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A schematic diagram illustrating the duplication and subsequent functional diversification of zebrafish igf1ra and igf1rb .
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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...
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... injected with control MOs were indistinguish- able from wild type embryos (Fig. 3A). 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 develop- mental retardation (Fig. 3A). Because injecting multi- ple targeting MOs is known to exert maximal effects ...
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... injected with control MOs were indistinguish- able from wild type embryos (Fig. 3A). 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 develop- mental retardation (Fig. 3A). Because injecting multi- ple targeting MOs is known to exert maximal effects (30,31), we injected both MOs for each receptor. Indeed, these embryos exhibited more severe pheno- types ( Fig. 3A) and were used for ...
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... injected with control MOs were indistinguish- able from wild type embryos (Fig. 3A). 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 develop- mental retardation (Fig. 3A). Because injecting multi- ple targeting MOs is known to exert maximal effects (30,31), we injected both MOs for each receptor. Indeed, these embryos exhibited more severe pheno- types ( 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 ...
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... and developmentally delayed (Fig. 3A). Likewise, injecting either ig f1rb MO 1 or ig f1rb MO 2 caused similar growth and develop- mental retardation (Fig. 3A). Because injecting multi- ple targeting MOs is known to exert maximal effects (30,31), we injected both MOs for each receptor. Indeed, these embryos exhibited more severe pheno- types ( 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 ...
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... these embryos exhibited more severe pheno- types ( 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 (1.5980.02 mM). Knockdown of either ig f1ra or ig f1rb significantly re- duced 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, ...
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... of either ig f1ra or ig f1rb significantly re- duced 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 retarda- tion, and developmental ...
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Citations
... For instance, zebrafish (unlike humans) do not possess bone marrow, with definitive hematopoiesis occurring instead in the kidney marrow [39,64]. In addition, zebrafish have undergone genome-wide duplication [65] (such as with insulin-like growth factor) [66], which potentially limits applicability of this model's results to other vertebrates. In addition, while research has supported the successful use of zebrafish as an exercise model [25], there are certainly exercise-associated research limitations that exist. ...
Osteoporosis (OP) is a degenerative disease characterized by reduced bone strength and increased fracture risk. As the global population continues to age, the prevalence and economic burden of osteoporosis can be expected to rise substantially, but there remain various gaps in the field of OP care. For instance, there is a lack of anti-fracture drugs with proven long-term efficacy. Likewise, though exercise remains widely recommended in OP prevention and management, data regarding the safety and efficacy for patients after vertebral fracture remain limited. This lack of evidence may be due to the cost and inherent difficulties associated with exercise-based OP research. Thus, the current research landscape highlights the need for novel research strategies that accelerate OP drug discovery and allow for the low-cost study of exercise interventions. Here, we outline an example of one strategy, the use of zebrafish, which has emerged as a potential model for the discovery of anti-osteoporosis therapeutics and study of exercise interventions. The strengths, limitations, and potential applications of zebrafish in OP research will be outlined.
... Due to whole genome duplication, fish have two subtypes of igf1r, igf1ra and igf1rb [14], and both of them play important biological functions. Igf1ra and igf1rb mRNA are highly expressed in gonads during vitellogenesis and spermatogonia proliferation in Pampus argenteus [15]. ...
Insulin-like growth factor-1 receptors (igf1rs) play important roles in regulating development , differentiation, and proliferation in diverse organisms. In the present study, subtypes of medaka igf1r, igf1ra, and igf1rb were isolated and characterized. RT-PCR results showed that igf1ra and igf1rb mRNA were expressed in all tissues and throughout embryogenesis. Using real-time PCR, the differential expression of igf1ra and igf1rb mRNA during folliculogenesis was observed. The results of in situ hybridization (ISH) revealed that both of them were expressed in ovarian fol-licles at different stages, and igf1rb was also expressed in theca cells and granulosa cells. In the testis, both igf1ra and igf1rb mRNA were highly expressed in sperm, while igf1rb mRNA was also obviously detected in spermatogonia. In addition, igf1ra mRNA was also present in Leydig cells in contrast to the distribution of igf1rb mRNA in Sertoli cells. Collectively, we demonstrated that differential igf1rs RNA expression identifies medaka meiotic germ cells and somatic cells of both sexes. These findings highlight the importance of the igf system in the development of fish gonads.
... The GH-IGF-I system is highly conserved throughout vertebrates and all its major players have orthologs in zebrafish, including IGF ligands, binding proteins, receptors and the intracellular signaling molecules (Duan, 1998;Pozios et al., 2001;Duan et al., 2003;Li et al., 2005;Wood et al., 2005;Schlueter et al., 2006;Kamei et al., 2008;Zhou et al., 2008;Wang et al., 2009;Zou et al., 2009;Dai et al., 2010;Zhong et al., 2011). According to the latest zebrafish genome version, (as of March 2020) GRCz11, there is only one gene coding for igfals (NCBI Gene ID: 570758; transcript XM_003198155.5). ...
... On the other hand, overexpression of igf-1 mRNA also resulted in dorsalization of zebrafish embryos (Eivers et al., 2004), while overexpression of a secreted form of IGF-1R (a truncated IGF-1R lacking both the transmembrane and intracellular domains) resulted in embryo ventralization, suggesting a role for igf-1 in dorso-ventral patterning during development (Table 1). Nevertheless, loss-of-function studies in zebrafish showed that suppression of IGF signaling by a dominant-negative IGF-IR or antisense morpholinos did not result in dorso-ventral patterning defects evidencing contrasting results depending on the technique used (Schlueter et al., 2006(Schlueter et al., , 2007. Both the toxic and off-target effects of MOs are well known and established (Eisen and Smith, 2008). ...
Mammalian acid-labile subunit (ALS) is a serum protein that binds binary complexes between Insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) extending their half-life and keeping them in the vasculature. Human ALS deficiency (ACLSD), due to homozygous or compound heterozygous mutations in IGFALS, leads to moderate short stature with reduced levels of IGF-I and IGFBP-3. There is only one corresponding zebrafish ortholog gene and it has not yet been studied. In this study we elucidate the role of igfals during zebrafish development. In zebrafish embryos igfals mRNA is expressed throughout development, mainly in the brain and subsequently also in the gut and swimbladder. To determine its role during development, we knocked down igfals gene product using morpholinos (MOs). Igfals morphant embryos displayed dorsalization in different degrees of severity, including a shortened trunk and loss of tail. Furthermore, co-injection of human IGFALS mRNA (hIGFALS) was able to rescue the MO-induced phenotype. Finally, overexpression of either hIGFALS or zebrafish igfals mRNA (zigfals) leads to ventralization of embryos including a reduced head and enlarged tail. These findings suggest that als plays an important role in dorso-ventral patterning during zebrafish development.
... There, conflicting data exists and igf1ra and igf1rb are reported to have largely overlapping expression domains, including the head region with the eyes, in either distinct or ubiquitous patterns (Li et al., 2014;Maures et al., 2002). When knocking down either receptor in zebrafish embryos, development is delayed, embryos are smaller than siblings and expression of marker genes such as rx1 is reduced (Schlueter et al., 2006). ...
Postembryonic stem cell niches are present throughout the vertebrate clade to facilitate development, homeostasis, regeneration and growth. While teleosts and amphibians display sustained stem cell activity in most organs after embryogenesis, higher vertebrates retain stem cell activity only in specific tissues. Despite these differences, similar challenges are imposed on all vertebrate organisms: new tissue has to be generated to expand or replace existent one while simultaneously ensuring integrity and functionality of the organ. Tight control of stem and progenitor cell proliferation is necessary to avoid aberrant growth such as in cancer. In the retina of the teleost medaka (Oryzias latipes), retinal stem (RSC) and progenitor (RPC) cells are located in the ciliary marginal zone (CMZ) and mediate postembryonic growth and neurogenesis. Since function and shape of the eye are intimately linked, the activity of RSCs and RPCs is tightly coordinated to establish proper cell type composition and number. In this thesis I addressed intrinsic and extrinsic regulation mechanisms of the RSC niche. I hypothesised that retinal growth underlies intrinsically active growth factor signaling, and that immune cells safeguard the RSC niche in homeostasis and injury. To analyse intrinsic regulation of RSC proliferation, I assessed the function of insulin-like growth factor (Igf) signaling in the CMZ using gain- and loss- of-function approaches. I found that Igf1 receptor over-activation increased cell cycle speed, RPC number and consequently retinal size, while simultaneously preserving the stereotypical retinal architecture. Strikingly, RSCs were not susceptible to mitogenic stimuli, indicating that RPC amplification is the determinant of retinal size and composition. To understand the extrinsic regulation of the RSC niche, I examined the interplay of immune cells and RSCs. I found that Ccl25b-positive RSCs are phagocytosed by Ccr9a-positive immune cells located in the CMZ. Ccl25b mutation abrogates reactivation of immune cells upon RSC injury, implicating Ccl25b–Ccr9a signaling in the immune–stem cell interaction during homeostatic surveillance and injury response. In summary, my results propose that accurate postembryonic growth and tissue integrity depend on both cell intrinsic and extrinsic mechanisms of growth control in the RSC niche of medaka.
... Proteins on the NSCs' surface and in the lysate revealed a decrease in IGF signaling with age ( Figures 4A and 4B), especially for the receptors IGF1R and insulin receptors. In zebrafish, two igf1r genes, igf1ra and igf1rb, are orthologous to the mammalian Igf1R and play complementary roles during development (Schlueter et al., 2006). Both were decreased on the surface fraction of aged brains. ...
In adult stem cell populations, recruitment into division is parsimonious and most cells maintain a quiescent state. How individual cells decide to enter the cell cycle and how they coordinate their activity remains an essential problem to be resolved. It is thus important to develop methods to elucidate the mechanisms of cell communication and recruitment into the cell cycle. We made use of the advantageous architecture of the adult zebrafish telencephalon to isolate the surface proteins of an intact neural stem cell (NSC) population. We identified the proteome of NSCs in young and old brains. The data revealed a group of proteins involved in filopodia, which we validated by a morphological analysis of single cells, showing apically located cellular extensions. We further identified an age-related decrease in insulin-like growth factor (IGF) receptors. Expressing IGF2b induced divisions in young brains but resulted in incomplete divisions in old brains, stressing the role of cell-intrinsic processes in stem cell behavior. : In this article, Chapouton and colleagues use the brain of the adult zebrafish to identify communication pathways in a native population of neural stem cells. They identify proteins expressed on the apical surfaces by a biotinylation technique and document the presence of filopodial extensions between cells. They further show the appearance of an abnormal mitotic response to IGF with age. Keywords: telencephalon, pallium, GFAP, radial glia, filopodia, lamellipodia, biotinylation, mass spectrometry, aging, neurogenesis, quiescence
... Both the α and β subunits are encoded in a single precursor cDNA. In zebrafish, two igf1r genes (igf1ra and igf1rb) are reportedly located on [55]. We found three genes encoding IGFR in the P. hypophthalmus genome, all of which are transmembrane proteins ( Table 2). ...
Background
The striped catfish, Pangasianodon hypophthalmus, is a freshwater and benthopelagic fish common in the Mekong River delta. Catfish constitute a valuable source of dietary protein. Therefore, they are cultured worldwide, and P. hypophthalmus is a food staple in the Mekong area. However, genetic information about the culture stock, is unavailable for breeding improvement, although genetics of the channel catfish, Ictalurus punctatus, has been reported. To acquire genome sequence data as a useful resource for marker-assisted breeding, we decoded a draft genome of P. hypophthalmus and performed comparative analyses.
Results
Using the Illumina platform, we obtained both nuclear and mitochondrial DNA sequences. Molecular phylogeny using the mitochondrial genome confirmed that P. hypophthalmus is a member of the family Pangasiidae and is nested within a clade including the families Cranoglanididae and Ictaluridae. The nuclear genome was estimated at approximately 700 Mb, assembled into 568 scaffolds with an N50 of 14.29 Mbp, and was estimated to contain ~ 28,600 protein-coding genes, comparable to those of channel catfish and zebrafish. Interestingly, zebrafish produce gadusol, but genes for biosynthesis of this sunscreen compound have been lost from catfish genomes. The differences in gene contents between these two catfishes were found in genes for vitamin D-binding protein and cytosolic phospholipase A2, which have lost only in channel catfish. The Hox cluster in catfish genomes comprised seven paralogous groups, similar to that of zebrafish, and comparative analysis clarified catfish lineage-specific losses of A5a, B10a, and A11a. Genes for insulin-like growth factor (IGF) signaling were conserved between the two catfish genomes. In addition to identification of MHC class I and sex determination-related gene loci, the hypothetical chromosomes by comparison with the channel catfish demonstrated the usefulness of the striped catfish genome as a marker resource.
Conclusions
We developed genomic resources for the striped catfish. Possible conservation of genes for development and marker candidates were confirmed by comparing the assembled genome to that of a model fish, Danio rerio, and to channel catfish. Since the catfish genomic constituent resembles that of zebrafish, it is likely that zebrafish data for gene functions is applicable to striped catfish as well.
... IGF-I is also viewed as a cardiac hormone since the IGF-I pathway promotes the development of cardiomyocytes and improves cardiac function by directly acting on cardiomyocytes [17]. Blockade of IGF-I-related pathways directly influences normal cardiac development and induces cardiac malformation and dysfunction [18][19][20]. ...
Background:
Tbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5.
Results:
Among the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group.
Conclusions:
These results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.
... Microinjections were performed 3 hours prior to LR injury, and MO uptake was confirmed via lissamine fluorescence prior to myectomy. The MO sequence for igf1ra and igf1rb genes was previously validated in zebrafish [32,33], and a standard MO targeting a human beta-globin intron mutation was used as control. Experiments were performed using 5 fish per experimental group, unless stated otherwise in the text and/or figure legend. ...
... However, due to the genome duplication in teleosts [43], two Igf1 receptor genes (igf1ra and igf1rb) have been described in zebrafish [44,45]. To further test whether Igf signaling plays a role in muscle regeneration, Igf1ra/b expression was knocked down using previously described translationblocking antisense MOs [32,33], which were delivered via microinjection followed by electroporation prior to myectomy. LR muscle regeneration at 7 dpi was measured as before ( Fig 1E) showing that knockdown of Igf1r expression effectively reduced LR muscle regeneration (Fig 1F). ...
Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.
... We have previously shown that low [Ca 2+ ] stress activates PI3K-Akt signaling in NaR cells exclusively in an IGF1R-dependent manner 25 . In zebrafish, there are two igf1r genes and two insr genes due to a teleost linage-specific genome duplication and these genes are expressed ubiquitously in embryonic and larval tissues 29,30 . To definitely show that these igf1r and insr genes are expressed in NaR cells and to determine whether their mRNA levels change in response to low [Ca 2+ ] stress, Tg(igfbp5a:GFP) larvae were transferred to normal and low [Ca 2+ ] solution, NaR cells were isolated by FACS-based cell sorting. ...
... In the zebrafish genome, there are two igf1r genes and two insr genes due to a teleost linage-specific genome duplication 29,30 . The results of this study showed that all 4 genes are expressed in NaR cells but their levels did not change under low [Ca 2+ ] stress. ...
Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca²⁺] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.
... In our study, we presented data that PI3K inhibition led to brain size decrease via regulating apoptosis in the brain region, and overexpressing Akt successfully rescued the reduction of brain size in zebrafish early embryogenesis. Combined with the consistent phenotypes of PI3K inhibition in Igf1 −/− mice (4) and IGF1R loss-of-function in zebrafish (35,36), these results illustrate that PI3K/Akt mediate the effects of trophic factors on brain development and is also essential for embryonic brain development in zebrafish. We also found a specific increase in brain size in zebrafish embryos overexpressing Akt1 mRNA at 24 hpf without displaying any ectopic expression. ...
Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis.
