Fernando M Mendive

Tom Van Loy

Sylvie Claeysen

Jeroen Poels

Michael Williamson

Frank Hauser

Cornelis J P Grimmelikhuijzen

Gilbert Vassart
email: gvassart@ulb.ac.be

Jozef Vanden Broeck
email: jozef.vandenbroeck@bio.kuleuven.ac.be

Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2

Bursicon is a neurohumoral agent responsible for tanning and hardening of the cuticle and expansion of the wings during the final phase of insect metamorphosis. Although the hormonal activity was described more than 40 years ago, the molecular nature of bursicon has remained elusive. We identify here Drosophila bioactive bursicon as a heterodimer made of two cystine knot polypeptides. This conclusion was reached in part from the unexpected observation that in the genome of the honey bee, the orthologs of the two Drosophila proteins are predicted to be fused in a single open reading frame. The heterodimeric Drosophila protein displays bursicon bioactivity in freshly eclosed neck-ligated flies and is the natural agonist of the orphan G protein-coupled receptor DLGR2.

Introduction

Bursicon was described more than 40 years ago as a bioactivity present in CNS (central nervous system) extracts and hemolymph of insects after eclosion, capable of rescuing flies from defective tanning and sclerotization of the cuticle caused by neck-ligation [START_REF] Fraenkel | Hormonal and nervous control of tanning in the fly[END_REF][START_REF] Fraenkel | Tanning in the adult fly: A new function of neurosecretion in the brain[END_REF][START_REF] Cottrell | The imaginal ecdysis of blowflies[END_REF][START_REF] Fraenkel | Properties of bursicon: An insect protein hormone that controls cuticular tanning[END_REF]. It is a key regulator of the final steps in the stereotyped developmental and behavioural program of ecdysis, i.e., the shedding of the remains of the old cuticle and the consequent maturation of the new one. Bursicon is believed to act downstream of a sequence of regulatory peptides, such as ecdysis triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP) [START_REF] Horodyski | Isolation and expression of the eclosion hormone gene from the tobacco hornworm, Manduca sexta[END_REF][START_REF] Zitnan | Identification of ecdysistriggering hormone from an epitracheal endocrine system[END_REF][START_REF] Baker | The hormonal coordination of behavior and physiology at adult ecdysis in Drosophila melanogaster[END_REF][START_REF] Fuse | Modulation of ecdysis in the moth Manduca sexta: The roles of the suboesophageal and thoracic ganglia[END_REF][START_REF] Park | Targeted ablation of CCAP neuropeptidecontaining neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior[END_REF][START_REF] Park | Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency[END_REF][START_REF] Clark | Neuroendocrine control of larval ecdysis behavior in Drosophila: Complex regulation by partially redundant neuropeptides[END_REF]. Bursicon bioactivity can be partially mimicked by cyclic AMP analogs [START_REF] Fraenkel | 30:50-cyclic AMP and hormonal control of puparium formation in the fleshfly Sarcophaga bullata[END_REF][START_REF] Baker | Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program[END_REF] and was recently shown to be involved in apoptotic removal of wing epidermal cells during the last step of metamorphosis into adult Drosophila [START_REF] Kimura | Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster[END_REF]. Purification of bioactive bursicon from nerve cords of the cockroach Periplaneta americana yielded a 30 kDa protein [START_REF] Kostron | Antisera against Periplaneta americana Cu,Zn-superoxide dismutase (SOD): Separation of the neurohormone bursicon from SOD, and immunodetection of SOD in the central nervous system[END_REF] from which several tryptic peptides were obtained and micro-sequenced [START_REF] Honegger | Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone[END_REF]. Comparison of these sequences with available genomic data identified a 522 nucleotide open reading frame (ORF) in Drosophila (CG13419) that codes for a cystine knot protein with close orthologs in the bee Apis mellifera and in the mosquito Anopheles gambiae [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF][START_REF] Riehle | Neuropeptides and peptide hormones in Anopheles gambiae[END_REF] (Fig. 1A ). The link between the Drosophila ORF and bursicon was established from the defective post-ecdysial behaviour of flies with loss-of-function mutations in the putative gene, and the corresponding decrease in bursicon bioactivity in mutant extracts [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF]. Accordingly, it was proposed that bursicon would be a homodimeric 30 kDa protein made of two 15 kDa CG13419 mature protomers [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF].

DLGR2 is a G protein-coupled receptor of Drosophila distantly related to the vertebrate glycoprotein hormone and relaxin receptors [START_REF] Eriksen | Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster[END_REF][START_REF] Nishi | Characterization of two fly LGR (leucine-rich repeat-containing, G proteincoupled receptor) proteins homologous to vertebrate glycoprotein hormone receptors: Constitutive activation of wild-type fly LGR1 but not LGR2 in transfected mammalian cells[END_REF]. It has been proposed as the receptor for bursicon because flies with loss-of-function mutations in DLGR2 display a phenotype (rickets rk -/-) which is similar to bursicon deficiency induced by neck-ligation. It consists in defective sclerotization and melanization of the cuticle, as well as abnormal wing expansion after adult eclosion. These mutants produce bioactive bursicon, and their phenotype cannot be rescued by bursicon injection [START_REF] Baker | Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program[END_REF].

In the present study we have used a combination of comparative genomic, biological and biochemical approaches to identify bioactive bursicon as a cystine knot heterodimeric protein and the natural agonist of DLGR2.

Methods

Similarity searches and protein alignments

Similarity searches of insect genomes and proteins were made on the NCBI platform (www.ncbi.nih.gov) by TBLASTN or BLASTP, respectively [START_REF] Altschul | Gapped BLAST and PSIBLAST: A new generation of protein database search programs[END_REF]. Multiple protein alignments were performed with CLUSTAL (www.clustalw.genome.jp) using default settings.

Protein predictions on WGS (whole-genome sequencing) DNA sequences

The bee, mosquito and silkworm genomes were analysed for the presence of Drosophila CG13419 or CG15284 orthologs with TBLASTN (Anopheles CG13419-like sequence was already present in the database; Accession No. AY735443). Because the N-termini are not conserved among species, the putative signal peptides could not be identified in the TBLASTN alignments. In consequence, each locus was individually screened for the presence of potential signal peptides [START_REF] Bendtsen | Improved prediction of signal peptides: SignalP 3.0[END_REF] located in frame in the same exon with CG13419 or CG15284 homologous sequences (ORF finder tool at the NCBI, www.ncbi.nih.gov/gorf; SignalP 3.0 Server; www.cbs.dtu.dk/services/SignalP/). In the case of the silkworm CG13419 ortholog, the putative signal peptide was identified in a predicted separate exon, located 56 base pairs upstream from the starting point of CG13419-like segment. Exon prediction was performed at www.fruitfly.org/seq_tools/splice.html. Bee, silkworm and mosquito predicted sequences were submitted to EMBL/GenBank/DDBJ databases. For accession numbers see legend of Fig. 1.

PCR, cloning and sequencing

Drosophila cDNAs, normalized to the housekeeping Rp49 (Ribosomal protein 49) cDNA, derived from different developmental stages and tissues ('Rapid-Scan™ Gene expression panel', Origene Technologies, Rockville, MD) were used as template to amplify bursicon α subunit (Bursα) and bursiconβ subunit (Bursβ) cDNAs by means of PCR with genespecific primers [Bursα (cg13419): fwd gcggatcctcgcagtcgggccgacgagatg; rev gcgaattcagtgctaaactgatgggggaatcta. They contain a 5′ BamHI and EcoRI restriction site (underlined), respectively. Annealing temperature 55 °C. Bursβ (cg15284): fwd atgcatgtccaggaactgctc; rev ttaacgtgtgaaatcgccaca. Annealing temperature 57 °C]. Both reactions were performed for 35 cycles. The resulting products were analysed by agarose gel electrophoresis, purified, cloned into pCR4-TOPO (Invitrogen, Carlsbad, CA) and sequenced (ABI Prism 310, Applied Biosystems, Foster City, CA). ORFs coding for the bursicon subunits were further subcloned into pcDNA3.1 (Invitrogen) for transfection of COS-7 cells.

Preparation of conditioned media

COS-7 cells growing in 35 mm culture dishes were transfected with 1 µg of plasmid DNA using the DEAE-dextran method [START_REF] Vlaeminck | Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist[END_REF]. The day after transfection, the culture medium (DMEM containing 10% Fetal Bovine Serum (FBS)) was replaced by the same medium with 1% FBS. Twenty-four hours later, the medium was recovered, centrifuged at 2000 × g to eliminate cellular debris, immediately frozen in liquid nitrogen and stored at -20 °C until use.

For dose-response curves and gel filtration chromatography, conditioned medium was concentrated using ultrafiltration devices with a 10 kDa MW cut-off (Amicon Ultra Centrifugal Filters, Millipore Corporation).

Stimulation of cAMP accumulation

Drosophila DLGR1, DLGR2 [START_REF] Eriksen | Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster[END_REF][START_REF] Hauser | Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals[END_REF] and human hLGR4 and hLGR5 cloned in pcDNA3 were transfected in COS-7 cells in the same way as described above. Forty-eight hours after transfection, expression of the various receptors was assayed by flow immunocytometry, using a panel of monoclonal antibodies (not shown). Cells were washed once with Krebs-Ringer-HEPES (KRH) buffer and incubated at 37 °C for 30 min. Buffer was then replaced by 1 ml of fresh KRH supplemented with 10% of conditioned medium and 25 µM rolipram (a cAMP phosphodiesterase inhibitor, Laboratoire Logeais, Paris, France). After a 30 min incubation, the level of intracellular cAMP was measured by RIA as described previously [START_REF] Vlaeminck | Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist[END_REF].

Gel filtration chromatography

One millilitre of 20× concentrated culture medium from cells co-expressing recombinant CG13419 and CG15284 proteins was applied onto a gel filtration column (HILoad™ 16/60 Superdex™ 75 prep Grade, Amersham Biosciences, Freiburg, Germany).

Calibrations and runs were made in Na3(PO4) 20 mM/NaCl 200 mM buffer. Eluates corresponding to sizes between 70 and 10 kDa were collected in 2 ml fractions and frozen immediately in liquid nitrogen. One hundred, or 5 µl of each fraction were assayed for cAMP stimulating activity on COS-7 cells expressing DLGR2, or in the bursicon bioassay, respectively.

Bursicon bioassay

Bursicon bioactivity was determined by means of the flesh fly cuticle tanning bioassay [START_REF] Fraenkel | Tanning in the adult fly: A new function of neurosecretion in the brain[END_REF]. Flies (Neobellieria bullata) were decapitated (or neck-ligated) after eclosion and kept at room temperature for 1.5 h in a humidified petri dish. This initial incubation period allowed for a pre-selection of flies (the vast majority) that did not display any endogenous bursicon activity after decapitation (or neck-ligation). Flies that remained pale were injected with 5 µl of solution (saline, CNS extract, 1 mM 8Br-cAMP, conditioned culture media or chromatographic fractions) and cuticular tanning was evaluated 3 h post-injection. Qualitative evaluation of tanning was as follows: flies do not tan: "Defective tanning"; only the abdomen tans: "Intermediate tanning"; both the abdomen and the thorax tan: "Full Tanning". The CNS extract was prepared from freshly eclosed flesh flies. CNS were dissected and homogenized in saline solution (127.7 mM NaCl, 4.7 mM KCl) and 0.25 CNS-equivalents were injected into each fly.

Results and discussion

Drosophila CG13419 has no Bursicon bioactivity and does not activate DLGR2

With the aim of establishing a direct experimental link between the cystine knot protein described by Dewey et al. [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF] (CG13419, hereafter Bursα) and bursicon, we transfected COS-7 cells with Bursα cDNA inserted in an expression plasmid. The conditioned medium was tested for bursicon bioactivity in freshly eclosed, neck-ligated N. bullata (see Fig. 2C and Section 2), and for its ability to stimulate cAMP production in COS-7 cells expressing DLGR2 (Fig. 2A). Contrary to what was suggested on the basis of genetic data by Dewey et al. [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF],

Bursα was devoid of bursicon bioactivity and incapable of activating DLGR2. Together with the evidence from loss-of-function mutations of Bursα [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF], these results indicated that a piece of information was lacking in the bursicon puzzle, most probably a dimerisation partner for Bursα.

Identification of a new Bursicon candidate

Honegger et al. [START_REF] Honegger | Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone[END_REF] reported a set of peptide sequences from purified bursicon preparations of the cockroach P. americana. It is from some of these peptides that CG13419 (Bursα) was identified in the Drosophila genome [START_REF] Dewey | Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[END_REF].

Using the sequence of one of these peptides which was initially considered as a contaminant in the bursicon preparation (peptide sequence: ESFLR, see Fig. 1), we found a new ORF in Drosophila encoding a 15 kDa cystine knot protein (CG15284, hereafter called Bursβ). We identified the corresponding orthologous sequences in mosquito (A. gambiae), silkworm (Bombyx mori) and honey bee (A. mellifera) genomes, and manually predicted the corresponding ORFs (Fig. 1A). The encoded proteins are 72%, 63% and 65% identical to Drosophila Bursβ, respectively.

In the honey bee Bursα and Bursβ are encoded in a single ORF

Whereas the fly, mosquito and silkworm CG15284-like (Bursβ) sequences are predicted to be of similar length (141, 153 and 137 residues, respectively), the bee sequence constitutes the aminoterminal portion of a larger conceptual protein (269 aminoacids, Fig. 1A). It harbours a second cystine knot domain at its C-terminus, suggesting that it originated from the intragenic duplication of an ancient motif. In Fig. 1B, we show both cystine knot domains of the bee protein and the two Drosophila Bursα and Bursβ proteins, aligned with the cystine knot consensus sequence (CTCK: C-terminal cystine knot). Unexpectedly, detailed inspection of this second cystine knot domain, revealed that it corresponds to the bee ortholog of Bursα (CG13419) (Fig. 1A). In the other available insect species, Bursα and Bursβ are clearly encoded in distinct loci. Considering that a multidomain protein, encoded in a single gene in one species, may correspond to a multimeric protein, with each subunit encoded by separate genes, in another species [START_REF] Enright | Protein interaction maps for complete genomes based on gene fusion events[END_REF][START_REF] Marcotte | Detecting protein function and protein-protein interactions from genome sequences[END_REF][START_REF] Sali | Functional links between proteins[END_REF], we hypothesized that Drosophila bioactive bursicon would be a heterodimer made of CG13419 and CG15284 subunits.

Co-expression of recombinant Drosophila Bursα and Bursβ generates bursicon bioactivity and DLGR2 stimulating activity

To test this hypothesis, Bursβ cDNA was cloned in the expression vector pcDNA3.1 and transfected in COS-7 cells, either alone or together with Bursα plasmid. Conditioned media were prepared from cultures of transfected cells and tested for their ability to stimulate cAMP accumulation in cells expressing DLGR2. Cells transfected with the cDNA of vertebrate or invertebrate orphan receptors belonging to the same subfamily of GPCRs (hLGR4, hLGR5 and DLGR1) [START_REF] Hsu | Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G proteincoupled, seven-transmembrane region[END_REF][START_REF] Hauser | Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals[END_REF] were used as controls. As can be seen in Fig. 2A, an 8-fold increase in cAMP production was induced in DLGR2-expressing cells, only when stimulated with conditioned medium from cells co-transfected with Bursα and Bursβ cDNAs. Stimulation with serial dilutions of the medium generated the expected sigmoid concentration-action curve (Fig. 2B).

The conditioned media from cells transfected with Bursα or Bursβ plasmids alone had no effect (Fig. 2A). No stimulation was observed neither with a mixture made of equal amounts of media from cells transfected separately with Bursα or Bursβ (not shown). This indicates that, in order to achieve DLGR2 agonistic activity, Bursα and Bursβ need to be co-expressed in the same cell. DLGR1, a Drosophila receptor for a still unknown ligand [START_REF] Hauser | Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals[END_REF], displayed strong constitutive activity [START_REF] Nishi | Characterization of two fly LGR (leucine-rich repeat-containing, G proteincoupled receptor) proteins homologous to vertebrate glycoprotein hormone receptors: Constitutive activation of wild-type fly LGR1 but not LGR2 in transfected mammalian cells[END_REF] but was not stimulated. Neither were the human hLGR4 and hLGR5 receptors (Fig. 2A).

The identity between bursicon and the material present in the medium of cells cotransfected with Drosophila Bursα and Bursβ cDNAs was established by microinjection in freshly eclosed, decapitated or neck-ligated Neobellieria (Sarcophaga) bullata flesh flies (Fig. 2C). Strong tanning of the cuticle was observed 3 h post-injection in flies injected with medium from COS-7 cells co-expressing Bursα and Bursβ. This response was also observed in flies injected with CNS extract derived from freshly eclosed flesh flies or with 8Br-cAMP, a membrane permeable cyclic AMP analog previously shown to mimic bursicon activity [START_REF] Baker | Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program[END_REF][START_REF] Kimura | Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster[END_REF]. However, no bursicon activity was observed in flies injected with saline, control medium or media derived from cells transfected with Bursα or Bursβ cDNA alone.

Drosophila Bursicon is a heterodimer made of Bursα and Bursβ cystine knot subunits

The molecular weight of the bioactive molecule present in the conditioned medium was estimated by size-exclusion chromatography (Fig. 3 ). Eluted fractions were evaluated for bursicon bioactivity and their capacity to stimulate cAMP accumulation in DLGR2 expressing cells. Both activities co-eluted in the 30-35 kDa region of the chromatogram (Fig. 3). These results are in agreement with the expected molecular weights of Bursα and Bursβ protomers (15.5 and 13.8 kDa, after signal peptide removal, respectively), with the size reported for bioactive hormone [START_REF] Kostron | Antisera against Periplaneta americana Cu,Zn-superoxide dismutase (SOD): Separation of the neurohormone bursicon from SOD, and immunodetection of SOD in the central nervous system[END_REF], and demonstrate the heterodimeric nature of bursicon.

Bursα and Bursβ mRNAs are expressed at the same developmental stages

The expression of Bursα and Bursβ transcripts in Drosophila melanogaster was explored by RT-PCR at various stages of development. Both transcripts were found in embryonic, larval and pupal stages, but not in the adult of the fruit fly (Fig. 4 ). This pattern of expression of Bursα and Bursβ transcripts during development correlates with the one previously reported for DLGR2 [START_REF] Eriksen | Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster[END_REF].

Conclusion

We have demonstrated that the molting neurohormone bursicon is a heterodimeric protein made of two cystine knot subunits, and the natural agonist of the previous orphan G protein-coupled receptor DLGR2. A similar situation exists in vertebrates, where the pituitary hormones FSH, LH and TSH are also heterodimeric cystine knot proteins acting on their respective leucine-rich repeats containing receptors. The orphan GPCRs LGR4, LGR5 and

LGR6 are putative orthologs of DLGR2 in vertebrates [START_REF] Hsu | The three subfamilies of leucine-rich repeat-containing G proteincoupled receptors (LGR): Identification of LGR6 and LGR7 and the signaling mechanism for LGR7[END_REF]. The knowledge of bursicon structure might help in the identification of candidate agonists for these orphan receptors, amongst the large family of cystine knot proteins. The two bursicon protomers, Bursα and Bursβ, are encoded by separate genes in Drosophila, Anopheles and Bombyx, but they are fused in a single gene in Apis. The sequence similarity between Bursα and Bursβ and their presence as a predicted single-chain protein in A. mellifera is a strong indication that they originated by duplication of (or in) an ancestral gene encoding a cystine knot protein.

From a practical point of view, our study makes it possible to develop screening assays for low molecular weight leucine-rich repeats containing G protein-coupled receptor 2 (LGR2) antagonists. These might qualify as effective novel agents for insect pest management.

Figure 1

 1 Figure 1 Alignments of insect bursicon proteins. (A) CG13419 and CG15284 orthologs (called Bursα and Bursβ, respectively) from mosquito (ag: Anopheles gambiae), silkworm (bm: Bombyx mori) and fruit fly (dm: Drosophila melanogaster) were aligned along the bee (am: Apis mellifera) single-chain multidomain bursicon. The tryptic peptides identified in bursicon preparations from the cockroach (pa: Periplaneta americana), a heterometabolic insect, are also aligned. Conserved Bursα and Bursβ sequences are shaded in grey and yellow, respectively. Residues identical among 3, 4, or 5 species are dark shaded, while residues identical between two species are pale shaded. Silkworm Bursα (BN000691) and Bursβ (BN000690), mosquito Bursβ (BN000689) and bee single-chain bursicon (BN000692) were manually predicted on available WGS DNA sequences (see Section 2 for details). Drosophila CG13419 and CG15284 cDNAs were cloned, sequenced and annotated in EMBL/GenBank/DDBJ databases (Accession Nos. AJ862523 and AJ862524, respectively). The putative signal peptides are shown at the left side on the top of the figure. (B) Alignment of Drosophila Bursα and Bursβ proteins, together with the Bursα and Bursβ domains from bee single-chain bursicon, and a consensus cystine knot-like domain (CTCK: C-terminal cystine knot) [30]. Conserved cysteine residues are highlighted. Other conserved residues are shaded in blue.

Figure 2

 2 Figure 2 Biological activities of Drosophila bursicon proteins. (A) Agonist activity on DLGR2. Conditioned media from control COS-7 cells (Basal) or cells expressing Bursα, Bursβ, or both proteins together (Bursαβ), were evaluated for their ability to induce cAMP accumulation in COS-7 cells transfected with Drosophila (DLGR1 and DLGR2) and human (hLGR4 and hLGR5) receptors (mock: cells transfected with empty plasmid). (B) Concentration-effect curve. Conditioned media were either diluted or concentrated (from 1/300 dilution to 30× concentration) to obtain serial concentrations of DLGR2 agonist activity. Error bars represent the range of duplicate determinations. (C) Bursicon bioassay. Saline, 8Br-cAMP, CNS extract or conditioned media from COS-7 cells transfected with empty plasmid (control medium) or from cells expressing Bursα, Bursβ, or both (Bursαβ) were injected into decapitated or neckligated, freshly eclosed flesh flies (Neobellieria bullata) to analyse their capacity to stimulate cuticular tanning. The number of flies tested per condition is given in parentheses. White, grey

Figure 3

 3 Figure 3 Molecular weight estimation. Twenty times concentrated conditioned medium, produced in COS-7 cells co-transfected with Bursα and Bursβ cDNAs, was size fractionated by gel filtration chromatography. Eluted fractions between 70 and 10 kDa were analysed for their ability to stimulate cAMP accumulation in COS-7 cells expressing DLGR2. Grey bars represent the cAMP value after subtraction of the cAMP level of non-stimulated DLGR2 cells. The chromatogram (blue line) corresponds to 280 nm absorbance curve of the proteins used as molecular weight markers (67 kDa: Albumin; 43 kDa: Ovalbumin; 35 kDa: β-Lactoglobulin; 25 kDa: Chymotrypsinogen; 13.7 kDa: Ribonuclease A). Numbers in abscissa indicate the volume eluted from the column (ml). Error bars represent the standard error of four independent cAMP values obtained from each fraction. The lower panel (neck-ligation bioassays) shows the effects of injecting the fractions in neck-ligated flies (-, no tanning response; +, intermediate tanning; ++, full tanning; n = 3 per fraction).

Figure 4

 4 Figure 4 Expression of Bursα and Bursβ transcripts in different developmental stages of the fruit fly, Drosophila melanogaster, as shown by PCR on normalized cDNA samples (see Section 2).Lanes 1-4: embryonic stages (0-4/4-8/8-12/12-24 h), lanes 5-7: larval stages 1-3, lane 8: pupal stage, lanes 9-10: adult head (males and females), lanes 11-12: adult body (males and females), M: marker (100 bp ladder).

Figure 1 Figure 2 Figure 3 Figure 4

 1234 Figure 1

Acknowledgements

We thank Katrien Backers and David Comuni for help with gel filtration chromatography, Sofie Van Soest for DNA sequencing, Roger Jonckers for rearing of flies and Julie Puttemans for photography. Supported by the "Belgian Programme on Interuniversity Poles of Attraction" (IUAP/PAI P5/30) initiated by the Belgian State, Prime Minister's Office Science Policy Programming. Also supported by grants from the "Fonds de la Recherche Scientifique Médicale", "Fonds National de la Recherche Scientifique", Francqui Foundation, "Fonds voor Wetenschappelijk Onderzoek" (FWO), Lundbeck Foundation, Novo Nordisk Foundation, and Carlsberg Foundation. Tom Van Loy obtained a PhD fellowship from the "Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen" (IWT). Jeroen Poels obtained a post-doctoral fellowship from the "Onderzoeksfonds KULeuven".


Bernard Elissalde

The circulation of concepts and how they are received by those involved in planning: the instance of the concepts of European Spatial Planning

Introduction

It is often recalled that the European Union has no legal competence in the area of territorial planning and development. At most, it has sector policies that have a spatial impact (such as the Common Agricultural Policy), which sometimes integrate European spatial planning objectives aiming to improve the balance of development potential across the EU into their concerns (e.g. the policy for trans-European networks, or the cohesion policy). However the EU does produce a certain number of outline documents (the European Spatial Development Perspective, Territorial Agenda, Territorial Agenda 2020), technical documents (evaluation of the cohesion policy via the regular publication of reports on cohesion), or applied research work on European spatial planning within the ESPON programme. These different initiatives, in addition to the development of discourse intended to orient action by the actors in spatial planning and development policies on infra-European scale, also mobilise a set of concepts or ideas which, even if their resonance may vary over time, progressively become key-words synthesising the orientations that the EU intends to set up as objectives in the domain under consideration.

The concepts and ideas generate debate intended to discuss their meanings and to define them for operational purposes. Generally speaking, the academic literature on European spatial planning converges on the fact that the concepts or notions proposed by the EU are defined in rather vague manner [START_REF] Tatzberger | Spatial Visions, Concepts and Metaphors: their Essential Role in European Spatial Development and Policy Discourse[END_REF]. This situation is seen as resulting from the setting in which European discourse is deployed, and the need for it to accommodate different contexts. The relative fuzziness of concepts, while giving the impression that there are common objectives and modes of action, enables them to be adapted to territorial settings that necessarily vary across the EU. The position is thus pragmatic, and what matters is not so much the strictness of the definition as what the concepts produce on the ground [START_REF] Abrahams | What "Is" Territorial Cohesion? What Does It "Do"?: Essentialist Versus Pragmatic Approaches to Using Concepts[END_REF]. This is referred to as their "generative capacity" (SHAW, [START_REF] Shaw | The concept of polycentricity in European spatial planning: Reflections on its interpretation and application in the practice of spatial planning[END_REF], that is to say their ability, at different territorial levels in the EU, to produce phenomena that are new in the conception and implementation of planning policies.

We do however need to distinguish specifically political, "conceptual" productions from productions that are grounded in academic reflection of a theoretical nature. The diffusion processes are different, even if the notion of the plasticity of the terms can apply in both instances.

Consequently, within the restricted scope of this paper, we intend to use a few examples of concepts or notions related to a theoretical construction later incorporated into political discourse, or conversely political notions subsequently incorporated into the scientific debate. In the second instance, we will use the concept of "territorial cohesion", which today is the key-concept synthesising the objective of the EU in the area of territorial planning and development. The concept of regional competitiveness is also a key element in European discourse, articulating the aim of enabling Europe to catch up in the area of economic development in the perspective of the Lisbon Strategy (2000) and the Europe 2020 strategy (2010) with the territorial dimension considered here. Alongside, the concept of polycentrism, prominent in the ESDP, which is a founding document in the planning approach to the European space, can be considered as a concept that enables the articulation of greater cohesion in the European space with the "imperative" of territorial economic development contained in the idea of regional competitiveness.

We therefore propose first of all to analyse the transfer of concepts from the scientific sphere to the EU political sphere, and second to analyse another process, which is the re-shaping of academic understanding by way of political productions at EU level. In this context, we will show how players in the area of spatial planning on infra-European scale confront this situation, using results from a survey on understandings of the phrases "regional competitiveness" and "territorial cohesion". We will also draw wider conclusions relating to the use of concepts in the area of spatial planning and development generally, and in Europe more specifically.

From the scientific to the political sphere

By definition, concepts generally derive from a particular theoretical field, even if this may not be explicit in the case of their emergence in European circles. It can be noted that in many cases, when a concept is taken up by the EU, there is a process of appropriation and re-use within another theoretical framework, i.e. political discourse, or in an empiricist approach, with a resulting proliferation of possible meanings. Texts circulate without their context, they do not carry with them the field by which they were produced, and the receivers, who belong to a different field of production, reinterpret them according to setting in which they find themselves [START_REF] Bourdieu | Les conditions sociales de la circulation internationale des idées[END_REF]. In the European sphere, change in the settings of discourse should replace the necessary disagreements of science by those of a political discourse with performance performative overtones. Instead, concepts are integrated into a consensus discourse. Two trends can be seen in the use of concepts deriving from the academic sphere: ensuring the credibility of messages setting out political lines of action, and the exploitation of academic debate on a concept so as to underline the polysemous nature of the term and legitimise the semantic blur that serves to broaden possible interpretations by the different players.

The idea of transfer supposes a process of "imports" entailing different operations, which can be summed up by the terms used by Bourdieu of "selection" and "reading" 1 (BOURDIEU, op.cit.) 2 . These operations rely on the implicit recognition of homologies between categories, 1

The word used by Bourdieu in French is "Lecture" which means understandings 2 3 enabling a process of comparison and identification, and also sufficient differentials for circulation, and possibly appropriation, to occur.

It is thus possible to propose the rough layout of the trajectory of a concept: a) its emergence in academic research, entailing a debate on the heuristic value of the concept, and then b) transfer to planning policies with a re-appropriation of meaning (often deviated from the first sense) in a different register (differing from the original setting). In the third stage c) applied research is required to validate the transfer and the re-use of the concept so as to legitimise new political orientations and the broader range of meanings.

1.1. The example of regional "competitiveness"

• The scientific debate

The general idea of competitiveness is not new, whether in the area of economics or in the references of the European Commission. In particular, there is a mention in the 1993 European Commission White Paper Growth, Competitiveness, Employment, but the two spheres of business economics and spatial planning have only recently come together. This concept of competitiveness, long-standing in business economics, was transferred to the area of spatial economics by M. [START_REF] Porter | The competitive advantage of nations[END_REF]. This author considers that if a country creates an economic environment providing favourable conditions for businesses (of international size) and if the said businesses become involved locally, this constitutes a competitive advantage for the country. This importance of the specific "environment" was synthesised via his famous "diamond" diagram. Porter combines the basic Marshall model with elements from his long-standing work on the competitive strategies of businesses. He claims that the most internationally competitive industries in a country invariably tend to group geographically in particular territories, forming clusters [START_REF] Porter | The competitive advantage of nations[END_REF][START_REF] Porter | Regions and the new economics of comptetition[END_REF], because according to him "the drivers of prosperity are increasingly subnational, based in cities and regions" [START_REF] Porter | Regions and the new economics of comptetition[END_REF]. The empirical delineation of the geographical boundaries of the clusters is unclear in Porter's work (ranging from a city-centre district to a county or to regions, or even to other countries). These clusters are at once the result of certain interactions, but they also contribute to reinforcing them, within what is known as the competition diamond. Porter considers that the geographical concentration of industries in a national economy plays an important part in the determination of the sectors that will have a competitive advantage at international level: "competitive advantages in global economy can be localised to a great extent and [...] they are derived from the concentrations of highly specialised skills and knowledge, institutions, rivalry and sophisticated buyers" (1998, p.5).

This homology between business and territory was widely criticised by P. KRUGMAN. This author considers that it is not possible to assimilate a territory (nation, region, etc.) to a business firm. Competition between businesses and that between regions cannot be compared. Poorly achieving businesses can relinquish a market according to their successfulness, but regions cannot leave their territory. On this basis, it is possible to pinpoint the main difference between competitiveness of a business and competitiveness of a region: businesses compete

The meanings provided are as follows: 1) selection: the choice of texts by an author intended for publication and comment, and 2) reading: the use of the work according to categories of thought, action and perception of the specific issues of the receiving field [START_REF] Bourdieu | Les conditions sociales de la circulation internationale des idées[END_REF] with one another and can improve their position on the market by ousting another company, or aggravate its situation, while regions can improve their situation without damaging the positions of other regions. Trade between countries is not a nought equation. The competitiveness of a nation or a region is thus not identical to that of a business. "The idea that welfare and economic performances of a State depend on the success on the global markets is a hypothesis and does not necessarily imply truth, moreover, the practical and empirical views have proved this hypothesis to be completely wrong" (1994, p. 30).

In contrast with the hypotheses of perfect competition and increasing economies of scale that are the basis of Ricardo's theory of comparative advantages, KRUGMAN considers that presentday industrial economies are characterised by situations of imperfect competition with increasing returns. One of the main contributions of Krugman's work is the idea that the understanding of a process of regional development is a prerequisite to understanding exchanges. This gives rise to a theory of unequal regional development involving retroactive processes: once established, an initial regional advantage can become cumulative. This opinion is backed up by later work by R. CAMAGNI (2005): regions are in direct competition with one another on the basis of a absolute advantage, not a comparative advantage which could enable each to sustain its level of development via functional specialisation.

These theoretical debates have drawn the attention of European agents on two points: • processes at present underway could lead to long-lasting divergences of regional growth rates, and economic integration will not necessarily produce convergence • The regional scale should be instated as the new basis for economic development on account of the greater impact of potential externalities on local economies (BRISTOL, 2005).

• Transfer operations between the academic world and the political sphere.

These different approaches were to be backed up by a report commissioned by the European Commission which served as an interface between debate in the academic sphere and EU bodies: A Study on the Factors of Regional Competitiveness, Report for the European Commission Directorate-General Regional Policy (2003). Its legitimacy was of course provided by its origin, Pr. Ronald L. MARTIN, University of Cambridge.

Martin's inventory reviews the different theoretical foundations of the concept of competitiveness in economics (neo-classical, Keynesian, the economy of development, the theory of endogenous growth). He then sets these theoretical trends against different conceptions of the region. From this is derived the idea that the concept of regional competitiveness can be defined via different aspects according to the theoretical framework chosen. The need at a given moment to use the word competitiveness results in a range of usages and meanings liable to accommodate all the options of local players. Thus three acceptations are obtained that are very different from one another: a) regional competitiveness implies the ability of a region to generate an adequate level of exports (towards another region or another country) to provide increasing levels of income and full employment for its citizens. However, the productivity of economic activities that are oriented locally is also of crucial importance, in particular because of the orientations of the large urban areas where consumer services develop b) regional competitiveness is also related to qualitative factors such as informal networks of knowledge, confidence, informal institutions, capital etc. c) regional competitiveness is a source of increasing profits obtained from scale economies, from businesses and from their interaction, and from broader capacities deriving from institutional and public entities in the region.

• The concept of regional competitiveness seen from the EU Following this accumulation of possible meanings, on the level of Europe the need to talk differently about regional development and the content of directives for the allocation of Structural Funds means that this polysemy is deployed across the outline documents, in the manner of the different "readings" in BOURDIEU's model cited above.

In the EU, the concept of regional competitiveness appeared in the European council in Berlin in 1999. Here there is reference to the "enhancement of competitiveness in regional economies". The rise of this concept as the main reference was then strengthened by the Lisbon Strategy in 2000, which is a policy orientation document aiming to make the EU "the most competitive and dynamic knowledge economy in the world by 2010". As before, to this objective were added sustainable growth and enhanced social cohesion. The reference to the idea of competitiveness applied to territories then became systematic in official texts. It is thus possible to cite the following: • The third report on Cohesion ( 2004) entitled Cohesion, competitiveness, employment and growth.

• The objective "regional competitiveness and employment" in regional policies in the period 2007-2013 • The Europe 2020 document which aims for "intelligent growth "developing an economy based on knowledge and innovation, "sustainable growth"promoting an economy that is more efficient in its use of resources, greener and more competitive, and "inclusive growth"encouraging an economy with a high employment rate favouring economic, social and territorial cohesion.

Beyond this progressive institutionalisation, the idea of regional competitiveness should be interpreted as an inflexion in European regional policy. In contrast to the earlier stance that was mainly compensatory in the use of regional policy funds, the introduction of the idea of competitiveness assumes that growth can be driven from inside by internal resources and the ability of territories to adapt. The emergence of the concept of regional competitiveness therefore needs to be repositioned in a context of reflection on the part of Community authorities on the most efficient means to restore dynamics to a European economy that is being outdistanced in the race to globalisation, considering that if the origins of the problems are global, their repercussions are local and regional.

One of the advantages of the present malleability of this model for the transfer of concepts to the EU is the absence of any apparent contradiction between the redistribution components of the regional policy and the recommendations concerning regional competitiveness. The orientations of the regional policy have no apparent difficulty articulating regional competitiveness and territorial cohesion, despite the fact that these two phrases relate to objectives that could appear difficult to reconcile 3 . 3For more details See JENSEN [START_REF] Jensen | Making European space: Mobility, Power and Territorial Identity[END_REF] on the question of the articulation of contradictions in European discourse on spatial planning.

The example of "polycentrism"

Unlike regional competitiveness, the concept of polycentrism, in its mobilisation by Community policies, does not entail any theoretical quarrel within the original discipline (geography or economy). The approach was extensive, and the supposed virtue of this syncretism was to ensure the widest possible diffusion of the objective underpinned by this concept.

The dichotomy polycentrism-monocentrism has long been linked to issues of urban morphology. Some of the discourse on the expected impact of technological innovations in terms of the organisation of space developed around the choice between pursuing concentration processes, or initiating de-concentration. The first examples of this discourse date back to the development of industry and the railway networks which gave rise to the formation of "conurbations" (GEDDES, 1915) later contrasted with the tendency to dispersion brought about by electricity and the automobile [START_REF] Mumford | Technics and Civilizations[END_REF]. In these early acceptations, the adjective "polycentric", or sometimes "polynuclear", only concerned the intra-urban space. For instance, as an alternative to the "concentric" model of [START_REF] Burgess | The City[END_REF], [START_REF] Harris | The nature of cities[END_REF] for their part presented a development model with a "polynuclear" structure. Several decades later, Paul CLAVAL in a synthesis on the urban phenomenon [START_REF] Claval | La logique des villes[END_REF] continued to restrict the theme of polycentrism to the interurban space, attributing the polycentric structure of certain urban areas to the rise of the automobile.

While the term polycentrism was initially almost exclusively used to describe intra-urban structures, its sematic field subsequently broadened considerably, entering the field of territorial planning and changing the scale on which it was applied. G. BAUDELLE (2005) has clearly retraced the history of this branching-out, both in its sematic evolution and in its international legitimisation. The origin of the new meaning appears to be Germany as early as 1991, in a study on European cities in which can be found the first image of the "bunch of grapes" used to represent the polycentric structure of the European urban system [START_REF] Kunzmann | The Pattern of Urbanisation in Western Europe 1960-1990[END_REF]. The idea was later returned to in strategy documents of the German federal planning Ministry relating to planning in the new Länder [START_REF] Faludi | The Making of the European Spatial Development Perspective[END_REF].

It is interesting to note that the phrase classically used to describe urban networks mainly focused on the notion of the "pole" (e.g. "growth poles" for the economist PERROUX), or on bipolar and multi-polar models focusing on the notions of attractiveness and regional driving forces. The understanding of urban networks and city systems in fact owes a lot to REILLY's gravitational model, or CHRISTALLER's central place model. The spatial application of this type of theory was developed on regional level around the concept of the "polarised region" [START_REF] Juillard | La région, essai de définition[END_REF] which postulates that in the contemporary world "it is cities that produce regions" (DUGRAND, 1963, p.3). K. KUNZMANN and M. WEGENER, aware of the advantages of polycentric organisation for their own country and its suitability for a federal political organisation, had the idea that this could be proposed as a model for the EU. Without explicitly using the term, the Community See JENSEN [START_REF] Jensen | Making European space: Mobility, Power and Territorial Identity[END_REF] on the question of the articulation of contradictions in European discourse on spatial planning.

document Europe 2000 took up the idea. The notion then circulated via the Dutch Planning Ministry and the Danish government when it held the EU presidency in 1992. The document, approved under the German Presidency by the Member States in Leipzig, mentions the concept in terms that have not varied, but which initially focused on the European scale.

By advocating a "balanced polycentric urban system", the ESDP consolidated the concept of polycentrism in interurban and urban-rural relationships ("development of a polycentric and balanced urban system"). This objective of the ESDP to lessen the weight of the "Pentagon" was later discussed and argued in the first report by ESPON: "At European level (macro), polycentricity is seen as a useful alternative model to enhance regional development more evenly across the European territory. A polycentric Europe is thus seen as an attractive alternative to a European space dominated by the Pentagon" (ESPON 1.1.1. final report, p. 3). The aim of the ESDP, avoiding any restriction to a Europe-centred viewpoint, was also to create "dynamic zones for worldwide economic integration evenly spread across EU territory" by envisaging the promotion of "poles" within the peripheral spaces of the EU. Polycentrism thus became a major element in the political project of rational planning on a European scale, so much so that HALL ( 2006) rather sarcastically entitled a paper Polycentricity: geographical phenomenon or Holy Grail? Finally, polycentrism in its broadest acceptation gives leverage to other ESDP orientations by creating a model that is consistent whatever the scale of analysis. Unlike territorial cohesion, reference to polycentrism enables the urban structuring of the whole European territory to be considered.

How does the shift occur from vocabulary produced by the political sphere to its application across the EU? The ESPON research programme funded by the European Commission was to act as an interface between planning projects and implementation. The ESPON 1.1.1. project in particular endeavoured to propose a normative framework for the term enabling fields of application to be fixed and outlined, along with the issues involved in terms of integration and planning: in the publications of this programme, polycentrism is operationalised on different scales, and concerns at once the morphological dimension, the functional dimension and the relational dimension of urban patterns.

The intermediate and final reports (The role, specific situation and potentials of urban areas as nodes in a polycentric development ESPON Project 1.1.1, third intermediate report, 2003;ESPON 1.1.1, Potentials for polycentric development in Europe, final report, 2004) retain the broadest possible acceptation of polycentrism, both to refer to the internal organisation of a metropolis and as a descriptor of a city system: "Our main strong position is that polycentrism cannot, and should not, be linked a priori to a poly-nuclear, and weakly hierarchical pattern, nor should it be drawn from a multidirectional pattern of relations between cities of a given territory. Poly-nuclearity and a weak hierarchy to the urban pattern are not prerequisites for polycentrism" (ESPON, 2003, p. 44); or again: "Spatial proximity is not a condition of polycentrism. Urban areas can cooperate and exchange even if they are not in close spatial proximity" (ESPON, 2003, p. 46). The new plasticity that has developed in this long-standing concept has enabled it to be operationalised on all scales, from the European regional to the national scale.

The processes at work have also been defined: "Polycentrism is not only the result of voluntary strategies and actions. Polycentrism [can] occur spontaneously as a product of historical, economic, or spatial patterns" (ESPON, 2003, p. 45). The authors stress the functional and relational aspects of the entities concerned by polycentrism by introducing the idea of the FUAs (Functional Urban Areas) to refer to local urban areas.

Polycentricity : two complementary aspects (morphological, relational) and two main processes (institutional and structural polycentricity)

source: ESPON, 2004 As noted earlier, shifts in meaning cannot be understood without reference to the "structure of the receiving field" proposed by BOURDIEU. These shifts can be reinterpreted in the setting of the recent enlargements of Europe (1995) and those planned for the future towards the countries of Central and Eastern Europe, justifying consideration of a strategy on European scale aiming to rebalance the Community space in relation to the "Pentagon" of the large metropolises. However, this political agenda does not explain the choice of the widest possible meanings for polycentrism. These broad definitions of forms, structures and processes enable debates and controversies on the legitimacy or otherwise of schools or currents of thought to be evacuated. The many definitions of the idea of polycentrism also remove the idea of a single model to be followed by all players in the different territorial collectives. Thus each can interpret and appropriate the concept according to context.

Finally, beyond the strategic dimension, the broad definition of the concept of polycentrism cannot be dissociated from urban dynamics themselves, and from the interpretations generated in the face of globalisation. The urban entity is in a sense interpreted as a spatial continuum alternating less dense centres and zones in the urban fabric, rather than in binary form (intra-or inter-urban). Concepts have emerged at the start of the 21 st century, such as that of the Polycentric Urban Region [START_REF] Davoudi | Polycentricitymodelling or determining reality?[END_REF] or the Global City-Region (SCOTT, 2001) or again the Mega-city Region (HALL, [START_REF] Hall | The polycentric metropolis[END_REF], introducing the idea of a recomposition of the urban space in the largest European and world metropolises, where the sprawl and ground cover end up creating a city system at regional level. Beyond the success of this term is the question of the dynamic trends of European cities, and their ability to organise territorial development.

Last but not least one should add the homologous dimension of the concept of polycentrism . Despite its apparent geographical simplicity, the concept also entails a symbolic dimension.

There is indeed a homology between a planning project referred to a polycentric model and the progressive construction of an enlarged European Union with a configuration that is at least decentralised. If not federal, the European project based on territorial equity, and the equality of all member states, andstruggling against inbalanced spatial patterns is in tune with the polycentric model 2. From the political to the scientific sphere: the example of "territorial cohesion".

The notion of "territorial cohesion" as viewed from the European Union

The notion of territorial cohesion follows on from the policy known as "economic and social cohesion of the European Union". This phrase refers back to one of the founding objectives of Europe, that is to say the reduction of disparities, gaps and imbalances between Member States and between European regions, in order to enable the instatement of a European market and improve quality of life for European citizens. This desire to achieve cohesion, here between territories, is related to the idea of a form of "spatial justice", and of a "European model of society" frequently restated in the different treaties.

It is from the Amsterdam Treaty in 1997 that the notion of territorial cohesion began to appear in major European texts. However the development came only after a long period of gestation. Before the Single European Act of 1986, which introduced the notion of "economic and social cohesion" 4 , there is no explicit reference to the notion of cohesion. Yet the idea is 4

The Single European Act, 1986: in order to promote harmonious development across the European Community as a whole, the Community is to develop and pursue its action aiming to strengthen economic and strongly rooted in the social project in which Europe was grounded, and the Treaty of Rome (1957), preparing the way for the European Union, already stressed the need to reduce disparities in development between regions, and for harmonious development across Member States.

It was in 1993, in the perspective of the intergovernmental Conference in Amsterdam, that an interest group, the Assembly of European Regions, (AER) decided to conduct a debate on the future of Europe. The report by the working group in charge of examining the impact of Community policies on territories was approved by this Assembly in a resolution voted unanimously in 1995. It described the considerable but unequal impact of European policies, and the risk of the collapse of territorial structure that could result. Consequently, for the future Treaty, it the inclusion of the notion of economic and social cohesion across territories by adding the notion of "territorial" cohesion. Two years later, reference was made to territorial cohesion in the Amsterdam Treaty, in an article relating to "services of general economic interest" and the part they play in the promotion of social and territorial cohesion on that scale 5 . This notion of territorial cohesion, which drew little notice at the time, was to become one of the main political objectives of the EU.

In 2001, following the European Spatial Development Perspective (1999) 6 , the second report on cohesion 7 gave particular prominence to this new idea. The third report on cohesion ( 2004) defines the relationship between economic and social cohesion on the one hand and territorial cohesion on the other. While economic and social cohesion remained the main objective of the EU, the advent of a borderless space and the construction of economic and monetary unity required European citizens to be able to have the same opportunities wherever they lived. From this point of view, the aim of territorial cohesion is to enable more harmonious economic development by fostering balanced development across European territories.

Generally, territorial cohesion provides a conceptual basis for the EU's regional policy, and it is in line with the drive to enhance economic and social cohesion. Finally, it also carries the idea of a community of destiny for Europeans, whereby bringing peoples closer to one another requires cohesion to be achieved between territories.

The phrase "territorial cohesion" enables the diversity of European societies and countries to be taken into account while at the same time endeavouring not to put a strain on links between territories, but rather to strengthen them. In this sense, territorial cohesion enables public intervention in territories to be sustained, in line with the EU aims for economic development. The reports on cohesion are documents that enable the follow-up of the implementation and the effects of regional policies. They also enable new perspectives for this policy to be sketched out.

It thus is relevant to the action of the EU in the area of spatial planning and development, even if this is not strictly one of the formal prerogatives of European bodies.

With regard to the implementation of policies, territories appear as privileged environments for articulating the sector policies of different players. This signals a specific conception based on the idea that the basis for development is to be found on territorial scales, arising from concrete modes of functioning, in particular the regions.

The territorial dimension of cohesion entails the following:

• the need to take account of the features specific to various types of territoryurban, rural, mountainous, coastal, insular, peripheral, exposed to natural hazards, etc.

• the need to take account of the territorial impact of other Community policies, so that they too can contribute to cohesion, or at least not go against it.

• the attachment of individuals to territories, for cultural or historical reasons. This territorial reference to belonging could also enable identification of what is specifically European compared to other continents.

Far from contradicting one another, these three approaches are complementary, although different categories of players tend to favour one over the others 8 . The first is particularly popular with associations of regions belonging to a given category 9 , while the second approach is advocated mainly by spatial development or environmental specialists wishing to promote integrated multi-sector strategies. In addition, reference to the territorial dimension enables individuals to identify their preferred territory for action: the Union for the Commission, the States for Member-States, or strategic development ensembles to which it seems relevant to adhere (for instance the "macro-regions"), or finally the regions. Thus the phrase assumes a multi-scalar approach that is valuable when defining and implementing European policies. It is indeed the EU territories as a whole that are concerned by cohesion, while alongside the territorial reference also introduces the idea of development that is suited to individual territories, in terms of endogenous potential, taking account of their diversity. Emphasis on these three points shows the flexible and adaptable nature of this notion.

Once established in European Union discourse, as described above, this notion was to be widely discussed and studied in the scientific field.

The different approaches to "territorial cohesion" in the academic field

Part of scientific activity in the human and social sciences consists in deconstructing the categories of political discourse in an attempt to reveal the universe of meaning in which, beyond the actual words, these notions find their place, and their political "usefulness" according to the political, economic and social issues of the moment.

8

See DOUCET, Philippe, 2006 "Territorial cohesion of tomorrow: a path to cooperation or competition?" European Planning Studies, 14, 10, November, pp1473-1485. 9

For instance, the Conference of Peripheral Maritime Regions (CPMR), the association of upland area delegates, Euro-cities, etc The field of spatial planning is no exception, but on account of its positioning between reflection and action, discussions on political notions take on particular forms reflecting several types of scientific stance. From this point of view, the notion of "territorial cohesion" which, as we have seen, derives from the European political sphere, is particularly interesting because it leads to a variety of scientific stances, which in various manners contribute to instating it as a relevant category for scientific reflection.

Thus, without setting out to exhaustively review all the different stances adopted by researchers, we propose to consider this aspect via a sort of typology of these various stances.

First of all, we propose to distinguish what we refer to here as the "exegete" stance. This approach consists at once in analysing the European context in which the notion is deployed, and in taking account of the evolution of political discourse on the notion itself. This approach is analytical, and also contributes to clarifying and enriching the meaning of the notion. In this perspective, territorial cohesion is interpreted both as the historical quest for more balanced development across the European space, and as a reference to the need to ensure greater coherence in political decisions on different levels. Thus territorial cohesion relates to one of the central issues in the implementation of sector policies in the EU, that is to say the multiscale coherence of sectorial action and policies (FALUDI, PEYRONY, 2001). In this way it is viewed as implicitly incorporating the concern to achieve better territorial governance of policies within the EU, in particular in spatial planning, an area in which the European authorities have no legal power. In addition, analysis based on the examination of the main elements of European discourse on spatial planning (in the Territorial Agenda) leads to their interpretation as a promotion of the territorial dimension of policies. According to this view, this type of analysis belongs to discourse in which territories are seen as relying on their own development potential (their territorial "capital") to ensure their own development, and, by a process of aggregation on European scale, to contribute to greater cohesion across the EU (WATERHOUT, 2011). Another type of stance, which we propose to refer to as "normative", consists in providing a definition of territorial cohesion based on proposals for concrete measures of what it might be. Here, the applied research conducted by ESPON is a particularly appropriate setting for this type of approach. Indeed, the studies commissioned are based on specifications that are negotiated on the political level, and this type of research provides the political sphere with a good opportunity to plead the legitimacy of its political categories. For instance, the ESPON project entitled Territorial Impact Package for Transport and Agricultural Policies (TIP TAP) proposes a model for the evaluation of territorial cohesion base on three general components (ESPON 2010): territorial quality, territorial efficiency and territorial identity. Each of these components takes account of a whole range of elements enabling a territory to be qualified, and making it possible, via measurements, to determine the relative position of each territory in relation to the objective of territorial cohesion. In this study, territorial cohesion provides a theoretical framework for the measurement of the impact of European sector policies. The three general components of territorial cohesion as defined by the project are broken down into criteria. For each criterion, indicators are formed to enable a quantitative measure. By ascending aggregation of the results for each indicator, and then for each criterion, and finally for each dimension of territorial cohesion, it is then possible to conclude on the effects of a given policy in the area of territorial cohesion according to evolution scenarios.

Another type of stance towards European concepts deriving from the political sphere can be described as the "Weber" stance. In this perspective, the issue is, in fine, to return the concept to its producer along with a proposal for an alternative interpretation. This can be called the Weber stance in the sense that it sets out to separate the academic sphere from the political sphere, at the same time addressing an alternative choice to the political sphere [START_REF] Denquin | TIP TAP Territorial Impact Package for Transports and Agricultural Policies[END_REF]. For instance, Claude GRASLAND and Grégory HAMEZ proposed two different measures of territorial cohesion in reference to the objectives set out in two major EU documents, the ESDP and the Lisbon Strategy (2000) (GRASLAND, HAMEZ, 2005). These authors show how the premises in these documents imply the choice of measurement variables that are combined into a single indicator of territorial cohesion. They therefore developed two different indicators for territorial cohesion by using, for the ESDP, GDP in purchasing power parity, unemployment rates, and the percentage of young people in the population, and, for the Lisbon strategy, GDP in purchasing power parity, along with the percentage of tertiary activity and the percentage of individuals with high qualifications. The measure is then implemented at Nuts 2 level (regional) yielding two measures of territorial cohesion that are relatively contrasted in spatial terms. In addition to pointing out that measures are shaped by the political and intellectual frameworks that produce them, they also propose an alternative to measures of spatial inequalities (and, by default, of the variable degree of cohesion between EU territories) in the form of a measure of spatial discontinuities.

Finally, researchers offer different definitions of the concept, or else they entrust political players with the task of choosing on the basis of scientific proposals.

Therefore, using an empirical survey conducted within an ESPON programme project, we propose to explore and analyse the way the concepts and notions produced at EU level are received by infra-European players.

Notions and concepts as seen by players on infra-European scales

The results presented here are derived from a survey conducted within an ESPON programme 10 project on the definition and use of notions and concepts in the field of European spatial planning among relevant players at national, regional and local levels. This analysis was conducted on the responses to a questionnaire addressed to these players in different European countries 11 . The aim was not to form a representative sample, but rather to use the fairly large number of responses (102) in a transnational sample (survey conducted in 8 European countries) to give a picture of the way notions and concepts were defined and used by the different players questioned.

The concept of regional competitiveness

Among the players questioned, regional competitiveness is interpreted in a wide range of definitions. Mainly, regional competitiveness is linked to the idea of competition between territories, and to that of the quest for a dominant position in the context of globalisation. It thus reflects a situation of competition between territories, on national and international scale: achieving regional competitiveness is seen as enabling a region to be "up to" the challenges of globalisation, and able to face competition with other regions in a certain number of fields. This attitude is backed up by the idea that regional competitiveness implies the emergence of areas of supremacy, which entails a struggle between regions pursuing the same objectives, leading inevitably to rivalry. It involves the ability of regions to enhance their performances and improve their ranking in the competition between regions, as well as their capacity for regional exchanges.

Further to this, the idea of competitiveness is also associated with the idea of attractiveness, and with that of development. Attractiveness is seen as being based on the qualifications and the competence of the labour force, and on the ability to attract foreign investment. There is thus an assimilation between regional competitiveness and regional attractiveness. This is thought to require a strengthening of regions in world competition, and the identification of complementary elements of between regions on national scale. It also entails the exploitation of comparative regional advantages. This strategy mobilises potential (economic, social, environmental etc.), and abilities (the ability to attract investments and businesses, reactivity towards change) that are specific to each region and should be used as efficiently as possible (sustainable and efficient use of endogenous resources for instance). Regional competitiveness is also defined by reference to the mobilisation of more specific strategies, such as the ability to anticipate economic change, to ensure cooperation among economic players so as to foster regional development, to provide infrastructures, and to issue direct aid.

A wider definition involves the identification of factors for regional competitiveness, such as those related to the initial economic and social structure (specialisation, cooperation among players, collective learning potential), geographical location (e.g. accessibility), and resources available, all of which provide information on the relative potential of regions in terms of competitiveness.

There is also a rather idealistic definition of regional competitiveness, linked to a development objective of generating high income and high standards of living. Here regional competitiveness is a means to achieve more balanced development and sustainable growth by reducing inequalities between regions and supporting the development of regions that are lagging.

The notion of territorial cohesion

The responses provided by interviewees showed the following main lines:

• territorial cohesion is seen as a principle and an objective. Its function is to ensure balanced development across territories (urban and rural areas) providing inhabitants with appropriate quality of life. There is a need to reduce social and economic disparities, with particular attention to the least developed territories • cohesion is one of the values of the EU. It is seen as promoting cooperation between peoples and territories via the improvement of territorial integration and the promotion of regional cooperation. Territorial cohesion is thus seen as materialising the notion of equity or fairness, transposed to the territorial dimension, and that of comparable living standards and equal access for inhabitants to basic services. Policies undertaken in the name of territorial cohesion therefore need to aim to organise forms of solidarity and levelling-out that are required to compensate for the drawbacks of the prevailing economic model and the risks of economic, social and spatial exclusion. Here practitioners underline the key role of public bodies (European as well as national and regional) in the redistribution process. The link between the notion of cohesion and the efficient use of funds for the regional policy is often underlined • a positive or even congratulatory view. Territorial cohesion is endowed with every virtue. It is thus a sort of ideal functioning of our living environment. The notion is linked to that of "harmonious" and "balanced" territorial development, viewed as fostering the sustainable development of all regions across the EU, and again promoting equal living standards across regions.

• territorial cohesion is also assimilated to a tool. It is seen as a tool to correct the errors of the economic system. It is an intervention and solidarity tool able to reduce inequalities between national economies. It is seen as able to combat social segregation and exclusion, which are risks that can occur at any territorial level. The emphasis is on the development of the least developed regions using structural funds. Thus the aim of territorial cohesion is seen as smoothing differences between regions by means of the different financial tools available.

• the evaluation of territorial cohesion. Practitioners, confronted with concrete demands, wonder how to assess this "territorial cohesion", and how to test the new measures of cohesion, via case studies taking account of quality of life and social relationships, access to facilities, economic productivity, accessibility, and mobility. Certain players suggest that territorial cohesion could be studied differently, not solely from a static viewpoint, in particular by taking account of new forms of organisation in certain territories (metropolitan urban areas, peri-urban zones, rural areas). Others underline the fact that the notion needs to be linked to other European concepts that practitioners and decision-makers have to deal with: what are the relationships between territorial cohesion and regional competitiveness, or polycentric development, for instance?

It must be conceded that the manners in which the concepts and notions under study here are used and defined on infra-European scale reflect interpretations that are fairly open and varied, and widely used, although it is not always possible to link these usages with European discourse per se. We can therefore wonder whether the diversity of definitions might not hamper the development of a convergent, operational interpretation in the setting of emergent European spatial planning. This question opens onto a wider debate on the value of possessing clear concepts to which infra-European players can refer when moving on to operational phases.

And

Polycentrism… there is not any reference to infranational (only 2 terms instead 3 as in the previous sections… it seems uncomplete!) 4. Are clear concepts required to shape European spatial planning?

Certain authors [START_REF] Markussen | Fuzzy Concepts, Scanty Evidence, Policy Distance: The Case for Rigour and Policy Relevance in Critical Regional Studies[END_REF] consider that fuzzy or ambiguous concepts are difficult to put into practice and compromise the overall coherence of action implemented in the field of spatial development. For a concept to be useful, it is thought to require a precise set of objectives, so that it can be understood in the same way by the different individuals using it (researchers, elected representatives, professionals, citizens etc.).

This debate is also found in European spatial planning. In 2007, the European commissioner for regional policy, Danuta HÜBNER, with the support of the European Parliament, called for a proposal for a clear, common understanding of the notion of territorial cohesion, so as to precisely, and to criticise them without any systematic reference to European conceptions. Thus it can be said that Europeanisation via the influence of key concepts cannot be reduced to a solely top-bottom mode. The ability of different players to offer general interpretations of selected notions can also be underlined.

It is however relevant to make a distinction between the operational viewpoint and the scientific and political viewpoints.

Indeed, from a scientific viewpoint, to restrict conceptions of European spatial planning to a strictly pragmatic approach precludes the possibility of considering intentions and modes of development of these notions at EU level. Here what is involved is not so much what notions and concepts actually produce as the way in which they are oriented towards certain explicit and implicit objectives, and the way in which they are conceived and diffused in the European debate. Ultimately a strictly pragmatic approach does not seem to us liable to identify the power balances within the EU that explain the emergence and diffusion of certain key notions in spatial planning and development for Europe.

The strictly pragmatic approach side-steps the question of political choice, always oriented according to the concerns and power balances of the moment. Consequently, we also consider it important to give consideration to the normative nature of these notions. To return to examples given in this paper, territorial cohesion offers a conceptual basis for EU action in spatial planning and development which enables specific action on this level to be legitimised. In contrast, the notion of regional competitiveness might appear as a form of "essentialisation" of economic competition between European regions.

Conclusion

The absence of legal powers in the EU in the area of spatial planning has led the European authorities to seek different forms of legitimacy, in particular via reliance on scientific research. This process is particularly prominent in the selection or proposal of concepts for exploration by research and diffusion in the public debate. The academic literature in the area of spatial planning in Europe often stresses the vague nature of the concepts mobilised. However this paper sets out to show that in fact the choice of concepts is related to their scientific construction, making it possible to play on the variety of possible interpretations, and that this situation is not linked to the blurred nature of the concepts themselves, but to the way they have been constructed. Thus the EU adopts concepts or circulates notions that are suited to the political and territorial situation of spatial planning (e.g. regional competitiveness), promotes, via research, certain conceptual acceptations suited to the context (e.g. polycentrism), or again launches political concepts into the debate, which researchers legitimise by appropriating them (e.g. territorial cohesion).

Finally, the value of concepts circulated by the EU towards infra-European, levels is that infra-national players find uses for them, because they can play on the plasticity of the notions and concepts involved. This connects to the pragmatic aspects observed in this analysis of the diffusion and mobilisation of concepts in European spatial planning. However this pragmatic approach should not obscure the genuinely formalised nature of the conceptual choices made on the political level in the EU.

  social cohesion. In particular, the Community aims to reduce gaps between the different regions, and the lagging behind interest are subject to public service obligations by Member States.6This document sets out the general aims fixed by the Member-States in the area of European spatial planning 7

  10 ESPON (European Observation Network for Territorial Development and Cohesion) is a programme of applied research funded by the EU and particularly concerned with spatial planning in Europe 11 This survey, conducted in 2011 within the ESPON 2013 programme was entitled Capitalisation and dissemination of ESPON concepts (CaDEC).

assist the EU in developing its political priorities 12 . This led to the publication in 2008 of the Green Paper on territorial cohesion which, using a public consultation involving, among others, infra-European, national, regional and local institutions, explored the different candidate views of the concept 13 . On the basis of empirical studies in the area of spatial planning in European territories, certain authors [START_REF] Faludi | The Making of the European Spatial Development Perspective[END_REF] consider, for their part, that differing notions play a part in the elaboration of plans for action in spite of their lack of conceptual clarity. For Andreas FALUDI, the notions mobilised by the EU have a creative potential within existing political frameworks, or for the elaboration of new policies. Likewise, David SHAW and Olivier SYKES consider that the concept they studied, polycentrism, "performs in different ways in different planning processes and contexts" (SHAW, SYKES, 2004, p.300). In a recent article, returning to the above debate, Gareth ABRAHAMS concludes that for these authors it is less important to determine exactly what a notion covers than to identify what results it produces (ABRAHAMS, 2013).

In the same article, the author extends the debate to planning in general, referring to the theoreticians of pragmatic planning, among whom authors such as Stanley M. STEIN and Thomas L. HARPER (2012). For the latter authors, the tools developed from normative 14 definitions impose sets of rules rather than encouraging innovative responses suited to settings that are always unique. These authors thus advocate concepts with a degree of fluidity, precisely enabling their use to be adjusted to the contexts in which they are implemented. This type of "pragmatic" approach has diffused today among theoreticians of planning such as Jean Hillier: "Drawing on the philosophy of Gilles DELEUZE, HILLIER suggests that we should not only ask what concepts "do" in the actual world around us, but we should also speculate what these concepts "might do" and how they "might" affect what other concepts, practices and material entities "do". This broader, speculative approach to pragmatism may prove useful to groups whose research is intended to consider how concepts affect policy-making practice now and in the future" (HILLIER in ABRAHAMS, p. 2). This point of view is coherent with the analysis of the results of our survey among players in spatial planning on infra-European level. Thus, without any claim to generalisation, the results presented show that European conceptions of spatial planning and territorial development are familiar to, and used by, the players interviewed. However, while there are indeed European influences in the ways in which these concepts are understood, the responses show the ability of the interviewees to appropriate these notions and to adapt them to their professional practice and to the territories in which they operate, and also to define and explore them more 12 HÜBNER, D. 2007. "Territorial cohesion: Towards a clear and common understanding of the concept". Speech delivered at the informal ministerial meeting on territorial cohesion and regional policy, Ponta Delgada, Azores, Portugal, November 23.


Eric Chevet
email: correspondance-eric.chevet@inserm.fr

AJP-Cell Physiology begins a Theme on "Cellular Processes in Tumour Metastasis -From Basic Research to Translation"

Cancers are so life-threatening not so much because of the ability of cancerous tumors to develop rapidly as by the risk of the spreading of tumor cells out of their originatation site. This proces is called metastasis. The term "metastasis" was proposed in 1829 in the context of cancer by Joseph Claude Recamier, a French physician who was the first to report anatomical observations in his book entitled "Recherche sur le traitement du cancer" that showed that the metastases came from the emigration of cancer cells out of the primary (breast) tumor and of their transplant into organs distant from the home of the primary cancer [START_REF] Recamier | Recherche sur le traitement du cancer[END_REF].

Metastases cause 90% of the patient mortality due to malignant carcinomas. Unfortunately, it should be noted that the prognosis of a patient with metastatic cancer is not much better now than it was ten or twenty years ago. Indeed, many patients, at the time of diagnosis, already have occult metastases (also known as micrometastases). These render the surgical or radiotherapeutic action ineffective, since it these treatments eradicate only the primary tumor and leave in place the secondary tumors. Later these may develop and current chemotherapeutic treatments are rarely effective. This explains the urgency to find new type(s) of chemotherapy targeted at metastatic disease and the need to better understand the biological mechanisms that are implemented during metastatic progression.

It is known that cancer cells can migrate locally into nearby non-tumoural tissue parenchyma, they can also spread to lymph nodes, tissues as well as to distant parts of the body. The cellular and molecular characteristics of metastatic cancer cells are somehow similar to those of the initial tumour cells but in the process of metastasis they are selected for their capacity to cope with non-native and stressful environments [START_REF] Scheel | Adaptation versus selection: the origins of metastatic behavior[END_REF]. Indeed, most of the time, spreading cancer cells die at some point in this process, but some of them are capable to form new tumors in other parts of the body and thus display the fittest and most adapted phenotype. Metastatic cancer cells can also remain inactive/dormant at a distant site for many years before they grow again, if at all (2). Carcinoma cells spread through the body in a series of steps. These steps include i) the growth into, or invasion, nearby non-tumoural tissue; ii) the capacity to slide through the walls of nearby lymph nodes or blood vessels, this process is called intravasation; iii) the ability of metastatic cells to travel through the lymphatic system or bloodstream to other parts of the body, this process is called circulation of the tumor cells; iv) adhesion to small blood vessels at a distant location and invasion of the blood vessel walls, this process is called extravasation; v) the formation of premetastatic lesions into the surrounding tissue, and formation of micrometastases; vi) colonization of the tissue and formation of new blood vessels to allow the metastatic tumor to grow [START_REF] Welch | Defining the Hallmarks of Metastasis[END_REF]. All these processes involve aberrant cell communication mechanisms that can be mediated at each stage by exposure of select proteins at the tumor cell surface (e.g. adhesion molecules), by the production of soluble materials (e.g. growth factors or chemokines), or by extracellular vesicles [START_REF] Lafitte | Roles of exosomes in metastatic colon cancer[END_REF].

A c c e p t e d m a n u s c r i p t

These aberrant cell communication mechanisms together with tumor cell intrinsic gain-of-functions (e.g. endoplasmic reticulum stress (4)) provide metastatic tumor cells with specific features that allow them to exert specific effects on neighboring cells in the tissue of origin, to evade the immune system, circulate in the blood stream and colonize the distant tissue. The first Review in this series addresses the roles of exosomes in metastatic tumour cell communications (3). The understanding and the characterization of the molecular mechanisms activated to drive metastasis will not only allow the identification of potential biomarkers that may predict at an early stage for instance the presence of premetastatic lesions but also lead to the design of novel effective therapeutics (1), thereby demonstrating the relevance of such translational approach.

We thank the authors for their excellent Reviews and believe that readers will find the Review articles in this Theme of interest. In addition, we hope that the ideas and results provided in the articles will stimulate experiments that address unanswered questions regarding cellular and molecular mechanisms controlling metastasis that may assist translational efforts to make tumour metastasis a non-life-threatening condition. look forward to receiving original manuscripts in response to the accompanying Call for Papers on "Cellular Processes in Tumour Metastasis -From Basic Research to Translation" which will start in October 2019.

Disclosures

EC is cofounder of Cell Stress Discoveries Ltd.