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Embryonic expression of Drosophila IMP in the developing CNS and PNS
Gene Expression Patterns 9 (2009) 138–143
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Embryonic expression of Drosophila IMP in the developing CNS and PNS Sidsel Kramshøj Adolph a,*, Robert DeLotto a, Finn Cilius Nielsen b, Jan Christiansen a a b
Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen N, Denmark Department of Clinical Biochemistry, University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
a r t i c l e
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Article history: Received 6 August 2008 Received in revised form 28 November 2008 Accepted 3 December 2008 Available online 11 December 2008 Keywords: IMP dIMP ZBP1 Vg1RBP IGF-II b-Actin CNS PNS Sensory organs Chordotonal organ External sensory organ Scolopale cell Sheath cell Drosophila RNA-binding protein
a b s t r a c t Drosophila IMP (dIMP) is related to the vertebrate RNA-binding proteins IMP1-3, ZBP1, Vg1RBP and CRD-BP, which are involved in RNA regulatory processes such as translational repression, localization and stabilization. The proteins are expressed in many fetal tissues, including the developing nervous system, and IMP up-regulation in solid tumors correlates with a high metastatic potential and poor prognosis. In this study, we used immunohistochemistry and live-imaging of an endogenous promoter-driven GFP-dIMP fusion protein to reveal the expression pattern of dIMP protein throughout embryogenesis. In the cellular blastoderm, immunoreactivity was seen in the entire cell-layer, where it was localized apically to the nucleus, and in the pole cells. Later, the GFP-dIMP fusion protein appeared in the developing central nervous system, both in the brain and in the ventral nerve cord. In the peripheral nervous system, immunoreactivity was detected in both neurons and accessory cells of chordotonal and external sensory organs. Ó 2008 Elsevier B.V. All rights reserved.
1. Results and discussion Drosophila melanogaster insulin-like growth factor II (IGF-II) mRNA-binding protein (dIMP) belongs to a family of RNA-binding proteins, comprising the human IMP1-3, chicken zipcode-binding protein 1 (ZBP1), murine c-myc coding region instability determinant binding protein (CRD-BP), and Xenopus Vg1 RNA-binding protein (Vg1RBP). During the last decade, different RNA targets such as IGF-II, b-actin, Vg1, c-myc, Tau and Semaphorin mRNAs, and the untranslated H19 RNA, have been identified. IMPs and their orthologues are implicated in post-transcriptional events such as translational inhibition, RNA localization and RNA stabilization (Yisraeli, 2005). The mammalian proteins are expressed in an oncofetal manner, with high levels in many fetal tissues and low levels in most adult tissues. Moreover, they are frequently up-regulated in transformed cell-lines (Leeds et al., 1997) and solid tumors where they are associated with a high metastatic potential and poor prognosis (Dimitriadis et al., 2007; Kato et al., 2007; Kobel et al., 2007; * Corresponding author. Tel.: +45 35322009; fax: +45 35322128. E-mail addresses: [email protected], [email protected] (S.K. Adolph). 1567-133X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2008.12.001
Sitnikova et al., 2008; Tessier et al., 2004). In the mouse, fetal expression is high in neuronal tissue, and IMP is also found in heart, lung, liver, intestine, epidermis and reproductive organs as well as in the snout, tail and limb buds (Hansen et al., 2004; Mori et al., 2001; Nielsen et al., 1999). In cultured neurons, IMPs are found in discrete particles in both axons and dendrites, and participate in the transport of mRNA to the growth cone in response to stimulation (Eom et al., 2003; Leung et al., 2006; Zhang et al., 2001). Moreover, Vg1RBP is necessary for migration of neural crest cells in the Xenopus embryo (Yaniv et al., 2003), and IMP1 and 3 are critical for invadopodia formation in cultured mammalian cells (Vikesaa et al., 2006). Recently, isolation of IMP granules revealed that the ribonucleoprotein (RNP) particles contain about 1% of the entire transcriptome (Jonson et al., 2007). Two dIMP proteins differing at the N-terminus are expressed from alternative promoters on the X chromosome (Fig. 1B). The two isoforms were recently reported to have different functions. SD-dIMP is functional in the oocyte, whereas RE-dIMP is active in motor neurons (Boylan et al., 2008). Both dIMP proteins contain four hnRNP K homology (KH) RNA-binding domains, but lack the two N-terminal RNA recognition motives (RRMs) present in verte-
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In this study, we describe the expression of dIMP protein throughout embryogenesis. In the cellular blastoderm, it was localized apically in the entire cell-layer, as well as in the pole cells. Later, it was found in the developing nervous system, both in the brain, the ventral nerve cord (VNC), and in the peripheral nervous system (PNS). In the latter, expression was detected in both neurons and accessory cells of sensory organs. 1.1. dIMP in the cellular blastoderm
Fig. 1. Schematic view of dIMP transcripts and proteins. (A) The modular architecture of dIMP and its vertebrate homologues. dIMP contains four KH domains and exhibits a glutamine(Q)-rich C-terminus, but it lacks the N-terminal RRMs. (B) Schematic view of the two different dIMP transcripts reported by Boylan and colleagues (Boylan et al., 2008). Boxes designate exons and lines refer to introns. Arrowheads mark translational initiation codons. The figure is an outline of the complex transcript pattern reported in FlyBase (Wilson et al., 2008). The GFPexon is only spliced into the transcripts encoding the RE polypeptide.
brate IMPs. Instead, the dIMP isoforms exhibit a glutamine-rich Cterminal tail that is absent in the vertebrate homologues (Fig. 1A). The dIMP protein is expressed in both the oocyte and in the nurse cells (Munro et al., 2006), but its expression has so far not been thoroughly described in the embryo. Maternal dIMP transcripts are ubiquitous in the early embryo, but at embryonic stage 5, the transcript almost disappears except in the pole cells. Later, zygotic expression of the dIMP transcript is seen in the central nervous system (CNS) and epidermis (Nielsen et al., 2000). In the third instar larvae, dIMP protein is found in moving RNPs in motor axons, and somewhat paradoxically, both the removal of dIMP protein and its increased expression give rise to severe locomotion defects such as an inability to fly, to crawl up the sides of the vial, or to straighten themselves up when falling over (Boylan et al., 2008). Examination of the synapses in third instar larvae revealed that loss-of-function and gain-offunction mutants exhibited decreased and increased number of boutons, respectively (Boylan et al., 2008). In the adult fly, the expression of GFP-dIMP fusion protein in four different protein trap lines is seen in both somatic cyst cells and pre-meiotic germ cells of Drosophila testis (Fabrizio et al., 2008). Two molecular studies of dIMP have been made previously, in which dIMP protein was shown to bind gurken and oskar transcripts (Geng and Macdonald, 2006; Munro et al., 2006). However, no abnormalities in gurken and oskar regulation were seen in oocytes lacking dIMP (Geng and Macdonald, 2006; Munro et al., 2006). Furthermore, dIMP has been identified in a gain-of-function screen, where third instar larvae overexpressing dIMP showed an abnormal branching pattern of the intersegmental nerve b (ISNb) (Kraut et al., 2001).
To elucidate the expression and localization pattern of dIMP protein in the cellular blastoderm, we immunostained dIMP with a polyclonal anti-dIMP rabbit antibody (Fig. 2). In stage 5 embryos, immunoreactivity was observed in the entire blastoderm celllayer. Staining was present throughout the cytoplasm, but it was stronger apically to the nucleus. To determine whether dIMP immunoreactivity could be seen in the nuclei, anti-dIMP and DAPI co-staining experiments were performed (Fig. 2 and Supplementary Fig. S1). No overlap was seen between the DAPI and the dIMP staining, showing that dIMP is cytoplasmic at steady-state. A coimmunostaining with the pole cell marker Vasa showed that dIMP immunoreactivity was also seen in the pole cells at the posterior end (Supplementary Fig. S2), implying that dIMP is expressed in the germline precursors similar to the murine homologue (Hammer et al., 2005). 1.2. GFP-dIMP expression Whilst studying dIMP immunostainings, we also saw staining of the VNC, but because the VNC develops rapidly around stages 14– 16 we decided to follow the development of live embryos to be able to clarify the precise timing of the VNC staining. Several gene trap projects have been performed in Drosophila, in which an exon encoding GFP is induced to jump into random positions in the genome. In one such line, called 126-1 (subsequently named GFP-dIMP flies), the GFP-exon is situated in the beginning of the dIMP gene (Fig. 1B), which corresponds to an intron present in the RE-dIMP transcript. Importantly, mutants without functional dIMP are semi-lethal (Boylan et al., 2008; Geng and Macdonald, 2006; Munro et al., 2006), whereas the GFP-dIMP flies are viable and fertile, suggesting that the fusion protein is functional. Moreover, the expression pattern of GFP-dIMP in 126-1 was identical to that observed when staining wild-type embryos with anti-dIMP antibody (Supplementary Fig. S3). 1.2.1. CNS Embryos from GFP-dIMP flies were followed by confocal microscopy from the blastoderm stage and throughout the rest of embryogenesis. From the blastoderm stage and until stage 13, fluo-
Fig. 2. dIMP expression in the blastoderm embryo. Lateral view of a stage 5 embryo stained with anti-dIMP antibody (anterior to the left, dorsal up). dIMP immunoreactivity is enhanced apically to the nucleus in the entire blastoderm cell-layer. At the right is shown an enlargement of the boxed area co-stained with DAPI. The scale bar is 50 lm in the overview and 20 lm in the enlargement.
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rescence was only seen in cells at the surface of the embryo as shown in Fig. 3A for stages 8, 10, and 12. At stage 14, GFP-dIMP fluorescence appeared in the developing VNC, and an embryo was followed live through stages 14, 15 and 16 (Fig. 3B). The fluorescence was maintained in the VNC at all three stages. At stage 16, when the longitudinal axons and commissures have formed, GFP-dIMP fluorescence seemed to be absent from the axons. To clarify this issue, a co-immunostaining with anti-dIMP and mAb BP102, which stains axons of the CNS (Seeger et al., 1993), was performed. As seen in Fig. 4, no overlap was observed, implying that dIMP is primarily localized to the cell body. When the embryonic brain was examined, GFP-dIMP was also expressed here (Fig. 3C and D). 1.2.2. Epidermis The GFP-dIMP fusion protein was broadly expressed in the epidermis covering the entire embryo as shown in Fig. 3E. DAPI costaining experiments with anti-dIMP antibody showed that dIMP was exclusively cytoplasmic at steady-state (Supplementary Fig. S1). 1.3. Expression in PNS
Fig. 3. Embryos expressing a GFP-dIMP fusion protein. (A) Lateral views of GFPdIMP embryos at stage 8, 10 and 12. Fluorescence is only seen in cells at the surface of the embryo at all three stages. (B) Ventral views: GFP-dIMP is emerging in the VNC at stage 14 (arrow) and is maintained throughout stages 15 and 16. (C and D) Dorsal view and lateral view, respectively, of stage 16 embryos showing that GFPdIMP is expressed in the brain (arrows). (E) Stage 16 – lateral view: GFP-dIMP is broadly expressed in the epidermis. Anterior is up – scale bars 50 lm.
dIMP has previously been reported to be present in moving particles in motor axons of third instar larvae (Boylan et al., 2008). To investigate the expression of dIMP in the embryonic PNS in greater detail, co-immunostaining experiments were performed on wildtype embryos with anti-dIMP antibody and known neuronal, muscle and accessory cell markers. Initially, an antibody directed against Fasciclin II (Fas II), which stains motor axons was employed (Grenningloh et al., 1991). As shown in Fig. 5A, no overlap between Fas II and dIMP in motor axons was observed, indicating that dIMP is not present in large amounts within motor axons. To examine whether dIMP is expressed in the sensory nervous system, the monoclonal mAb 22C10 antibody that binds to the microtubuleassociated protein Futsch was used. mAb 22C10 detects several motor- and interneurons throughout development, and at the end of embryogenesis all sensory neurons are revealed (Hummel et al., 2000). As seen in Fig. 5B, dIMP immunoreactivity was present in mAb 22C10 positive cells in the periphery, indicating that dIMP is expressed in sensory neurons. Importantly, dIMP protein was also expressed in surrounding non-neuronal cells that were not stained by mAb 22C10 (Fig. 5C). To reveal the identity of the non-neuronal dIMP-positive cells, a muscle marker that binds muscle Myosin was used initially. As seen in Fig. 5D, no obvious overlap was observed, suggesting that dIMP is not extensively expressed in muscles. Next, an antibody raised against Prospero, which stains accessory cells of external sensory organs (the sheath cells, Fig. 5E) and chordotonal sensory organs (scolopale cells, Fig. 5E), was employed (Doe et al., 1991). An overlap between dIMP and Prospero immunoreactivities was seen in both sheath cells and
Fig. 4. dIMP is not present in the axons of the VNC. Co-immunostaining of the VNC of a stage 16 embryo with anti-dIMP antibody (green) and mAb BP102 (red), which stains the axons of the CNS. Ventral view – anterior up. Scale bars 20 lm.
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Fig. 5. Co-immunostaining with anti-dIMP antibody and different markers in the periphery. Ventral or lateral/ventral views of wild-type stage 16 embryos coimmunostained with anti-dIMP antibody (green) and various markers (red). Anterior is up. The boxed area in the overview shown at the left is enlarged to the right. (A) mAb 1D4-Fas II: dIMP immunoreactivity does not overlap with Fasciclin II immunoreactivity in peripheral motor axons (arrowhead). (B and C) mAb 22C10-Futsch: dIMP immunoreactivity overlaps with Futsch immunoreactivity in cell bodies of peripheral sensory neurons (arrowheads in B), whereas Futsch-negative cells around the neurons are dIMP-positive (arrowhead in C). (D) mAb FMM5-muscle Myosin: No overlap between dIMP immunoreactivity and muscle Myosin immunoreactivity is observed. (E) mAb MR1A-Prospero: Overlap between Prospero and dIMP immunoreactivities is found in the scolopale cells of the chordotonal sensory organs (arrowheads in box 1) and in the sheath cells of the external sensory organs (arrowheads in box 2). The scale bar is 50 lm in the overviews and 20 lm in the enlargements.
scolopale cells. Both cell-types are in proximity to the neuron in their sensory organ, and therefore these cells could be candidates
for the non-neuronal dIMP-positive cells seen in the mAb 22C10/ anti-dIMP co-immunostainings (Fig. 5C).
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1.4. Discussion In the cellular blastoderm, dIMP protein was expressed in all cells including the germline precursors, and it was apically localized in the cell-layer. The IMP family is known to participate in mRNA localization, so a putative function of dIMP at the blastoderm stage could be connected to the localization of pair-rule mRNAs (Wilkie and Davis, 2001). Pair-rule transcripts exhibit an apical localization similar to the one seen for dIMP at the blastoderm stage, so although they are expressed in stripes individually, pair-rule transcripts are found in most of the blastoderm cell-layer. The expression of dIMP in pole cells is reminiscent of the murine expression of IMP in embryonic gonadal cells (Hammer et al., 2005), but the functional role of the mammalian homologues in reproductive tissues is an unresolved issue. Throughout development, dIMP protein was expressed at high levels in ectodermal-derived tissues such as the epidermis and the nervous system. Abundant expression was seen in the VNC but dIMP protein was not detected in the neuropil or in motor axons in the periphery, implying a major localization to cell bodies. However, this does not exclude that low levels of dIMP protein were present in axonal particles in the embryonic motor axon as reported for the third instar larva (Boylan et al., 2008). The mammalian IMP1 has been described in axonal RNP particles, where it co-localized with HuD (an ELAV family member) and RasGAP-associated endoribonuclease G3BP (Atlas et al., 2004), but the majority of IMP1 protein and the overlap with HuD and G3BP were found in the cell body. In the PNS, dIMP protein was found both in neurons and in accessory cells of sensory organs. External sensory organs exist in several distinct forms, and they can sense mechanical or chemical stimuli or function as proprioceptors, whereas chordotonal organs are mainly proprioceptors in the larvae (Kernan, 2007). Sensory organs arise from sensory organ precursors (SOPs) that give rise to both the neuron and all accessory cells of a particular sensory organ, through a series of asymmetric cell divisions. A progeny of the SOP called pIIIb divides to produce the neuron and either the sheath cell or the scolopale cell, depending on the type of organ (Lai and Orgogozo, 2004). The accessory cells are part of the architecture of the sensory organs, and both the sheath cell and the scolopale cell enwrap the dendrites of the sensory neuron. Moreover, the sheath cell produces an extracellular matrix, termed the dendritic sheath, surrounding the neuron, so when a sensory bristle is stimulated the matrix is compressed (Kernan, 2007; Lai and Orgogozo, 2004). The presence of dIMP protein in both neurons and accessory cells suggests a function for dIMP in receiving or processing sensory stimuli. The phenotype of the dIMP mutant described by Boylan and colleagues (Boylan et al., 2008), which exhibited severe locomotion defects, was interpreted to be due to a smaller number of synapses at the neuromuscular junction, but faulty sensory apparatus may also cause severe locomotion defects (Suster and Bate, 2002). Taken together, the broad expression of dIMP protein in both the CNS and in neurons and accessory cells of the PNS, combined with the severe locomotion defects of mutant flies, suggests a fundamental role for dIMP in establishing or maintaining the Drosophila nervous system.
mic insertion site followed by sequencing was used to verify the exact position of the GFP insertion site. Moreover, a Southern blot with a probe hybridizing to the GFP-exon was carried out to ensure that the trap was inserted once in the genome, and a western analysis using anti-dIMP antibody confirmed that the fusion protein was expressed. Flies were kept on standard medium at 25 °C. 2.2. GFP live-imaging Embryos were allowed to develop until stage 14, dechorionized for two minutes in 2% sodium hypochlorit in water, transferred to siliconized chambers and mounted in Dulbecco’s phosphate buffered saline (GIBCO). Live embryos were examined by confocal Zeiss 510 Confocor 2 microscopy. 2.3. Polyclonal antibody raised against dIMP Recombinant dIMP protein was expressed in Escherichia coli from a Novagene pET28a vector containing the dIMP coding region from EST SD7045. The His-tag was used to purify the protein on nickel-agarose beads from Sigma. Rabbits were immunized with recombinant dIMP protein four times, and the serum examined by western blotting of embryo extracts and recombinant dIMP protein. Serum was diluted 40,000 times and reacted against a 70 kDa protein (Supplementary Fig. S4). 2.4. Immunohistochemistry Wild-type embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (Dulbecco’s Phosphate Buffered Saline, GIBCO) and stained with anti-dIMP polyclonal antibody (1:8000) alone or with one of the following antibodies: polyclonal goat anti-Vasa (1:50 from Santa Cruz Biotechnology, Inc.), mAb antiFutsch 22C10 (1:100 from Developmental Studies Hybridoma Center, University Iowa), mAb anti-Prospero MR1A (1:100 from Developmental Studies Hybridoma Center, University Iowa), mAb anti-Fasciclin II 1D4 (1:10 from Developmental Studies Hybridoma Center, University Iowa), mAb BP102 (1:100 from Developmental Studies Hybridoma Center, University Iowa), or mAb anti-muscle Myosin FMM5 (1:8, a gift from Daniel P. Kiehart). Secondary antibodies used were anti-mouse Alexa FluorÒ 555, anti-goat Alexa FluorÒ 555 and anti-rabbit Alexa FluorÒ 488 from Molecular Probes (1:1000). DAPI (40 ,6-diamidino-2-phenylindole) was added to the mounting media in a concentration of 1 lg/ml. Immunostainings were visualized in a confocal Zeiss LSM510 microscope. Controls were carried out without the addition of primary antibodies and no staining was observed. Acknowledgements We thank Yvonne DeLotto and Lena Bjørn Johansson for technical assistance and Daniel Kiehart for the FMM5 antibody. This work was supported by the Danish Natural Science Research Council and the Lundbeck Foundation. Appendix A. Supplementary data
2. Experimental procedures
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gep.2008.12.001.
2.1. Fly stocks
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Embryonic expression of Drosophila IMP in the developing CNS and PNS Sidsel Kramshøj Adolph a,*, Robert DeLotto a, Finn Cilius Nielsen b, Jan Christiansen a a b
Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen N, Denmark Department of Clinical Biochemistry, University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 6 August 2008 Received in revised form 28 November 2008 Accepted 3 December 2008 Available online 11 December 2008 Keywords: IMP dIMP ZBP1 Vg1RBP IGF-II b-Actin CNS PNS Sensory organs Chordotonal organ External sensory organ Scolopale cell Sheath cell Drosophila RNA-binding protein
a b s t r a c t Drosophila IMP (dIMP) is related to the vertebrate RNA-binding proteins IMP1-3, ZBP1, Vg1RBP and CRD-BP, which are involved in RNA regulatory processes such as translational repression, localization and stabilization. The proteins are expressed in many fetal tissues, including the developing nervous system, and IMP up-regulation in solid tumors correlates with a high metastatic potential and poor prognosis. In this study, we used immunohistochemistry and live-imaging of an endogenous promoter-driven GFP-dIMP fusion protein to reveal the expression pattern of dIMP protein throughout embryogenesis. In the cellular blastoderm, immunoreactivity was seen in the entire cell-layer, where it was localized apically to the nucleus, and in the pole cells. Later, the GFP-dIMP fusion protein appeared in the developing central nervous system, both in the brain and in the ventral nerve cord. In the peripheral nervous system, immunoreactivity was detected in both neurons and accessory cells of chordotonal and external sensory organs. Ó 2008 Elsevier B.V. All rights reserved.
1. Results and discussion Drosophila melanogaster insulin-like growth factor II (IGF-II) mRNA-binding protein (dIMP) belongs to a family of RNA-binding proteins, comprising the human IMP1-3, chicken zipcode-binding protein 1 (ZBP1), murine c-myc coding region instability determinant binding protein (CRD-BP), and Xenopus Vg1 RNA-binding protein (Vg1RBP). During the last decade, different RNA targets such as IGF-II, b-actin, Vg1, c-myc, Tau and Semaphorin mRNAs, and the untranslated H19 RNA, have been identified. IMPs and their orthologues are implicated in post-transcriptional events such as translational inhibition, RNA localization and RNA stabilization (Yisraeli, 2005). The mammalian proteins are expressed in an oncofetal manner, with high levels in many fetal tissues and low levels in most adult tissues. Moreover, they are frequently up-regulated in transformed cell-lines (Leeds et al., 1997) and solid tumors where they are associated with a high metastatic potential and poor prognosis (Dimitriadis et al., 2007; Kato et al., 2007; Kobel et al., 2007; * Corresponding author. Tel.: +45 35322009; fax: +45 35322128. E-mail addresses: [email protected], [email protected] (S.K. Adolph). 1567-133X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2008.12.001
Sitnikova et al., 2008; Tessier et al., 2004). In the mouse, fetal expression is high in neuronal tissue, and IMP is also found in heart, lung, liver, intestine, epidermis and reproductive organs as well as in the snout, tail and limb buds (Hansen et al., 2004; Mori et al., 2001; Nielsen et al., 1999). In cultured neurons, IMPs are found in discrete particles in both axons and dendrites, and participate in the transport of mRNA to the growth cone in response to stimulation (Eom et al., 2003; Leung et al., 2006; Zhang et al., 2001). Moreover, Vg1RBP is necessary for migration of neural crest cells in the Xenopus embryo (Yaniv et al., 2003), and IMP1 and 3 are critical for invadopodia formation in cultured mammalian cells (Vikesaa et al., 2006). Recently, isolation of IMP granules revealed that the ribonucleoprotein (RNP) particles contain about 1% of the entire transcriptome (Jonson et al., 2007). Two dIMP proteins differing at the N-terminus are expressed from alternative promoters on the X chromosome (Fig. 1B). The two isoforms were recently reported to have different functions. SD-dIMP is functional in the oocyte, whereas RE-dIMP is active in motor neurons (Boylan et al., 2008). Both dIMP proteins contain four hnRNP K homology (KH) RNA-binding domains, but lack the two N-terminal RNA recognition motives (RRMs) present in verte-
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In this study, we describe the expression of dIMP protein throughout embryogenesis. In the cellular blastoderm, it was localized apically in the entire cell-layer, as well as in the pole cells. Later, it was found in the developing nervous system, both in the brain, the ventral nerve cord (VNC), and in the peripheral nervous system (PNS). In the latter, expression was detected in both neurons and accessory cells of sensory organs. 1.1. dIMP in the cellular blastoderm
Fig. 1. Schematic view of dIMP transcripts and proteins. (A) The modular architecture of dIMP and its vertebrate homologues. dIMP contains four KH domains and exhibits a glutamine(Q)-rich C-terminus, but it lacks the N-terminal RRMs. (B) Schematic view of the two different dIMP transcripts reported by Boylan and colleagues (Boylan et al., 2008). Boxes designate exons and lines refer to introns. Arrowheads mark translational initiation codons. The figure is an outline of the complex transcript pattern reported in FlyBase (Wilson et al., 2008). The GFPexon is only spliced into the transcripts encoding the RE polypeptide.
brate IMPs. Instead, the dIMP isoforms exhibit a glutamine-rich Cterminal tail that is absent in the vertebrate homologues (Fig. 1A). The dIMP protein is expressed in both the oocyte and in the nurse cells (Munro et al., 2006), but its expression has so far not been thoroughly described in the embryo. Maternal dIMP transcripts are ubiquitous in the early embryo, but at embryonic stage 5, the transcript almost disappears except in the pole cells. Later, zygotic expression of the dIMP transcript is seen in the central nervous system (CNS) and epidermis (Nielsen et al., 2000). In the third instar larvae, dIMP protein is found in moving RNPs in motor axons, and somewhat paradoxically, both the removal of dIMP protein and its increased expression give rise to severe locomotion defects such as an inability to fly, to crawl up the sides of the vial, or to straighten themselves up when falling over (Boylan et al., 2008). Examination of the synapses in third instar larvae revealed that loss-of-function and gain-offunction mutants exhibited decreased and increased number of boutons, respectively (Boylan et al., 2008). In the adult fly, the expression of GFP-dIMP fusion protein in four different protein trap lines is seen in both somatic cyst cells and pre-meiotic germ cells of Drosophila testis (Fabrizio et al., 2008). Two molecular studies of dIMP have been made previously, in which dIMP protein was shown to bind gurken and oskar transcripts (Geng and Macdonald, 2006; Munro et al., 2006). However, no abnormalities in gurken and oskar regulation were seen in oocytes lacking dIMP (Geng and Macdonald, 2006; Munro et al., 2006). Furthermore, dIMP has been identified in a gain-of-function screen, where third instar larvae overexpressing dIMP showed an abnormal branching pattern of the intersegmental nerve b (ISNb) (Kraut et al., 2001).
To elucidate the expression and localization pattern of dIMP protein in the cellular blastoderm, we immunostained dIMP with a polyclonal anti-dIMP rabbit antibody (Fig. 2). In stage 5 embryos, immunoreactivity was observed in the entire blastoderm celllayer. Staining was present throughout the cytoplasm, but it was stronger apically to the nucleus. To determine whether dIMP immunoreactivity could be seen in the nuclei, anti-dIMP and DAPI co-staining experiments were performed (Fig. 2 and Supplementary Fig. S1). No overlap was seen between the DAPI and the dIMP staining, showing that dIMP is cytoplasmic at steady-state. A coimmunostaining with the pole cell marker Vasa showed that dIMP immunoreactivity was also seen in the pole cells at the posterior end (Supplementary Fig. S2), implying that dIMP is expressed in the germline precursors similar to the murine homologue (Hammer et al., 2005). 1.2. GFP-dIMP expression Whilst studying dIMP immunostainings, we also saw staining of the VNC, but because the VNC develops rapidly around stages 14– 16 we decided to follow the development of live embryos to be able to clarify the precise timing of the VNC staining. Several gene trap projects have been performed in Drosophila, in which an exon encoding GFP is induced to jump into random positions in the genome. In one such line, called 126-1 (subsequently named GFP-dIMP flies), the GFP-exon is situated in the beginning of the dIMP gene (Fig. 1B), which corresponds to an intron present in the RE-dIMP transcript. Importantly, mutants without functional dIMP are semi-lethal (Boylan et al., 2008; Geng and Macdonald, 2006; Munro et al., 2006), whereas the GFP-dIMP flies are viable and fertile, suggesting that the fusion protein is functional. Moreover, the expression pattern of GFP-dIMP in 126-1 was identical to that observed when staining wild-type embryos with anti-dIMP antibody (Supplementary Fig. S3). 1.2.1. CNS Embryos from GFP-dIMP flies were followed by confocal microscopy from the blastoderm stage and throughout the rest of embryogenesis. From the blastoderm stage and until stage 13, fluo-
Fig. 2. dIMP expression in the blastoderm embryo. Lateral view of a stage 5 embryo stained with anti-dIMP antibody (anterior to the left, dorsal up). dIMP immunoreactivity is enhanced apically to the nucleus in the entire blastoderm cell-layer. At the right is shown an enlargement of the boxed area co-stained with DAPI. The scale bar is 50 lm in the overview and 20 lm in the enlargement.
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rescence was only seen in cells at the surface of the embryo as shown in Fig. 3A for stages 8, 10, and 12. At stage 14, GFP-dIMP fluorescence appeared in the developing VNC, and an embryo was followed live through stages 14, 15 and 16 (Fig. 3B). The fluorescence was maintained in the VNC at all three stages. At stage 16, when the longitudinal axons and commissures have formed, GFP-dIMP fluorescence seemed to be absent from the axons. To clarify this issue, a co-immunostaining with anti-dIMP and mAb BP102, which stains axons of the CNS (Seeger et al., 1993), was performed. As seen in Fig. 4, no overlap was observed, implying that dIMP is primarily localized to the cell body. When the embryonic brain was examined, GFP-dIMP was also expressed here (Fig. 3C and D). 1.2.2. Epidermis The GFP-dIMP fusion protein was broadly expressed in the epidermis covering the entire embryo as shown in Fig. 3E. DAPI costaining experiments with anti-dIMP antibody showed that dIMP was exclusively cytoplasmic at steady-state (Supplementary Fig. S1). 1.3. Expression in PNS
Fig. 3. Embryos expressing a GFP-dIMP fusion protein. (A) Lateral views of GFPdIMP embryos at stage 8, 10 and 12. Fluorescence is only seen in cells at the surface of the embryo at all three stages. (B) Ventral views: GFP-dIMP is emerging in the VNC at stage 14 (arrow) and is maintained throughout stages 15 and 16. (C and D) Dorsal view and lateral view, respectively, of stage 16 embryos showing that GFPdIMP is expressed in the brain (arrows). (E) Stage 16 – lateral view: GFP-dIMP is broadly expressed in the epidermis. Anterior is up – scale bars 50 lm.
dIMP has previously been reported to be present in moving particles in motor axons of third instar larvae (Boylan et al., 2008). To investigate the expression of dIMP in the embryonic PNS in greater detail, co-immunostaining experiments were performed on wildtype embryos with anti-dIMP antibody and known neuronal, muscle and accessory cell markers. Initially, an antibody directed against Fasciclin II (Fas II), which stains motor axons was employed (Grenningloh et al., 1991). As shown in Fig. 5A, no overlap between Fas II and dIMP in motor axons was observed, indicating that dIMP is not present in large amounts within motor axons. To examine whether dIMP is expressed in the sensory nervous system, the monoclonal mAb 22C10 antibody that binds to the microtubuleassociated protein Futsch was used. mAb 22C10 detects several motor- and interneurons throughout development, and at the end of embryogenesis all sensory neurons are revealed (Hummel et al., 2000). As seen in Fig. 5B, dIMP immunoreactivity was present in mAb 22C10 positive cells in the periphery, indicating that dIMP is expressed in sensory neurons. Importantly, dIMP protein was also expressed in surrounding non-neuronal cells that were not stained by mAb 22C10 (Fig. 5C). To reveal the identity of the non-neuronal dIMP-positive cells, a muscle marker that binds muscle Myosin was used initially. As seen in Fig. 5D, no obvious overlap was observed, suggesting that dIMP is not extensively expressed in muscles. Next, an antibody raised against Prospero, which stains accessory cells of external sensory organs (the sheath cells, Fig. 5E) and chordotonal sensory organs (scolopale cells, Fig. 5E), was employed (Doe et al., 1991). An overlap between dIMP and Prospero immunoreactivities was seen in both sheath cells and
Fig. 4. dIMP is not present in the axons of the VNC. Co-immunostaining of the VNC of a stage 16 embryo with anti-dIMP antibody (green) and mAb BP102 (red), which stains the axons of the CNS. Ventral view – anterior up. Scale bars 20 lm.
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Fig. 5. Co-immunostaining with anti-dIMP antibody and different markers in the periphery. Ventral or lateral/ventral views of wild-type stage 16 embryos coimmunostained with anti-dIMP antibody (green) and various markers (red). Anterior is up. The boxed area in the overview shown at the left is enlarged to the right. (A) mAb 1D4-Fas II: dIMP immunoreactivity does not overlap with Fasciclin II immunoreactivity in peripheral motor axons (arrowhead). (B and C) mAb 22C10-Futsch: dIMP immunoreactivity overlaps with Futsch immunoreactivity in cell bodies of peripheral sensory neurons (arrowheads in B), whereas Futsch-negative cells around the neurons are dIMP-positive (arrowhead in C). (D) mAb FMM5-muscle Myosin: No overlap between dIMP immunoreactivity and muscle Myosin immunoreactivity is observed. (E) mAb MR1A-Prospero: Overlap between Prospero and dIMP immunoreactivities is found in the scolopale cells of the chordotonal sensory organs (arrowheads in box 1) and in the sheath cells of the external sensory organs (arrowheads in box 2). The scale bar is 50 lm in the overviews and 20 lm in the enlargements.
scolopale cells. Both cell-types are in proximity to the neuron in their sensory organ, and therefore these cells could be candidates
for the non-neuronal dIMP-positive cells seen in the mAb 22C10/ anti-dIMP co-immunostainings (Fig. 5C).
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1.4. Discussion In the cellular blastoderm, dIMP protein was expressed in all cells including the germline precursors, and it was apically localized in the cell-layer. The IMP family is known to participate in mRNA localization, so a putative function of dIMP at the blastoderm stage could be connected to the localization of pair-rule mRNAs (Wilkie and Davis, 2001). Pair-rule transcripts exhibit an apical localization similar to the one seen for dIMP at the blastoderm stage, so although they are expressed in stripes individually, pair-rule transcripts are found in most of the blastoderm cell-layer. The expression of dIMP in pole cells is reminiscent of the murine expression of IMP in embryonic gonadal cells (Hammer et al., 2005), but the functional role of the mammalian homologues in reproductive tissues is an unresolved issue. Throughout development, dIMP protein was expressed at high levels in ectodermal-derived tissues such as the epidermis and the nervous system. Abundant expression was seen in the VNC but dIMP protein was not detected in the neuropil or in motor axons in the periphery, implying a major localization to cell bodies. However, this does not exclude that low levels of dIMP protein were present in axonal particles in the embryonic motor axon as reported for the third instar larva (Boylan et al., 2008). The mammalian IMP1 has been described in axonal RNP particles, where it co-localized with HuD (an ELAV family member) and RasGAP-associated endoribonuclease G3BP (Atlas et al., 2004), but the majority of IMP1 protein and the overlap with HuD and G3BP were found in the cell body. In the PNS, dIMP protein was found both in neurons and in accessory cells of sensory organs. External sensory organs exist in several distinct forms, and they can sense mechanical or chemical stimuli or function as proprioceptors, whereas chordotonal organs are mainly proprioceptors in the larvae (Kernan, 2007). Sensory organs arise from sensory organ precursors (SOPs) that give rise to both the neuron and all accessory cells of a particular sensory organ, through a series of asymmetric cell divisions. A progeny of the SOP called pIIIb divides to produce the neuron and either the sheath cell or the scolopale cell, depending on the type of organ (Lai and Orgogozo, 2004). The accessory cells are part of the architecture of the sensory organs, and both the sheath cell and the scolopale cell enwrap the dendrites of the sensory neuron. Moreover, the sheath cell produces an extracellular matrix, termed the dendritic sheath, surrounding the neuron, so when a sensory bristle is stimulated the matrix is compressed (Kernan, 2007; Lai and Orgogozo, 2004). The presence of dIMP protein in both neurons and accessory cells suggests a function for dIMP in receiving or processing sensory stimuli. The phenotype of the dIMP mutant described by Boylan and colleagues (Boylan et al., 2008), which exhibited severe locomotion defects, was interpreted to be due to a smaller number of synapses at the neuromuscular junction, but faulty sensory apparatus may also cause severe locomotion defects (Suster and Bate, 2002). Taken together, the broad expression of dIMP protein in both the CNS and in neurons and accessory cells of the PNS, combined with the severe locomotion defects of mutant flies, suggests a fundamental role for dIMP in establishing or maintaining the Drosophila nervous system.
mic insertion site followed by sequencing was used to verify the exact position of the GFP insertion site. Moreover, a Southern blot with a probe hybridizing to the GFP-exon was carried out to ensure that the trap was inserted once in the genome, and a western analysis using anti-dIMP antibody confirmed that the fusion protein was expressed. Flies were kept on standard medium at 25 °C. 2.2. GFP live-imaging Embryos were allowed to develop until stage 14, dechorionized for two minutes in 2% sodium hypochlorit in water, transferred to siliconized chambers and mounted in Dulbecco’s phosphate buffered saline (GIBCO). Live embryos were examined by confocal Zeiss 510 Confocor 2 microscopy. 2.3. Polyclonal antibody raised against dIMP Recombinant dIMP protein was expressed in Escherichia coli from a Novagene pET28a vector containing the dIMP coding region from EST SD7045. The His-tag was used to purify the protein on nickel-agarose beads from Sigma. Rabbits were immunized with recombinant dIMP protein four times, and the serum examined by western blotting of embryo extracts and recombinant dIMP protein. Serum was diluted 40,000 times and reacted against a 70 kDa protein (Supplementary Fig. S4). 2.4. Immunohistochemistry Wild-type embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (Dulbecco’s Phosphate Buffered Saline, GIBCO) and stained with anti-dIMP polyclonal antibody (1:8000) alone or with one of the following antibodies: polyclonal goat anti-Vasa (1:50 from Santa Cruz Biotechnology, Inc.), mAb antiFutsch 22C10 (1:100 from Developmental Studies Hybridoma Center, University Iowa), mAb anti-Prospero MR1A (1:100 from Developmental Studies Hybridoma Center, University Iowa), mAb anti-Fasciclin II 1D4 (1:10 from Developmental Studies Hybridoma Center, University Iowa), mAb BP102 (1:100 from Developmental Studies Hybridoma Center, University Iowa), or mAb anti-muscle Myosin FMM5 (1:8, a gift from Daniel P. Kiehart). Secondary antibodies used were anti-mouse Alexa FluorÒ 555, anti-goat Alexa FluorÒ 555 and anti-rabbit Alexa FluorÒ 488 from Molecular Probes (1:1000). DAPI (40 ,6-diamidino-2-phenylindole) was added to the mounting media in a concentration of 1 lg/ml. Immunostainings were visualized in a confocal Zeiss LSM510 microscope. Controls were carried out without the addition of primary antibodies and no staining was observed. Acknowledgements We thank Yvonne DeLotto and Lena Bjørn Johansson for technical assistance and Daniel Kiehart for the FMM5 antibody. This work was supported by the Danish Natural Science Research Council and the Lundbeck Foundation. Appendix A. Supplementary data
2. Experimental procedures
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gep.2008.12.001.
2.1. Fly stocks
References
The 126-1 GFP-dIMP line, obtained from SzegedFly, was created in Alain Debec’s laboratory using the protein trap vector described by Xavier Morin (Morin et al., 2001). PCR amplification of the geno-
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