GENERAL ENDOCRINOLOGY Epidermal Growth Factor Receptor in the Prawn Macrobrachium rosenbergii: Function and Putative Signaling Cascade Omri Sharabi, Tomer Ventura, Rivka Manor, Eliahu D. Aflalo, and Amir Sagi Department of Life Sciences and the National Institute of Biotechnology in the Negev (O.S., R.M., E.D.A., A.S.), Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel; and Faculty of Science, Health, Education, and Engineering (T.V.), GeneCology Research Centre, the University of the Sunshine Coast, Queensland 4558, Australia Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene is expressed in most tested tissues. The role of Mr-EGFR was revealed by temporarily silencing the transcript through weekly injections of double-stranded Mr-EGFR RNA. Such treatment resulted in a significant reduction in growth and a delay in the appearance of a male secondary sexual characteristic, namely the appendix masculina. An additional function of Mr-EGFR was revealed with respect to eye development. Although the optic ganglion appeared to have retained its normal morphology, Mr-EGFR-silenced individuals developed abnormal eyes that presented irregular organization of the ommatidia, reflected by unorganized receptor cells occupying large areas of the dioptric portion and by a shortened crystalline tract layer. (Endocrinology 154: 3188 –3196, 2013) I n earlier work, we identified and characterized an insulin-like hormone in the giant freshwater prawn, Macrobracium rosenbergii. This hormone, termed Mr-IAG, was found to determine sexual differentiation and to affect growth of the animal (1). Recently it was shown that the insulin signaling pathway interconnects with the epidermal growth factor (EGF) signaling pathway (2). Epidermal growth factor receptors (EGFRs) comprise an important and highly conserved receptor group that forms part of the EGF signal transduction pathway. Widely found in metazoans ranging from yeast to mammals, with detailed information having been primarily gathered from a few model organisms, EGFRs have yet to be identified in crustaceans. EGFR is a transmembrane glycoprotein belonging to the tyrosine kinase receptor family. After activation by a variety of ligands, such as EGF and insulin (3), plasma membrane-localized monomeric tyrosine kinase receptors dimerize and autophosphorylate their cytoplasmic domains. This triggers the tyrosine kinase activity of the receptors that in turn stimulates a signal transduction cascade, leading to changes in cellular physiology and/or patterns of gene expression (4). ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received March 19, 2013. Accepted June 26, 2013. First Published Online July 3, 2013 Abbreviations: aa, amino acids; AM, appendix masculina; ds, double-stranded; EGF, epidermal growth factor; EGFR, EGF receptor; Mr-EGFR, transcript encoding M. rosenbergii EGFR; PL, post-larvae; RB, Remebee; SEM, scanning electron microscopy; SMART, Simple Modular Architecture Research Tool; t, time. 3188 endo.endojournals.org Endocrinology, September 2013, 154(9):3188 –3196 doi: 10.1210/en.2013-1259 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/en.2013-1259 Although EGFR functions similarly in vertebrates and invertebrates at the structural and cellular levels, with both inducing cell proliferation and differentiation, distinct phenotypic effects of receptor activity are seen in the 2 groups. EGFR plays crucial roles during vertebrate development, with mutations inducing defects in multiple organs, such as the brain, lung, skin, heart, and eye, and in many instances death (4). Likewise, EGFR is also a major component controlling invertebrate development and has been extensively studied in model organisms, such as Drosophila melanogaster (5), Caenorhabditis elegans (6), and recently the cricket Gryllus bimaculatus (7). Such studies have shown the EGFR to assume more than 30 distinct roles during invertebrate development, including those related to dorsoventral development, oogenesis, and spermatogenesis, eye development, and growth. In addition, research on invertebrate model organisms has shown that many physiological processes are influenced by the EGF pathway, including growth, development, reproduction, and aging. The EGFR-mediated cascade thus plays major roles in invertebrate life history regulation (8) and is thought to be important for the early development of M. rosenbergii, possibly intersecting with the Mr-IAG system that regulates sexual differentiation (9). Indeed, the connection of a similar group of receptors, the insulin-like receptors, to masculine sexual differentiation was demonstrated in a vertebrate (10). To gain further insight into the role of the EGFR during invertebrate sexual development, a transcriptome library prepared from early developmental stages of the prawn M. rosenbergii recently established in our laboratory using next-generation sequencing techniques (11) was used to mine for EGFR-related transcripts. Among the EGFR-related transcripts identified, several were found upstream of the receptor, encoding potential ligands, whereas genes involved in the cellular signal transduction cascade of the EGFR pathway were identified downstream of the receptor. To elucidate the function of EGFR, the application of long double-stranded (ds) RNA was performed similarly to our previous work in the prawn (1, 12). Although, the mechanism by which the long dsRNA is internalized into crustacean cells is not fully understood, it is hypostasized that the dsRNA travels via the circulating hemolymph to distant tissues (13, 14). Our findings on the function of M. rosenbergii EGFR represent, to the best of our knowledge, the first such report from any crustacean. Upon long-term silencing of M. rosenbergii EGFR expression from the early post-larvae (PL) stage, a significant reduction in growth, delayed development of an external sexual characteristic, and abnormalities in eye development were observed. endo.endojournals.org 3189 Materials and Methods Animals M rosenbergii PL16 (postlarvae, 16 d from metamorphosis) males were produced from a ‘neo-female’ animal and maintained at Ben-Gurion University of the Negev, as previously described (12). These individuals were used to study the effects of dsMrEGFR treatment. Mature male and female individuals, progeny of normal females, were used for tissue specificity assessment. Bioinformatics analyses Genes encoding putative components of a generalized diagram of the EGF signal transduction cascade were excavated, based on the report by Borisov et al (2), as were potential EGFR ligands, including Mr-IAG (1) and 4 D. melanogaster EGFR ligands (5). A key word-based search of our transcript library (11) yielded several sequences, which then served as queries in the BLAST searches designed to reveal the similarity between our hypothetical sequences and those homologues found in other organisms. To obtain the deduced protein sequence, full length MrEGFR cDNA was computationally translated using the ExPASy Proteomics Server (http://web.expasy.org/translate/),and the most likely frame was selected (ie, the first 5⬘ to 3⬘ frame). Conserved domains were identified in the putative Mr-EGFR protein using Simple Modular Architecture Research Tool (SMART) (15). Using ClustalW (16), multiple sequence alignment of the predicted Mr-EGFR sequence was performed with 2 representative arthropod members of the EGFR family, namely from D. melanogaster (GenBank accession number AAM70919) and Apis mellifera (UniprotKB accession number P0cCY46). Spatial expression pattern of Mr-EGFR The spatial expression pattern of Mr-EFGR was examined by RT-PCR. Total RNA was isolated from the male testis, hepatopancreas, androgenic gland, thoracic-ganglia, sperm duct, heart, and tail muscle from 3 mature males. Two of the males were in the intermolt stage and one at postmolt. RNA was also extracted from the ovary, hepatopancreas, thoracic-ganglia, heart, and tail muscle of 3 mature females, all at the intermolt stage, as previously described (12). All tissue sampling was done after the animals were anesthetized on ice for 10 minutes. First-strand cDNA was synthesized by reverse transcription using the Verso cDNA kit (Thermo Fisher Scientific, Epsom, Surrey) with 1 ␮g of total RNA. Specific Mr-EGFR forward (Mr-EGFR-1F; nucleotides 88 –111, 5⬘-GCAAGGACAAAGGGAAAATATGC-3⬘) and reverse (Mr-EGFR-R; nucleotides 576 –599, 5⬘-CAGGATTTGGAGCACTGACAAGT-3⬘) primers were used for PCR amplification, as previously described (12), except that 37 cycles and an annealing temperature of 60°C was used here. Mr-actin, serving as a positive control, was amplified as previously described (1). PCR products were separated on a 1.2% agarose gel, visualized by ethidium bromide staining, and photographed under UV illumination by a gel image documentation system (Bio Imaging system; Chemi Genius, Syngene, Cambridge, United Kingdom). Preparation of dsRNA Two PCR products were prepared using a T7 promoter anchor (T7P; 5⬘-TAATACGACTCACTATAGGG-3⬘) attached to The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. 3190 Sharabi et al Prawn EGFR Function and Putative Cascade one primer for each product. The primers used for generating the template for sense-strand RNA synthesis were (5⬘-T7P-GAAAGATAGTGGTGCCTGCGTTA-3⬘) as forward primer and (5⬘CTTTTCCCCAGCAACCTTCATTA-3⬘) as reverse primer. Primers used for generating the template for antisense strand RNA synthesis were 5⬘-GAAAGATAGTGGTGCCTGCGTTA-3⬘ as forward primer and 5⬘-T7P-CTTTTCCCCAGCAACCTTCATTA-3⬘. PCR amplicons were separated by electrophoresis on a 1.2% agarose gel, visualized with ethidium bromide and UV light, excised from the gel, and purified with a HiYield Gel/PCR DNA fragments extraction kit (RBC Bioscience, New Taipei City, Taiwan). dsRNA of the Mr-EGFR open reading frame (650 nucleotides from positions 918 to 1567 was prepared using a TranscriptAid T7 high yield transcription kit (Fermentas UAB, Vilnius, Lithuania), according to the manufacturer’s instructions. The 2 strands were hybridized by heating to 70°C for 15 minutes and to 65°C for 15 minutes, followed by incubation at room temperature for 30 minutes. RNA was quantified and diluted to 1 ␮g/␮L and quality was assessed on an agarose gel. The dsRNA was maintained at ⫺80°C until used. In vivo Mr-EGFR silencing Ninety-five PL16 male individuals, progeny of a ‘neo-female’ (17), were collected and divided into 2 groups. Each individual was injected on a weekly basis. Seventy-one individuals were injected with dsMr-EGFR (5 ␮g/g body weight). Twenty-four individuals were injected with Remebee (RB), a dsRNA formulated to silence Israeli acute paralysis virus affecting bees (18), supplemented by Beeologics (Rehovot, Israel; 5 ␮g/g body weight). Body weight was monitored weekly up to the 13th week. One day after the 13th injection, 6 individuals from each treatment were taken for Mr-EGFR transcript level analysis by real-time RT-PCR, whereas the remaining individuals were kept under the weekly injection regimen and the development of the appendix masculina (AM) was monitored until the point that all control individuals have developed the AM (week 25). From week 22 to week 25, remaining individuals of the treatment group were monitored for molt events weekly, and compared to control animals at the same age, size, and conditions, in order to study the molting rate/growth interrelationship. Endocrinology, September 2013, 154(9):3188 –3196 quantitative Mr-18S reverse (5⬘-TACCCCCGGAACTCAAAGA-3⬘) with the above-mentioned mix and the Universal ProbeLibrary Probe 152 kit (Roche). Reactions were performed using the ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, California). Eye histology and scanning electron microscopy (SEM) Eyes from one individual of each group were removed at the base of the eye stalk. Left eye samples were fixed in modified Carnoy’s II for 48 hours at 4°C and dehydrated gradually through a series of increasing alcohol concentrations. The tissues were cleared and embedded in Paraplast (Kendall, Mansfield, Massachusetts), according to conventional procedures. Sections (5 ␮m thick) were mounted onto silane-coated slides (MenzelGläser, Braunschweig, Germany), stained with hematoxylin and eosin, and observed under a light microscope. Right eye stalks were prepared as previously described by Bitan et al (19) for SEM. Statistical analyses The effects of dsRNA injection on relative transcript levels, as revealed by the real-time RT-PCR, were statistically analyzed by the nonparametric Mann-Whitney U test using Statistica 6.1 Relative quantitation of Mr-EGFR expression levels RNA was extracted from the testes of silenced individuals after 13 injections of dsMr-EGFR and from control individuals injected with Remebee (Beeologics). Total RNA was isolated with the EZ-RNA total RNA isolation kit (Biological Industries, Beit Haemek, Israel), according to the manufacturer’s instructions. First-strand cDNA was synthesized using the Verso cDNA kit (Thermo Fisher Scientific) with 1 ␮g of total RNA. Relative quantification of Mr-EGFR transcript levels was achieved using the Mr-EGFR quantitative PCR forward (5⬘-GAAAGAAAATACGCTCACCTTG-3⬘) and Mr-EGFR quantitative PCR reverse (5⬘-AGTCACCTCTTGGACGTTGC-3⬘) primers together with the FastStart Universal probe master (Rox; Roche Diagnostics, Penzberg, Germany) and Universal ProbeLibrary Probe 50 (Roche) kits. Mr-18S (GenBank accession number GQ131934), serving as a normalizing gene, was also quantified by means of real-time RT-PCR using primers quantitative Mr18S forward (5⬘-CCCTAAACGATGCTGACTAGC-3⬘) and Figure 1. A general EGF cell signaling cascade. Pathway components for which transcripts were found in the M. rosenbergii transcript library were annotated and are shown in gray. Solid lines with arrows show the activation of proteins and lipids by tyrosine phosphorylation. Dotted lines represent direct protein-protein and protein-lipid interactions. Lines with blunt ends reflect inhibition. [Reproduced from N. Borisov et al: Systems-level interactions between insulin-EGF networks amplify mitogenic signaling. Mol Syst Biol. 5:256, 2009 (2), with permission. ©The Endocrine Society.] The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/en.2013-1259 endo.endojournals.org software (StatSoft, Tulsa, Oklahoma). The effects of dsRNA injection on body weight was statistically analyzed by the MannWhitney U test, whereas to evaluate effects on molt frequency, a ␹2 test comparing molt event numbers per group per week, calculated as the percentage of the number of individuals per group, was performed. To evaluate the effects of dsRNA injection (binary explanatory variable dsEGFR injection equals 1 and dsRB equals 0) on the rate of AM development, a Cox proportional hazards regression model (20) was used. The model is expressed by the equation ␮(t; z) ⫽ ␮0(t)exp (␴␤izi), where ␮(t,z) represents AM development and ␮0(t) corresponds to the baseline AM development rate that can change over time (t). The regression coefficient to be estimated, ␤i, represents the independent effect of dsRNA injection on AM development. Exp ␤ represent the expected change in AM developmental rate after the treatment. Analysis was performed using S-PLUS 2000 software (Mathsoft, Needham, Massachusetts). Results Identification of putative components of the MrEGF signaling cascade In our M. rosenbergii postembryonic transcriptomic library (11), transcripts putatively involved in the EGF signaling cascades were found (Figure 1), including EGFR, Mr-IAG, and 4 other potential EGFR ligands. Three of the ligands (Spitz, Gurken, and Keren) showed high similarity to the sequence of one contig (identical domain structures), whereas a fourth ligand, Argos, showed high similarity to another contig (Table 1). All downstream EGF cascade genes listed by Borisov et al (2) were also found in our library (see Supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Table 1. 3191 Sequence of the Mr-EGFR transcript and the deduced protein Efforts focused on that transcript possessing high similarity (as high as 60%) to known EGFRs in the GenBank database and thus termed M rosenbergii epidermal growth factor receptor (Mr-EGFR). Mr-EGFR (accession number NCBI SRA STUDY PRJNA174197) is 6864 nucleotides long, with a predicted open reading frame encoding a 1461-amino acid (aa) translation product, a 138-bp 5⬘untranslated region and a 2340 bp 3⬘-untranslated region ending with a poly A tail (Supplemental Figure 1). The best BlastX hit for Mr-EGFR was a predicted EGFR of the leaf cutter bee Megachile rotundata (GenBank accession number XP_003708039), sharing 60% similarity. Examination of the deduced amino acid sequence of Mr-EGFR revealed several conserved domains, namely a signal peptide (aa 1–23), 2 ligand-binding domains (aa 63–174; E-value ⫽ 3.7e⫺25, and aa 354 – 482; E-value ⫽ 2.1e⫺25), 7 Furin-like domains (aa 191–230, 234 –278, 500 –550, 553–597, 599 – 655, 672–717, and 721–776, E-values between 2.23 and 3.4e⫺7), a transmembrane domain (aa 804 – 826), a tyrosine kinase domain (aa 872–1128; E-value ⫽ 2.58e⫺131), and 2 low-complexity domains (aa 1191–1213 and 1412–1429) (Figure 2). Multiple sequence alignment of the 2 ligand-binding domains of Mr-EGFR and the D melangostar and A mellifera EGFRs computed by the ClustalW algorithm revealed a high degree of similarity (Figure 3A). SMART representation of the 3 full sequences demonstrated the highly similar domain patterns of Mr-EGFR and D melanogaster EGFR, with lesser similarity between Mr-EGFR and A mellifera EGFR being noted (Figure 3B). The ex- Putative Homologues of Potential Ligands of Mr-EGFR Encoded in the M rosenbergii Transcript Library Best 3 BLASTP Results Transcript GenBank Accession Number Description Accession E-Value Identity Mr-IAG ACJ38227 Insulin-like androgenic gland specific factor (Macrobrachium rosenbergii) Insulin-like androgenic gland factor (Macrobrachium nipponense) Insulin-like androgenic gland factor (Macrobrachium lar) TGF-␣ (fragment) (Gryllus bimaculatus) Protein spitz, putative (fragment) (Ixodes scapularis) Putative uncharacterized protein (Pediculus humanus corporis) Conserved hypothetical protein (Pediculus humanus corporis) Predicted protein giant-lens-like (Acyrthosiphon pisum) Predicted: similar to argos (Tribolium castaneum) ACJ38227 2e-124 100% AGB56976 6 e-99 81% BAJ78349 1e-82 70% D7RNZ2_GRYBI 1e-39 80.91% B7P9D6_IXOSC 1 e-35 72.22% E0W2V4_PEDHC 2e-35 73.64% Mr-Spitz; Mr-Keren; Mr-Gurken Mr-Argos National Center for Biotechnology Information (Bethesda, MD) SRA study PRJNA174197 National Center for Biotechnology Information SRA study PRJNA174197 XP_002429035 5 e-51 74% XP_001948865 2e-44 71% XP_970857 6e-42 67% SMART Predicted Domains Conserveed insulin-like cysteine pattern EGF, transmembrane Argos GeneBank accession numbers refer to sequences found in the M. rosenbergii transcript library. E values represent the similarity between the best 3 Blastp results to the putative homologues from the transcription library. The conserved domains are based on SMART results of the putative proteins. The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. 3192 Sharabi et al Prawn EGFR Function and Putative Cascade Endocrinology, September 2013, 154(9):3188 –3196 The spatial expression pattern of Mr-EGFR RT-PCR showed that in adult M. rosenbergii, the Mr-EGFR transcript is expressed in the gonads and thoracic ganglia of both male and female animals. Amplification was also observed in the heart, hepatopancreas, and tail muscles of both males and females and also in the male sperm duct (represented in Figure 4 by a male and female in the intermolt stage. All trials showed a similar expression pattern). No expression was detected in the androgenic gland. Mr-EGFR silencing and effects on growth, molt, and secondary sexual characteristics Real-time RT-PCR performed using RNA extracted from the testes of treated males showed significant reduction (⬃98%) in Mr-EGFR transcript levels, as compared with the control group (Mann-Whitney U test, P ⬍ .001; Figure 5). Mr-EGFR silencing resulted in a significant inhibition of accumulated body weight, as compared with the control group, from the sixth injection at PL50 (Mann-Whitney U test, P ⬍ 0.001; Figure 6A). The average body Figure 2. The deduced amino acid sequence Mr-EGFR. Beginning at the N terminus, one finds a weight of control group individuals signal peptide (bold, double underlined), 2 ligand-binding domains (bold and italicized, gray was 870.4 ⫾ 76.4 mg, whereas the background), 7 interspersed Furin-like domains (bold, dashed underline), a transmembrane average body weight of injected indomain (bold, italicized), a tyrosine kinase domain (gray boxes), and 2 low-complexity domains dividuals was 319.2 ⫾ 28.2 mg on (bold and italicized, dashed-dot underline). the 13th week of the experiment. Molting frequency was unaffected tracellular portions of the proteins (left of the transmem2 brane domain indicated by a white rectangle representing by the silencing (␹ test, t ⫽ 0.04, df ⫽ 3, P ⫽ .985) that domain) included a signal peptide (pentagonal arrows (Figure 6B). The percentage of molting events per week in the Mr-EGFR and the D. melanogaster EGFR se- was similar in both the control and silenced groups quences) followed by the 2 ligand-binding domains (gray (76.3% and 76.6%, respectively). The development of rectangles), separated by 2 Furin-like domains and fol- AM, a male-specific secondary sexual characteristic, lowed by 5 additional Furin-like domains (circles). The was also delayed in the Mr-EGFR-silenced group. In the intracellular portion of the protein (right of the transmem- control group, the AM appeared as early as PL57, brane domain) contains a tyrosine kinase domain (penta- whereas in the Mr-EGFR-silenced group, the first AM gon) followed by the 2 unstructured domains (inverted appeared only at PL84. This delay became significant triangles), found near the C terminus of the receptor. Thus, (Cox proportional hazards regression model, P ⬍ .05) the Mr-EGFR, D. melangoster EGFR, and A. mellifera by PL99, when compared with the control group. By EGFR sequences share the same number and order of re- PL106, all individuals in the control group had developed the AM, whereas in the Mr-EGFR-silenced group, ceptor domains. The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/en.2013-1259 endo.endojournals.org Figure 3. A, Multiple sequence alignment of the first and second Mr-EGFR ligand-binding domains with 2 arthropod EGFR ligand-binding domains. M rosenbergii EGFR (Mr-EGFR), Apis mellifera EGFR (Am-EGFR), and Drosophila melangostar EGFR (Dm-EGFR) were aligned with fully conserved residues (indicated by an asterisk), residues with strongly similar properties (indicated by a colon), and residues with weakly similar properties (indicated by a period) indicated. B, SMART algorithm representation of predicted EGFR domains in arthropod proteins. Signal peptide (arrow), ligand-binding domains (rectangles), repeats of Furin-like cysteine-rich domains (circles), a transmembrane domain (white rectangle), a tyrosine kinase domain (pentagram), and low-complexity domains (triangles) are portrayed. 3193 sized control individual (Figure 7, middle panels). Histological sections show that the cornea, crystalline cones, and crystalline tracts are present in silenced individuals (Figure 7, left bottom panel), although they were reduced to about half the size of the normal tracts in the intact eye (Figure 7, left top panel). In addition, the rhabdoms seem denser and thicker and appear to have much more surrounding pigmentation, as compared with a normal eye. The 3 ganglia regions in the eye stalk, ie, the medulla terminalis, medulla interna, and medulla externa, appear to be normal in the eyes of silenced individuals (Figure 7, left panels). The 3-dimensional surface of the eye was examined by SEM, which revealed that the shape of the facets on the surface of the optic portion was deformed. Whereas in the normal eye the facets have a rectangular shape, the eye facets of Mr-EGFR-silenced animals exhibited irregular shapes (Figure 7, right panels). only 47.6% of the animals had developed the AM by this point (Figure 6C). Effects of Mr-EGFR silencing on eye development In Mr-EGFR-silenced prawns, the optic portion of the eye was about half the size of a normal eye of an equally Figure 4. Mr-EGFR expression in various mature M. rosenbergii male and female tissues. Mr-EGFR cDNA is expressed in all tissue samples, excluding the AG, as shown by RT-PCR (top panel). cDNA for M. rosenbergii ␤-actin (bottom panel) served as a positive control. AG, androgenic gland; He, heart; HP, hepatopencreas; Mus, muscle; Ov, ovary; SD, sperm duct; Tes, testis; TG, thoracic ganglia. Discussion One of the transcripts found in the transcriptomic library of juvenile M. rosenbergii prawns (11), termed Mr-EGFR, represents the first gene of the EGFR family to be found in crustaceans. In the same library, we also detected transcripts representing most of the genes involved in the general EGF signaling cascade, as proposed by Borisov et al (2) (see Figure 1), including the gene encoding ERK, the MAPK. ERK is the ultimate enzyme in the cascade that targets many proteins whose activation eventually leads to cell proliferation and differentiation (21). Borisov et al (2) suggested that many of these genes are shared with the insulin-signaling cascade. The insulin-like androgenic gland hormone discovered in M. rosenbergii (1) was found to control sexual differentiation and to sustain male sexual characteristics and was suggested to be a driving force behind bimodal growth in this species, in which males grow faster and reach higher weights than do females (1, 9). It has been known for several years that insulin can be mitogenic (22), whereas EGF can evoke metabolic responses (23). Both share many The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. 3194 Sharabi et al Prawn EGFR Function and Putative Cascade Endocrinology, September 2013, 154(9):3188 –3196 Figure 5. Transcript levels affected by silencing of Mr-EGFR using dsRNA in male postlarvae. Silencing of Mr-EGFR was confirmed using real-time RT-PCR. The control group was injected with Remebee (Beeologics), an exogenous dsRNA. Asterisks represent statistically significant differences (Mann-Whitney U test, P ⬍ .01). downstream components in their networks and were found to regulate each other’s pathway of signal transduction (2). In our library of juvenile M. rosenbergii transcripts, we found many components of the insulin and EGF signal transduction networks suggested by Borisov et al (2) as possibly interacting. However, from the results presented here, it seems that in the prawn, interactions between the insulin (Mr-IAG) and EGF cascades are likely not extensive because Mr-EGFR is expressed in both adult males and females and in all tissues but the androgenic gland, whereas Mr-IAG is produced and expressed exclusively in males and in the androgenic gland (1). Emlen et al (24) suggested that the insulin and EGF pathways are involved with the development and evolution of secondary sexual characteristic in beetles. Similarly, our study demonstrated delayed development of the AM in the MrEGFR-silenced group. However, this seems to be related to growth rather than reproduction because no shifting of reproductive system development, as noted upon silencing of Mr-IAG, was observed (12, 25). Without further study, the involvement of EGFR in sexual differentiation cannot be completely ruled out. Indeed, the role of EGFR in M rosenbergii seems to be mostly related to the control of growth because silencing of the gene slowed mass accumulation in young males rather than frequency of molt events. These results agree with the findings of studies demonstrating the involvement of EGFR in body size regulation in arthropods, such as D. melanogaster, A. mellifera, and the cricket Gryllus bimaculatus (5, 7, 26). With respect to possible Mr-EGFR ligands, Mr-IAG, mentioned above, is a member of the insulin-like family Figure 6. Effects of Mr-EGFR dsRNA injection on growth, molt, and appearance of appendices masculinae in young M. rosenbergii males. A, Body weight of Mr-EGFR-dsRNA-injected (E) and dsRB-RNA injected (F) groups. Statistically significant differences (asterisks) were observed from the first measurement onward (Mann-Whitney U test, P ⬍ .001). Weekly injections started 16 days after metamorphosis. B, A 4-week comparison of individuals molted (percentage) per week in silenced vs control prawns was conducted (␹2 test, P ⫽ .829). C, Cumulative appearance of the appendix masculina as a percentage of the population in Mr-EGFR-dsRNA-injected (E) and dsRB-RNA-injected (F) groups. *, Statistically significant differences (Cox proportional hazards test, P ⬍ .05). and has been proven to be a growth regulator in M. rosenbergii (1). This is further implied through the suggested cross talk between the 2 cascades (2) and reports on the The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/en.2013-1259 endo.endojournals.org 3195 which are the square shape of the facets, unlike the hexagonal shape in aposition eyes (31), and the fact that the rhabdoms are separated from the crystalline cones by considerable distances (31–33). These distances were found to be significantly shorter in the eyes of Mr-EGFR-silenced M. rosenbergii. Most of the research on the involvement of EGFR in regard to eye development in arthropods has been performed on Drosophila, which possess aposition eyes much like prawn larva but unlike superposition eyes of postlarval and adult prawns. Aposition eyes do not contain crystalline tract or a clear zone, Figure 7. Effect of Mr-EGFR silencing on eye development in M. rosenbergii. A, Shown are a and the crystalline cones are in conhistological section (left panel), an image (middle panel), and a SEM image (right panel) of a tact with the rhabdoms (34, 35). normal eye. B, Shown are a histological section from a Mr-EGFR-silenced individual (left panel), as are an eye image (middle panel) and a SEM image (right panel). cor, cornea; crc, crystalline Thus, studies on the role of EGFR in cones; crt, crystalline tract; Fct, facets; mee, medulla externa; mei, medulla interna; met, medulla prawn eye development could not be terminalis; rha, rhabdomes. readily compared with studies on their fly counterparts, including the possible effects of insulin on EGFR (27, 28). However, the major effects seen in our study on the arrangement of the above differences in the mode of action and expression rhabdoms and their shape as well as on the proliferation suggest that Mr-IAG is probably not a ligand of Mr- of pigment cells in the prawn eye. Facets of prawn larvae, EGFR. Other than Mr-IAG, our next-generation sequenc- which are hexagonal, transform into the adult square form ing library includes transcripts similar to 4 growth and during metamorphosis (33, 36). Normally, crustacean development regulator ligands known to interact with D. eyes grow by adding new ommatidias each successive molt melanogaster EGFR, as described by Shilo (5). Three of (37). It is possible that during the period of the injections these ligands (Spitz, Keren, and Gurken) were mapped to and the silencing of Mr-EGFR, the process of forming and the same M rosenbergii transcript from our library be- adding new ommatidias was negatively affected; having cause all three are TGF-␣ homologues. The fourth ligand said that, we do not know whether there was any cell loss. identified (Argos) was similar to a different transcript, in To the best of our knowledge, this study represents the agreement with it being a D. melanogaster EGFR inhibitor first report of an EGFR, its upstream ligands, and its and it following a different trafficking and processing downstream putative signaling cascade in Decapoda. Our pathway from the other 3 ligands (5). We did not identify silencing of this gene revealed 3 morphological effects (eye a fifth ligand mentioned by Shilo (5), termed Vein, in our development, delayed AM development, and total body library. Also, the functions of the above-mentioned li- size), whereas in other arthropods, such as A. mellifera and gands in the context of the EGF cascade need to be further D. melanogaster, many more phenotypes were affected (5, elaborated. 26). As in insects, EGFR might also play an important role In arthropods, EGFR is known to play an important in early developmental stages in crustaceans. Because our role in the recruitment of almost all cell types involved in experiment was conducted at later developmental stages, the development of the ommatidium (29). Moreover, it is likely that Mr-EGFR silencing in crustacean larvae EGFR inhibition in Drosophila prevented differentiation and during the metamorphosis will elucidate additional of cone and pigment cells, as well as receptor cells, in the roles. eye (30). Similarly, our study in crustaceans demonstrated a major effect of Mr-EGFR silencing related to eye morAcknowledgments phology. Decapods from the family Palaemonidae have what is known as superposition eyes, which are charac- We thank Dr Ofer Ovadia for his help with the statistical analysis terized by certain anatomical features, the most notable of and Ms Yaara Lazar for her assistance with the molecular stud- The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 February 2014. at 00:48 For personal use only. No other uses without permission. . All rights reserved. 3196 Sharabi et al Prawn EGFR Function and Putative Cascade Endocrinology, September 2013, 154(9):3188 –3196 ies. We also thank Beeologics for their donation of Remebee dsRNA. 16. Address all correspondence and requests for reprints to: Amir Sagi, PhD, Department of Life Sciences and the National Institute for Biotechnology, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel. E-mail: sagia@bgu.ac.il. This work was supported by grants from Tiran, Ltd through BGNegev and National Institute of Biotechnology in the Negev. Disclosure Summary: The authors have nothing to disclose. 17. 18. 19. References 1. Ventura T, Manor R, Aflalo ED, et al. Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis. Endocrinology. 2009; 150(3):1278 –1286. 2. Borisov N, Aksamitiene E, Kiyatkin A, Legewie S, et al. Systemslevel interactions between insulin-EGF networks amplify mitogenic signaling. Mol Syst Biol. 2009;5:256. 3. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103(2):211–225. 4. 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