Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling

Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling Abstract Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as “catch-up” growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1–mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia. Fetal growth and developmental timing are cooperatively defined by genetic and epigenetic factors. Inadequacies of some conditions lead to developmental arrest or intrauterine growth restriction (IUGR) in human fetuses (1). Intriguingly, once an unfavorable condition is removed, most stunted animals restart growth with an accelerated progression rate, which is referred to as catch-up growth (2–4). Although catch-up growth is important for the accidentally stunted animal to regain its size and compete with nonstunted ones, it may not always be beneficial in humans, as recent epidemiological data showed that catch-up growth after IUGR was often associated with adult-onset disorders or unfavorable outcomes during growth (1, 5, 6). Because changes in such an anomalistic growth pattern seem to be a key for deciphering the cause of future pathogenesis of infants with IUGR, we especially need to increase our understanding of the molecular and cellular bases of the catch-up phenomenon. Insulin/insulinlike growth factor (Igf) signaling (IIS) is a major hormonal pathway facilitating embryonic growth in a wide range of metazoans (7–9). The insulin and Igf ligands bind to the insulin receptor (Ir) or type 1 Igf receptor (Igf1r), which triggers activation of the receptor tyrosine kinases and subsequent tyrosine phosphorylation of specific substrates, the Ir substrates (Irs; Irs1 through Irs4) (10). Among the multiple irs genes, irs1 is known to be uniquely responsible for normal growth and development in studies using knockout mouse models (11), and the signaling cascade activates a number of cellular behaviors (i.e., cell survival, proliferation, differentiation, and migration) via several major downstream pathways, such as the phosphoinositide 3-kinase‒Akt pathway and Ras-Raf-Mek-Erk1/2 pathway (8). Importantly, Irs is known to be very sensitive to variable physiological/environmental conditions, and the Irs proteins tend to fluctuate their expression levels (and to coordinate IIS activities) according to the confronting situation or immediate stressors (12–15). These facts imply that irs1 may be important for consolidating animal growth and development not only under undisturbed conditions but also under abnormal or variable circumstances. Hypoxia is a major external factor causing IUGR in the human fetus (16). Pregnancy at higher altitudes often induces relative hypoxic intrauterine conditions and results in risks of miscarriage or small-for-gestational-age fetuses (17). Intrauterine hypoxia may also be induced by compression/coiling of the umbilical cord or abnormal placental functions (18, 19); however, these risk factors are always eliminated after birth, and these infants are likely to take a catch-up trajectory. It is thought that hypoxia-induced growth retardation is in part due to the blunted Igf action through the hypoxia-induced overexpression of an inhibitory IGF-binding protein (Igfbp1) in the fetus (9). Previous studies have shown that the Igfbp1-mediated hypoxic response is found not only in humans but also in fish models (20). Despite the strong link between oxygen supply and IIS-dependent embryonic growth, the molecular and cellular bases of hypoxia-/reoxygenation-induced growth alteration remains largely elusive. Studies using rodent models present technical complexities for observing intrauterine specimens and conducting hypoxia experiments. Zebrafish are especially well suited for studying relationships between oxygen conditions and embryonic growth because of their fast and conserved development, the availability of established experimental methods, and the ease of manipulating environmental oxygen levels independent from the mother organism (3, 21). In zebrafish embryos, hypoxia caused growth delay, but subsequent reoxygenation induced catch-up growth (3). In this study, we investigated the role of Irs1-mediated growth signaling in hypoxia-/reoxygenation-induced catch-up growth using the zebrafish model. Materials and Methods Chemicals and reagents Chemicals and reagents were purchased from Wako (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan) unless noted otherwise. DNA ligase and restriction endonucleases were purchased from Promega (Madison, WI). For reverse transcription polymerase chain reaction (RT-PCR), Trizol reagent, M-MLV reverse transcription, and oligonucleotide primers were purchased from Invitrogen Life Technologies (Carlsbad, CA). The translation block antisense-morpholino oligo (MO) was purchased from Gene Tools, LLC (Philomath, OR). Experimental animals Adult wild-type zebrafish (Danio rerio) were maintained at ∼28°C on a 14-hour light: 10-hour dark cycle and fed twice daily. Embryos were generated from natural crosses, and embryos were raised in standard rearing solution at 28.5°C. At the time of use, embryos were anesthetized in tricaine mesylate (ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich Japan, Tokyo, Japan), and all experiments were conducted in accordance with guidelines approved by the Graduate School of Agriculture and Life Sciences at The University of Tokyo and the Guide for the Care and Use of Laboratory Animals prepared by Kanazawa University. RT-PCR Total RNA was isolated from adult fish and embryos. After DNase treatment, 2.5 μg total RNA was reverse-transcribed to single-strand complementary DNA (cDNA) using an engineered M-MLV reverse transcriptase (SuperScript™ II Reverse Transcriptase, Invitrogen Life Technologies) following the manufacturer’s instructions. RT-PCR for zebrafish irs1 was performed with a set of primers described in Supplemental Table 1. The level of β-actin messenger RNA (mRNA) was also measured. Whole-mount in situ hybridization Embryos used for whole-mount in situ hybridization were raised in embryo-rearing solution with 0.003% (weight-to-volume ratio) 2-phenylthiourea to inhibit pigmentation. Partial cDNA fragment of zebrafish Irs1 (786 bps) was cloned and used for in vitro transcription with either T7- or T3-RNA polymerase to generate digoxigenin-labeled complementary RNA probes according to the manufacturer’s instructions. Other complementary RNA probes for various developmental marker genes were similarly prepared. Hybridization was carried out following the standard method (22). Images were captured using an Olympus SZ61 microscope (Tokyo, Japan) with a Canon iVIS HF M52 camera (Tokyo, Japan) or BZ9000 microscope (Keyence, Tokyo, Japan). Biochemical characterization of zebrafish Irs1 protein Recombinant FLAG-tagged zebrafish Irs1 and rat IRS1 proteins were expressed in human embryonic kidney 293T cells. Briefly, cells were cultured in high glucose Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and nonessential amino acids (Nissui, Tokyo, Japan). Cells were transfected with each expression plasmid by using polyethylenimine (Polyscience Inc., Warrington, PA). One day after transfection, cells were incubated with serum-free medium for 12 hours. One hour before IGF-I stimulation, the serum-starved cells were exposed to the low-molecular-weight compound inhibitor of IR/IGF-1R, BMS754807 (1.0 μM). Then, in the last 5 minutes, cells were treated with 100 ng/mL of IGF-I and collected in lysis buffer with protease inhibitor cocktails and phosphatase inhibitor mix. Cell lysate was subjected to immunoprecipitation as previously described (23). The protein concentration was quantified using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and subjected to immunoblotting. Microinjection experiment The translational block antisense MO against zebrafish Irs1 mRNA (irs1 MO: 5′-ACAGAAAAATTGCAGGATCGGAAGT-3′) and the standard control MO (ctr MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′) were designed and synthesized by Gene Tools, LLC. The previously validated antisense MOs against mRNAs for Prdm1a (prdm1a MO: 5′-TGGTGTCATACCTCTTTGGAGTCTG-3′) and Sox10 (sox10 MO: 5′-ATGCTGTGCTCCTCCGCCGACATCG-3′) were similarly prepared (24, 25). The zebrafish Irs1 5′-UTR cDNA was amplified and subcloned into pCS2+Venus plasmid, and the plasmid was used for in vitro mRNA transcription (mMessage mMachine; Ambion, Austin, TX) to prepare the capped Venus mRNA harboring the irs1 MO target sequence (5′-Irs1UTR-Venus). All MOs were injected into embryos at the same dose (4 ng/embryo). The full-length open reading frame of zebrafish Irs1 cDNA was amplified with high-fidelity polymerase (Pyrobest; TaKaRa, Shiga, Japan) and used for capped RNA synthesis. The capped RNAs encoding Venus, N terminus FLAG-tagged zebrafish Irs1, constitutive active-Akt [N-terminal myristoylation signal–attached mouse Akt1 (myrAkt)], and constitutively active-H-Ras (HRasV12) were also prepared using the mMessage mMachine kit. Capped RNAs for Venus (250 pg/embryo), FLAG-zebrafish Irs1 (1000 pg/embryo), myrAkt (20 pg/embryo), HRasV12 (5 pg/embryo), and MOs were injected into one- or two-cell stage embryos, and they were kept at 28.5°C until sampling. Hypoxia and reoxygenation Hypoxic water was prepared by bubbling pure nitrogen gas into the embryo-rearing solution. The oxygen concentration was measured using a dissolved oxygen meter (ProODO; YSI Nanotech Japan, Kawasaki, Japan). The dissolved oxygen content in the hypoxic water was set at 0.6 ± 0.2 mg/L O2 (6% to 10% oxygen content, as the oxygen level in the normoxic water is set at 100%: approximately 8.0 mg/L O2). In hypoxia/reoxygenation experiments, all embryos were kept under normoxia until 24 hours postfertilization (hpf). Embryos developed under constant normoxia were termed Norm. Embryos transferred to hypoxia from 24 to 36 hpf were termed Hypo. The embryos exposed to hypoxia from 24 to 36 hpf and then put back to normoxic water were termed Reoxy (hypoxia to normoxia). Growth-level measurement and relative growth rate calculation The growth level of an embryo was determined by measuring the head-trunk angle (HTA) (3, 20, 21). Growth rate was driven by the formula (dy/dt) = (yn−y0)/(tn−t0), with y0 = HTA at the initial time-point (t0) and yn = HTA at the end time point (tn). The growth rates were shown as relative values of the control group. Immunoblot analysis Immunoblot analyses were performed as previously described (3, 23). The antibodies used for the Akt and Erk1/2 western blottings were purchased from Cell Signaling Technology (CST-Japan, Tokyo). The antiphospho-Akt antibody (9271 for phosphorylation at Ser473) was used at a 1:500 dilution, and total-Akt (9272), antiphospho-Erk1/2 (9101 for phosphorylation at Thr202 and Tyr204), and total-Erk1/2 (137F5) were used at a 1:1000 dilution according to the instruction manuals. An equal amount of protein was used for the immunoblot analysis. Tubulin antibody (2148; CST-Japan) was purchased and used at indicated dilutions per manufacturer’s instruction. Whole-mount immunostaining Whole-mount immunostaining was conducted by using anti-zebrafish Sox10 (AS-55651s, AnaSpec), anti‒active caspase-3 (559565; BD PharmingenTM), antiphospho-Akt (S473) DE9 XP (4060; CST-Japan), and antimyosin heavy chain (05-716; Millipore, Darmstadt, Germany) according to the instruction manuals. For the double staining, either Alexa-488 or -546 conjugated second-antibody (Thermo Fisher Scientific, Pittsburgh, PA) was used. Alternatively, antibodies were biotinylated by EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions, and the biotinylated antibodies were detected by the Alexa-488 conjugated streptavidin (Streptavidin, Alexa Fluor™ 488 conjugate, Thermo Fisher Scientific). The Alexa-488‒conjugated cleaved-active caspase-3 (D175) antibody (9669S; CST-Japan) and the Alexa-488‒conjugated antiphospho-Akt antibody (S473) (4071S; CST-Japan) were also used for double-staining experiments (Table 1). Table 1. Antibodies Used Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Monoclonal or Polyclonal; Species Raised in  Dilution Used  RRID   phospho-Akt1/2/3    Phospho-Akt (Ser473) antibody  CST-Japan, Tokyo, 9271  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.  1:500 dilution  AB_329825  Akt1/2/3    Akt antibody  CST-Japan, Tokyo, 9272  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt.  1:1000 dilution  AB_329827  Phospho-p44/42 MAPK (Erk1/2)    Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody  CST-Japan, Tokyo, 9101  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.  1:1000 dilution  AB_331646  p44/42 MAPK (Erk1/2)    p44/42 MAPK (Erk1/2) (137F5) rabbit mAb  CST-Japan, Tokyo, 4695  Monoclonal antibody is produced by immunizing rabbit with a synthetic peptide corresponding to residues near the C-terminus of rat p44 MAP kinase.  1:1000 dilution  AB_390779  Tubulin    α/β-Tubulin antibody  CST-Japan, Tokyo, 2148  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the sequence of human α- and β-tubulin.  1:100 dilution  AB_2288042  Sox10    Anti-SOX-10 (IN), Z-FISH®  AnaSpec, Fremont, CA, AS-55651s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the intermediate region of zebrafish Sox10 protein (GenBank accession #NP_571950.1).  1:100 dilution  AB_10631841  Active caspase-3    Purified rabbit anti‒active caspase-3  BD Biosciences, San Jose, CA, 559565  Monoclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the human active caspase-3 fragment.  1:250 dilution  AB_397274  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb  CST-Japan, Tokyo, 4060  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:200 dilution  AB_2315049  Phospho-histone H3    Phospho-Histone H3 (Ser10) (D2C8) XP® rabbit mAb  CST-Japan, Tokyo, 3377P  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3.  1:500 dilution  AB_1549592  Active caspase-3    Cleaved caspase-3 (Asp175) antibody (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 9669s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3.  1:50 dilution  AB_2069869  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 4071s  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:50 dilution  AB_1031106  Myosin heavy chain    Anti-myosin heavy chain antibody, clone A4.1025  Millipore, Darmstadt, Germany, 05-716  Monoclonal antibody is produced by immunizing mouse.  1:100 dilution  AB_309930  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488  Thermo Fisher Scientific, Pittsburgh, PA, R37116  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2556544  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11035  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534093  Mouse IgG (H+L)    Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11003  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534089  Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Monoclonal or Polyclonal; Species Raised in  Dilution Used  RRID   phospho-Akt1/2/3    Phospho-Akt (Ser473) antibody  CST-Japan, Tokyo, 9271  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.  1:500 dilution  AB_329825  Akt1/2/3    Akt antibody  CST-Japan, Tokyo, 9272  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt.  1:1000 dilution  AB_329827  Phospho-p44/42 MAPK (Erk1/2)    Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody  CST-Japan, Tokyo, 9101  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.  1:1000 dilution  AB_331646  p44/42 MAPK (Erk1/2)    p44/42 MAPK (Erk1/2) (137F5) rabbit mAb  CST-Japan, Tokyo, 4695  Monoclonal antibody is produced by immunizing rabbit with a synthetic peptide corresponding to residues near the C-terminus of rat p44 MAP kinase.  1:1000 dilution  AB_390779  Tubulin    α/β-Tubulin antibody  CST-Japan, Tokyo, 2148  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the sequence of human α- and β-tubulin.  1:100 dilution  AB_2288042  Sox10    Anti-SOX-10 (IN), Z-FISH®  AnaSpec, Fremont, CA, AS-55651s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the intermediate region of zebrafish Sox10 protein (GenBank accession #NP_571950.1).  1:100 dilution  AB_10631841  Active caspase-3    Purified rabbit anti‒active caspase-3  BD Biosciences, San Jose, CA, 559565  Monoclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the human active caspase-3 fragment.  1:250 dilution  AB_397274  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb  CST-Japan, Tokyo, 4060  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:200 dilution  AB_2315049  Phospho-histone H3    Phospho-Histone H3 (Ser10) (D2C8) XP® rabbit mAb  CST-Japan, Tokyo, 3377P  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3.  1:500 dilution  AB_1549592  Active caspase-3    Cleaved caspase-3 (Asp175) antibody (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 9669s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3.  1:50 dilution  AB_2069869  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 4071s  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:50 dilution  AB_1031106  Myosin heavy chain    Anti-myosin heavy chain antibody, clone A4.1025  Millipore, Darmstadt, Germany, 05-716  Monoclonal antibody is produced by immunizing mouse.  1:100 dilution  AB_309930  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488  Thermo Fisher Scientific, Pittsburgh, PA, R37116  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2556544  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11035  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534093  Mouse IgG (H+L)    Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11003  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534089  Abbreviations: H+L, heavy and light chain; IgG, immunoglobulin G; mAb, monoclonal antibody; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; RRID, Research Resource Identifier. View Large Immunohistochemistry of cryosections The embryos fixed in 4% paraformaldehyde were rinsed and immersed in 30% sucrose at 4°C overnight. Then, the 30% sucrose-immersed embryos were transferred to the optimal cutting temperature (O.C.T.) compound/30% sucrose mixture (O.C.T compound: 30% sucrose = 2:1) at 4°C for another overnight. The specimens were embedded in O.C.T. compound and solidified on dry ice. The 20-μm-thick cryosections were prepared with cryostat (HM505N, Zeiss-MICROM; Microedge Instruments, Osaka, Japan). The sections were attached to the MAS-coated glass-slides (MATSUNAMI, Osaka, Japan) and subjected to immunohistochemistry procedure using antizebrafish Sox10 antibody and anti‒active caspase-3 antibody as described in the whole-mount immunostaining. The immunostained sections were counterstained with Hoechst33342, and the fluorescent images were pictured. The immunopositive cells were counted by using ImageJ software. Whole-mount terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining Whole-mount terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit, TMR red (Sigma-Aldrich Japan) according to the instruction manual. The embryos fixed by 4% paraformaldehyde were dehydrated in a graded ethanol series (50%, 70%, 90%, 100%) and permeabilized by acetone at −20°C for 10 minutes. The embryos were rinsed in phosphate-buffered saline (PBS)(-) and the embryos were further permeabilized by 0.1% Triton X-100, 0.1% sodium citrate in PBS(-) for 15 minutes at room temperature. After two rinses in PBS(-), the specimens were incubated with the labeling solution of the In Situ Cell Death Detection Kit at 37°C for 1 hour. After the TUNEL-labeling reaction, all samples were counterstained with Hoechst33342. Quantitative RT-PCR Quantitative RT-PCR was performed using SYBR® Green Realtime PCR Master Mix-Plus (Toyobo, New York, NY) and Applied Biosystems Step One™ Real-time PCR system (Applied Biosystems, Japan, Tokyo) as described previously (3, 26). Primers used are listed in Supplemental Table 1. The specificity of the PCR was verified by denaturing curve analysis, and the PCR products were analyzed by electrophoresis to determine whether a single product predominated. Small molecular compounds The chemical inhibitor for IR/IGF-1R kinase domains, BMS754807 (CAS#1001350-96-4), was obtained from Selleck Chemicals (Boston, MA) and used at 2.0 μM. Leflunomide (CAS#75706-12-6), a potent inhibitor of neural crest stem cell proliferation (27), was purchased from Sigma-Aldrich (St. Louis, MO) and used at 1.0 μM. General caspase inhibitor, zVADfmk, was purchased from BD Biosciences and used at 50 μM. Statistics Statistical significance between two groups was analyzed by the Student t test. Significance among multiple groups was determined by one-way analysis of variance, followed by the Tukey multiple comparison tests. Calculations were performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA), and significance was accepted at P < 0.05. Results Identification and functional characterization of zebrafish Irs1 First, we characterized the structure and biochemical features of zebrafish Irs1. We found a single irs1 gene (ENSDARG00000054087) in the zebrafish database (GRCz9) in Ensembl (http://asia.ensembl.org/Danio_rerio/Info/Index). The domain structure of the deduced zebrafish Irs1 protein in comparison with human IRS1 is shown in Fig. 1A. As found in IRS proteins in other species, the deduced primary sequence of zebrafish Irs1 contains a highly conserved pleckstrin-homology domain (residues 16 to 112) and phosphotyrosine-binding (PTB) domain (residues 142 to 246). The Ser-rich region is adjacent to the PTB domain in zebrafish Irs1, and the sequence is also highly conserved in IRS1 in other species (Fig. 1B). Although the Ser-rich region sequence is also found in IRS2, it is not positioned in the same region as in Irs1/IRS1 (next to the PTB domain in Irs1/IRS1, the C-terminal region in IRS2). To assess if this zebrafish Irs was correctly classified as Irs1, we further performed phylogenetic analysis using full-length amino acid sequences. As a result, the zebrafish Irs1 was grouped into the IRS1 clade with high confidence (Fig. 1C). Second, the full-length zebrafish Irs1 coding sequence was overexpressed in human embryonic kidney 293T cells as an N-terminal FLAG-tagged protein. The recombinant protein was detected as an approximately 130- to 140-kDa protein in immunoblot analysis (Fig. 1D; lysate, IB: FLAG). Under serum-starved conditions, the expressed zebrafish Irs1 showed a basal level of tyrosine phosphorylation, and this was greatly increased on IGF-I stimulation (IGF-I, 5 minutes). Importantly, IGF-I‒induced tyrosine phosphorylation was impaired by the specific IR/IGF-1R inhibitor (BMS-754807), demonstrating that the IGF1R-dependent tyrosine phosphorylation of Irs1 (Fig. 1D; IP, IB: FLAG or 4G10). Comparable results were obtained in the control experiments expressing rat IRS1. These data indicate that the identified zebrafish Irs1 encodes a functional Igf1r substrate. Figure 1. View largeDownload slide Identification and characterization of zebrafish Irs1. (A) A schematic illustration comparing major domains and motifs in zebrafish Irs1 and human IRS1. Pleckstrin homology (PH) domain, PTB domain, and serine-rich region are shown. (B) Amino acid identities of conserved domains and regions between zebrafish Irs1 and IRS1 in other vertebrate species. (C) Phylogenetic tree of the IRS proteins. Amino acid sequences of full-length IRSs were analyzed using the neighbor-joining method. The rooted phylogenetic tree with branch lengths is shown. (D) Functional characterization of zebrafish Irs1 in human embryonic kidney 293T cells. FLAG-tagged rat IRS1 and zebrafish Irs1 were expressed, and the cells were treated with or without the specific IR/IGF-1R tyrosine kinase inhibitor BMS-754807 and were stimulated with IGF-I (100 ng/mL) for 5 minutes. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed using denoted antibodies. Figure 1. View largeDownload slide Identification and characterization of zebrafish Irs1. (A) A schematic illustration comparing major domains and motifs in zebrafish Irs1 and human IRS1. Pleckstrin homology (PH) domain, PTB domain, and serine-rich region are shown. (B) Amino acid identities of conserved domains and regions between zebrafish Irs1 and IRS1 in other vertebrate species. (C) Phylogenetic tree of the IRS proteins. Amino acid sequences of full-length IRSs were analyzed using the neighbor-joining method. The rooted phylogenetic tree with branch lengths is shown. (D) Functional characterization of zebrafish Irs1 in human embryonic kidney 293T cells. FLAG-tagged rat IRS1 and zebrafish Irs1 were expressed, and the cells were treated with or without the specific IR/IGF-1R tyrosine kinase inhibitor BMS-754807 and were stimulated with IGF-I (100 ng/mL) for 5 minutes. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed using denoted antibodies. Temporal and spatial expression of the irs1 gene RT-PCR analysis showed that zebrafish Irs1 mRNA was expressed throughout embryogenesis, suggesting it is a maternally deposited transcript (Fig. 2A). In agreement with the RT-PCR data, Irs1 mRNA was indeed detected throughout embryogenesis by whole-mounted in situ hybridization analysis (Fig. 2B, a‒h). The Irs1 mRNA was expressed in a broad range of developing tissues, including brain (forebrain, midbrain, hindbrain), eye, mandibular arch, and trunk muscle (somite) in pharyngula-stage embryos (Fig. 2B, c and d) at 24 hpf. The hybridization signal was specific, as indicated by the lack of intense signals using the corresponding sense RNA probe in the same batch of embryos (Fig. 2B, c′ and d′). In advanced stages (at 2 to 10 days after fertilization), the predominant expression domains were brain, pectoral fin, pharyngeal arch, thyroid primordium, sensory hair cells, olfactory bulb, and gill arch, with a reduced but still detectable expression in brain and trunk muscle (Fig. 2B, e‒h). In adult fish, Irs1 mRNA was detected in most tissues examined in both males and females, and basically no obvious sex difference was found, except the male liver had relatively lower expression levels (Fig. 2C). Figure 2. View largeDownload slide Temporal and spatial expression patterns of zebrafish irs1. (A) RT-PCR analysis of irs1 expression during zebrafish embryogenesis. Developmental stages are shown at the top in hpf. (B) Whole-mount in situ hybridization analysis. (a) Embryos of two-cell stage, (b) 50% to 75% epiboly, (c, d, c′, and d′) pharyngula stage, (e) hatching (48 hpf), and (f‒h) hatched (72 to 240 hpf) embryos were analyzed. Shown in (a)–(h) are embryos probed with the antisense riboprobe. In c′ and d′, embryos probed with sense riboprobe are shown. a‒g, c′, and d′ are lateral views with the head to the left, and h is a ventral view of the embryo in g. Irs1 mRNA signals were detected in various developing cells and tissues, and arrowheads indicate typical domains with intense signals. The chronological age of each sample is shown in the upper right corner of the panel. Scale bar = 100 μm. (C) RT-PCR analysis of irs1 in adult tissues. Three males or females were used to prepare total RNA, and the pooled RNA from each tissue was used to prepare cDNA for RT-PCR analysis. Ba, branchial (gill) arch; Ey, eye; Fb, forebrain; Hb, hindbrain; Hc, lateral sensory hair cell; Ma, mandibular arch; Mb, midbrain; Ob, olfactory bulb; Pa, pharyngeal arch; Pf, pectoral fin; Sc, anterior sensory hair cell; Sm, somite (trunk muscle); Tp, thyroid primordia. Figure 2. View largeDownload slide Temporal and spatial expression patterns of zebrafish irs1. (A) RT-PCR analysis of irs1 expression during zebrafish embryogenesis. Developmental stages are shown at the top in hpf. (B) Whole-mount in situ hybridization analysis. (a) Embryos of two-cell stage, (b) 50% to 75% epiboly, (c, d, c′, and d′) pharyngula stage, (e) hatching (48 hpf), and (f‒h) hatched (72 to 240 hpf) embryos were analyzed. Shown in (a)–(h) are embryos probed with the antisense riboprobe. In c′ and d′, embryos probed with sense riboprobe are shown. a‒g, c′, and d′ are lateral views with the head to the left, and h is a ventral view of the embryo in g. Irs1 mRNA signals were detected in various developing cells and tissues, and arrowheads indicate typical domains with intense signals. The chronological age of each sample is shown in the upper right corner of the panel. Scale bar = 100 μm. (C) RT-PCR analysis of irs1 in adult tissues. Three males or females were used to prepare total RNA, and the pooled RNA from each tissue was used to prepare cDNA for RT-PCR analysis. Ba, branchial (gill) arch; Ey, eye; Fb, forebrain; Hb, hindbrain; Hc, lateral sensory hair cell; Ma, mandibular arch; Mb, midbrain; Ob, olfactory bulb; Pa, pharyngeal arch; Pf, pectoral fin; Sc, anterior sensory hair cell; Sm, somite (trunk muscle); Tp, thyroid primordia. Loss of zebrafish irs1 expression blunted reoxygenation-induced catch-up growth Loss-of-function experiments using antisense MO were then conducted. The validation results of MO are shown in Supplemental Fig. 1. The levels of embryonic growth were measured by the head-trunk angle (HTA) (Fig. 3A), which is highly correlated with body length. Representative growth patterns under different oxygen conditions and the experimental design are shown in Fig. 3B and 3C. Changes in HTA and representative embryos are shown in Fig. 3D and 3E, respectively. Under constant normoxia (Norm), the irs1 MO embryos showed moderate growth delay (Fig. 3D and 3E; Norm). Under hypoxia, the growth of both irs1 MO embryos and ctr MO embryos was similarly slowed down (Fig. 3D and 3E; hypoxia group at 36 hpf). Upon returning to normoxia from hypoxia (Reoxy), the ctr MO embryos showed robust acceleration of their growth to catch up with the Norm embryos; however, the irs1 MO embryos failed to do so (Fig. 3D and 3E; Reoxy). The loss of Irs1 expression significantly reduced growth rate in the Reoxy period, and the reduction was greater than that in the Norm period (Fig. 3F). Figure 3. View largeDownload slide Irs1 was required for reoxygenation-induced catch-up growth in the zebrafish embryo. Schematic illustrations of (A) the head-trunk angle, (B) representative growth patterns, and (C) experimental design. (D) Changes in head-trunk angle. Data are mean ± standard deviation; n = 9 to 23. (E) Representative embryos of Norm, Hypo, and Reoxy. The head-trunk angles are shown. Scale bar = 500 μm. (F) Analysis of relative growth rates during indicated developmental periods. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Figure 3. View largeDownload slide Irs1 was required for reoxygenation-induced catch-up growth in the zebrafish embryo. Schematic illustrations of (A) the head-trunk angle, (B) representative growth patterns, and (C) experimental design. (D) Changes in head-trunk angle. Data are mean ± standard deviation; n = 9 to 23. (E) Representative embryos of Norm, Hypo, and Reoxy. The head-trunk angles are shown. Scale bar = 500 μm. (F) Analysis of relative growth rates during indicated developmental periods. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Disturbed irs1 expression significantly blunted catch-up growth with attenuated IIS not during but before the catch-up period Next, the activation of IIS was tested. Representative growth patterns and sampling timings are shown in Fig. 4A. The loss of Irs1 expression attenuated phosphorylation levels of both Akt and Erk1/2 in prepharyngula embryos (8 to 12 hpf) before hypoxic exposure and in later pharyngula embryos under hypoxia (36 hpf). In contrast, in more advanced developmental stages, the phosphorylation levels of Akt and Erk1/2 were hardly changed by irs1 MO under both Norm (Norm at 36 to 38 hpf) and Reoxy (Reoxy at 44 to 48 hpf) conditions (Fig. 4B). These data demonstrate that Irs1 is responsible for maintaining IIS during very early stages of development and during severe hypoxic conditions in advance of catch-up growth in the current experiment, implying the importance of early embryonic IIS for later catch-up growth. Figure 4. View largeDownload slide Irs1-mediated insulin/Igf signaling was sufficient for catch-up growth. (A) Schematic illustration of experimental design; the sampling timing is indicated by red circles. (B) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2. (C) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2 of embryos at the prepharyngula stage under normoxia (12 hpf) and the pharyngula stage under hypoxia (36 hpf). (D) Changes in head-trunk angle in the indicated groups. Data are mean ± standard deviation; n = 8 to 20. (E) Relative growth rate. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Figure 4. View largeDownload slide Irs1-mediated insulin/Igf signaling was sufficient for catch-up growth. (A) Schematic illustration of experimental design; the sampling timing is indicated by red circles. (B) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2. (C) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2 of embryos at the prepharyngula stage under normoxia (12 hpf) and the pharyngula stage under hypoxia (36 hpf). (D) Changes in head-trunk angle in the indicated groups. Data are mean ± standard deviation; n = 8 to 20. (E) Relative growth rate. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Irs1-mediated IIS was important for embryonic catch-up growth To test whether attenuated IIS before catch-up growth is a causation of significant growth loss in irs1 MO embryos during Reoxy conditions, IIS was force-activated in irs1 MO embryos. Capped RNA molecules were coinjected with irs1 MO. The MO-resistant Irs1 RNA (Flag-Irs1) restored the reduced activation of IIS by Irs1 MO injection (Fig. 4C; Irs1 RNA). Similarly, the coinjection of mryAkt RNA with Irs1 MO also resulted in overcoming Irs1 MO-induced reduction of IIS levels (Fig. 4C; myrAkt RNA). Importantly, blunted catch-up growth in Irs1 MO-injected embryos was apparently rescued by coinjection of MO-resistant Irs1 RNA or myrAkt RNA during Reoxy conditions (Fig. 4D), and this was demonstrated by significantly regained relative growth rate of the Reoxy group fish as shown in Fig. 4E. Similar results were obtained in HRasV12 RNA coinjection experiments (Supplemental Fig. 2). These data strongly suggest that blunted IIS before reoxygenation is one of the major reasons for failed catch-up growth caused by the loss of irs1 expression. Disturbed irs1 expression reduced dlx2-expressing cells under hypoxic conditions To gain insight into cellular events that occurred before and during catch-up growth, we examined changes in cell proliferation in constant normoxia, hypoxia, and reoxygenation. Although there was a slight tendency toward decreased phospho-histone H3-positive cells in irs1 MO fish under reoxygenation conditions, the change was modest, and we failed to see any significant difference between the phospho-histone H3-positive cell density of irs1 MO fish and that of ctr MO fish in all three conditions (Supplemental Fig. 3A and 3B). Next, cell death was examined by active caspase-3 staining. We found that the numbers of active caspase-3‒positive cells were increased mainly in the anterior half of irs1 MO embryos under hypoxia (32 hpf) (Fig. 5A; α active caspase-3). We also examined the expression of specific genes (dlx2, ntl, emx1, and pax2a) and found reduced dlx2 expression in irs1 MO embryos under prolonged hypoxia (36 hpf) (Fig. 5A; dlx2, domains i to iv). The irs1 MO failed to cause any major changes in expression of either dlx2 or other genes under Norm conditions (Supplemental Fig. 4A). Under hypoxia, except for dlx2, we did not see any major changes in ntl and pax2a expression in irs1 MO embryos; trunk muscle labeled by myosin-heavy chain staining was also comparable between ctr MO embryos and irs1 MO embryos under hypoxia. Only a minor cell death rate was found in the anterior trunk in the irs1 MO-injected embryo (Supplemental Fig. 4B). Figure 5. View largeDownload slide Knockdown of Irs1 led to increased cell death in NCCs under hypoxia. (A) Cell death and dlx2 expression pattern under hypoxia. (a) The lateral view. (b) The dorsal view of the anterior region of embryos. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 250 μm. (B) Quantitative RT-PCR analysis of NCC marker genes in irs1 MO embryos under hypoxia. Each transcript level was normalized to β-actin expression level, and the values were represented as relative abundance to the value in ctr MO embryos. Data are shown as mean ± standard deviation, two to four independent experiments. (C) Whole-mount double-label immunostaining results. The area of fish body is traced by the dashed line. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 125 μm. (D) Double-label immunostaining of section specimens. The cross sections of NCC-enriched dorsal regions were prepared, and the immunostaining was conducted on glass slides. The emergency rate of active caspase-3‒positive Sox10-labeled cells was analyzed. Data are mean ± standard deviation; n = 4 to 12. The area of fish body is traced by the dashed line. Scale bar = 25 μm. (E) Effects of irs1 knockdown on the phosphorylation level of Akt in NCCs. Embryos were subjected to immunostaining using denoted antibodies. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. (F) Whole-mount double-label immunostaining of active caspase-3 and Sox10 of hypoxia-treated irs1 MO embryos with or without coinjection of myrAkt RNA. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. *P < 0.05; **P < 0.01. A, anterior; D, dorsal; L, left; Nt, neural tube; OtV, otic vesicle; P, posterior; R, right; V, ventral; Yk, yolk. Figure 5. View largeDownload slide Knockdown of Irs1 led to increased cell death in NCCs under hypoxia. (A) Cell death and dlx2 expression pattern under hypoxia. (a) The lateral view. (b) The dorsal view of the anterior region of embryos. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 250 μm. (B) Quantitative RT-PCR analysis of NCC marker genes in irs1 MO embryos under hypoxia. Each transcript level was normalized to β-actin expression level, and the values were represented as relative abundance to the value in ctr MO embryos. Data are shown as mean ± standard deviation, two to four independent experiments. (C) Whole-mount double-label immunostaining results. The area of fish body is traced by the dashed line. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 125 μm. (D) Double-label immunostaining of section specimens. The cross sections of NCC-enriched dorsal regions were prepared, and the immunostaining was conducted on glass slides. The emergency rate of active caspase-3‒positive Sox10-labeled cells was analyzed. Data are mean ± standard deviation; n = 4 to 12. The area of fish body is traced by the dashed line. Scale bar = 25 μm. (E) Effects of irs1 knockdown on the phosphorylation level of Akt in NCCs. Embryos were subjected to immunostaining using denoted antibodies. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. (F) Whole-mount double-label immunostaining of active caspase-3 and Sox10 of hypoxia-treated irs1 MO embryos with or without coinjection of myrAkt RNA. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. *P < 0.05; **P < 0.01. A, anterior; D, dorsal; L, left; Nt, neural tube; OtV, otic vesicle; P, posterior; R, right; V, ventral; Yk, yolk. Irs1 was required for maintaining the neural crest cell population under hypoxic conditions The dlx2 gene is expressed in the mesenchyme of developing pharyngula arches, and these cells were derived from neural crest cells (NCCs). We then analyzed the expression of several NCC-specific genes (crestin, sox10, sox9b, foxd3, tfap2a, mitfa, ngfr, and pdgfrb) and found that they were prone to decrease in irs1 MO embryos under hypoxia (Fig. 5B). Furthermore, Sox10 and active caspase-3 double-positive signals in irs1 MO embryos were prominent under hypoxia, which was more noticeable than under Norm conditions (Fig. 5C; Supplemental Fig. 5A). Indeed, the immunohistochemistry analysis of the cryosections revealed that the emergence of active caspase-3‒positive cells within the Sox10-positive NCCs was significantly increased in the irs1 MO-injected embryos under prolonged hypoxia compared with that of ctr MO-injected embryos and with that of the irs1 MO-injected embryos under constant normoxia (Fig. 5D; Supplemental Fig. 5B). To test the IIS activation level, the approximately stage-matched embryos under constant normoxia and hypoxia were used for immunostainings of phospho-Akt and Sox10 (Fig. 5E). Under normoxic conditions, the phospho-Akt signal was slightly reduced in Sox10-positive cells of irs1 MO embryos. On the other hand, under prolonged hypoxic conditions, the phosphorylation level of Akt was clearly diminished in Sox10-positive cells by irs1 MO (Fig. 5E; hypoxia, irs1 MO). Importantly, the forced-activation of Akt-signaling by coinjection of myrAkt RNA prevented this (Fig. 5F), suggesting that the reduced Irs1-Akt signaling was deleterious for NCCs under hypoxia. NCCs played indispensable roles in embryonic catch-up growth Because NCCs play considerable roles in embryonic development and because the loss of Irs1 induces massive cell death in NCCs under hypoxia and in the failed catch-up growth phenotype under Reoxy conditions, the importance of NCCs for embryonic catch-up growth was further examined by using small-molecular-weight chemicals and knockdown of genes whose expression is required for NCC development. Wild-type embryos pretreated with leflunomide (Fig. 6A), a potent inhibitor of neural crest stem cell renewal (27), did not change the growth level under hypoxia, but the growth acceleration during Reoxy conditions was significantly hindered (Fig. 6B and 6C). In addition, knockdown of prdm1a and sox10, both of which are required for NCC development (28), clearly blunted growth acceleration in Reoxy (Fig. 6D). Figure 6. View largeDownload slide Loss of the NCC population inhibited catch-up growth, and suppression of cell-death rescued catch-up growth of irs1 MO embryos. (A) Schematic illustration of the mode of action of leflunomide and the outline of the experiment. (B) Changes in head-trunk angles of embryos treated with leflunomide. Data are mean ± standard deviation; n = 8 to 19. (C) Relative growth rate of drug-treated embryos during the indicated developmental periods. Data are mean ± standard deviation, three independent experiments.(D) Relative growth rate of MO-injected embryos. Data are mean ± standard deviation, three independent experiments. (E) Outline of the zVADfmk experiment. (F) Representative result of TUNEL staining. The prominent TUNEL signals (red) are indicated by arrows. The specimens were counterstained with Hoechst33342 (blue). The area of fish body is traced by the dashed line. Scale bar = 250 μm. (G) Relative growth rate of zVADfmk-treated embryos during reoxygenation periods. Data are mean ± standard deviation, two independent experiments. *P < 0.05. DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide. Figure 6. View largeDownload slide Loss of the NCC population inhibited catch-up growth, and suppression of cell-death rescued catch-up growth of irs1 MO embryos. (A) Schematic illustration of the mode of action of leflunomide and the outline of the experiment. (B) Changes in head-trunk angles of embryos treated with leflunomide. Data are mean ± standard deviation; n = 8 to 19. (C) Relative growth rate of drug-treated embryos during the indicated developmental periods. Data are mean ± standard deviation, three independent experiments.(D) Relative growth rate of MO-injected embryos. Data are mean ± standard deviation, three independent experiments. (E) Outline of the zVADfmk experiment. (F) Representative result of TUNEL staining. The prominent TUNEL signals (red) are indicated by arrows. The specimens were counterstained with Hoechst33342 (blue). The area of fish body is traced by the dashed line. Scale bar = 250 μm. (G) Relative growth rate of zVADfmk-treated embryos during reoxygenation periods. Data are mean ± standard deviation, two independent experiments. *P < 0.05. DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide. To test whether the loss of NCCs under hypoxia is a reason for the failed catch-up growth by irs1 knockdown, MO-injected embryos were treated with the pan-caspase inhibitor zVADfmk (Fig. 6E). The zVADfmk treatment of irs1 MO embryos resulted in clear reduction of TUNEL-positive apoptotic cells (Fig. 6F) and significant growth regain under Reoxy conditions (Fig. 6G). Discussion Irs1 is known as a key molecule for animal growth and development in the rodent model (29). Despite the evolutionarily conserved IIS pathway from worms to primates, one of the most crucial nodes in this pathway, the receptor substrates, was less extensively studied in nonmammalian vertebrates (30). In this study, zebrafish Irs1 was identified and first characterized as a functional Igf1r substrate (Fig. 1) Also, both embryonic and adult expression profiles of the irs1 gene in the fish model were first investigated (Fig. 2). The developmental expression of the zebrafish irs1 gene is especially key for embryonic growth, because we found that the loss of irs1 expression significantly blunted growth rate (Fig. 3D and 3F). This demonstrated that a nonmammalian Irs1 is evidently tyrosine phosphorylated in an Igf1r activity-dependent manner and is required for body growth in the developing animal. The major finding in this study is that irs1 is indispensable for catch-up. Blockade of irs1 expression markedly inhibited catch-up growth. Unexpectedly, however, it did not cause any obvious changes in activation levels of Akt and Erk1/2 during catch-up growth (Fig. 4B; 48-hpf Reoxy); instead, both Akt and Erk1/2 signals in the earlier stage (Fig. 4B; 8-hpf Norm) were greatly diminished by Irs1 MO. Also, hypoxia reduced IIS, and the loss of irs1 expression further reduced it (Fig. 4B; 36-hpf Hypo). Once environmental oxygen is reduced, the hypoxia-inducible Igfbp1 systemically limits Igf action (20, 31), and it is one of the reasons for IUGR (20). Even in such cases, however, “minimal” IIS must be maintained to secure the survival of important cellular populations via increasing cellular sensitivity to limited Igf-ligand, which likely occurred at the level of Irs1 in the current study. Indeed, the phospho-Akt signal was weaker but still detectable in stunted embryos under hypoxia and was further reduced by the loss of irs1 expression. Moreover, we found increased irs1 expression under hypoxia (data not shown). In a mammalian cell culture model, reduced IIS increased the IRS level, but increased IIS conversely reduced it (14), which led to the efficient use of a limited amount of activated receptor to maintain basal IIS or prevent the overactivation of IIS. Given that the zebrafish Irs1 is an important Ir/Igf1r substrate (Fig. 1D) and IIS confers cell survival, the loss of Irs1-mediated IIS would reduce cellular viability, and further hypoxia-induced Igfbp1-mediated attenuation of IIS may aggravate reduced viability of embryonic cells. The active caspase-3‒positive cells were more pronounced after irs1 knockdown under hypoxia than under constant normoxia, suggesting that Irs1 is indispensable for the survival of certain sets of cells under hypoxia. Massive apoptosis occurred in the anterior dorsal domain, and dlx2 expression was reduced in hypoxia-exposed irs1 MO-injected embryos (Fig. 5A). The dlx2 expresses in the mesenchyme of developing pharyngula arches, and these cells were derived from the anterior population of the NCCs (32). Therefore, we inferentially focused on changes in NCCs in this study. Accordingly, clear reduction of NCC-marker gene expression and increased cell death in Sox10-positive cells in the anterior region of embryos were found in irs1 MO-injected embryos under hypoxia (Fig. 5B‒5D). These data indicated the NCCs were impaired in Irs1 knocked-down embryos under hypoxic conditions. Indeed, IIS mediates survival and proliferation of stem cells including NCCs (33–36), and the forced activation of Akt in irs1 MO-injected embryos considerably restored NCC viability under hypoxia (Fig. 5F). Because IIS is known to be a key requirement for the culture of stem cells including NCCs (35), our results strongly suggest that Irs1 maintains survival of NCCs during hypoxia, which leads to reoxygenation-induced catch-up growth. Because Irs1 mRNA was ubiquitously detected (Fig. 2), it is still uncertain whether Irs1 guaranteed NCC survival in a “cell-autonomous” or “non‒cell-autonomous” manner. NCC-specific control of IIS is needed to interpret this issue in future studies. Embryos subjected to the chemical or genetic depletion of NCCs failed to show catch-up growth without modifying Irs1 expression (Fig. 6A‒6D). Conversely, the antiapoptotic chemical zVADfmk, which potentially blocked apoptotic loss of NCCs under hypoxic conditions, significantly restored catch-up growth in irs1 MO-injected embryos (Fig. 6G). In agreement with the facts that (1) the Irs1 knockdown increased cell death in NCCs under hypoxia and (2) the loss of NCCs led to blunted catch-up growth in reoxygenated conditions, data from the zVADfmk experiment suggest that Irs1 is required for the maintenance or survival of NCCs under severe hypoxic stress conditions, and the cells are key for future achievement of catch-up growth after stressor remits. The NCCs become various types of cells, such as cartilage, cranial bones, connective tissues, muscles, glia, neurons, and pigment cells (37, 38). It is likely that the NCCs and their derivatives change their behaviors or fates in response to the severe stressors, and this may influence body growth after the removal of stressors. The molecular and cellular changes in NCCs during the stunting and catch-up periods need to be investigated in the future. In conclusion, we found Irs1 plays a pivotal role in mediating limited IIS in early stages of embryos [Fig. 7(①)]. The Irs1-mediated IIS, such as phosphoinositide 3-kinase‒Akt signaling, is essential for NCC survival, especially under severely hypoxic conditions [Fig. 7(②)]. The NCCs are crucial for the current fish model of catch-up growth, though the clarification of its role(s) requires further research [Fig. 7(③)]. Obesity, cardiovascular diseases, insulin resistance, delayed mental development, and other unfavorable physiological outcomes are often found in individuals who experienced severe growth delay and catch-up growth in early stages of life. Currently unknown molecular and cellular changes in NCCs during IUGR/catch-up growth, which are not necessarily responsible for the change in apparent body growth, may provide an important clue for deciphering the underlying mechanisms of such pathogenesis. Figure 7. View largeDownload slide A proposed model. The Irs1-mediated IIS in developing embryos is important for NCC survival under severe hypoxia (①, ②), and the NCCs are indispensable for reoxygenation-induced catch-up growth (③). Figure 7. View largeDownload slide A proposed model. The Irs1-mediated IIS in developing embryos is important for NCC survival under severe hypoxia (①, ②), and the NCCs are indispensable for reoxygenation-induced catch-up growth (③). Abbreviations: Abbreviations: cDNA complementary DNA hpf hours postfertilization HTA head-trunk angle Hypo embryos transferred to hypoxia from 24 to 36 hours postfertilization Igf insulinlike growth factor Igf1r type 1 Igf-receptor Igfbp1 inhibitory IGF-binding protein IIS insulin/insulinlike growth factor signaling Ir insulin receptor Irs insulin receptor substrate IUGR intrauterine growth restriction MO morpholino oligo mRNA messenger RNA myrAkt N-terminal myristoylation signal–attached mouse Akt1 NCC neural crest cell Norm constant normoxia O.C.T. optimal cutting temperature PBS phosphate-buffered saline PTB phosphotyrosine binding RT-PCR reverse transcription polymerase chain reaction TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling Acknowledgments We thank Drs. Daisuke Yamanaka and Takashi Shibano (The University of Tokyo) for invaluable discussions and Dr. Susan Hall (The University of North Carolina at Chapel Hill) for proofreading. Financial Support: This work was partially supported by Grants-in-Aid (A)(2) 16208028, (A) 22248030, (A) 25252047, and (S) 25221204l; Challenging Exploratory Research Grants 20658065, 25660210, and 15K14840l; the core- to-core program from the Japan Society for the Promotion of Science (JSPS) and Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (25006A) (to S.-I.T.), and Grant-in-Aid (B) 243801521 (to F.H.). This work was also partially supported by Grants-in-Aid for Young Scientists (Start-up) 23880008, (B) 25850220, and (B) 15K18799 (to H.K.) from JSPS. H.K. was supported by National Institute of Genetics, Cooperative Research Program Grants 2012-B11, 2013-A28, and 2014-A26. Disclosure Summary: The authors have nothing to disclose. References 1. Saenger P, Czernichow P, Hughes I, Reiter EO. Small for gestational age: short stature and beyond. Endocr Rev . 2007; 28( 2): 219– 251. Google Scholar CrossRef Search ADS PubMed  2. 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Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling

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Endocrine Society
Copyright
Copyright © 2018 Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
D.O.I.
10.1210/en.2017-00847
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Abstract

Abstract Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as “catch-up” growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1–mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia. Fetal growth and developmental timing are cooperatively defined by genetic and epigenetic factors. Inadequacies of some conditions lead to developmental arrest or intrauterine growth restriction (IUGR) in human fetuses (1). Intriguingly, once an unfavorable condition is removed, most stunted animals restart growth with an accelerated progression rate, which is referred to as catch-up growth (2–4). Although catch-up growth is important for the accidentally stunted animal to regain its size and compete with nonstunted ones, it may not always be beneficial in humans, as recent epidemiological data showed that catch-up growth after IUGR was often associated with adult-onset disorders or unfavorable outcomes during growth (1, 5, 6). Because changes in such an anomalistic growth pattern seem to be a key for deciphering the cause of future pathogenesis of infants with IUGR, we especially need to increase our understanding of the molecular and cellular bases of the catch-up phenomenon. Insulin/insulinlike growth factor (Igf) signaling (IIS) is a major hormonal pathway facilitating embryonic growth in a wide range of metazoans (7–9). The insulin and Igf ligands bind to the insulin receptor (Ir) or type 1 Igf receptor (Igf1r), which triggers activation of the receptor tyrosine kinases and subsequent tyrosine phosphorylation of specific substrates, the Ir substrates (Irs; Irs1 through Irs4) (10). Among the multiple irs genes, irs1 is known to be uniquely responsible for normal growth and development in studies using knockout mouse models (11), and the signaling cascade activates a number of cellular behaviors (i.e., cell survival, proliferation, differentiation, and migration) via several major downstream pathways, such as the phosphoinositide 3-kinase‒Akt pathway and Ras-Raf-Mek-Erk1/2 pathway (8). Importantly, Irs is known to be very sensitive to variable physiological/environmental conditions, and the Irs proteins tend to fluctuate their expression levels (and to coordinate IIS activities) according to the confronting situation or immediate stressors (12–15). These facts imply that irs1 may be important for consolidating animal growth and development not only under undisturbed conditions but also under abnormal or variable circumstances. Hypoxia is a major external factor causing IUGR in the human fetus (16). Pregnancy at higher altitudes often induces relative hypoxic intrauterine conditions and results in risks of miscarriage or small-for-gestational-age fetuses (17). Intrauterine hypoxia may also be induced by compression/coiling of the umbilical cord or abnormal placental functions (18, 19); however, these risk factors are always eliminated after birth, and these infants are likely to take a catch-up trajectory. It is thought that hypoxia-induced growth retardation is in part due to the blunted Igf action through the hypoxia-induced overexpression of an inhibitory IGF-binding protein (Igfbp1) in the fetus (9). Previous studies have shown that the Igfbp1-mediated hypoxic response is found not only in humans but also in fish models (20). Despite the strong link between oxygen supply and IIS-dependent embryonic growth, the molecular and cellular bases of hypoxia-/reoxygenation-induced growth alteration remains largely elusive. Studies using rodent models present technical complexities for observing intrauterine specimens and conducting hypoxia experiments. Zebrafish are especially well suited for studying relationships between oxygen conditions and embryonic growth because of their fast and conserved development, the availability of established experimental methods, and the ease of manipulating environmental oxygen levels independent from the mother organism (3, 21). In zebrafish embryos, hypoxia caused growth delay, but subsequent reoxygenation induced catch-up growth (3). In this study, we investigated the role of Irs1-mediated growth signaling in hypoxia-/reoxygenation-induced catch-up growth using the zebrafish model. Materials and Methods Chemicals and reagents Chemicals and reagents were purchased from Wako (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan) unless noted otherwise. DNA ligase and restriction endonucleases were purchased from Promega (Madison, WI). For reverse transcription polymerase chain reaction (RT-PCR), Trizol reagent, M-MLV reverse transcription, and oligonucleotide primers were purchased from Invitrogen Life Technologies (Carlsbad, CA). The translation block antisense-morpholino oligo (MO) was purchased from Gene Tools, LLC (Philomath, OR). Experimental animals Adult wild-type zebrafish (Danio rerio) were maintained at ∼28°C on a 14-hour light: 10-hour dark cycle and fed twice daily. Embryos were generated from natural crosses, and embryos were raised in standard rearing solution at 28.5°C. At the time of use, embryos were anesthetized in tricaine mesylate (ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich Japan, Tokyo, Japan), and all experiments were conducted in accordance with guidelines approved by the Graduate School of Agriculture and Life Sciences at The University of Tokyo and the Guide for the Care and Use of Laboratory Animals prepared by Kanazawa University. RT-PCR Total RNA was isolated from adult fish and embryos. After DNase treatment, 2.5 μg total RNA was reverse-transcribed to single-strand complementary DNA (cDNA) using an engineered M-MLV reverse transcriptase (SuperScript™ II Reverse Transcriptase, Invitrogen Life Technologies) following the manufacturer’s instructions. RT-PCR for zebrafish irs1 was performed with a set of primers described in Supplemental Table 1. The level of β-actin messenger RNA (mRNA) was also measured. Whole-mount in situ hybridization Embryos used for whole-mount in situ hybridization were raised in embryo-rearing solution with 0.003% (weight-to-volume ratio) 2-phenylthiourea to inhibit pigmentation. Partial cDNA fragment of zebrafish Irs1 (786 bps) was cloned and used for in vitro transcription with either T7- or T3-RNA polymerase to generate digoxigenin-labeled complementary RNA probes according to the manufacturer’s instructions. Other complementary RNA probes for various developmental marker genes were similarly prepared. Hybridization was carried out following the standard method (22). Images were captured using an Olympus SZ61 microscope (Tokyo, Japan) with a Canon iVIS HF M52 camera (Tokyo, Japan) or BZ9000 microscope (Keyence, Tokyo, Japan). Biochemical characterization of zebrafish Irs1 protein Recombinant FLAG-tagged zebrafish Irs1 and rat IRS1 proteins were expressed in human embryonic kidney 293T cells. Briefly, cells were cultured in high glucose Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and nonessential amino acids (Nissui, Tokyo, Japan). Cells were transfected with each expression plasmid by using polyethylenimine (Polyscience Inc., Warrington, PA). One day after transfection, cells were incubated with serum-free medium for 12 hours. One hour before IGF-I stimulation, the serum-starved cells were exposed to the low-molecular-weight compound inhibitor of IR/IGF-1R, BMS754807 (1.0 μM). Then, in the last 5 minutes, cells were treated with 100 ng/mL of IGF-I and collected in lysis buffer with protease inhibitor cocktails and phosphatase inhibitor mix. Cell lysate was subjected to immunoprecipitation as previously described (23). The protein concentration was quantified using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and subjected to immunoblotting. Microinjection experiment The translational block antisense MO against zebrafish Irs1 mRNA (irs1 MO: 5′-ACAGAAAAATTGCAGGATCGGAAGT-3′) and the standard control MO (ctr MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′) were designed and synthesized by Gene Tools, LLC. The previously validated antisense MOs against mRNAs for Prdm1a (prdm1a MO: 5′-TGGTGTCATACCTCTTTGGAGTCTG-3′) and Sox10 (sox10 MO: 5′-ATGCTGTGCTCCTCCGCCGACATCG-3′) were similarly prepared (24, 25). The zebrafish Irs1 5′-UTR cDNA was amplified and subcloned into pCS2+Venus plasmid, and the plasmid was used for in vitro mRNA transcription (mMessage mMachine; Ambion, Austin, TX) to prepare the capped Venus mRNA harboring the irs1 MO target sequence (5′-Irs1UTR-Venus). All MOs were injected into embryos at the same dose (4 ng/embryo). The full-length open reading frame of zebrafish Irs1 cDNA was amplified with high-fidelity polymerase (Pyrobest; TaKaRa, Shiga, Japan) and used for capped RNA synthesis. The capped RNAs encoding Venus, N terminus FLAG-tagged zebrafish Irs1, constitutive active-Akt [N-terminal myristoylation signal–attached mouse Akt1 (myrAkt)], and constitutively active-H-Ras (HRasV12) were also prepared using the mMessage mMachine kit. Capped RNAs for Venus (250 pg/embryo), FLAG-zebrafish Irs1 (1000 pg/embryo), myrAkt (20 pg/embryo), HRasV12 (5 pg/embryo), and MOs were injected into one- or two-cell stage embryos, and they were kept at 28.5°C until sampling. Hypoxia and reoxygenation Hypoxic water was prepared by bubbling pure nitrogen gas into the embryo-rearing solution. The oxygen concentration was measured using a dissolved oxygen meter (ProODO; YSI Nanotech Japan, Kawasaki, Japan). The dissolved oxygen content in the hypoxic water was set at 0.6 ± 0.2 mg/L O2 (6% to 10% oxygen content, as the oxygen level in the normoxic water is set at 100%: approximately 8.0 mg/L O2). In hypoxia/reoxygenation experiments, all embryos were kept under normoxia until 24 hours postfertilization (hpf). Embryos developed under constant normoxia were termed Norm. Embryos transferred to hypoxia from 24 to 36 hpf were termed Hypo. The embryos exposed to hypoxia from 24 to 36 hpf and then put back to normoxic water were termed Reoxy (hypoxia to normoxia). Growth-level measurement and relative growth rate calculation The growth level of an embryo was determined by measuring the head-trunk angle (HTA) (3, 20, 21). Growth rate was driven by the formula (dy/dt) = (yn−y0)/(tn−t0), with y0 = HTA at the initial time-point (t0) and yn = HTA at the end time point (tn). The growth rates were shown as relative values of the control group. Immunoblot analysis Immunoblot analyses were performed as previously described (3, 23). The antibodies used for the Akt and Erk1/2 western blottings were purchased from Cell Signaling Technology (CST-Japan, Tokyo). The antiphospho-Akt antibody (9271 for phosphorylation at Ser473) was used at a 1:500 dilution, and total-Akt (9272), antiphospho-Erk1/2 (9101 for phosphorylation at Thr202 and Tyr204), and total-Erk1/2 (137F5) were used at a 1:1000 dilution according to the instruction manuals. An equal amount of protein was used for the immunoblot analysis. Tubulin antibody (2148; CST-Japan) was purchased and used at indicated dilutions per manufacturer’s instruction. Whole-mount immunostaining Whole-mount immunostaining was conducted by using anti-zebrafish Sox10 (AS-55651s, AnaSpec), anti‒active caspase-3 (559565; BD PharmingenTM), antiphospho-Akt (S473) DE9 XP (4060; CST-Japan), and antimyosin heavy chain (05-716; Millipore, Darmstadt, Germany) according to the instruction manuals. For the double staining, either Alexa-488 or -546 conjugated second-antibody (Thermo Fisher Scientific, Pittsburgh, PA) was used. Alternatively, antibodies were biotinylated by EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions, and the biotinylated antibodies were detected by the Alexa-488 conjugated streptavidin (Streptavidin, Alexa Fluor™ 488 conjugate, Thermo Fisher Scientific). The Alexa-488‒conjugated cleaved-active caspase-3 (D175) antibody (9669S; CST-Japan) and the Alexa-488‒conjugated antiphospho-Akt antibody (S473) (4071S; CST-Japan) were also used for double-staining experiments (Table 1). Table 1. Antibodies Used Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Monoclonal or Polyclonal; Species Raised in  Dilution Used  RRID   phospho-Akt1/2/3    Phospho-Akt (Ser473) antibody  CST-Japan, Tokyo, 9271  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.  1:500 dilution  AB_329825  Akt1/2/3    Akt antibody  CST-Japan, Tokyo, 9272  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt.  1:1000 dilution  AB_329827  Phospho-p44/42 MAPK (Erk1/2)    Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody  CST-Japan, Tokyo, 9101  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.  1:1000 dilution  AB_331646  p44/42 MAPK (Erk1/2)    p44/42 MAPK (Erk1/2) (137F5) rabbit mAb  CST-Japan, Tokyo, 4695  Monoclonal antibody is produced by immunizing rabbit with a synthetic peptide corresponding to residues near the C-terminus of rat p44 MAP kinase.  1:1000 dilution  AB_390779  Tubulin    α/β-Tubulin antibody  CST-Japan, Tokyo, 2148  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the sequence of human α- and β-tubulin.  1:100 dilution  AB_2288042  Sox10    Anti-SOX-10 (IN), Z-FISH®  AnaSpec, Fremont, CA, AS-55651s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the intermediate region of zebrafish Sox10 protein (GenBank accession #NP_571950.1).  1:100 dilution  AB_10631841  Active caspase-3    Purified rabbit anti‒active caspase-3  BD Biosciences, San Jose, CA, 559565  Monoclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the human active caspase-3 fragment.  1:250 dilution  AB_397274  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb  CST-Japan, Tokyo, 4060  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:200 dilution  AB_2315049  Phospho-histone H3    Phospho-Histone H3 (Ser10) (D2C8) XP® rabbit mAb  CST-Japan, Tokyo, 3377P  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3.  1:500 dilution  AB_1549592  Active caspase-3    Cleaved caspase-3 (Asp175) antibody (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 9669s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3.  1:50 dilution  AB_2069869  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 4071s  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:50 dilution  AB_1031106  Myosin heavy chain    Anti-myosin heavy chain antibody, clone A4.1025  Millipore, Darmstadt, Germany, 05-716  Monoclonal antibody is produced by immunizing mouse.  1:100 dilution  AB_309930  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488  Thermo Fisher Scientific, Pittsburgh, PA, R37116  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2556544  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11035  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534093  Mouse IgG (H+L)    Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11003  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534089  Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Monoclonal or Polyclonal; Species Raised in  Dilution Used  RRID   phospho-Akt1/2/3    Phospho-Akt (Ser473) antibody  CST-Japan, Tokyo, 9271  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.  1:500 dilution  AB_329825  Akt1/2/3    Akt antibody  CST-Japan, Tokyo, 9272  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt.  1:1000 dilution  AB_329827  Phospho-p44/42 MAPK (Erk1/2)    Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody  CST-Japan, Tokyo, 9101  Polyclonal antibodies are produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.  1:1000 dilution  AB_331646  p44/42 MAPK (Erk1/2)    p44/42 MAPK (Erk1/2) (137F5) rabbit mAb  CST-Japan, Tokyo, 4695  Monoclonal antibody is produced by immunizing rabbit with a synthetic peptide corresponding to residues near the C-terminus of rat p44 MAP kinase.  1:1000 dilution  AB_390779  Tubulin    α/β-Tubulin antibody  CST-Japan, Tokyo, 2148  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to the sequence of human α- and β-tubulin.  1:100 dilution  AB_2288042  Sox10    Anti-SOX-10 (IN), Z-FISH®  AnaSpec, Fremont, CA, AS-55651s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the intermediate region of zebrafish Sox10 protein (GenBank accession #NP_571950.1).  1:100 dilution  AB_10631841  Active caspase-3    Purified rabbit anti‒active caspase-3  BD Biosciences, San Jose, CA, 559565  Monoclonal antibodies are produced by immunizing rabbit with a synthetic peptide of the human active caspase-3 fragment.  1:250 dilution  AB_397274  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb  CST-Japan, Tokyo, 4060  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:200 dilution  AB_2315049  Phospho-histone H3    Phospho-Histone H3 (Ser10) (D2C8) XP® rabbit mAb  CST-Japan, Tokyo, 3377P  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3.  1:500 dilution  AB_1549592  Active caspase-3    Cleaved caspase-3 (Asp175) antibody (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 9669s  Polyclonal antibodies are produced by immunizing rabbit with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3.  1:50 dilution  AB_2069869  Phospho-Akt 1/2/3    Phospho-Akt (Ser473) (D9E) XP® rabbit mAb (Alexa Fluor® 488 Conjugate)  CST-Japan, Tokyo, 4071s  Monoclonal antibody is produced by immunizing rabbit with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.  1:50 dilution  AB_1031106  Myosin heavy chain    Anti-myosin heavy chain antibody, clone A4.1025  Millipore, Darmstadt, Germany, 05-716  Monoclonal antibody is produced by immunizing mouse.  1:100 dilution  AB_309930  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488  Thermo Fisher Scientific, Pittsburgh, PA, R37116  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2556544  Rabbit IgG (H+L)    Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11035  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534093  Mouse IgG (H+L)    Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546  Thermo Fisher Scientific, Pittsburgh, PA, A-11003  Polyclonal antibodies are produced by immunizing goat.  1:250 dilution  AB_2534089  Abbreviations: H+L, heavy and light chain; IgG, immunoglobulin G; mAb, monoclonal antibody; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; RRID, Research Resource Identifier. View Large Immunohistochemistry of cryosections The embryos fixed in 4% paraformaldehyde were rinsed and immersed in 30% sucrose at 4°C overnight. Then, the 30% sucrose-immersed embryos were transferred to the optimal cutting temperature (O.C.T.) compound/30% sucrose mixture (O.C.T compound: 30% sucrose = 2:1) at 4°C for another overnight. The specimens were embedded in O.C.T. compound and solidified on dry ice. The 20-μm-thick cryosections were prepared with cryostat (HM505N, Zeiss-MICROM; Microedge Instruments, Osaka, Japan). The sections were attached to the MAS-coated glass-slides (MATSUNAMI, Osaka, Japan) and subjected to immunohistochemistry procedure using antizebrafish Sox10 antibody and anti‒active caspase-3 antibody as described in the whole-mount immunostaining. The immunostained sections were counterstained with Hoechst33342, and the fluorescent images were pictured. The immunopositive cells were counted by using ImageJ software. Whole-mount terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining Whole-mount terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit, TMR red (Sigma-Aldrich Japan) according to the instruction manual. The embryos fixed by 4% paraformaldehyde were dehydrated in a graded ethanol series (50%, 70%, 90%, 100%) and permeabilized by acetone at −20°C for 10 minutes. The embryos were rinsed in phosphate-buffered saline (PBS)(-) and the embryos were further permeabilized by 0.1% Triton X-100, 0.1% sodium citrate in PBS(-) for 15 minutes at room temperature. After two rinses in PBS(-), the specimens were incubated with the labeling solution of the In Situ Cell Death Detection Kit at 37°C for 1 hour. After the TUNEL-labeling reaction, all samples were counterstained with Hoechst33342. Quantitative RT-PCR Quantitative RT-PCR was performed using SYBR® Green Realtime PCR Master Mix-Plus (Toyobo, New York, NY) and Applied Biosystems Step One™ Real-time PCR system (Applied Biosystems, Japan, Tokyo) as described previously (3, 26). Primers used are listed in Supplemental Table 1. The specificity of the PCR was verified by denaturing curve analysis, and the PCR products were analyzed by electrophoresis to determine whether a single product predominated. Small molecular compounds The chemical inhibitor for IR/IGF-1R kinase domains, BMS754807 (CAS#1001350-96-4), was obtained from Selleck Chemicals (Boston, MA) and used at 2.0 μM. Leflunomide (CAS#75706-12-6), a potent inhibitor of neural crest stem cell proliferation (27), was purchased from Sigma-Aldrich (St. Louis, MO) and used at 1.0 μM. General caspase inhibitor, zVADfmk, was purchased from BD Biosciences and used at 50 μM. Statistics Statistical significance between two groups was analyzed by the Student t test. Significance among multiple groups was determined by one-way analysis of variance, followed by the Tukey multiple comparison tests. Calculations were performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA), and significance was accepted at P < 0.05. Results Identification and functional characterization of zebrafish Irs1 First, we characterized the structure and biochemical features of zebrafish Irs1. We found a single irs1 gene (ENSDARG00000054087) in the zebrafish database (GRCz9) in Ensembl (http://asia.ensembl.org/Danio_rerio/Info/Index). The domain structure of the deduced zebrafish Irs1 protein in comparison with human IRS1 is shown in Fig. 1A. As found in IRS proteins in other species, the deduced primary sequence of zebrafish Irs1 contains a highly conserved pleckstrin-homology domain (residues 16 to 112) and phosphotyrosine-binding (PTB) domain (residues 142 to 246). The Ser-rich region is adjacent to the PTB domain in zebrafish Irs1, and the sequence is also highly conserved in IRS1 in other species (Fig. 1B). Although the Ser-rich region sequence is also found in IRS2, it is not positioned in the same region as in Irs1/IRS1 (next to the PTB domain in Irs1/IRS1, the C-terminal region in IRS2). To assess if this zebrafish Irs was correctly classified as Irs1, we further performed phylogenetic analysis using full-length amino acid sequences. As a result, the zebrafish Irs1 was grouped into the IRS1 clade with high confidence (Fig. 1C). Second, the full-length zebrafish Irs1 coding sequence was overexpressed in human embryonic kidney 293T cells as an N-terminal FLAG-tagged protein. The recombinant protein was detected as an approximately 130- to 140-kDa protein in immunoblot analysis (Fig. 1D; lysate, IB: FLAG). Under serum-starved conditions, the expressed zebrafish Irs1 showed a basal level of tyrosine phosphorylation, and this was greatly increased on IGF-I stimulation (IGF-I, 5 minutes). Importantly, IGF-I‒induced tyrosine phosphorylation was impaired by the specific IR/IGF-1R inhibitor (BMS-754807), demonstrating that the IGF1R-dependent tyrosine phosphorylation of Irs1 (Fig. 1D; IP, IB: FLAG or 4G10). Comparable results were obtained in the control experiments expressing rat IRS1. These data indicate that the identified zebrafish Irs1 encodes a functional Igf1r substrate. Figure 1. View largeDownload slide Identification and characterization of zebrafish Irs1. (A) A schematic illustration comparing major domains and motifs in zebrafish Irs1 and human IRS1. Pleckstrin homology (PH) domain, PTB domain, and serine-rich region are shown. (B) Amino acid identities of conserved domains and regions between zebrafish Irs1 and IRS1 in other vertebrate species. (C) Phylogenetic tree of the IRS proteins. Amino acid sequences of full-length IRSs were analyzed using the neighbor-joining method. The rooted phylogenetic tree with branch lengths is shown. (D) Functional characterization of zebrafish Irs1 in human embryonic kidney 293T cells. FLAG-tagged rat IRS1 and zebrafish Irs1 were expressed, and the cells were treated with or without the specific IR/IGF-1R tyrosine kinase inhibitor BMS-754807 and were stimulated with IGF-I (100 ng/mL) for 5 minutes. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed using denoted antibodies. Figure 1. View largeDownload slide Identification and characterization of zebrafish Irs1. (A) A schematic illustration comparing major domains and motifs in zebrafish Irs1 and human IRS1. Pleckstrin homology (PH) domain, PTB domain, and serine-rich region are shown. (B) Amino acid identities of conserved domains and regions between zebrafish Irs1 and IRS1 in other vertebrate species. (C) Phylogenetic tree of the IRS proteins. Amino acid sequences of full-length IRSs were analyzed using the neighbor-joining method. The rooted phylogenetic tree with branch lengths is shown. (D) Functional characterization of zebrafish Irs1 in human embryonic kidney 293T cells. FLAG-tagged rat IRS1 and zebrafish Irs1 were expressed, and the cells were treated with or without the specific IR/IGF-1R tyrosine kinase inhibitor BMS-754807 and were stimulated with IGF-I (100 ng/mL) for 5 minutes. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed using denoted antibodies. Temporal and spatial expression of the irs1 gene RT-PCR analysis showed that zebrafish Irs1 mRNA was expressed throughout embryogenesis, suggesting it is a maternally deposited transcript (Fig. 2A). In agreement with the RT-PCR data, Irs1 mRNA was indeed detected throughout embryogenesis by whole-mounted in situ hybridization analysis (Fig. 2B, a‒h). The Irs1 mRNA was expressed in a broad range of developing tissues, including brain (forebrain, midbrain, hindbrain), eye, mandibular arch, and trunk muscle (somite) in pharyngula-stage embryos (Fig. 2B, c and d) at 24 hpf. The hybridization signal was specific, as indicated by the lack of intense signals using the corresponding sense RNA probe in the same batch of embryos (Fig. 2B, c′ and d′). In advanced stages (at 2 to 10 days after fertilization), the predominant expression domains were brain, pectoral fin, pharyngeal arch, thyroid primordium, sensory hair cells, olfactory bulb, and gill arch, with a reduced but still detectable expression in brain and trunk muscle (Fig. 2B, e‒h). In adult fish, Irs1 mRNA was detected in most tissues examined in both males and females, and basically no obvious sex difference was found, except the male liver had relatively lower expression levels (Fig. 2C). Figure 2. View largeDownload slide Temporal and spatial expression patterns of zebrafish irs1. (A) RT-PCR analysis of irs1 expression during zebrafish embryogenesis. Developmental stages are shown at the top in hpf. (B) Whole-mount in situ hybridization analysis. (a) Embryos of two-cell stage, (b) 50% to 75% epiboly, (c, d, c′, and d′) pharyngula stage, (e) hatching (48 hpf), and (f‒h) hatched (72 to 240 hpf) embryos were analyzed. Shown in (a)–(h) are embryos probed with the antisense riboprobe. In c′ and d′, embryos probed with sense riboprobe are shown. a‒g, c′, and d′ are lateral views with the head to the left, and h is a ventral view of the embryo in g. Irs1 mRNA signals were detected in various developing cells and tissues, and arrowheads indicate typical domains with intense signals. The chronological age of each sample is shown in the upper right corner of the panel. Scale bar = 100 μm. (C) RT-PCR analysis of irs1 in adult tissues. Three males or females were used to prepare total RNA, and the pooled RNA from each tissue was used to prepare cDNA for RT-PCR analysis. Ba, branchial (gill) arch; Ey, eye; Fb, forebrain; Hb, hindbrain; Hc, lateral sensory hair cell; Ma, mandibular arch; Mb, midbrain; Ob, olfactory bulb; Pa, pharyngeal arch; Pf, pectoral fin; Sc, anterior sensory hair cell; Sm, somite (trunk muscle); Tp, thyroid primordia. Figure 2. View largeDownload slide Temporal and spatial expression patterns of zebrafish irs1. (A) RT-PCR analysis of irs1 expression during zebrafish embryogenesis. Developmental stages are shown at the top in hpf. (B) Whole-mount in situ hybridization analysis. (a) Embryos of two-cell stage, (b) 50% to 75% epiboly, (c, d, c′, and d′) pharyngula stage, (e) hatching (48 hpf), and (f‒h) hatched (72 to 240 hpf) embryos were analyzed. Shown in (a)–(h) are embryos probed with the antisense riboprobe. In c′ and d′, embryos probed with sense riboprobe are shown. a‒g, c′, and d′ are lateral views with the head to the left, and h is a ventral view of the embryo in g. Irs1 mRNA signals were detected in various developing cells and tissues, and arrowheads indicate typical domains with intense signals. The chronological age of each sample is shown in the upper right corner of the panel. Scale bar = 100 μm. (C) RT-PCR analysis of irs1 in adult tissues. Three males or females were used to prepare total RNA, and the pooled RNA from each tissue was used to prepare cDNA for RT-PCR analysis. Ba, branchial (gill) arch; Ey, eye; Fb, forebrain; Hb, hindbrain; Hc, lateral sensory hair cell; Ma, mandibular arch; Mb, midbrain; Ob, olfactory bulb; Pa, pharyngeal arch; Pf, pectoral fin; Sc, anterior sensory hair cell; Sm, somite (trunk muscle); Tp, thyroid primordia. Loss of zebrafish irs1 expression blunted reoxygenation-induced catch-up growth Loss-of-function experiments using antisense MO were then conducted. The validation results of MO are shown in Supplemental Fig. 1. The levels of embryonic growth were measured by the head-trunk angle (HTA) (Fig. 3A), which is highly correlated with body length. Representative growth patterns under different oxygen conditions and the experimental design are shown in Fig. 3B and 3C. Changes in HTA and representative embryos are shown in Fig. 3D and 3E, respectively. Under constant normoxia (Norm), the irs1 MO embryos showed moderate growth delay (Fig. 3D and 3E; Norm). Under hypoxia, the growth of both irs1 MO embryos and ctr MO embryos was similarly slowed down (Fig. 3D and 3E; hypoxia group at 36 hpf). Upon returning to normoxia from hypoxia (Reoxy), the ctr MO embryos showed robust acceleration of their growth to catch up with the Norm embryos; however, the irs1 MO embryos failed to do so (Fig. 3D and 3E; Reoxy). The loss of Irs1 expression significantly reduced growth rate in the Reoxy period, and the reduction was greater than that in the Norm period (Fig. 3F). Figure 3. View largeDownload slide Irs1 was required for reoxygenation-induced catch-up growth in the zebrafish embryo. Schematic illustrations of (A) the head-trunk angle, (B) representative growth patterns, and (C) experimental design. (D) Changes in head-trunk angle. Data are mean ± standard deviation; n = 9 to 23. (E) Representative embryos of Norm, Hypo, and Reoxy. The head-trunk angles are shown. Scale bar = 500 μm. (F) Analysis of relative growth rates during indicated developmental periods. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Figure 3. View largeDownload slide Irs1 was required for reoxygenation-induced catch-up growth in the zebrafish embryo. Schematic illustrations of (A) the head-trunk angle, (B) representative growth patterns, and (C) experimental design. (D) Changes in head-trunk angle. Data are mean ± standard deviation; n = 9 to 23. (E) Representative embryos of Norm, Hypo, and Reoxy. The head-trunk angles are shown. Scale bar = 500 μm. (F) Analysis of relative growth rates during indicated developmental periods. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Disturbed irs1 expression significantly blunted catch-up growth with attenuated IIS not during but before the catch-up period Next, the activation of IIS was tested. Representative growth patterns and sampling timings are shown in Fig. 4A. The loss of Irs1 expression attenuated phosphorylation levels of both Akt and Erk1/2 in prepharyngula embryos (8 to 12 hpf) before hypoxic exposure and in later pharyngula embryos under hypoxia (36 hpf). In contrast, in more advanced developmental stages, the phosphorylation levels of Akt and Erk1/2 were hardly changed by irs1 MO under both Norm (Norm at 36 to 38 hpf) and Reoxy (Reoxy at 44 to 48 hpf) conditions (Fig. 4B). These data demonstrate that Irs1 is responsible for maintaining IIS during very early stages of development and during severe hypoxic conditions in advance of catch-up growth in the current experiment, implying the importance of early embryonic IIS for later catch-up growth. Figure 4. View largeDownload slide Irs1-mediated insulin/Igf signaling was sufficient for catch-up growth. (A) Schematic illustration of experimental design; the sampling timing is indicated by red circles. (B) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2. (C) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2 of embryos at the prepharyngula stage under normoxia (12 hpf) and the pharyngula stage under hypoxia (36 hpf). (D) Changes in head-trunk angle in the indicated groups. Data are mean ± standard deviation; n = 8 to 20. (E) Relative growth rate. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Figure 4. View largeDownload slide Irs1-mediated insulin/Igf signaling was sufficient for catch-up growth. (A) Schematic illustration of experimental design; the sampling timing is indicated by red circles. (B) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2. (C) Immunoblot analysis of the phosphorylation levels of Akt and Erk1/2 of embryos at the prepharyngula stage under normoxia (12 hpf) and the pharyngula stage under hypoxia (36 hpf). (D) Changes in head-trunk angle in the indicated groups. Data are mean ± standard deviation; n = 8 to 20. (E) Relative growth rate. Data are mean ± standard deviation, three independent experiments. *P < 0.05. Irs1-mediated IIS was important for embryonic catch-up growth To test whether attenuated IIS before catch-up growth is a causation of significant growth loss in irs1 MO embryos during Reoxy conditions, IIS was force-activated in irs1 MO embryos. Capped RNA molecules were coinjected with irs1 MO. The MO-resistant Irs1 RNA (Flag-Irs1) restored the reduced activation of IIS by Irs1 MO injection (Fig. 4C; Irs1 RNA). Similarly, the coinjection of mryAkt RNA with Irs1 MO also resulted in overcoming Irs1 MO-induced reduction of IIS levels (Fig. 4C; myrAkt RNA). Importantly, blunted catch-up growth in Irs1 MO-injected embryos was apparently rescued by coinjection of MO-resistant Irs1 RNA or myrAkt RNA during Reoxy conditions (Fig. 4D), and this was demonstrated by significantly regained relative growth rate of the Reoxy group fish as shown in Fig. 4E. Similar results were obtained in HRasV12 RNA coinjection experiments (Supplemental Fig. 2). These data strongly suggest that blunted IIS before reoxygenation is one of the major reasons for failed catch-up growth caused by the loss of irs1 expression. Disturbed irs1 expression reduced dlx2-expressing cells under hypoxic conditions To gain insight into cellular events that occurred before and during catch-up growth, we examined changes in cell proliferation in constant normoxia, hypoxia, and reoxygenation. Although there was a slight tendency toward decreased phospho-histone H3-positive cells in irs1 MO fish under reoxygenation conditions, the change was modest, and we failed to see any significant difference between the phospho-histone H3-positive cell density of irs1 MO fish and that of ctr MO fish in all three conditions (Supplemental Fig. 3A and 3B). Next, cell death was examined by active caspase-3 staining. We found that the numbers of active caspase-3‒positive cells were increased mainly in the anterior half of irs1 MO embryos under hypoxia (32 hpf) (Fig. 5A; α active caspase-3). We also examined the expression of specific genes (dlx2, ntl, emx1, and pax2a) and found reduced dlx2 expression in irs1 MO embryos under prolonged hypoxia (36 hpf) (Fig. 5A; dlx2, domains i to iv). The irs1 MO failed to cause any major changes in expression of either dlx2 or other genes under Norm conditions (Supplemental Fig. 4A). Under hypoxia, except for dlx2, we did not see any major changes in ntl and pax2a expression in irs1 MO embryos; trunk muscle labeled by myosin-heavy chain staining was also comparable between ctr MO embryos and irs1 MO embryos under hypoxia. Only a minor cell death rate was found in the anterior trunk in the irs1 MO-injected embryo (Supplemental Fig. 4B). Figure 5. View largeDownload slide Knockdown of Irs1 led to increased cell death in NCCs under hypoxia. (A) Cell death and dlx2 expression pattern under hypoxia. (a) The lateral view. (b) The dorsal view of the anterior region of embryos. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 250 μm. (B) Quantitative RT-PCR analysis of NCC marker genes in irs1 MO embryos under hypoxia. Each transcript level was normalized to β-actin expression level, and the values were represented as relative abundance to the value in ctr MO embryos. Data are shown as mean ± standard deviation, two to four independent experiments. (C) Whole-mount double-label immunostaining results. The area of fish body is traced by the dashed line. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 125 μm. (D) Double-label immunostaining of section specimens. The cross sections of NCC-enriched dorsal regions were prepared, and the immunostaining was conducted on glass slides. The emergency rate of active caspase-3‒positive Sox10-labeled cells was analyzed. Data are mean ± standard deviation; n = 4 to 12. The area of fish body is traced by the dashed line. Scale bar = 25 μm. (E) Effects of irs1 knockdown on the phosphorylation level of Akt in NCCs. Embryos were subjected to immunostaining using denoted antibodies. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. (F) Whole-mount double-label immunostaining of active caspase-3 and Sox10 of hypoxia-treated irs1 MO embryos with or without coinjection of myrAkt RNA. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. *P < 0.05; **P < 0.01. A, anterior; D, dorsal; L, left; Nt, neural tube; OtV, otic vesicle; P, posterior; R, right; V, ventral; Yk, yolk. Figure 5. View largeDownload slide Knockdown of Irs1 led to increased cell death in NCCs under hypoxia. (A) Cell death and dlx2 expression pattern under hypoxia. (a) The lateral view. (b) The dorsal view of the anterior region of embryos. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 250 μm. (B) Quantitative RT-PCR analysis of NCC marker genes in irs1 MO embryos under hypoxia. Each transcript level was normalized to β-actin expression level, and the values were represented as relative abundance to the value in ctr MO embryos. Data are shown as mean ± standard deviation, two to four independent experiments. (C) Whole-mount double-label immunostaining results. The area of fish body is traced by the dashed line. The major dlx2-expression domains (i–vi) are indicated by arrows. The body axis is shown in the inset. Scale bar = 125 μm. (D) Double-label immunostaining of section specimens. The cross sections of NCC-enriched dorsal regions were prepared, and the immunostaining was conducted on glass slides. The emergency rate of active caspase-3‒positive Sox10-labeled cells was analyzed. Data are mean ± standard deviation; n = 4 to 12. The area of fish body is traced by the dashed line. Scale bar = 25 μm. (E) Effects of irs1 knockdown on the phosphorylation level of Akt in NCCs. Embryos were subjected to immunostaining using denoted antibodies. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. (F) Whole-mount double-label immunostaining of active caspase-3 and Sox10 of hypoxia-treated irs1 MO embryos with or without coinjection of myrAkt RNA. The area of fish body is traced by the dashed line. The body axis is shown in the inset. Pictures are taken at a lateral view with the head to the left. Scale bar = 125 μm. *P < 0.05; **P < 0.01. A, anterior; D, dorsal; L, left; Nt, neural tube; OtV, otic vesicle; P, posterior; R, right; V, ventral; Yk, yolk. Irs1 was required for maintaining the neural crest cell population under hypoxic conditions The dlx2 gene is expressed in the mesenchyme of developing pharyngula arches, and these cells were derived from neural crest cells (NCCs). We then analyzed the expression of several NCC-specific genes (crestin, sox10, sox9b, foxd3, tfap2a, mitfa, ngfr, and pdgfrb) and found that they were prone to decrease in irs1 MO embryos under hypoxia (Fig. 5B). Furthermore, Sox10 and active caspase-3 double-positive signals in irs1 MO embryos were prominent under hypoxia, which was more noticeable than under Norm conditions (Fig. 5C; Supplemental Fig. 5A). Indeed, the immunohistochemistry analysis of the cryosections revealed that the emergence of active caspase-3‒positive cells within the Sox10-positive NCCs was significantly increased in the irs1 MO-injected embryos under prolonged hypoxia compared with that of ctr MO-injected embryos and with that of the irs1 MO-injected embryos under constant normoxia (Fig. 5D; Supplemental Fig. 5B). To test the IIS activation level, the approximately stage-matched embryos under constant normoxia and hypoxia were used for immunostainings of phospho-Akt and Sox10 (Fig. 5E). Under normoxic conditions, the phospho-Akt signal was slightly reduced in Sox10-positive cells of irs1 MO embryos. On the other hand, under prolonged hypoxic conditions, the phosphorylation level of Akt was clearly diminished in Sox10-positive cells by irs1 MO (Fig. 5E; hypoxia, irs1 MO). Importantly, the forced-activation of Akt-signaling by coinjection of myrAkt RNA prevented this (Fig. 5F), suggesting that the reduced Irs1-Akt signaling was deleterious for NCCs under hypoxia. NCCs played indispensable roles in embryonic catch-up growth Because NCCs play considerable roles in embryonic development and because the loss of Irs1 induces massive cell death in NCCs under hypoxia and in the failed catch-up growth phenotype under Reoxy conditions, the importance of NCCs for embryonic catch-up growth was further examined by using small-molecular-weight chemicals and knockdown of genes whose expression is required for NCC development. Wild-type embryos pretreated with leflunomide (Fig. 6A), a potent inhibitor of neural crest stem cell renewal (27), did not change the growth level under hypoxia, but the growth acceleration during Reoxy conditions was significantly hindered (Fig. 6B and 6C). In addition, knockdown of prdm1a and sox10, both of which are required for NCC development (28), clearly blunted growth acceleration in Reoxy (Fig. 6D). Figure 6. View largeDownload slide Loss of the NCC population inhibited catch-up growth, and suppression of cell-death rescued catch-up growth of irs1 MO embryos. (A) Schematic illustration of the mode of action of leflunomide and the outline of the experiment. (B) Changes in head-trunk angles of embryos treated with leflunomide. Data are mean ± standard deviation; n = 8 to 19. (C) Relative growth rate of drug-treated embryos during the indicated developmental periods. Data are mean ± standard deviation, three independent experiments.(D) Relative growth rate of MO-injected embryos. Data are mean ± standard deviation, three independent experiments. (E) Outline of the zVADfmk experiment. (F) Representative result of TUNEL staining. The prominent TUNEL signals (red) are indicated by arrows. The specimens were counterstained with Hoechst33342 (blue). The area of fish body is traced by the dashed line. Scale bar = 250 μm. (G) Relative growth rate of zVADfmk-treated embryos during reoxygenation periods. Data are mean ± standard deviation, two independent experiments. *P < 0.05. DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide. Figure 6. View largeDownload slide Loss of the NCC population inhibited catch-up growth, and suppression of cell-death rescued catch-up growth of irs1 MO embryos. (A) Schematic illustration of the mode of action of leflunomide and the outline of the experiment. (B) Changes in head-trunk angles of embryos treated with leflunomide. Data are mean ± standard deviation; n = 8 to 19. (C) Relative growth rate of drug-treated embryos during the indicated developmental periods. Data are mean ± standard deviation, three independent experiments.(D) Relative growth rate of MO-injected embryos. Data are mean ± standard deviation, three independent experiments. (E) Outline of the zVADfmk experiment. (F) Representative result of TUNEL staining. The prominent TUNEL signals (red) are indicated by arrows. The specimens were counterstained with Hoechst33342 (blue). The area of fish body is traced by the dashed line. Scale bar = 250 μm. (G) Relative growth rate of zVADfmk-treated embryos during reoxygenation periods. Data are mean ± standard deviation, two independent experiments. *P < 0.05. DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide. To test whether the loss of NCCs under hypoxia is a reason for the failed catch-up growth by irs1 knockdown, MO-injected embryos were treated with the pan-caspase inhibitor zVADfmk (Fig. 6E). The zVADfmk treatment of irs1 MO embryos resulted in clear reduction of TUNEL-positive apoptotic cells (Fig. 6F) and significant growth regain under Reoxy conditions (Fig. 6G). Discussion Irs1 is known as a key molecule for animal growth and development in the rodent model (29). Despite the evolutionarily conserved IIS pathway from worms to primates, one of the most crucial nodes in this pathway, the receptor substrates, was less extensively studied in nonmammalian vertebrates (30). In this study, zebrafish Irs1 was identified and first characterized as a functional Igf1r substrate (Fig. 1) Also, both embryonic and adult expression profiles of the irs1 gene in the fish model were first investigated (Fig. 2). The developmental expression of the zebrafish irs1 gene is especially key for embryonic growth, because we found that the loss of irs1 expression significantly blunted growth rate (Fig. 3D and 3F). This demonstrated that a nonmammalian Irs1 is evidently tyrosine phosphorylated in an Igf1r activity-dependent manner and is required for body growth in the developing animal. The major finding in this study is that irs1 is indispensable for catch-up. Blockade of irs1 expression markedly inhibited catch-up growth. Unexpectedly, however, it did not cause any obvious changes in activation levels of Akt and Erk1/2 during catch-up growth (Fig. 4B; 48-hpf Reoxy); instead, both Akt and Erk1/2 signals in the earlier stage (Fig. 4B; 8-hpf Norm) were greatly diminished by Irs1 MO. Also, hypoxia reduced IIS, and the loss of irs1 expression further reduced it (Fig. 4B; 36-hpf Hypo). Once environmental oxygen is reduced, the hypoxia-inducible Igfbp1 systemically limits Igf action (20, 31), and it is one of the reasons for IUGR (20). Even in such cases, however, “minimal” IIS must be maintained to secure the survival of important cellular populations via increasing cellular sensitivity to limited Igf-ligand, which likely occurred at the level of Irs1 in the current study. Indeed, the phospho-Akt signal was weaker but still detectable in stunted embryos under hypoxia and was further reduced by the loss of irs1 expression. Moreover, we found increased irs1 expression under hypoxia (data not shown). In a mammalian cell culture model, reduced IIS increased the IRS level, but increased IIS conversely reduced it (14), which led to the efficient use of a limited amount of activated receptor to maintain basal IIS or prevent the overactivation of IIS. Given that the zebrafish Irs1 is an important Ir/Igf1r substrate (Fig. 1D) and IIS confers cell survival, the loss of Irs1-mediated IIS would reduce cellular viability, and further hypoxia-induced Igfbp1-mediated attenuation of IIS may aggravate reduced viability of embryonic cells. The active caspase-3‒positive cells were more pronounced after irs1 knockdown under hypoxia than under constant normoxia, suggesting that Irs1 is indispensable for the survival of certain sets of cells under hypoxia. Massive apoptosis occurred in the anterior dorsal domain, and dlx2 expression was reduced in hypoxia-exposed irs1 MO-injected embryos (Fig. 5A). The dlx2 expresses in the mesenchyme of developing pharyngula arches, and these cells were derived from the anterior population of the NCCs (32). Therefore, we inferentially focused on changes in NCCs in this study. Accordingly, clear reduction of NCC-marker gene expression and increased cell death in Sox10-positive cells in the anterior region of embryos were found in irs1 MO-injected embryos under hypoxia (Fig. 5B‒5D). These data indicated the NCCs were impaired in Irs1 knocked-down embryos under hypoxic conditions. Indeed, IIS mediates survival and proliferation of stem cells including NCCs (33–36), and the forced activation of Akt in irs1 MO-injected embryos considerably restored NCC viability under hypoxia (Fig. 5F). Because IIS is known to be a key requirement for the culture of stem cells including NCCs (35), our results strongly suggest that Irs1 maintains survival of NCCs during hypoxia, which leads to reoxygenation-induced catch-up growth. Because Irs1 mRNA was ubiquitously detected (Fig. 2), it is still uncertain whether Irs1 guaranteed NCC survival in a “cell-autonomous” or “non‒cell-autonomous” manner. NCC-specific control of IIS is needed to interpret this issue in future studies. Embryos subjected to the chemical or genetic depletion of NCCs failed to show catch-up growth without modifying Irs1 expression (Fig. 6A‒6D). Conversely, the antiapoptotic chemical zVADfmk, which potentially blocked apoptotic loss of NCCs under hypoxic conditions, significantly restored catch-up growth in irs1 MO-injected embryos (Fig. 6G). In agreement with the facts that (1) the Irs1 knockdown increased cell death in NCCs under hypoxia and (2) the loss of NCCs led to blunted catch-up growth in reoxygenated conditions, data from the zVADfmk experiment suggest that Irs1 is required for the maintenance or survival of NCCs under severe hypoxic stress conditions, and the cells are key for future achievement of catch-up growth after stressor remits. The NCCs become various types of cells, such as cartilage, cranial bones, connective tissues, muscles, glia, neurons, and pigment cells (37, 38). It is likely that the NCCs and their derivatives change their behaviors or fates in response to the severe stressors, and this may influence body growth after the removal of stressors. The molecular and cellular changes in NCCs during the stunting and catch-up periods need to be investigated in the future. In conclusion, we found Irs1 plays a pivotal role in mediating limited IIS in early stages of embryos [Fig. 7(①)]. The Irs1-mediated IIS, such as phosphoinositide 3-kinase‒Akt signaling, is essential for NCC survival, especially under severely hypoxic conditions [Fig. 7(②)]. The NCCs are crucial for the current fish model of catch-up growth, though the clarification of its role(s) requires further research [Fig. 7(③)]. Obesity, cardiovascular diseases, insulin resistance, delayed mental development, and other unfavorable physiological outcomes are often found in individuals who experienced severe growth delay and catch-up growth in early stages of life. Currently unknown molecular and cellular changes in NCCs during IUGR/catch-up growth, which are not necessarily responsible for the change in apparent body growth, may provide an important clue for deciphering the underlying mechanisms of such pathogenesis. Figure 7. View largeDownload slide A proposed model. The Irs1-mediated IIS in developing embryos is important for NCC survival under severe hypoxia (①, ②), and the NCCs are indispensable for reoxygenation-induced catch-up growth (③). Figure 7. View largeDownload slide A proposed model. The Irs1-mediated IIS in developing embryos is important for NCC survival under severe hypoxia (①, ②), and the NCCs are indispensable for reoxygenation-induced catch-up growth (③). Abbreviations: Abbreviations: cDNA complementary DNA hpf hours postfertilization HTA head-trunk angle Hypo embryos transferred to hypoxia from 24 to 36 hours postfertilization Igf insulinlike growth factor Igf1r type 1 Igf-receptor Igfbp1 inhibitory IGF-binding protein IIS insulin/insulinlike growth factor signaling Ir insulin receptor Irs insulin receptor substrate IUGR intrauterine growth restriction MO morpholino oligo mRNA messenger RNA myrAkt N-terminal myristoylation signal–attached mouse Akt1 NCC neural crest cell Norm constant normoxia O.C.T. optimal cutting temperature PBS phosphate-buffered saline PTB phosphotyrosine binding RT-PCR reverse transcription polymerase chain reaction TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling Acknowledgments We thank Drs. Daisuke Yamanaka and Takashi Shibano (The University of Tokyo) for invaluable discussions and Dr. Susan Hall (The University of North Carolina at Chapel Hill) for proofreading. 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EndocrinologyOxford University Press

Published: Apr 1, 2018

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