Gene Expression Patterns 6 (2006) 772–776 www.elsevier.com/locate/modgep

Tenectin, a novel extracellular matrix protein expressed during Drosophila melanogaster embryonic development Ste´phane Fraichard, Anne-Laure Bouge, Isabelle Chauvel, Herve´ Bouhin

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UMR CNRS 5548, ‘‘De´veloppement, Communication Chimique’’, 6 Bd Gabriel, 21000 Dijon, France Received 23 November 2005; received in revised form 5 January 2006; accepted 19 January 2006 Available online 28 February 2006

Abstract During Drosophila embryonic development, various morphogenetic processes require the remodeling of the extracellular matrix. In a previous study, we have identified and characterized a cDNA encoding a novel putative extracellular matrix protein named tenebrin, in the beetle Tenebrio molitor. Here, we examine the expression of the Drosophila ortholog, referred to as Tenectin (Tnc), during embryonic development. Tnc is expressed in the majority of tissues of neuroectodermic origin such as hindgut, foregut, tracheal system, anal plate, and CNS. In the CNS, the Tnc transcript is restricted to a few cells, whereas the protein is located in the dorsal part of the axonal tracts. In the hindgut and the trachea, Tnc protein is expressed on the apical pole of the cells. Tnc is an extracellular matrix protein secreted in a polarized way in different organs of Drosophila embryos. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Development; Drosophila; Embryo; Tenectin; Extracellular matrix; Developmental hormones; Whole-mount in situ hybridization; Hindgut; Foregut; Small intestine; CNS; Axonal tracts; Midline; Anal plate; Spiracles; Trachea; Morphogenic process; Epithelial cells; Apical secretion; Neuroectodermic

During embryogenesis, morphogenetic movements are induced and/or controlled by different molecules, such as growth factors or hormones. In holometabolous insect as Drosophila, two types of hormones, ecdysteroids and juvenile hormones, control development. These hormones play a very important role during embryogenesis in various morphogenic processes such as cell movements and cuticle deposition (Chavez et al., 2000) and during the post-embryonic development, in histoblasts proliferation, imaginal discs differentiation or larval cells apoptosis (Bodenstein, 1965; Fristrom and Fristrom, 1993; Robinow et al., 1993; Truman et al., 1994). Moreover, during various morphogenetic processes the cells will change their shape and move, which requires the remodeling of the extracellular matrix (ECM). In a previous work, we have characterized a cDNA encoding a novel putative ECM protein named Tenebrin,

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Corresponding author. Tel.: +33 380 396 302; fax: +33 380 396 289. E-mail address: [email protected] (H. Bouhin).

1567-133X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.01.007

whose expression is regulated by both developmental hormones (Royer et al., 2004). The deduced protein sequence contains typical extracellular matrix protein features, including a signal peptide, internal repeats, a RGD tripeptide sequence (a motif known to bind integrins), and c-type von Willebrand Factor domains which are typically involved in protein–protein interactions. The Drosophila ortholog has been identified (CG13648) and named Tenectin (Tnc). The cytogenetic map position of this gene is at 96B15. In this paper, we characterize the expression pattern of Tnc during embryogenesis and we demonstrate that Tnc is a novel extracellular matrix protein. 1. Results and discussion 1.1. Expression of Drosophila Tnc during the embryogenesis Whole-mount in situ hybridization of Drosophila embryos was used to examine the temporal and spatial expression patterns of Tnc during embryogenesis using

S. Fraichard et al. / Gene Expression Patterns 6 (2006) 772–776

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Fig. 1. Tnc RNA localization in Drosophila embryos as detected by whole-mount in situ hybridizations. (A) At stage 5, Tnc is not detected. (B) In stage 11 embryos, Tnc is expressed first in the proctodeum (PR) and weakly in the CNS. (C) At stage 12, Tnc persists in the hindgut (HG) and in the CNS and appears in the posterior spiracles placodes (PSP). Lateral view (D) and dorsal view (E) of stage 14 embryo. Tnc is strongly expressed in the anterior part of the hindgut, the anal plate (AP) and the posterior spiracles (PS), and more weakly in the foregut (FG), the CNS, the sensory organs of the clypeolabrum (SOC), and in longitudinal tracheal trunks (LTT). (F) In stage 16, Tnc expression is restricted to the CNS.

two different antisense RNA probes (Fig. 1); both gave the same result. Labeled sense RNA probes were used as control and did not show specific labeling (data not shown). We did not detect any maternal transcript in early stages (Fig. 1A). At the onset of germband retraction, Tnc transcript is expressed first in the proctodeum and weakly in the central nervous system (CNS) (Fig. 1B). As germband retraction proceeds (Fig. 1C), Tnc expression persists in the hindgut and in the CNS and appears in the placode of the posterior spiracles. By stage 14 (Figs. 1D and E), Tnc expression appears in the sensory organs of the clypeolabrum, the foregut, and the dorsal longitudinal tracheal trunks. High levels of Tnc transcript were found in the anterior part of the hingut, anal plate, posterior spiracles, and in a few cells of the CNS. At the end of embryogenesis, Tnc expression is restricted to the CNS (Fig. 1F). As shown by Fig. 2, the transcript expression pattern was in agreement with the protein distribution.

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1.2. Tnc is secreted to the apical surface of the hindgut and tracheal cells From the onset of gastrulation until the end of embryogenesis, Tnc is detected in the hindgut. The Drosophila hindgut is composed of three morphologically distinct regions along its antero-posterior axis: small intestine, large intestine, and rectum. The large intestine is further subdivided into a ventral and a dorsal domain. A one-cell-wide domain, which was designated as ‘‘border cells’’ (Takashima and Murakami, 2001), forms at the anterior and posterior borders of the large intestine and separates the dorsal and ventral domains of the large intestine (Fig. 3A). After stage 14, the border cells are distinguished by marked expression of the transmembrane proteins Crumbs (Crb) (Takashima and Murakami, 2001; Tepass et al., 1990). During embryogenesis, Tnc RNA transcript is detected first in all domains of the hindgut. After stage 14, Tnc expression is restricted to the anterior part of the hindgut. To determine the precise

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Fig. 2. (A) Comparison of the distribution of Tnc protein and Tnc transcript in stage 14 embryos (same abbreviations as Fig. 1). (B) To control the specificity of the anti-sera, Df(3R)ED6220 homozygous embryos were stained with the anti-Tnc sera. No staining was detected (a stage 14 embryo is shown).

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1.3. Expression of Tenectin in the embryonic central nervous system

Fig. 3. (A) Schematic representation of the spatial organization of the hindgut domains of Drosophila embryo. Just after the junction of the posterior end of the midgut (MG) and the Malpighian tubules (MT) is localized the most anterior domain of the hindgut, the small intestine (SI). The small intestine is followed by the large intestine (LI) and the rectum (REC), which opens to the anal slit that is surrounded by the anal pads (AP). A one-cell-wide domain named border cells (BC) defines the anterior and posterior borders of the large intestine and subdivides it into a ventral and a dorsal domain. (B) Tnc is expressed in the small intestine. Confocal microscopy image of the hindgut in stage 14/15 embryo double stained for Tnc transcript (green) and Crbs protein (red).

During embryogenesis, Tnc is expressed in the central nervous system. The CNS is composed of neuromeres which are divided in three distinct regions: the two lateral regions (neuro-ectodermic origin) which are separated by the midline region (meso-ectodermic origin) (Goodman and Doe, 1993). The lateral regions are composed of progenitor cells (neuroblasts, glioblasts, and neuroglioblasts) which generate neurones and glial cells that will occupy the rest of the CNS. By stage 12, Tnc mRNA is first detected in lateral cells of the CNS with a typical chevron-like pattern (Fig. 5). Later, Tnc is expressed in lateral and midline cells and finally, Tnc is only found in midline cells of the CNS but Tnc mRNA does not colocalize with the glial cells marker Repo (data not shown). Comparatively, Tnc protein is detected at stage 12 in lateral cells of the CNS. Then, Tnc is first expressed in longitudinal axon tracts and later in longitudinal and commissural axon tracts (Fig. 5). To confirm this point, we compared the expression of Tnc with the axon membrane protein BP102 (Fig. 6). Tnc and BP102 are colocalized (ventral view) in the same areas. However, Tnc is detected on the periphery of the axon tracts and more specifically in the dorsal part of the CNS and around some midline cells (sagittal view). This confirms and demonstrates that Tnc is secreted in the extracellular matrix in the CNS. 1.4. Conclusions

localization of Tnc RNA during the last embryonic stages, we compared the expression of Tnc RNA with that of the Crb protein. This revealed that Tnc transcription is limited to the small intestine (Fig. 3B). In addition, the exam of the Tnc protein pattern by confocal microscopy in the hindgut shows that its secretion is polarized. Analysis of Tnc deduced protein sequence reveals the presence of a putative signal peptide sequence suggesting that Tnc protein could be secreted. To confirm this hypothesis double staining between Tnc and the two membrane proteins Crb and Fasciclin III was performed (Fig. 4B). In epithelial cells, Crb is located in the sub-apical complex and takes part in the establishment and the maintenance of cell polarity (Klebes and Knust, 2000; Tepass et al., 1990) (Fig. 4A). Fasciclin III is a transmembrane glycoprotein localized basolaterally in epithelial cells (Klebes and Knust, 2000). Tnc is detected in the lumen of the hindgut which corresponds to the apical surface of the intestinal cells. Moreover, Tnc is also detected in the lumen of tracheal trunks, detectable by the 2A12 antibody which recognizes an unknown luminal component (Ikeya and Hayashi, 1999) (Fig. 4B). So, Tnc is secreted to the apical surface of the hindgut and tracheal cells.

Our findings show that in Drosophila, Tnc is expressed in a complex and dynamic pattern, in different neuroectodermic tissues. In addition, Tnc protein is secreted in the extracellular matrix in a polarized way to the apex of the hindgut and tracheal cells and in the dorsal part of the CNS. Moreover, given that the Tenebrio orthologous gene presents a clear hormonal gene regulation, Tnc appears as a good candidate to study the relationships between the remodeling of the ECM and the developmental hormones during embryogenesis. 2. Materials and methods 2.1. Whole-mount in situ hybridization and immunohistochemistry Whole-mount in situ hybridization was performed using a variation of the protocol described by (Tautz and Pfeifle, 1989). To prepare the tenectin in situ RNA probes, two distinct cDNA encoding regions were PCR-amplified (5 0 -GACAATTCCCGAAATCTC CA/5 0 -CAGCATCCTGAGGAGACACA and 5 0 -GATAACCAGGTC TCATTCTC/5 0 -TCCGGAGAGTGGTAGGGCACG) and cloned in pGEM-T easy vector (Promega). Digoxigenin-labeled sense and antisense riboprobes were prepared by in vitro transcription using T7 and SP6 polymerase, respectively, using the Roche Dig RNA labeling system.

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Fig. 4. Tnc protein is located in the ECM and is secreted apically by the hindgut and tracheal cells. (A) Schematic representation of the transversal epithelial tubes structure (i.e., hindgut, trachea). (B) Confocal microscopy images of the hindgut and longitudinal tracheal trunk in stage 14 embryos. Hindguts are double stained for Tnc protein, Crumbs and Fasciclin III (Fas III), whereas longitudinal tracheal trunk is double stained for Tnc and 2A12. Tnc protein is secreted in the lumen of the hindgut and of the trachea.

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Tnc protein Fig. 5. Comparative distribution of Tnc mRNA and protein in the CNS of Drosophila embryos. At stage 12, the distribution of Tnc RNA and protein is similar. The expression is located in the lateral cells of the CNS. In stage 14 Tnc transcript is expressed weakly in lateral cells and strongly in midline cells. Comparatively, at stage 14 Tnc protein is detected in the longitudinal axon tracts (arrow). At stage 16, Tnc transcript is restricted to the midline cells whereas Tnc protein is expressed in the longitudinal and commissural axonal tracts (arrowhead).

Fig. 6. Tnc is secreted in the dorsal part of the CNS. Ventral and sagittal of Drosophila CNS embryos (stage 15/16), double stained for BP102 (red) and Tnc protein (green). In the ventral view, Tnc protein is detected in the longitudinal and commissural axon tracts. The sagittal optical sections (dorsal is up) were realized according the dash line observed in the ventral view. Tnc protein is detected in the dorsal part of the CNS and around some midline cells (arrowhead).

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2.2. Tenectin antibodies and immunohistochemistry Two polyclonal antibodies were raised and purified by Eurogenetec (Brussel, Belgium). Two rabbits were immunized against two different synthetic peptides corresponding, respectively, to the N- and C-terminal parts of Tnc (APVQEYTEIQQYSEGC and CPQDSDK TPSSEAPQD). The two unpurified sera gave the same labeling and only one of them was further used. This serum was purified against each of the two peptides of Tnc. The two purified sera gave the same result, whatever the peptide used for the purification. The preimmune sera did not show any staining. Moreover, the two anti-sera were tested against homozygous embryos for a deficiency of the Tnc region (Df(3R) ED6220, Dros Del project). For immunohistochemistry, wild-type embryos fixed in 4% paraformaldehyde were stained with the following primary antibodies: mouse anti-BP102 (1:100; Developmental Studies Hybridoma Center, University Iowa), mouse anti-Crumbs (1:25, provided by E. Knust), mouse anti-Fasciclin III (1:100; Developmental Studies Hybridoma Center, University Iowa), mouse 2A12 (1:10; Developmental Studies Hybridoma Center, University Iowa), and anti-Tenectin (1:4000). Detection of the different primary antibodies was carried out using alkaline phosphatase anti-rabbit (1:50, Sigma), Alexa 594 anti-mouse (1:200, Molecular Probe), and Alexa 488 anti-rabbit (1:50, Molecular Probe).

Acknowledgements We thank Jean-Philippe Charles and Juan Galceran for critical comments on the manuscript. We thank Developmental Studies Hybridoma Center, University Iowa, for providing antibodies and Dros Del project, University of Cambridge, for providing the deficiency line. This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the University of Burgundy, and grants from the French ministry of Research and Education and from the Fondation pour la Recherche Medicale (FRM). References Bodenstein, D., 1965. The embryonic development of Drosophila. In: Demerec, M. (Ed.), Biology of Drosophila. Hafner, New York, pp. 275–277.

Chavez, V.M., Marques, G., Delbecque, J.P., Kobayashi, K., Hollingsworth, M., Burr, J., Natzle, J.E., O’Connor, M.B., 2000. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127, 4115– 4126. Fristrom, D.F., Fristrom, J.W., 1993. The metamorphic development of the adult epidermis. In: Bate, M., Martinez Arias, A. (Eds.), The Development of Drosophila melanogaster, vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 843– 897. Goodman, C.S., Doe, C.Q., 1993. Embryonic development of Drosophila central nervous system. In: Bate, M., Arias, A.M. (Eds.), The development of Drosophila melanogaster, vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1131– 1206. Ikeya, T., Hayashi, S., 1999. Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea. Development 126, 4455–4463. Klebes, A., Knust, E., 2000. A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Curr. Biol. 10, 76–85. Robinow, S., Talbot, W.S., Hogness, D.S., Truman, J.W., 1993. Programmed cell death in the Drosophila CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform. Development 119, 1251–1259. Royer, V., Hourdry, A., Fraichard, S., Bouhin, H., 2004. Characterization of a putative extracellular matrix protein from the beetle Tenebrio molitor: hormonal regulation during metamorphosis. Dev. Genes Evol. 214, 115–121. Takashima, S., Murakami, R., 2001. Regulation of pattern formation in the Drosophila hindgut by wg, hh, dpp, and en. Mech. Dev. 101, 79– 90. Tautz, D., Pfeifle, C., 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85. Tepass, U., Theres, C., Knust, E., 1990. Crumbs encodes an EGFlike protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61, 787– 799. Truman, J.W., Talbot, W.S., Fahrbach, S.E., Hogness, D.S., 1994. Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development. Development 120, 219–234.