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Dynamic expression of d-CdGAPr, a novel Drosophila melanogaster gene encoding a GTPase activating protein
Mechanisms of Development 94 (2000) 267±270
www.elsevier.com/locate/modo
Gene expression pattern
Dynamic expression of d-CdGAPr, a novel Drosophila melanogaster gene encoding a GTPase activating protein Thierry Sagnier, AureÂlie Grienenberger, Marie-Christine Mariol, HeÂleÁne BeÂrenger, Jacques Pradel, Yacine Graba* Laboratoire de GeÂneÂtique et Physiologie du DeÂveloppement, Institut de Biologie du DeÂveloppement de Marseille, CNRS/INSERM/Universite de la MeÂditerraneÂe, Parc Scienti®que de Luminy, Case 907, 13288 Marseille Cedex 9, France Received 20 January 2000; received in revised form 14 February 2000; accepted 14 February 2000
Abstract Small GTPases of the rho family function as signal transducer for extra-cellular stimuli to control cytoskeletal re-organization and a variety of other cellular processes including adhesion, proliferation and transcriptional regulation (Hall, A., 1998. RhoGTPases and the actin cytoskeleton. Science 279, 509±514). Usually widely expressed, their activities are tightly controlled by conformational changes induced by hydrolysis of the GTP bound molecule (Bourne H.R., Sanders D.A., 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125±132). Conversion of GTP to GDP relies on a rho intrinsic GTPase domain that requires GTPase activating proteins (GAPs) for potent activity (Lamarche, N., Hall. A., 1994. GAPs for rho-related GTPases. Trends Genet. 10, 436±440). Here we report on the identi®cation of a novel Drosophila GAP gene, d-CdGAPr, encoding a protein related to mammalian CdGAPs. The gene is expressed throughout development as well as in adults. Spatio-temporal transcription pattern of d-CdGAPr during embryogenesis is highly dynamic. Abundant in the pre-blastoderm embryo prior to the onset of zygotic transcription, messengers accumulate at the blastoderm posterior pole after cellularisation. During gastrulation and subsequent development, all cells accumulate low levels of d-CdGAPr RNA, while a few territories transiently display stronger expression. Sites of preferential expression include the posterior pole of the early cellular blastoderm, the neuro-ectoderm prior to neuroblast delamination, rows of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments and a ring of epidermal cells at the posterior end of the embryo. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Drosophila; Rho GTPases; GTPase activating protein/GAP; CdGAP
1. Cloning d-CdGAPr was cloned starting from a P element insertion (Pw 1 5) mapped on the second chromosome at cytological location 38A (Fig. 1A). Following plasmid rescue and screening of gridded genomic and cDNA libraries (see methods), several overlapping cDNAs were isolated. The longest one (29G3) was sequenced. The intron/exon structure of the transcriptional unit was established from comparison of 29G3 sequence with that of genomic DNA available from BDGP (Fig. 1A). Used to probe a developmental Northern blot, 29G3 revealed a major transcript of 7.5 kb present at all stages analyzed (embryos, third instar larvae, and adults; Fig. 1B). This indicates that 29G3, which is 7496 bp long, most certainly constitutes a complete copy of the transcript. The Northern blot also revealed a smaller and less * Corresponding author. Tel.: 133-491-269-613; fax: 133-491-820-682. E-mail address: [email protected] (Y. Graba).
abundant 3 kb transcript speci®cally present in adult males. This male speci®c transcript might result from alternative splicing and/or the use of another promoter for transcriptional initiation.
2. Sequence analysis The cDNA sequence de®nes an open reading frame (ORF) starting at position 109 and predicts a 1843 amino acid protein. Several stop codons precede and follow the ORF, and the sequence surrounding the ®rst in frame ATG matches the consensus for translation start (Cavener, 1987). A search of protein sequence databases revealed that the putative protein encoded by 29G3 presents a 150 amino acid conserved block that correspond to a GTPase activating domain. Within this domain, strongest homologies (55±62% identity and 71±76% similarity) were observed with two human sequences and with the mouse CdGAP protein
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00291-4
268
T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
(Fig. 2A), that has been shown to function as a regulator of Cdc42 and Rac to control actin re-organization (Lamarche et al., 1998). Remarkably, activating domains from previously identi®ed Drosophila GAPs show rather weak identity/similarity scores to d-CdGAPr: the best score was observed with that of Rotund rac GAP (31/48%; Agnel et al., 1992). Outside the GTPase activating domain, a few proteins display signi®cant homologies with d-CdGAPr. The strongest homologies were found with mouse CdGAP downstream of the domain and with the Caenorhabditis elegans breakpoint cluster region protein (Bcr) upstream of it (Fig. 2B). No signi®cant homology was observed with any of the identi®ed Drosophila GAPs outside of the GTPase activating domain. 3. Embryonic expression pattern Whole-mount in situ hybridization revealed a dynamic spatio-temporal expression pattern of d-CdGAPr during embryogenesis. Important maternal contribution is visible in pre-blastoderm embryos, and vanishes by the end of the cellularisation process (Fig. 3A). At that stage, transcripts are exclusively found at the posterior pole, but not within the pole cells (Fig. 3B). Starting at early gastrulation, low level of ubiquitous zygotic expression occurs, while sites of preferential expression appear soon after. These sites include the cephalic furrow and neuro-ectoderm prior to neuroblast delamination (Fig. 3C,D) as well as scattered
cells within each segment of germ band extended embryos that most certainly correspond to PNS precursor cells (Fig. 3E). By the end of germ band retraction, d-CdGAPr transcription occurs at high rate in a row of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments, and in ventral cell clusters that presumably correspond to chordotonal organ precursors (Fig. 3F). Slightly later, more dorsally located cell clusters likely corresponding to chordotonal precursors also start to express the gene (Fig. 3G). By the end of embryogenesis, strong d-CdGAPr transcription occurs at the posterior end of the embryo in a ring of epidermal cells and in two laterally located cell clusters (Fig. 3H). The regulation of Drosophila GTPases Rap1 and Ras1 activities has been proposed to be controlled by temporarily and spatially restricted expression of GAPs (Chen et al., 1997). The dynamic expression of d-CdGAPr extends this observation.
4. Methods 4.1. Flies and in situ hybridization Oregon R was used as standard. The Pw 1 5 line has been generated by mobilization of a PlacW. Whole-mount in situ hybridization using 29G3 RNA probes was as in O'Neil and Bier (1994), except that embryos were ®rst ®xed in 4% formaldehyde and that xylene washes were omitted.
Fig. 1. (A) The d-CdGAPr locus. The site of P element insertion (Pw 1 5) is shown on the genomic restriction map (R: EcoRI; Sl: SalI; Sc: SacI; X: XbaI). l 4A9, l 94H6 and l 20G8 are three genomic recombinant phages isolated by probing a gridded EMBL3 Drosophila genomic library with Pw 1 5 plasmidrescued DNA. Filled bars in the intron/exon structure indicate the coding sequence of the 29G3 cDNA. B) Northern blot analysis. Poly (A1) RNA of 0±2 h embryos, third instar larvae, adult males and females were probed with the 7.5 kb 29G3 cDNA (upper panel) and with rp49 as a loading control. Sizes of the 2 d-CdGAPr transcripts are indicated on the left.
T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
269
Fig. 2. (A) Cladogram of d-CdGAPr and the closest mammalian, C. elegans and Drosophila GTPase activating domain sequences. Numbers in brackets indicate identity/similarity scores. (B) d-CdGAPr alignment with the mouse CdGAP and C. elegans Bcr sequences. Black and grey shadings indicate identical and similar amino acids, respectively. The GTPase activating domain is underlined.
4.2. Molecular biology Cloning, library screenings and Northern blot hybridization was performed according to standard procedures. Plasmid rescue following EcoRI digestion allowed to isolate a 3kb EcoRI fragment containing genomic DNA ¯anking the site of insertion. This DNA was used to screen a Drosophila genomic library (EMBL3, Stratagene) gridded in the laboratory. Three phages were recovered, and subsequently used to isolate several overlapping cDNAs from an embryonic cDNA library (Brown and Kafatos, 1988) gridded in the laboratory. Searches for homologies were performed using Blast programs and sequences alignment done using PIMA 1.4 (Infobiogen). The cladogram of the GTPase activating domains was generated with Treeview after ClustalW analysis. Accession numbers for sequences used in this study are the following: mouse CdGAP: AF151363; C. elegans Bcr: Z498888; human b2 chimerin: P52757; human AB033030: AB033030; human AC002398: AC002398; rat b2 chimerin: B53764; Drosophila Rotund: P40809; Drosophila Rlip: AF 037470; Drosophila genomic
sequence at 38A5-B4: AC0149777. The d-CdGAPr sequence has been deposited to GenBank under the accession number AF218776. Acknowledgements We thank Assia Bousteila for her contribution to the early part of this study. This work was supported by the CNRS and grants from ªl'Association pour la Recherche contre le Cancer (ARC)º and ªLa Ligue Nationale Contre le Cancer (LNCC)º. References Agnel, M., Roder, L., Vola, C., Grif®n-Shea, R., 1992. A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase activating (racGAP) proteins. Mol. Cell. Biol. 12, 5111±5122. Brown, N.H., Kafatos, F.C., 1988. Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425±437. Cavener, D.R., 1987. Comparison of the consensus sequence ¯anking trans-
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T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
Fig. 3. d-CdGAPr expression pattern during Drosophila embryonic development. All embryos are anterior to the left and ventral side down except for (D) and (H) which are ventral and dorsal views, respectively. (A) Pre-blastoderm embryo. (B) Cellular blastoderm. (C±D) Early germ band extended embryos. In (D) the bracket marks the neuro-ectoderm. (E) Late germ band extended embryo. The arrow points to cells that most certainly correspond to PNS precursors. (F±H) Germ band retracted embryos. Arrowheads in (F) point toward rows of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments (also visible in G). Arrows point to group of cells that presumably correspond to chordotonal organ precursors. In (H), the arrow indicates a ring of epidermal cells at the posterior end of the embryo and arrowheads point to two strongly expressing cell clusters. lational start sites in Drosophila and vertebrates. Nucl. Acids. Res. 15, 1353±1361. Chen, F., Barkett, M., Ram, K.T., Quintanilla, A., Hariharan, I.K., 1997. Biological characterisation of Drosophila Rapgap1, a GTPase activating protein for Rap1. Proc. Natl. Acad. Sci. USA 94, 12485±12490.
Lamarche, N., Hall, A., 1998. CdGAP, a novel proline-rich GTPase activating protein for Cdc42 and Rac. J. Biol. Chem. 273, 29172± 29177. O'Neil, J.W., Bier, E., 1994. Double label in situ hybridisation using biotin and digoxigenin tagged RNA probed. Biotechniques 870, 874±875.
www.elsevier.com/locate/modo
Gene expression pattern
Dynamic expression of d-CdGAPr, a novel Drosophila melanogaster gene encoding a GTPase activating protein Thierry Sagnier, AureÂlie Grienenberger, Marie-Christine Mariol, HeÂleÁne BeÂrenger, Jacques Pradel, Yacine Graba* Laboratoire de GeÂneÂtique et Physiologie du DeÂveloppement, Institut de Biologie du DeÂveloppement de Marseille, CNRS/INSERM/Universite de la MeÂditerraneÂe, Parc Scienti®que de Luminy, Case 907, 13288 Marseille Cedex 9, France Received 20 January 2000; received in revised form 14 February 2000; accepted 14 February 2000
Abstract Small GTPases of the rho family function as signal transducer for extra-cellular stimuli to control cytoskeletal re-organization and a variety of other cellular processes including adhesion, proliferation and transcriptional regulation (Hall, A., 1998. RhoGTPases and the actin cytoskeleton. Science 279, 509±514). Usually widely expressed, their activities are tightly controlled by conformational changes induced by hydrolysis of the GTP bound molecule (Bourne H.R., Sanders D.A., 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125±132). Conversion of GTP to GDP relies on a rho intrinsic GTPase domain that requires GTPase activating proteins (GAPs) for potent activity (Lamarche, N., Hall. A., 1994. GAPs for rho-related GTPases. Trends Genet. 10, 436±440). Here we report on the identi®cation of a novel Drosophila GAP gene, d-CdGAPr, encoding a protein related to mammalian CdGAPs. The gene is expressed throughout development as well as in adults. Spatio-temporal transcription pattern of d-CdGAPr during embryogenesis is highly dynamic. Abundant in the pre-blastoderm embryo prior to the onset of zygotic transcription, messengers accumulate at the blastoderm posterior pole after cellularisation. During gastrulation and subsequent development, all cells accumulate low levels of d-CdGAPr RNA, while a few territories transiently display stronger expression. Sites of preferential expression include the posterior pole of the early cellular blastoderm, the neuro-ectoderm prior to neuroblast delamination, rows of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments and a ring of epidermal cells at the posterior end of the embryo. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Drosophila; Rho GTPases; GTPase activating protein/GAP; CdGAP
1. Cloning d-CdGAPr was cloned starting from a P element insertion (Pw 1 5) mapped on the second chromosome at cytological location 38A (Fig. 1A). Following plasmid rescue and screening of gridded genomic and cDNA libraries (see methods), several overlapping cDNAs were isolated. The longest one (29G3) was sequenced. The intron/exon structure of the transcriptional unit was established from comparison of 29G3 sequence with that of genomic DNA available from BDGP (Fig. 1A). Used to probe a developmental Northern blot, 29G3 revealed a major transcript of 7.5 kb present at all stages analyzed (embryos, third instar larvae, and adults; Fig. 1B). This indicates that 29G3, which is 7496 bp long, most certainly constitutes a complete copy of the transcript. The Northern blot also revealed a smaller and less * Corresponding author. Tel.: 133-491-269-613; fax: 133-491-820-682. E-mail address: [email protected] (Y. Graba).
abundant 3 kb transcript speci®cally present in adult males. This male speci®c transcript might result from alternative splicing and/or the use of another promoter for transcriptional initiation.
2. Sequence analysis The cDNA sequence de®nes an open reading frame (ORF) starting at position 109 and predicts a 1843 amino acid protein. Several stop codons precede and follow the ORF, and the sequence surrounding the ®rst in frame ATG matches the consensus for translation start (Cavener, 1987). A search of protein sequence databases revealed that the putative protein encoded by 29G3 presents a 150 amino acid conserved block that correspond to a GTPase activating domain. Within this domain, strongest homologies (55±62% identity and 71±76% similarity) were observed with two human sequences and with the mouse CdGAP protein
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00291-4
268
T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
(Fig. 2A), that has been shown to function as a regulator of Cdc42 and Rac to control actin re-organization (Lamarche et al., 1998). Remarkably, activating domains from previously identi®ed Drosophila GAPs show rather weak identity/similarity scores to d-CdGAPr: the best score was observed with that of Rotund rac GAP (31/48%; Agnel et al., 1992). Outside the GTPase activating domain, a few proteins display signi®cant homologies with d-CdGAPr. The strongest homologies were found with mouse CdGAP downstream of the domain and with the Caenorhabditis elegans breakpoint cluster region protein (Bcr) upstream of it (Fig. 2B). No signi®cant homology was observed with any of the identi®ed Drosophila GAPs outside of the GTPase activating domain. 3. Embryonic expression pattern Whole-mount in situ hybridization revealed a dynamic spatio-temporal expression pattern of d-CdGAPr during embryogenesis. Important maternal contribution is visible in pre-blastoderm embryos, and vanishes by the end of the cellularisation process (Fig. 3A). At that stage, transcripts are exclusively found at the posterior pole, but not within the pole cells (Fig. 3B). Starting at early gastrulation, low level of ubiquitous zygotic expression occurs, while sites of preferential expression appear soon after. These sites include the cephalic furrow and neuro-ectoderm prior to neuroblast delamination (Fig. 3C,D) as well as scattered
cells within each segment of germ band extended embryos that most certainly correspond to PNS precursor cells (Fig. 3E). By the end of germ band retraction, d-CdGAPr transcription occurs at high rate in a row of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments, and in ventral cell clusters that presumably correspond to chordotonal organ precursors (Fig. 3F). Slightly later, more dorsally located cell clusters likely corresponding to chordotonal precursors also start to express the gene (Fig. 3G). By the end of embryogenesis, strong d-CdGAPr transcription occurs at the posterior end of the embryo in a ring of epidermal cells and in two laterally located cell clusters (Fig. 3H). The regulation of Drosophila GTPases Rap1 and Ras1 activities has been proposed to be controlled by temporarily and spatially restricted expression of GAPs (Chen et al., 1997). The dynamic expression of d-CdGAPr extends this observation.
4. Methods 4.1. Flies and in situ hybridization Oregon R was used as standard. The Pw 1 5 line has been generated by mobilization of a PlacW. Whole-mount in situ hybridization using 29G3 RNA probes was as in O'Neil and Bier (1994), except that embryos were ®rst ®xed in 4% formaldehyde and that xylene washes were omitted.
Fig. 1. (A) The d-CdGAPr locus. The site of P element insertion (Pw 1 5) is shown on the genomic restriction map (R: EcoRI; Sl: SalI; Sc: SacI; X: XbaI). l 4A9, l 94H6 and l 20G8 are three genomic recombinant phages isolated by probing a gridded EMBL3 Drosophila genomic library with Pw 1 5 plasmidrescued DNA. Filled bars in the intron/exon structure indicate the coding sequence of the 29G3 cDNA. B) Northern blot analysis. Poly (A1) RNA of 0±2 h embryos, third instar larvae, adult males and females were probed with the 7.5 kb 29G3 cDNA (upper panel) and with rp49 as a loading control. Sizes of the 2 d-CdGAPr transcripts are indicated on the left.
T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
269
Fig. 2. (A) Cladogram of d-CdGAPr and the closest mammalian, C. elegans and Drosophila GTPase activating domain sequences. Numbers in brackets indicate identity/similarity scores. (B) d-CdGAPr alignment with the mouse CdGAP and C. elegans Bcr sequences. Black and grey shadings indicate identical and similar amino acids, respectively. The GTPase activating domain is underlined.
4.2. Molecular biology Cloning, library screenings and Northern blot hybridization was performed according to standard procedures. Plasmid rescue following EcoRI digestion allowed to isolate a 3kb EcoRI fragment containing genomic DNA ¯anking the site of insertion. This DNA was used to screen a Drosophila genomic library (EMBL3, Stratagene) gridded in the laboratory. Three phages were recovered, and subsequently used to isolate several overlapping cDNAs from an embryonic cDNA library (Brown and Kafatos, 1988) gridded in the laboratory. Searches for homologies were performed using Blast programs and sequences alignment done using PIMA 1.4 (Infobiogen). The cladogram of the GTPase activating domains was generated with Treeview after ClustalW analysis. Accession numbers for sequences used in this study are the following: mouse CdGAP: AF151363; C. elegans Bcr: Z498888; human b2 chimerin: P52757; human AB033030: AB033030; human AC002398: AC002398; rat b2 chimerin: B53764; Drosophila Rotund: P40809; Drosophila Rlip: AF 037470; Drosophila genomic
sequence at 38A5-B4: AC0149777. The d-CdGAPr sequence has been deposited to GenBank under the accession number AF218776. Acknowledgements We thank Assia Bousteila for her contribution to the early part of this study. This work was supported by the CNRS and grants from ªl'Association pour la Recherche contre le Cancer (ARC)º and ªLa Ligue Nationale Contre le Cancer (LNCC)º. References Agnel, M., Roder, L., Vola, C., Grif®n-Shea, R., 1992. A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase activating (racGAP) proteins. Mol. Cell. Biol. 12, 5111±5122. Brown, N.H., Kafatos, F.C., 1988. Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425±437. Cavener, D.R., 1987. Comparison of the consensus sequence ¯anking trans-
270
T. Sagnier et al. / Mechanisms of Development 94 (2000) 267±270
Fig. 3. d-CdGAPr expression pattern during Drosophila embryonic development. All embryos are anterior to the left and ventral side down except for (D) and (H) which are ventral and dorsal views, respectively. (A) Pre-blastoderm embryo. (B) Cellular blastoderm. (C±D) Early germ band extended embryos. In (D) the bracket marks the neuro-ectoderm. (E) Late germ band extended embryo. The arrow points to cells that most certainly correspond to PNS precursors. (F±H) Germ band retracted embryos. Arrowheads in (F) point toward rows of epidermal cells in the most posterior part of thoracic and ®rst abdominal segments (also visible in G). Arrows point to group of cells that presumably correspond to chordotonal organ precursors. In (H), the arrow indicates a ring of epidermal cells at the posterior end of the embryo and arrowheads point to two strongly expressing cell clusters. lational start sites in Drosophila and vertebrates. Nucl. Acids. Res. 15, 1353±1361. Chen, F., Barkett, M., Ram, K.T., Quintanilla, A., Hariharan, I.K., 1997. Biological characterisation of Drosophila Rapgap1, a GTPase activating protein for Rap1. Proc. Natl. Acad. Sci. USA 94, 12485±12490.
Lamarche, N., Hall, A., 1998. CdGAP, a novel proline-rich GTPase activating protein for Cdc42 and Rac. J. Biol. Chem. 273, 29172± 29177. O'Neil, J.W., Bier, E., 1994. Double label in situ hybridisation using biotin and digoxigenin tagged RNA probed. Biotechniques 870, 874±875.