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* Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA; and
Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois, USA
1Correspondence: Department of MCDB, University of Michigan, Kraus Natural Science Bldg., Ann Arbor, MI 48109, USA. E-mail: cduan{at}umich.edu
SPECIFIC AIMS
RECENT STUDIES HAVE shown that zebrafish have two distinct insulin-like growth factor type 1 receptor (IGF1R) genes, ig f1ra and ig f1rb; however, their relationship and their functions are unknown. The aim of this study was to test the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event that led to their functional divergence.
PRINCIPAL FINDINGS
1. Genomic structure and chromosomal locations of zebrafish igf1ra and igf1rb
The zebrafish ig f1ra spans 151.5 kb in the genome and has 21 exons. The zebrafish ig f1rb spans 156.5 kb and has 22 exons. Comparisons of exon number and length of the two zebrafish genes with those of the human IGF1R gene indicated a high degree of cross-species conservation. ig f1ra is located on zebrafish linkage group (LG) 18, while ig f1rb is located on LG 7. Further analysis suggested that ig f1rb and six other zebrafish genes located on LG 7 have orthologs on human chromosome 15. Similarly, ig f1ra and four other zebrafish genes located on LG 18 also have orthologs on human chromosome 15. Therefore, the two zebrafish ig f1rs are encoded by distinct genetic loci believed to evolve from a common ancestral IGF1R locus, and there is strong syntenic correspondence between the two zebrafish ig f1r genes and the human IGF1R gene.
2. Both igf1ra and igf1rb are required for proper zebrafish embryonic growth, development, and survival
To elucidate the functional relationship between igf1ra and igf1rb, we knocked down each individually or in combination using an antisense MO-based targeted gene knockdown approach. Two distinct antisense MOs were designed for each target gene, and their efficacy and specificity in targeting their respective ig f1r gene product were validated. Two gene-specific mis-sense MOs were used as controls. Knockdown of ig f1ra or ig f1rb resulted in embryos that were smaller and more developmentally delayed than control MO-injected embryos (Fig. 1
A). These embryos survived to 48 hpf, and 
50% were alive at 72 hpf but none beyond 96 hpf. At 24 hpf, the mean body length and somite number of ig f1ra and ig f1rb MO-injected embryos were significantly reduced (Fig. 1B, C
). When both ig f1ra and ig f1rb were knocked down by injecting a combination of all four targeting ig f1r MOs, all embryos died by 30 hpf. At 24 hpf, their body size and developmental rate (somite number) were significantly smaller than those of the single knockdown group (Fig. 1B, C
), suggesting the two genes play similar and likely additive roles in regulating embryonic growth, developmental rate, and survival.
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3. Overlapping functions of igf1ra and igf1rb in regulating embryonic tissue growth and differentiation
To determine the roles of ig f1ra and ig f1rb in organogenesis, we analyzed the impact of knocking down ig f1ra or ig f1rb on eye, inner ear, and heart formation and differentiation. Knockdown of either ig f1ra or ig f1rb caused similar reductions in the expression levels of rx1 mRNA, a retina-specific homoebox gene. There was also a similar absence of differentiated retinal ganglion cells in the retina and sensory hair cells in the otic vesicles in ig f1ra and ig f1rb MO-injected embryos. Knockdown of ig f1ra or ig f1rb individually also caused a similar degree of delay in the timing of heart morphogenesis and reduction of differentiated cardiac tissue. These results indicated that ig f1ra and ig f1rb play largely similar roles in eye, inner ear, and heart development.
4. 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 the somitic myotome. 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. 2
A). In the ig f1ra MO-injected group, 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 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.
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The lack of spontaneous muscle contractility would imply defects in muscle differentiation, motoneuron innervation, or both. Whole mount in situ hybridization analysis showed that both ig f1ra and ig f1rb MO-injected embryos exhibited robust mRNA expression of myoD (Fig. 2B
, upper panels) and myogenin (data not shown) at 24 hpf, indicating normal somitogenesis. F59 staining of fast and slow muscle indicated that knockdown of either ig f1ra or ig f1rb caused a reduction in muscle differentiation and the degree of reduction was comparable (Fig. 2B
, middle panels). Therefore, although muscle differentiation was impaired, it is not likely the underlying cause of the difference observed in spontaneous muscle contractility. These results led us to hypothesize alternately that ig f1rb may play a greater or more specific role in promoting motoneuron differentiation. This idea was tested by staining embryos with an antibody against SV2, which labels motoneurons and their axons. As shown in Fig. 2B
, axons from the caudal primary motoneurons (CaP) of control MO-injected embryos clearly innervated the muscle and were well organized; however, CaP axons in ig f1ra and ig f1rb MO-injected embryos were significantly impaired, but the effect was more pronounced in the ig f1rb MO-injected embryos. The ig f1rb MO-injected embryos had 4.5 ± 0.46 CaP axons, significantly less than the 8.4 ± 0.8 axons in ig f1ra MO-injected embryos, and the 9.0 ± 0.0 axons in control MO-injected embryos (p < 0.0001). These data suggest that while both Igf1ra and Igf1rb are important for muscle differentiation, Igf1rb plays a greater or more specific role in promoting motoneuron innervation.
CONCLUSIONS AND SIGNIFICANCE
In this study we provide evidence that zebrafish Igf1ra and Igf1rb are encoded by two distinct genetic loci believed to have evolved from a common ancestral locus. Additionally, we show a strong syntenic correspondence between the two zebrafish ig f1r genes and the human IGF1R gene, suggesting that zebrafish ig f1ra and ig f1rb likely originated from a gene duplication event that occurred during teleost evolution. This conclusion is consistent with the notion that zebrafish and other fishes in the killifish lineage may have experienced an additional gene duplication event during evolution, a theory derived from studies of the Hox gene family in zebrafish and Fugu.
Gene duplication is thought to be the primary source of new genes. Permanent preservation of both duplicates requires divergent functions, but deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. 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 significantly smaller, about half that of their wild-type siblings, and exhibited significantly reduced somite number, suggesting developmental delay. These phenotypes are consistent with those reported in human patients and Ig f1r null mice. When a single ig f1r, 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 resulted in similar reductions in body size, development rate, 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 similar roles in zebrafish development.
Despite these apparent overlapping functions, ig f1ra and ig f1rb 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. 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 greater or more specific role in promoting motoneuron innervation of the myotome. The mechanism underlying the functional differences in spontaneous muscle contractility and motoneuron differentiation between zebrafish Igf1ra and Igf1rb is not yet clear. 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. 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 their cytoplasmic regions. 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.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3882fje
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