Article Figures & Data
Figures
- Figure 1.
Expression of candidate neuronal IEGs in the brain of cycloheximide pretreated crickets 30 min after PTX injection. A, PTX-induced neuronal hyperexcitability in the cricket. Crickets show seizure-like behavior ∼2 min after PTX injection. 1 h before PTX/vehicle injection, 20 mM cycloheximide was injected to block de novo protein synthesis. B–G, Expression of (B) Gryllus fra total transcript, (C) fra-A isoform, (D) fra-B isoform, (E) jra, (F) egr-B, and (G) hr38 in the brains of cycloheximide pretreated crickets 30 min after injection of vehicle (5% DMSO in saline) or PTX. Expression levels of each target gene were normalized with that of Gryllus ef1α gene (Fig. 1-1). RT-qPCR analyses were performed on eight biological replicates. Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Asterisks denote statistical significance (*, p < 0.05). See Table 3 for the details of statistical analysis. See Figs. 1-2, 1-3, 1-4, and 1-5 for the structures of the encoded proteins of candidate neuronal IEGs.
- Figure 2.
Expression characteristics of Gryllus egr-B in the cricket brain. A, B, Expression time course of Gryllus egr-B after PTX injection in the brains of (A) cycloheximide- and (B) saline-pretreated crickets. C, Expression time course of Gryllus egr-B pre-mRNA in the brain of cycloheximide-pretreated crickets after PTX injection. D, E, Behaviorally evoked expression of Gryllus egr-B in the brain of crickets 1 h after (D) feeding of sucrose solution and (E) agonistic interaction. RT-qPCR analyses were performed on eight biological replicates. The expression levels were normalized to the mean of those of naïve animals (baseline expression level). Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Asterisks donate statistical significance to the control (0 min after PTX injection; A–C) or to the naïve animals (D, E; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). See Table 3 for the details of statistical analysis. See Figs. 2-1, 2-2, and 1-5 for the expression characteristics of other candidate neuronal IEGs.
- Figure 3.
Gene regulatory regions of the insect/crustacean egr-B homologs. A, Putative core promoter regions of basal insect and crustacean egr-B homologs share a high-level sequence similarity. The upstream sequences of insect/crustacean egr-B homologs are aligned with the core promoter region of Gryllus egr-B. The conserved bases are marked with asterisks under the alignment. Cis-regulatory elements and sequence motifs that are conserved are indicated above the alignment. CRE, cAMP-responsive element; SRE, serum response element; Inr, initiator element; DPE, downstream promoter element. B, Sequence logo representation of the conserved motifs in the core promoter region of insect egr-B homologs. The sequence logo of the GAGA motif was generated by multiple alignment of the upstream sequences of polyneopteran egr-B homologs. The other sequence logos were generated by multiple alignment of the upstream sequences of insect egr-B homologs. The positions of conserved motifs are indicated by black bars under the logo. C, Schematic representation of the gene regulatory regions of insect/crustacean egr-B homologs. The genomic regions were aligned to the position of the +1 site of Gryllus egr-B or the 5′-end of the putative core promoter region. The red bars indicate genomic regions aligned in Fig. 3A. Positions of transcription factor binding sites predicted using the LASAGNA-Search 2.0 program (score >8.0) are indicated by arrowheads. The phylogenetic relationship of insect/crustacean species is indicated as a phylogram tree. AP-1, activator protein 1; CREB, cAMP response element-binding protein; C/EBP, CCAAT-enhancer-binding protein; MEF2, myocyte enhancer factor 2; NF-AT, nuclear factor of activated T-cells; SRF, serum response factor. See Table 2 for the details of genomic sequences used for promoter analysis. See Fig. 3-1 and Table 3-1 for the structural conservations of the transcription factors used for the binding site prediction.
- Figure 4.
Phylogenetic footprinting revealed conserved cis-regulatory modules in the upstream regions of polyneopteran egr-B homologs. A, mVISTA plot of the upstream regions of polyneopteran egr-B homologs based on MLAGAN alignment using the upstream region of Gryllus egr-B as a reference sequence. Positions of potential transcription factor binding sites in the upstream region of Gryllus egr-B are indicated by arrowheads (Fig. 3B). The horizontal and vertical axes of the plot represent the position in the sequences and the percentage identity, respectively. Two conserved cis-regulatory modules (CRMs; CRM-800 and CRM-400) and the conserved core promoter region are shaded blue and red on the plot, respectively. B, Nucleotide sequence alignments of two conserved CRMs (CRM-800 and CRM-400) found in the upstream region of polyneopteran egr-B homologs. The conserved bases are marked with asterisks under the alignment. Cis-regulatory elements conserved among most of the sequences are indicated above the alignment. Black bars under the alignments indicate sequence motifs conserved across species where no transcription factor is assigned. AP-1, binding site for activator protein 1; AP-4, binding site for activating enhancer binding protein 4; ATF2, binding site for activating transcription factor 2; CDP/Cut, binding site for CCAAT-displacement protein/cut homeobox; C/EBP, binding site for C/EBP; CRE, cAMP-responsive element; SRE, serum response element. See Table 2 for the details of genomic sequences used for promoter analysis.
- Figure 5.
IEG promoter-driven transgenic reporter system in the cricket brain. A, Flowchart of the experimental procedures to establish the IEG reporter line. See the Materials and Methods section for detail. B, Schematic representation of the piggyBac transgenic vector for the IEG promoter-driven transgenic reporter system. The vector harbors the expression cassette of EYFPnls:PEST driven by the Gryllus egr-B promoter. 3xP3-mCherry was used as a visible selection marker. A gypsy insulator sequence (gyp) was inserted between two expression cassettes. ARE, AU-rich element; LTR, long terminal repeat. Ci, Schematic representation of the piggyBac insertion in the IEG reporter line. A 5629-bp insertion was inserted into the piggyBac donor TTAA site (highlighted in red). To conduct genotyping PCR, two primers, line19_fw and line19_rv, were designed at the 5′ and 3′ flanking region of the insertion sites, respectively. Cii, The nucleotide sequence of the genomic region flanking the piggyBac insertion in the IEG reporter line. The piggyBac donor TTAA site is highlighted in red. The positions of the annealing site of primers for genotyping PCR are indicated by white arrows under the sequence. D, Basal mRNA expressions of EYFPnls:PEST and Gryllus egr-B in the brain of naïve IEG reporter line. E, Expression time course of (Ei) EYFPnls:PEST and (Eii) Gryllus egr-B in the brain of the IEG reporter line after PTX injection. RT-qPCR analyses were performed on eight biological replicates. The expression levels were normalized to the mean of those of naïve animals (baseline expression level). Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Asterisks donate statistical significance to the control (naïve animals; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). F, Correlation plot between the expression levels of EYFPnls:PEST and Gryllus egr-B in the brains of the IEG reporter line. The data from PTX-injected crickets (n = 75; black circles), vehicle pre-injected crickets (n = 8; gray circles), and naïve crickets (n = 8; white circles) were plotted. See Table 3 for the details of statistical analysis.
- Figure 6.
PTX-induced reporter protein expression in the brain of the IEG reporter line. Distribution of the reporter protein (EYFPnls:PEST) in the brain of the IEG reporter line was examined by whole-mount fluorescent immunohistochemistry. A, B, Frontal views of the supraesophageal ganglion stained with anti-GFP antibody. A, EYFP immunoreactivity was only observed in the cells is indicated by white arrowheads 6 h after vehicle injection. B, EYFP immunoreactivity was observed throughout the ganglion 6 h after PTX injection. C, D, Ventral views of the subesophageal ganglion stained with anti-GFP antibody. C, EYFP immunoreactivity was not observed 6 h after vehicle injection. D, EYFP immunoreactivity was observed throughout the ganglion 6 h after PTX injection. Dorsoventral (D-V) or rostrocaudal (R-C) axes were indicated. Scale bars represent 200 µm. See Movies 1 and 2 for the full stack of optical sections of the supraesophageal ganglia shown in A and B.
- Figure 7.
Sucrose feeding-evoked reporter protein expression in the DUM neurons of the IEG reporter line. A, Dorsal view of the subesophageal ganglion of the Hokudai WT strain stained with anti-Gryllus Tdc2 antibody. The outline of the ganglion is surrounded by the white dotted line. The depth of the cells is color coded as indicated in the inset. Rostrocaudal (R-C) axis was indicated. Scale bar represents 200 µm. See Fig. 7-1 for the octopamine biosynthesis pathway and the structures of the Tdc proteins in insects. See Fig. 7-2 for the frontal view of the supraesophageal ganglion and the ventral view of the subesophageal ganglion stained with anti-Gryllus Tdc2 antibody. B, Schematic drawing of the positions and numbers of the cell bodies of three DUM clusters (DUM1, DUM2, DUM3) on the dorsal side of the subesophageal ganglion. C, Double fluorescent immunostaining confirmed that the DUM neurons contain octopamine. The DUM neurons were stained with the anti-Gryllus Tdc2 antibody (green) and anti-octopamine antibody (magenta). Scale bar represents 50 µm. D, Distribution of the reporter protein (EYFPnls:PEST) in the DUM neurons of the IEG reporter line before and 6 h after feeding of sucrose solution (n = 4 each). The DUM neurons were stained with the anti-Gryllus Tdc2 antibody (green) and anti-GFP antibody (magenta). The cell bodies of Gryllus Tdc2 immunoreactive DUM neurons are surrounded by the white dotted line. The DUM neurons with nuclear EYFP immunoreactivity are indicated by white arrowheads. Scale bar represents 50 µm.
Tables
Forward primer Reverse primer Degenerate primers egr 5′-GGA GTN CAR CTN GCH GAR TA-3′ 5′-GAN CGC ATG CAD ATN CGR CAY TG-3′ 5′-ACS AGN AAR GGN CAY GAR AT-3′ 5′-TTN AGR TGN ACY TTN GCR TG-3′ 5′-CCA RTG YCG NAT HTG CAT GCG-3′ hr38 5′-AAC CGC TGC CAR TTY TGC-3′ 5′-AGA AGC TCC TGR TCR TNG C-3′ tdc2 5′-TCG AGT ACG CSG AYT CKT TCA ACA C-3′ 5′-GGA TCR CTS ACC ATN CGN ACG AAG AA-3′ Primers for full-length ORF amplification fra-A 5′-CGC GGG AGT AAG GAC GTG-3′ 5′-CCC CAT TGT CCA AAT CCT CC-3′ fra-B 5′-GGC GGC TTG TGT GTT TGT G-3′ 5′-CCC CAT TGT CCA AAT CCT CC-3′ jra 5′-GAC GGT CGC GGA GAG TC-3′ 5′-GAT CTC ATA TGT ATA TGC ATG TGT TCA C-3′ egr-B 5′-TTC ATT CAT AAA AGT GTT GTA GAG CG-3′ 5′-ATA TAT ACG AAT CGA GGA GAA CAC-3′ tdc1 5′-CAT CTG GCG TTC GCT C-3′ 5′-CGC AGT CCC AGA AGA G-3′ tdc2 5′-CGA CGC CCG ACG ACA TTC G-3′ 5′-CCG GCT CGT ATG TTG TGT GG-3′ Primers for RT-qPCR fra total transcripts 5′-GGA CGG CCT CAA TTC GGG-3′ 5′-GGA TTC CAC CTC GCA CTG C-3′ fra-A 5′-CCT GCC TTC ATC TGC GTA CG-3′ 5′-GTC TCA CTG GGC GAA ACG TG-3′ fra-B 5′-GGC GGC TTG TGT GTT TGT G-3′ 5′-GGA TTC CAC CTC GCA CTG C-3′ jra 5′-GAG CGG ACG GTT GTG TTA GG-3′ 5′-GCA GTT GCG TAC CAT CTA AAT CC-3′ egr-B (for initial expression analysis) 5′-GAC CTA GGC GTC GAA CCC-3′ 5′-GTT CCA AGG ATC CTG TGA TGG G-3′ egr-B 5′-GTT TGG AAA CGC TGA GCC C-3′ 5′-CCT GAC GCT GTA GAG GCA C-3′ egr-B pre-mRNA 5′-GTG ACA CAT GTA ATT GGC GTA AC-3′ 5′-CAA TTC CTC GGG TTC CAA GG-3′ hr38 5′-CCA ACC TCG ACT ATT CAC AGT ATC-3′ 5′-CCG GAA TCT TAT CAG CAA ACG TG-3′ hr38 pre-mRNA 5′-GAA GCA TCT ACT CCA GTC TCA TAA TAG-3′ 5′-GTA GGC TCA CGA TAC TGG AAA TG-3′ EYFPnls:PEST 5′-CGA GGA GCT GTT CAC CGG-3′ 5′-GGT GCA GAT GAA CTT CAG GG-3′ β-actin 5′-CGT AAA CTC AAC TAC TAA CCA TGT GC-3′ 5′-GCC CTG GGT GCA TCA TCG-3′ ef1α 5′-CGA CTC CGG TAA ATC TAC GAC C-3′ 5′-CAC CCA GGC ATA CTT GAA AGA AC-3′ rpl32 5′-CGC CCA GTT TAT CGT CCA AC-3′ 5′-GCC TGC GAA CTC TGT TGT C-3′ Primers used to amplify the core promoter regions of orthopteran egr-B homologs 5′-GTT ACG TCA TTT GAC GTC A-3′ 5′-GTC CCA TAT TTG GAA GTC G-3′ 5′-GTT ACG TCA TTT TGA CGT CA-3′ 5′-CAM CAS TTT TAT GAA TGA AG-3′ Primers used to amplify the genomic DNA fragment upstream to the coding sequence of the orthopteran egr-B homologs Gryllus bimaculatus 5′-CAG GGG TTG TTT ATT CGC CG-3′ 5′-CTG TGA TGG GAG GCG GTT CAA C-3′ Acheta domesticus 5′-AAA TTC GAA AGC CTT GAC AGT GG-3′ 5′-ACG ATG GAC GAG CGT CGT G-3′ Gampsocleis buergeri 5′-ATG TTC CCC CTC CAT GCC AG-3′ 5′-ACA TGC TGA CGC GCA ACA C-3′ Locusta migratoria 5′-CAG TGT TGC CAG CCT CC-3′ 5′-CCG ACG AGT ACA GGC AGT C-3′ Species GenBank ID Genomic region targeted for TFBS prediction Position of the conserved core promoter Position of the +1 site Position of CRM-800 Position of CRM-400 Drosophila melanogaster NT_033777.2 Base 13945983 to 13948265 Base 13947783 Apis mellifera NC_007084.3 Base 6690515 to 6692794 Base 6691064** Tribolium castaneum NC_007417.3 Base 2995987 to 2998141 Base 2997452 to 2997620(169 bp) Acyrthosiphon pisum ABLF02030506.1 Base 25286 to 24071 Base 25155 to 24964(192 bp) Pediculus humanus NW_002987224.1 Base 14669 to 12120 Base 12809 to 12649(161 bp) Blattella germanica JPZV01078734.1 Base 44,303 to 41,904 Base 42626 to 42446(181 bp) Base 43369 to 43205 (165 bp) Base 42891 to 42809 (83 bp) Zootermopsis nevadensis AUST01012629.1 Base 2539 to 150 Base 954 to 753 (197 bp) Base 1667 to 1493 (83 bp) Base 1248 to 1170 (79 bp) Gryllus bimaculatus LC215247* Base 1 to 2574 Base 1518 to 1711 (194 bp) Base 1668** Base 786 to 937 (152 bp) Base 1235 to 1316 (82 bp) Acheta domesticus LC215248* Base 1 to 2781 Base 2113 to 2306 (194 bp) Base 1358 to 1509 (152 bp) Base 1830 to 1911 (82 bp) Gampsocleis buergeri LC215249* Base 1 to 2098 Base 1223 to 1413 (191 bp) Base 498 to 652 (155 bp) Base 945 to 1024 (80 bp) Locusta migratoria LC215250* Base 1 to 1909 Base 1166 to 1404 (239 bp) Daphnia pulex ACJG01000376.1 Base 1585904 to 1583495 Base 1584357 to 1584191(167 bp) Figure Experimental conditions Statistical test 1B–G Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Crickets were injected with 20 mM cycloheximide. 1 h later, 5 mM PTX or vehicle were injected to the crickets. 30 min later, brains were dissected for RNA extraction (n = 8 in each group). Gryllus fra total vehicle vs. Gryllus fra total PTX, U = 23; Gryllus fra-A vehicle vs. Gryllus fra-A PTX, U = 24, Gryllus fra-B vehicle vs. Gryllus fra-B PTX, U = 15; Gryllus jra vehicle vs. Gryllus jra PTX, U = 32; Gryllus egr-B vehicle vs. Gryllus egr-B PTX, U = 11; Gryllus hr38 vehicle vs. Gryllus hr38 PTX, U = 29, Mann-Whitney U test. 2A, B Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Crickets were injected with (A) 20 mM cycloheximide or (B) saline. 1 h later, 5 mM PTX was injected to the crickets. Brains were dissected for RNA extraction before PTX injection (0 min), or 15, 30, 45, 60, 90, or 120 min after PTX injection (n = 8 in each group). Effect of pre-treatment: F(1,98) = 0.9604, p = 0.9004; Effect of time: F(6,98) = 9.034, p < 0.0001; interaction: F(6,98) = 0.3634, p = 0.9004, Two-way ANOVA, Dunnett’s post-hoc test. 2C Same as in Fig. 2A. H = 29.21, p < 0.0001, Kruskal-Wallis test, Dunn’s post-hoc test. 2D Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Cricket were fed with 0.5 M sucrose solution 3 times with 5 min intervals. Brains were dissected for RNA extraction before feeding (naïve), or 1 h after feeding (n = 8 in each group). t = 3.051, df = 8.491, Welch’s unpaired t-test. 2E Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Weight-matched crickets were introduced into an arena to interact for 5 min, then re-isolated. Brains were dissected for RNA extraction before interaction (naïve), or 1 h after start of interaction (n = 8 in each group). H = 12.62, p < 0.01, Kruskal-Wallis test, Dunn’s post-hoc test. 5D Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Then, brains were dissected for RNA extraction (n = 8 in each group). t = 15.95 df = 7.094, Welch’s unpaired t-test. 5E Adult male crickets 1 week after the imaginal molt were isolated for 3 days. 5 mM PTX was injected to the crickets. Brains were dissected for RNA extraction before PTX injection (0 min), or 15, 30, 45, 60, 90, or 120 min after PTX injection (n = 8 in each group). H = 25.87, p < 0.001, Kruskal-Wallis test, Dunn’s post-hoc test. 5F Same as in Fig. 5C. H = 39.42, p < 0.0001, Kruskal-Wallis test, Dunn’s post-hoc test. 5G Pooled expression data of EYFPnls:PEST and Gryllus egr-B in the brain within 120 min after PTX injection (including data presented in Fig. 5C, D), 60 min after vehicle injection, and naïve crickets were analyzed (n = 72, 8, and 8, respectively). r = 0.8269, p < 0.0001, Pearson’s correlation analysis. 2-1A, E, I Same as in Fig. 2A. Gryllus fra-A, F(6,49) = 3.529, p < 0.001; Gryllus fra-B, F(6,49) = 3.327, p < 0.01; Gryllus jra, F(6,49) = 5.305, p < 0.001, One-way ANOVA, Dunnett’s post-hoc test. 2-1B, F, J Pooled expression data of Gryllus fra-A, fra-B, jra and egr-B in the brain of cycloheximide pretreated crickets within 120 min after PTX injection (including data presented in Figs. 2-1A, E, and I and naïve crickets were analyzed (n = 48 and 8, respectively). Gryllus fra-A vs. egr-B, r = 0.7856, p < 0.0001; Gryllus fra-B vs. egr-B, r = 0.5250, p < 0.0001; Gryllus jra vs. egr-B, r = 0.6222, p < 0.0001, Pearson’s correlation analysis. 2-1C, G, K Same as in Fig. 2D. Gryllus fra-A, U = 13; Gryllus fra-B, U = 7; Gryllus jra, U = 22, Mann-Whitney U test. 2-1D, H, L Same as in Fig. 2E. Gryllus fra-A, H = 10.24, p < 0.05; Gryllus fra-B, H = 13.09, p < 0.01; Gryllus jra, H = 2.284, p = 0.5156, Kruskal-Wallis test, Dunn’s post-hoc test. 1-5B, C Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Crickets were injected with (B) 20 mM cycloheximide or (C) saline. 1 h later, 5 mM PTX was injected to the crickets. Brains were dissected for RNA extraction before PTX injection (0 min), or 15, 30, 45, 60, 90, or 120 min after PTX injection (n = 8 in each group). Effect of pre-treatment: F(1,98) = 2.060, p = 0.1544; Effect of time: F(6,98) = 14.57, p < 0.0001; interaction: F(6,98) = 0.7991, p = 0.5729, Two-way ANOVA, Šidák’s post-hoc test. 1-5D Same as in Fig. 2A, B. Effect of pre-treatment: F(1,98) = 0.5631, p = 0.4548; Effect of time: F(6,98) = 16.11, p < 0.0001; interaction: F(6,98) = 1.733, p = 0.1212, Two-way ANOVA, Šidák’s post-hoc test. 1-5E Pooled expression data of Gryllus hr38 and Gryllus egr-B in the brain of saline and cycloheximide pretreated crickets within 120 min after PTX injection (including data presented in Figs. 2A, B and Figs. 1-5B, C), and naïve crickets were analyzed (n = 48, 48, and 8, respectively). Saline pretreated crickets: r = 0.8498, p < 0.0001; cycloheximide pretreated crickets: r = 0.8446; p < 0.0001; all data included: r = 0.8498, p < 0.0001, Pearson’s correlation analysis 1-5F Same as in Fig. 2D. t = 2.501, df = 13.18, Welch’s unpaired t-test. 1-5G Same as in Fig. 2E. H = 17.61, p < 0.001, Kruskal-Wallis test, Dunn’s post-hoc test. 2-2 Adult male crickets 1 week after the imaginal molt were isolated for 3 days. Crickets were received injection of PTX, forskolin, TPA, SNAP, anisomycin, A23187, or vehicle. Brains were dissected for RNA extraction 60 min after injection (PTX, n = 8; the other treatments, n = 16). Gryllus fra-A, H = 62.94, p < 0.0001; Gryllus fra-B, H = 40.22, p < 0.0001; Gryllus jra, H = 56.30, p < 0.0001; Gryllus egr-B, H = 39.41, p < 0.0001; Gryllus hr38, H = 67.07, p < 0.0001, Kruskal-Wallis test, Dunn’s post-hoc test.
Movies
- Movie 1.
EYFP immunoreactivity in the optical sections of the supraesophageal ganglion of IEG reporter line 6 h after vehicle injection.
- Movie 2.
EYFP immunoreactivity in the optical sections of the supraesophageal ganglion of IEG reporter line 6 h after PTX injection stained with anti-GFP antibody.
Figure 1-1
Gryllus ef1α is the most stable internal control for RT-qPCR analysis in the cricket brain. Validation of housekeeping genes as reference genes for RT-qPCR expression analysis in the cricket brain. A, Expression levels of three housekeeping genes (Gryllus β-actin, ef1α, and rpl32) in the brain of wild-type adult crickets used for initial expression analysis (n = 16 including the PTX-injected and vehicle-injected crickets [n = 8, respectively]; see Fig. 1). The expression level of each housekeeping gene was normalized to the geometric mean of three housekeeping genes (GMβ-actin/ef1α/rpl32; Vandesompele et al., 2002). Box plots indicate 25th to 75th percentile ranges and central values, and ‘+’ indicates mean. Error bars indicate 5th to 95th percentile ranges. In the cricket brain, Gryllus β-actin and ef1α genes were expressed at the same levels, but Gryllus rpl32 was expressed more weakly than other two housekeeping genes. Among three housekeeping genes, the coefficient of variation (CV) of Gryllus ef1α was the lowest (CV of Gryllus β-actin/GMβ-actin/ef1α/rpl32, 20.99%; CV of Gryllus ef1α/GMβ-actin/ef1α/rpl32, 8.85%; CV of Gryllus rpl32/GMβ-actin/ef1α/rpl32, 22.5%), indicating Gryllus ef1α is the most stably expressed in the cricket brain. A, Gene expression stability of three housekeeping genes calculated using the Normfinder algorithm (Andersen et al., 2004). NormFinder algorithm supported that Gryllus ef1α is the most stable housekeeping gene (stability value of 0.036). The stability value of Gryllus ef1α was lower than that of the best combination pair (Gryllus β-actin and ef1α; stability value of 0.058). Download Figure 1-1, EPS file.
Figure 1-2
Gryllus fra gene encodes two protein isoforms closely related to insect and vertebrate Fos/Fra homologs. A, Protein domain structures of Gryllus Fra proteins and Fos/Fra homologs of other species. Conserved domains and sequences important for transcriptional regulation are indicated by color boxes. DBD, DNA-binding domain; HOB, homology box; Leu zip, Leucine zipper domain; TBM, TATA-binding protein (TBP)-binding motif. Gryllus fra-A encodes a 382-amino-acid protein containing the DBD, Leu zip domains and the C-terminal regulatory domain. Vertebrate c-Fos proteins contain N-terminal and C-terminal transactivation domains and a repression domain at its C-terminus. The core motif of the N-terminal transactivation domain (HOB1-N) is well conserved among vertebrate Fra proteins, but not conserved in insect Fra proteins. Instead, insect Fra proteins contain sequence motifs which might play important roles in transactivation function: Drosophila Kayak-A isoform contains a δ-like motif (Ciapponi et al., 2001), and Drosophila Kayak-D/F isoforms contain glutamine-rich regions in the isoform-specific N-terminal region. Gryllus Fra-A isoform and its related insect Fra proteins contain an acidic patch structurally resembles the acidic activation domains of many eukaryotic transcriptional activators such as Gal4, VP16, p53 and EcR-B1 (Cress and Triezenberg, 1991; Ruden, 1992; Regier et al., 1993; Watanabe et al., 2010). In addition, the T/P-rich region is conserved in most insect Fra proteins. Gryllus fra-B encodes an N-terminal truncated 284-amino-acid protein. B, Sequence alignment of the N-terminal region of Gryllus Fra-A and insect Fra/Kayak isoforms, and the C-terminal regulatory domain of Fos/Fra proteins. The conserved residues are marked with asterisks above the alignments. The amino acid residues are represented in the default color scheme of ClustalX. Positions of conserved domains/motifs were indicated by bars under the alignments. GenBank IDs of proteins are following: Apis mellifera Kayak-X1, XP_006564216; Bombyx mori Kayak-X2, XP_004921825; Drosophila melanogaster Kayak-A, NP_001027579; D. melanogaster Kayak-B, NP_001027578; D. melanogaster Kayak-D, NP_001027580; D. melanogaster Kayak-F, NP_001027577; Homo sapience c-Fos, NP_005243; H. sapience Fra1, NP_005429: H. sapience Fra2, NP_005244; H. sapience Fos-B, NP_006723; Tribolium castaneum Kayak-C, NP_001164294. Download Figure 1-2, EPS file.
Figure 1-3
Gryllus jra gene encodes a protein closely related to insect and vertebrate Jun/Jra homologs. A, Protein domain structures of Gryllus Jra protein and Jun/Jra homologs of other species. Conserved domains and sequences important for transcriptional regulation are indicated by color boxes. DBD, DNA-binding domain; HOB; homology box; Leu zip, Leucine zipper domain. B, Sequence alignment of the conserved domains for transcriptional regulation (δ domain and HOB motifs). The conserved residues are marked with asterisks above the alignments. The amino acid residues are represented in the default color scheme of ClustalX. Positions of conserved domains/motifs were indicated by bars under the alignment. GenBank IDs of proteins are following: D. melanogaster Jra, NP_476586; H. sapience c-Jun, NP_002219; H. sapience Jun-B, NP_002220; H. sapience Jun-D, NP_005345. Download Figure 1-3, EPS file.
Figure 1-4
Gryllus egr gene encodes a protein closely related to insect and vertebrate Egr-1 homologs. A, Protein domain structures of Gryllus Egr-B protein and Egr homologs of other species. Conserved domains and sequences important for transcriptional regulation are indicated by color boxes. Three C2H2-type zinc finger domains, as well as a nuclear localization signal (NLS) and a potential acetylation site (Ac), were highly conserved across vertebrate and invertebrate Egr homologs. On the other hand, we found low sequence conservation in the repressor domain between Egr homologs of insect and other species. The WW binding motif (PPxY, where x = any amino acid), which involved in protein–protein interaction with the Yes kinase-associated protein 1 (Zagurovskaya et al., 2009), was conserved across vertebrate and invertebrate Egr homologs. B, Sequence alignment of the C-terminal region of Egr proteins. The conserved residues are marked with asterisks above the alignment. Positions of functional domains important for DNA-binding, protein localization, and transcriptional regulation are indicated by bars under the alignment. C, Sequence alignment of the N-terminal region of insect/crustacean Egr-B proteins. The N-termini of insect/crustacean Egr-B proteins were highly conserved (conserved N-terminal motif 1; consensus sequence: MIM(D/E)FΨ(D/E)TL, where Ψ = bulky hydrophobic residues). Another conserved motif was found at residues from 105 to 140 of Gryllus Egr (conserved N-terminal motif 2). These two conserved motifs were only found in the N-terminal region of the insect/crustacean Egr-B proteins, but not in the vertebrate Egr homologs. Another Egr isoform (Egr-A or Stripe-A) found in several insect species (e.g. fruit flies and honeybees) contains an N-terminal extension with polyglutamine stretch (Ugajin et al., 2016). The conserved residues are marked with asterisks above the alignment. The amino acid residues are represented in the default color scheme of ClustalX. The positions of conserved motifs were indicated by bars under the alignment. GenBank IDs of proteins are following: Acyrthosiphon pisum Egr, XP_001943786; Anoplophora glabripennis Egr-B, XP_018579268; Apis mellifera Egr-B (AmEgr variant III), ANS58852; Aplysia californica Egr-1-like, NP_001268725; Calliphora vicina Stripe-B, AAZ95459; D. melanogaster Stripe-B, NP_732289; Tribolium castaneum Egr, XP_015837968l; Mus musculus Egr-1, NP_031939. The N-terminal sequences of Homarus americanus Egr-B, Periplaneta americana Egr-B, and Procambarus clarkii Egr-B were deduced from following transcriptome shotgun assembly sequences: GEBG01017003.1, GEIF01013459.1, and GBEV01045599.1, respectively. Download Figure 1-4, EPS file.
Figure 1-5
Molecular cloning and expression characteristics of Gryllus hr38 in the cricket brain. A, Comparison of the amino acid sequences of Gryllus hr38 deduced from its partial cDNA and its corresponding part of hr38 homologs in other insects. The conserved residues are marked with asterisks above the alignments. The amino acid residues are represented in the default color scheme of ClustalX. The domain structure of Drosophila DHR38-D is represented above the alignment. GenBank IDs of proteins are following: D. melanogaster DHR38 isoform D (DHR38-D), NP_001163024; Bombyx mori HR38, P49870.1; Tribolium castaneum HR38, XP_008194320.1; Apis mellifera HR38, XP_016773251.1; Zootermopsis nevadensis HR38, KDR09534.1. B, C, Expression of Gryllus hr38 after PTX injection in the brains of (B) cycloheximide- and (C) saline-pretreated crickets. Asterisks indicate statistical significance to control (0 min after PTX injection). In both pretreatment groups, Gryllus hr38 reached a maximum 60–90 min after PTX injection (∼60-fold and ∼15-fold up-regulation relative to the naïve animals and to the control (0 min after PTX injection), respectively) and remained at a high level 120 min after PTX injection. The expression kinetics of Gryllus hr38 was not affected by blockade of de novo protein synthesis (two-way ANOVA, Effect of pre-treatment: F(1,98) = 0.2142, p = 0.6445; Effect of time: F(6,98) = 14.45, p < 0.0001; interaction: F(6,98) = 0.5547, p = 0.7652). D, Expression time course of the pre-mRNA of Gryllus hr38 in the brain of cycloheximide- and saline-pretreated crickets after PTX-injection. Asterisks and daggers indicate statistical significance to control (0 min after PTX injection) within each pre-treatment group (***, p < 0.001; ††††, p < 0.0001). In both pretreatment groups, the expression of Gryllus hr38 pre-mRNA was significantly elevated 30 min after PTX injection (200∼300-fold up-regulation relative to the naïve animals), and rapidly decreased to near baseline level by 60–90 min after injection. The expression kinetics of Gryllus hr38 pre-mRNA was not affected by blockade of de novo protein synthesis (two-way ANOVA, Effect of pre-treatment: F(1,98) = 0.5631, p = 0.4548; Effect of time: F(6,98) = 16.11, p < 0.0001; interaction: F(6,98) = 1.733, p = 0.1212). E, Correlation plot between the expression levels of Gryllus hr38 and Gryllus egr-B in the brains of cycloheximide-pretreated crickets. The data from the cycloheximide- and PTX-injected crickets (n = 48; black circles), cycloheximide pretreated crickets (n = 8; gray circles), and naïve crickets (n = 8; white circles) were plotted. F, G, Behaviorally evoked expression of Gryllus hr38 in the brain of crickets 1 h after (F) sucrose feeding and (G) agonistic interaction. The expression levels were normalized to the mean of those of the naïve animals (baseline expression level). An asterisk indicates statistical significance between the indicated groups. RT-qPCR analyses were performed on eight biological replicates. Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Asterisks donate statistical significance to the control (0 min after PTX injection; B, C) or to the naïve animals (F, G; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). See Table 3 for the details of statistical analysis.Download Figure 1-5, EPS file
Figure 2-1
Expression characteristics of bZip transcription factor genes in the cricket brain. Expression characteristics of (A–D) Gryllus fra-A (E–H) fra-B, and (I–K) jra. A, E, I, Expression time course of Gryllus fra-A, fra-B, and jra after PTX-injection in the brains of cycloheximide-pretreated crickets. The expression levels were normalized to the mean of those of the naïve animals (baseline expression level). The expressions of Gryllus fra-A and fra-B reached a maximum 60 min after PTX injection (fra-A, ∼3.5-fold and ∼2.5-fold up-regulation relative to the naïve animals and to the control (0 min after PTX injection), respectively; fra-B, ∼2-fold up-regulation relative to both the naïve animals and to the control (0 min after PTX injection), whereas that of Gryllus jra reached a maximum 45 min after PTX injection (∼6-fold and ∼2-fold up-regulation relative to the naïve animals and to the control, 0 min after PTX injection, respectively). B, F, J, Correlation plot between the expression levels of Gryllus fra-A, fra-B, and jra with Gryllus egr-B in the brains of cycloheximide-pretreated crickets. The data from the cycloheximide and PTX-injected crickets (n = 48; yellow circles) and naïve crickets (n = 8; black circles) were plotted. Behaviorally evoked expression of Gryllus fra-A, fra-B, and jra in the brain of crickets 1 h after (C, G, K) sucrose feeding and (D, H, L) agonistic interaction. The expression levels were normalized to the mean of those of the naïve animals (baseline expression level). RT-qPCR analyses were performed on eight biological replicates. Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Asterisks donate statistical significance to the control (0 min after PTX injection; A, E, I) or to the naïve animals (C, D, G, H, K, L; *, p < 0.05; **, p < 0.01). See Table 3 for the details of statistical analysis. Download Figure 2-1, EPS file.
Figure 2-2
Pharmacologically induced expression of neuronal IEGs in the cricket brain. Pharmacologically induced expression of (A) Gryllus fra-A, (B) fra-B, (C) jra, (D) egr-B, and (E) hr38 in the brain of crickets. Following activators were used to stimulate intracellular signaling pathways: forskolin, an activator for adenylyl cyclases; 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator for protein kinase C; anisomycin, an activator for c-Jun N-terminal kinases; S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor; and a calcium-selective ionophore A23187. PTX was used as a positive control. The expression levels were normalized to the mean of those of the naïve animals (baseline expression level). Injection of 200 µM TPA resulted in significant increases in the expression levels of all neuronal IGEs (A–D). Injection of 200 µM forskolin and 200 µM A23187 resulted in significant increases in the expression levels of fra-A and jra, respectively. No obvious change was observed with any drug other than TPA in the expression levels of Gryllus fra-B and egr-B (B and D). The expression level of Gryllus hr38 was drastically affected by stimulation of intracellular signaling pathways (E). RT-qPCR analyses were performed on 8 or 16 biological replicates. Box plots indicate the 25th to 75th percentile ranges and central values. Error bars indicate the 5th to 95th percentile ranges. The “+” denotes the mean. Letters above the plots (a and b) indicate statistical significance (p < 0.05) to the naïve control and vehicle control, respectively. See Table 3 for the details of statistical analysis. Download Figure 2-2, EPS file.
Figure 3-1
Sequence comparison of the DNA-binding domain of stimulus-regulated transcription factors. The amino acid sequence of the DNA-binding domain of stimulus-regulated transcription factors of mouse and several insect species were aligned. (A) Fos family, (B) Jun family, (C) ATF2, (D) ATF3, (E) ATF4/5, (F) ATF6, (G) large Maf family, (H) small Maf family, (I) CREB1 family, (J) insect CREB-B family, (K) C/EBPs except for C/EBPγ and C/EBPζ, (L) C/EBPγ, (M) Egr family, (N) NFAT family, (O) MEF2 family, and (P) SRF. The residues for protein–DNA interaction were highly conserved in Jun family proteins, large/small Maf family proteins, CREB-like proteins, C/EBPs except for C/EBPγ, MEF2, and SRF (see A, G–K, and O–P). One or few substations were detected in Fos family proteins, Egr and NFAT homologs (see A, M, and N). Extensive substitutions were found in the DBD of ATF4/5, ATF6, and C/EBPγ (see B and F). A lineage-specific occurrence of substitutions in the DBD of insect ATF2 homologs (see C). That is, extensive substitutions were detected in ATF2 homologs of Drosophila, Apis, and Tribolium (“advanced” holometabolous insects), whereas the amino acid sequence of the ATF2 DBD is conserved between mouse and Zootermopsis (a “basal” hemimetabolous insect). The conserved residues are marked with asterisks above the alignments. Residues important for nucleotide binding were indicated by black circles under the alignments. Red circles indicate the residues important for nucleotide binding where amino acid substitutions were found in most insect homologs. Residues important for protein–protein interaction (i.e. dimerization) were indicated by white circles under the alignments. The amino acid residues are represented in the default color scheme of ClustalX. GenBank IDs of proteins used for sequence comparison are listed in Table 3-1. Download Figure 3-1, EPS file.
Table 3-1
List of stimulus-regulated transcription factors (TFs) in mammals and their homologs in insects. Download Table 3-1, DOC file.
Figure 7-1
Molecular cloning of Gryllus Tdc genes. A, Octopamine biosynthesis pathway in the insect brain. Tdc catalyzes the first step of octopamine biosynthesis by converting L-tyrosine into tyramine. B, C, Insect Tdc proteins contain subtype-specific C-terminal extensions. Comparison of the amino acid sequences of the C-terminal extensions of (B) Gryllus Tdc1 and (C) Tdc2 with their corresponding parts of Tdc homologs in other insects. The conserved residues are marked with asterisks above the alignments. The amino acid residues are represented in the default color scheme of ClustalX. Positions of the C-terminal portion of the catalytic domains were indicated by bars under the alignment. GenBank IDs of proteins are following: D. melanogaster Tdc1, NP_610226; D. melanogaster Tdc2, NP_724489; A. aegypti Tdc1, XP_001656851; A. aegypti Tdc2, XP_001656857; T. castaneum Tdc1, XP_972728; T. castaneum Tdc2, XP_972688. Download Figure 7-1, EPS file.
Figure 7-2
Distribution of the Tdc2-expressing neurons in the cricket brain. A, B, Frontal (A) view of the supraesophageal ganglion and ventral (B) view of the subesophageal ganglion stained with anti-Gryllus Tdc2 antibody. The outlines of the ganglia are indicated by the white dotted lines. The depth of the cells is color coded as indicated in the inset. Dorsoventral (D-V) or rostrocaudal (R-C) axes were indicated. Scale bars represent 200 µm. Download Figure 7-2, EPS file.
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