injected embryos survived to 48 hpf and ~50% were alive at 72 hpf but none beyond 96 hpf. At 24 hpf, the mean body lengths of ig f1ra MO and ig f1rb MO-injected embryos were 0.95 ± 0.03 mM and 1.00 ± 0.02 mM, respectively (Fig. 3B ). These values were significantly smaller compared to that of control MO-injected embryos (1.598±0.02 mM). Knockdown of either ig f1ra or ig f1rb significantly reduced somite number, which is a quantitative indicator of the developmental rate in zebrafish before 24hpf (19) . Compared to 29.97 ± 0.20 somites in control MO-injected embryos, ig f1ra MO-injected embryos had only 20.14 ± 0.28 somites, and ig f1rb MO-injected embryos had only 19.54 ± 0.48 somites (Fig. 3C ). According to this criterion, embryos at 24 hpf in ig f1ra and ig f1rb MO-injected groups were developmentally equivalent to control embryos at ~18 hpf. These results indicate that knockdown of either zebrafish ig f1ra or ig f1rb resulted in embryonic lethality, growth retardation, and developmental delay.


Figure 3
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  		Figure 3. Knockdown of ig f1ra and/or ig f1rb reduces embryonic growth and developmental rate. A) Morphology of 24 hpf embryos injected with ig f1ra MO 1, ig f1ra MO 2, ig f1ra MO 1 + 2, ig f1rb MO 1, ig f1rb MO 2, ig f1rb MO 1 + 2, all four ig f1r MOs (ig f1rab MO), or control MOs. Scale bar = 250 µm. B) Body length and C) somite number of embryos injected with either ig f1ra MOs (ig f1ra MO 1+MO 2), ig f1rb MOs (ig f1rb MO 1+2), ig f1rab MOs (all four ig f1r MOs), or control MOs. Results are from three independent microinjection experiments, each with 15 embryos per group. *P < 0.0001 compared with the control MO-injected group, and # P < 0.001 compared with that of the ig f1ra or ig f1rb MO-injected group.

To determine the effect of ablating both ig f1r genes simultaneously, we injected a combination of all four targeting MOs. All injected embryos died by 30 hpf. At 24 hpf, their mean body length was 0.854 ± 0.02 mM, significantly smaller than those of the single knockdown group (P < 0.001). Likewise, knockdown of ig f1ra and ig f1rb together resulted in a further reduction in somite number (16.02±0.31), which is significantly smaller than those of the single knockdown group (P < 0.001). These data indicate that Igf1ra and Igf1rb play overlapping but nonredundant roles in regulating embryonic growth, developmental rate, and survival.

Overlapping roles of zebrafish Igf1ra and Igf1rb in eye, inner ear, and heart development
To investigate the functional roles of the zebrafish Igf1rs, we analyzed the impact of knocking down ig f1ra or ig f1rb on organogenesis of the eye, inner ear, and heart. As shown in Fig. 4 A, knockdown of either ig f1ra or ig f1rb markedly reduced the expression levels of rx1 mRNA, a retina-specific homoebox gene. Neurogenesis in the zebrafish retina begins with the differentiation of the retinal ganglion cells (RGCs) at around 30 hpf (32) . By 48 hpf, RGC axons occupy the inner layer of the retina and converge to form a single bundle of axons as they pass out of the retina (33 , 34) . Staining of cryosections of control, ig f1ra, and ig f1rb MO-injected embryos at 48 hpf with an RGC-specific antibody (Zn5) revealed a general absence of differentiated RGCs in the retina (Fig. 4A ). Similarly, there was a notable reduction in the expression domain of claudin a, an otic vesicle (inner ear) marker, in ig f1ra and ig f1rb MO-injected embryos (Fig. 4B ). In developing zebrafish otic vesicles, differentiated sensory hair cells are detectable by 36 hpf, and these cells can be labeled by anti-Tubulin staining (35) . Analysis of 48 hpf ig f1ra and ig f1rb MO-injected embryos revealed a lack of differentiated sensory hair cells (Fig. 4B ). Additionally, the protrusions in the epithelium, which mark the beginning of semicircular canal formation, were also absent in ig f1ra and ig f1rb MO-injected embryos (Fig. 4B ). Since knockdown of ig f1ra and ig f1rb together caused early lethality, these analyses could not be performed in these embryos.


Figure 4
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  		Figure 4. Knockdown of ig f1ra or ig f1rb causes similar defects in eye, inner ear, and heart development. A) Expression of rx1 mRNA in 24 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs (upper panels). Scale bar = 100 µm. Differentiated retinal ganglion cells (red, Zn5 staining, arrowhead) in 48 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs. The sections were counter stained with sytox (green, nuclear staining). Dorsal views are shown with anterior up. Scale bar = 50 µm. Similar results were obtained in two independent microinjection experiments. B) Expression of claudin a mRNA in 24 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs (upper panels). Differentiated hair cells labeled by anti-Tubulin antibody staining (red, arrowhead) in 48 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs. The sections were counter stained with sytox (green, nuclear staining). Epithelium protrusions (arrow), which mark the beginning of semicircular canal formation, were also absent in ig f1ra and ig f1rb MO-injected embryos. Lateral views are shown with anterior to the left and dorsal up. Scale bars = 50 µm. Similar results were obtained in two independent microinjection experiments. C) Expression of nkx2.5 mRNA in 24 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MO (upper panels). Note the tubular heart in the control MO-injected embryo (arrowhead), compared to the shallow cone structure of the heart tissue in ig f1ra and ig f1rb MO-injected embryos (arrows). Dorsal views are shown with anterior up. Scale bar = 100 µm. Differentiated cardiac cells (red, labeled by F59 staining) in 48 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs. The sections were counter stained with sytox (green, nuclear staining). Ventricles in ig f1ra and ig f1rb MO-injected embryos failed to form distinct chambers. Lateral views are shown with anterior to the left and dorsal up. Scale bar = 50 µm. Similar results were obtained in three independent experiments. D) Heart rates (bpm = beat per minute) in 48 hpf embryos injected with control MOs, igf1ra MOs or ig f1rb MOs. Results were obtained from three independent microinjection experiments, each with 15 embryos per group. *P < 0.001.

We also detected a delay in heart morphogenesis caused by depleting of Igf1ra or Igf1rb. In situ hybridization analysis of nkx2.5 expression, a heart-specific homeobox gene, indicated that hearts in 24 hpf control MO-injected embryos developed into tubular structures. In contrast, the ventricular tissue in ig f1ra and ig f1rb MO-injected embryos remained shallow cones, resembling WT embryos at ~18 hpf. Immunostaining using F59 revealed a notable reduction in heart size in ig f1ra and ig f1rb MO-injected embryos (Fig. 4C ). The ventricles of ig f1ra and ig f1rb MO-injected embryos failed to form distinct chambers as observed in control MO-injected embryos. There were also changes in heart function (Fig. 4D ). While the mean heart rate of control MO-injected embryos was 120.5 ± 1.88 bpm (beats per minute), the heart rates of ig f1ra and ig f1rb MO-injected embryos were significantly lower (79.8±9.16 bpm and 72.3±7.51 bpm). Taken together, these results indicated that ig f1ra and ig f1rb play largely similar roles in eye, inner ear, and heart development.

Igf1rb plays a greater role in spontaneous muscle contractility and motoneuron development.
During the course of the study, we noticed a marked difference in spontaneous contractile activity among the experimental groups. Wild-type zebrafish embryos spontaneously contract their tail muscles at 24 hpf as their motoneuron axons innervate their somitic myotomes (36) . By 48 hpf, this spontaneous contractility ceases and embryos exhibit rhythmic bouts of swimming. Control MO-injected embryos were indistinguishable from their wild-type siblings, 100% exhibiting spontaneous muscle contractions at 24 hpf and 0% at 48 hpf (Fig. 5 A). In ig f1ra MO-injected embryos, 69.0 ± 3.07% of the embryos at 24 hpf and 91.5 ± 0.54% at 48 hpf displayed spontaneous contractility, indicating that depletion of ig f1ra delayed or prolong the timing of this behavior. In contrast, few of the ig f1rb MO-injected embryos exhibited spontaneous muscle contractility at either 24 hpf (8.65±0.60%) or 48 hpf (5.5±0.75%), suggesting that depletion of ig f1rb either abolished this behavior or caused a greater delay.


Figure 5
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  		Figure 5. Igf1rb plays a greater role in regulating spontaneous muscle contractility and motoneuron formation. A) Percent of embryos exhibiting spontaneous muscle contractility at 24 (filled bar) or 48 hpf (open bar) injected with control MOs, ig f1ra MOs, or ig f1rb MOs. Results were obtained from three independent microinjection experiments, with 45 embryos per group. *P < 0.0001 between either ig f1ra or ig f1rb MO-injected embryos (24 and 48 hpf) and control MO-injected embryos (24hpf). B) Expression of myoD mRNA in 24 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs. Scale bar = 250 µm. Differentiated staining slow and fast muscles (labeled by F59 staining, red) of 48 hpf embryos injected with control MOs, ig f1ra MOs, or ig f1rb MOs. The sections were counter stained with sytox (green, nuclear staining). Scale bar = 20 µm. Analysis of CaP motoneuron axons in 48 hpf control, ig f1ra, and ig f1rb MO-injected embryos stained with anti-SV2, a synaptic vesicle protein expressed in motoneuron axons. Lateral views are shown with anterior to the left and dorsal up. Scale bar = 100 µm. Similar results were obtained in three independent microinjection experiments.

The lack of spontaneous muscle contractility would imply defects in muscle differentiation, motoneuron innervation, or both. To determine whether knockdown of ig f1ra or ig f1rb caused defects in somite formation, in situ hybridization was performed using probes for myoD and myogenin expression. Both ig f1ra and ig f1rb MO-injected embryos showed robust mRNA expression of myoD (Fig. 5B ) and myogenin (data not shown) at 24 hpf, indicating no defects in somitogenesis. Immunostaining of 48 hpf embryos using F59 (labeling both fast and slow muscle at this stage) indicated that knockdown of either ig f1ra or ig f1rb caused a reduction in differentiated slow and fast muscle. The degree of reduction, however, was comparable (Fig. 5B ). Therefore, although muscle differentiation was impaired, it is not likely the underlying cause of the difference observed with spontaneous muscle contractility. These results led us to hypothesize alternately that ig f1rb may perhaps be more important for promoting motoneuron differentiation. To test this idea, 48 hpf old control, ig f1ra, and ig f1rb MO-injected embryos were stained with an Ab against SV2, which is a synaptic vesicle membrane protein expressed in motoneurons and their axons. As shown in Fig. 5B , axons of CaP motoneurons in control MO-injected embryos clearly innervated the muscle and were well organized, but CaP axons in ig f1ra and ig f1rb MO-injected embryos were significantly impaired. In particular, ig f1rb MO-injected embryos exhibited a reduced number of CaP motoneuron axons. To quantify this effect, we measured the number of CaP axons innervating the somites above the yolk sac extension. There were only 4.5 ± 0.46 axons detected in ig f1rb MO-injected embryos. This value was significantly less than the 8.4 ± 0.8 axons in ig f1ra MO-injected embryos (n=8, P <0.0001), which was comparable to control MO-injected embryos (9.0±0.0 axons). These data suggest that while both ig f1ra and ig f1rb are important for muscle differentiation, Igf1rb plays a more specific or greater role in promoting motoneuron innervation.

DISCUSSION

It is well established that IGF1R-mediated signaling is essential for normal organismal survival, growth, and development in mammals (1 2 3 4) . The accumulated evidence to date suggests that the major components of the Igf signaling system in teleosts are similar to those in mammals (37) . However, there are several key differences. During mammalian fetal development, the mannose 6-phosphate/ type 2 Igf receptor (M6P/IGF2R) has an important function as a biological sink to prevent tissue overgrowth stimulated by IGF2 (3) . Comparative studies indicate that the mannose 6-phosphate receptors of nonmammalian species do not possess the capacity to bind IGF2 with high-affinity (37) . In contrast to the presence of a single IGF1R gene in mammals, zebrafish and other teleost species have two genes structurally related to the human IGF1R (18 , 38) . These two receptors are orthologous to the human IGF1R gene phylogenetically, and they both bind to IGFs, but not insulin (18) . In this study, we provide evidence that zebrafish Igf1ra and Igf1rb are encoded by distinct genetic loci believed to have evolved from a common ancestral locus. There is strong syntenic correspondence between the two zebrafish ig f1r genes and the human IGF1R gene, suggesting that zebrafish ig f1ra and ig f1rb originated from a gene duplication event that occurred during teleost evolution. The high degree of conservation in the exon number and length also supports this idea. A previous study has also identified two cDNAs encoding two structurally distinct insulin receptor genes (18) . These findings are consistent with the proposal that zebrafish and other ray-finned fishes may have experienced an additional gene duplication event during evolution, a theory derived from studies of the Hox gene family in zebrafish and Fugu (39) .

The finding that two ig f1r genes are present in zebrafish raised the interesting question regarding their functional relationship. Gene duplication is thought to be the primary source of new genes (40) and evolution by gene duplication has emerged as a general principal of biological evolution, evident in a number of sequenced genomes ranging from Bacteria to humans (41) . Permanent preservation of both duplicates requires divergent functions, but deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its versatility, amenability to manipulation, and because it possess a large number of duplicated genes. In this study, we explored the power of the zebrafish model and determined the functional relationship of the duplicated zebrafish ig f1r genes. Knockdown of zebrafish ig f1ra and ig f1rb together resulted in 100% lethality by 30 hpf. These embryos were also significantly smaller in body size, about half that of their wild type siblings. They also had significantly reduced somite number, suggesting delayed temporal development. These phenotypes are consistent with those reported in human patients, Ig f1r null mice, and insulin receptor mutant Drosophila (5 , 6 , 42) , suggesting that the role of IGF1R-mediated signaling in growth, development, and survival is conserved across a wide range of species. When either ig f1ra or ig f1rb was knocked down, the embryos could survive beyond 48 hpf, but with greatly increased mortality rates thereafter. Knockdown of ig f1ra or ig f1rb individually also resulted in similar growth retarded and developmentally delayed phenotypes. Additionally, knockdown of ig f1ra or ig f1rb resulted in a similar reduction of differentiated RGCs, sensory hair cells in the inner ear, and skeletal muscle. Heart development and growth were also impaired in these embryos. These results suggest that the duplicated zebrafish ig f1r genes play largely overlapping roles in zebrafish development.

Despite these apparent overlapping functions, Igf1ra or Igf1rb are not strictly redundant, but appear to play additive roles, because embryos lacking both receptors are more severely growth and developmentally retarded than embryos lacking either receptor alone. It also appears that Igf1ra and Igf1rb have evolved divergent functions. While knockdown of either ig f1rb or ig f1ra caused a comparable reduction in muscle differentiation, knockdown of ig f1rb, but not ig f1ra, caused a failure of embryos to exhibit spontaneous muscle contractility. Further analysis indicated that Igf1rb plays a more specific or greater role in promoting motoneuron innervation of the myotome. Two dominant hypotheses have been proposed regarding the rules governing the functional divergence after gene duplication. The neofunctionalization hypothesis argues that after duplication one daughter gene retains the ancestral function while the other acquires new functions (40) . The duplication-degeneration-complementation (DDC) hypothesis asserts that the functions of the ancestral gene are partitioned between the duplicated genes, such that the duplicate genes complement each other by jointly performing the necessary subfunctions of the ancestral gene (43) . Our findings concerning the functions of the duplicated zebrafish ig f1r genes cannot yet be adequately explained by either theory, but are more consistent with recent emerging evidence suggesting that neither subfunctionalization nor neofunctionalization alone can explain the functional evolution of duplicated genes. It has been proposed that a large portion of duplicated genes undergo rapid subfunctionalization followed by prolonged and substantial neofunctionalization (44) . Therefore, it is possible that both mechanisms governing duplicated gene evolution have contributed to the current functional state of the two distinct zebrafish ig f1rs.

The mechanism underlying the functional differences in spontaneous muscle contractility and motoneuron maturation/axon extension between zebrafish Igf1ra and Igf1rb is not yet clear. However, there are precedents for two or more isoforms of a growth factor receptor exerting different functions. For example, the two mammalian platelet-derived growth factor (PDGF) receptors (PDGFR), PDGF{alpha}R and PDGFßR, are thought to be products of a gene duplication event predating the divergence of nonjawed vertebrates and jawed vertebrates (45 , 46) . The PDGF{alpha}R and PDGFßR genes exhibit different spatio-temporal expression patterns, and display distinct ligand binding properties. While PDGF{alpha}R binds, and is activated by all forms of PDGF (AA, BB and AB), PDGFßR is activated exclusively by PDGF-BB (45 , 46) . Furthermore, exchanging the intracellular signaling domains of the mouse PDGF{alpha}R and PDGFßR, caused differences in the abilities of these chimeric receptors to mediate sustained MAP kinase activation, resulting in varying degrees of vascular disease (45 , 47) . Differences in ligand binding affinities, gene expression patterns, and/or signal transduction mechanisms may account for the functional specificity of the two zebrafish ig f1r genes. Previous studies have demonstrated that both zebrafish Igf1ra and Igf1rb bind Igfs and that the two zebrafish genes display similar spatial expression patterns during early development, although there are temporal differences in their relative abundance (18) . In particular, higher levels of Igf1rb were observed in early embryonic stages (18) . It was also noted that the two zebrafish Igf1rs are only 70% identical to each other, with the divergent residues spread throughout the molecules, and there are significant differences in the cytoplasmic regions of the two Igf1rs. It is possible that these divergent sequences may confer different signaling capacities between the two receptors. Further studies focusing on determining whether the duplicated zebrafish Igf1rs possess similar or different cellular distribution patterns and/or signaling properties will be needed to elucidate the molecular mechanisms underlying their functions.

ACKNOWLEDGMENTS

This study was supported in part by NSF IBN 0110864 to C.D. We thank Drs. A.W. Wood and J. Zhang for thoughtful comments on earlier drafts of this manuscript. We would also like to thank Drs. M. Ekker, F. Stockdale, R. Kollmar, S. Lyons, P. Raymond, and S.J. Du for kindly providing reagents for this work.

Received for publication November 15, 2005. Accepted for publication January 9, 2006.

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