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The transduction signalling protein G0 during embryonic development of Drosophila melanogaster
CellularSignallingVol. 3 No. 4, pp. 341-352, 1991. Printedin Great Britain.
0898-6568/91 $3.00+,00 © 1991PergamonPressplc
T H E T R A N S D U C T I O N S I G N A L L I N G P R O T E I N G OD U R I N G E M B R Y O N I C D E V E L O P M E N T OF D R O S O P H I L A M E L A N O G A S T E R ALBERTO GUILLI~N, MICHEL SI~MI~RIVA,* JOEL BOCKAERT a n d VINCENT HOMBURGERt
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier Cedex 5, France and *Laboratoire de G6n6tique et Biologic Cellulaires, CNRS, 13288 Marseille Cedex 9, France (Received 22 February 1991; and accepted 10 March 1991)
A~traet--G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the • subunit of the mammalian Go protein. The Drosophila Go~ protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h of embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go~ protein was also examined in several neurogenic mutants. The Go~ protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.
Key words: Go protein, central nervous system, axonogenesis, Drosophila melanogaster embryonic development.
INTRODUCTION
cellular signalling systems including calcium channels [12-14], potassium channels [15] and phospholipase C [16]. A Go-like protein has been characterized in the nervous system of Drosophila melanogaster adults [17]. Moreover, two c D N A clones coding for an ~ subunit of a Go protein have been isolated from the fruit fly [18-20]. ln situ hybridization to Drosophila adults combined with immunohistochemical studies have revealed the presence of transcripts and Go protein throughout the optic lobes and central brain and in the thoracic and abdominal ganglia [21,22]. The G o protein present in the CNS of Drosophila adults appear to be involved in learning and memory mechanisms [17]. In order to gain a better understanding of the physiological role of the Go protein and as a preliminary approach, we undertook the biochemical characterization of a Go protein in D. melanogaster embryos and its localization
GUANINE nucleotide-binding regulatory proteins (G proteins) are a family of heterotrimeric proteins (0e//y) that couple receptors for extracellular signals to several intracellular effector systems [1-4]. In mammals, the Go protein, a member of this family that is a substrate for the pertussis toxin, is primarily expressed in the nervous system [5-7], a n d is concentrated in plasma membranes of cultured primary neurons, especially at regions of cell-cell contact [8]. The functional role of this protein remains as yet unclear, although it can interact with a number of cell surface receptors such as GABA B [9], muscarinic [10] and ~z-adrenergic receptors [11], and may affect a variety of intratAuthor to whom correspondence should be addressed at: Centre CNRS-INSERM de Pharmacologie-Endocrinologic, Rue de la Cardonille, 34094 Montpellier Cedex 5, France. Telephone: 33-67142936. Fax: 33-67542432. CELLS 3:4-F
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during embryonic development in the wild-type and some neurogenic mutants. The intermediate level of complexity of D. melanogaster and its amenability to genetic analysis make this organism particularly interesting for studying the possible role of the Go protein in the development of the nervous system. Some recent evidence suggests that G proteins and second messenger systems could be related to axonal growth and guidance. It has been described that the Go protein in mammals can be regulated by the intracellular growth cone protein GAP-43 [23], whose expression and localization correlate with axonal growth [24]. With respect to the axonal guidance and targeting, a genetic interaction has been shown in Drosophila between the fasciclin I gene encoding a neural cell adhesion molecule and the Abelson gene which encodes a tyrosine kinase [25]. It has also been proposed that neural cell adhesion molecules in vertebrates could regulate second messenger systems via pertussis toxin-sensitive G proteins [26]. D a t a presented in this report are discussed in relation to Drosophila CNS development and the possible role of Go in axonogenesis.
MATERIALS AND METHODS Drosophila stocks D. melanogaster adults and embryos were from the wild-type Canton-S and were raised at 25°C under standard laboratory conditions. Neurogenic mutant strains used in this study were kindly provided by Dr Campos-Ortega and were as follows: marez3 cn bw/Cyo, Df(3)DI FX3/TMI and Df(3)E(spl)/TM1 (R3251) [27]. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Preparation of membrane fractions and pertussis toxin-catalysed AD P-ribosylation
Membrane preparations from adult heads or embryos, dechorionated as described below, were obtained as previously reported [17]. ADP-ribosylation of membrane preparations with 0.4 #g pertussis toxin (List Biological Labs) and 2.8/tCi [ct-32P]NAD (sp. act. 800 Ci/mmol; New England Nuclear) for 90 min and electrophoretical separation of ADP-ribosylated substrates were performed according to Guill6n et al. [17] and Brabet et al. [8], respectively.
Two-dimensional electrophoretic analysis
Two-dimensional analysis of pertussis-toxin ADPribosylated substrates in membranes from embryos (50 #g of protein) consisted of isoelectrofocussing and SDS-PAGE in the first and second dimension, respectively. Isoelectric focussing was performed in accordance with Guill6n et al. [17]. Vertical isoelectrofocussing tube gels were laid onto the top of 1.5mm-thick 10% polyacrylamide slab gels to further separate proteins in the second dimension. Production of antisera and purification of antibodies
Antibodies against the peptide ANNLRGCGLY from the carboxy-terminal sequence of the Go, protein were used. The peptide was covalently linked to thyroglobulin according to Goldsmith et al. [29] and then injected in New Zealand White rabbits (HY 278, Elevage Scientifique des Dombes, France). Antibody purification was performed in accordance with Brabet et al. [8]. IgG fractions were precipitated with (NH4)2SO4 and antibodies were purified by affinity chromatography, firstly on Affi-gel l0 (Bio-Rad) linked to thyroglobulin and then on Affi-gel l0 linked to the ANNLRGCGLY peptide. Immunoprecipitation and immunoblotting
Immunoprecipitation of pertussis toxin-labelled substrates and immunoblotting experiments were carried out as previously described [17]. In immunoprecipitation assays, 20/~g of anti-peptide antibody were added to ultracentrifugation supernatant after membrane solubilization with 1% cholate. In order to assess the specificity of the immunoprecipitation, controls were performed in the presence of the anti-peptide antibody and a 10-fold excess of peptide or in the presence of 20 #g of purified IgG fraction from non-immune serum. In immunoblotting experiments, after electrotransfer onto nitrocellulose sheets of the membrane proteins previously separated by SDS-PAGE, blots were incubated overnight with 9 #g/ml of anti-peptide antibody. The antibody reaction was detected by incubation for 90 min with 250,000cpm/ml [~25I]Protein A (sp. act. 2-10 mCi/mg; New England Nuclear). Immunohistochemistry
After collection, embryos were dechorionated in 50% chlorox and rinsed with 0.1% Triton X-100. Vitelline membranes were removed as described by Mitchison and Sedat [30]. Embryos were blocked in PBS containing 0.1% Triton X-100 (PBST) and 10% heat-inactivated lamb serum (PBSTL) for 2 to 3 h at room temperature and then incubated overnight at
Go and embryonicdevelopmentin Drosophila 4°C with antibodies in PBSTL. The afffinity-purified anti-Go, antiserum was routinely used at 9/~g/ml and the primary antibody was detected as follows using Vectastain kits (Vector Labs). Embryos were washed four times for 15 min in PBSTL, then incubated for 1 h at room temperature with biotinylated goat antirabbit antibody in PBSTL. After four washes in PBST, embryos were incubated for 30 min with preformed avidin-horseradish peroxidase (HRP) complexes in PBST. Visualization of the HRP activity was as described by the manufacturer. In immunofluorescence experiments, fluorescently labelled anti-horseradish peroxidase antibodies (Cappel) were used at a 1/100 dilution in PBSTL.
Protein determination Protein concentration was determined according to the method of Lowry et al. [31], using BSA as standard. RESULTS
Characterization of a Go~ protein in embryos Particulate preparations obtained from D. melanogaster overnight (0-18h) embryos contained only one substrate ADP-ribosylated with pertussis toxin and [~-32p]NAD as observed after protein separation by SDS-PAGE (Fig. IA, lane 1). This labelled band, which did not correspond to a major protein as deduced from gels stained with Coomassie blue (data not shown), migrated with an apparent molecular weight of 39,000-40,000 and corresponded to the only ADP-ribosylated protein in membranes from D. melanogaster adult heads (Fig. 1A, lane 2). The same electrophoretical conditions allow the resolution of three pertussis toxin substrates (39,000, 40,000 and 41,000 molecular weight) in neuronal membranes from mammals [8,32,33]. We have previously demonstrated that the substrate at 39,000-40,000 molecular weight in heads of adult flies corresponds to the ~ subunit of a Go-like protein [17]. An affinity-purified polyclonal antibody raised against the carboxyterminal sequence (ANNLRGCGLY) of vertebrate [34] and fruit fly [18-20] Go, was tested by immunoblotting on membrane proteins from overnight embryos (Fig. 1B, lane 1) and from adult heads (Fig. I B, lane 2); the latter used as
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a control. In both preparations, the antipeptide antibody detected a single band at 39,000-40,000 molecular weight. In order to determine whether the pertussis toxin-labelled substrate in embryos corresponded to the protein detected by immunoblotting, immunoprecipitation experiments were carried out (Fig. 2). Membrane proteins from embryos or adult heads were ADP-ribosylated with pertussis toxin and [~-32p]NAD, and extracted in cholate. Embryo and adult head extracts resolved by SDS-PAGE are shown in Fig. 2A, lane 1 and Fig. 2B, lane 5, respectively. After incubation of the radiolabelled extracts with the anti-peptide antibody, a single band at 39,000-40,000 molecular weight was immunoprecipitated in both embryo and adult extracts (Fig. 2A, lane 2 and Fig. 2B, lane 6, respectively). In both cases, no band was immunoprecipitated when the antibody was preincubated with the cognate peptide (Fig. 2A, lane 3 and Fig. 2B, lane 7). In addition, a nonimmune antiserum failed to precipitate the radiolabelled protein in both extracts (Fig. 2A, lane 4 and Fig. 2B, lane 8). These results strongly suggest that the ADP-ribosylated substrate at 39,000-40,000 molecular weight in membranes from D. melanogaster embryos corresponds to a Go,-like protein.
Expression of the Go, protein at different stages of embryonic development We have studied the pertussis toxin-catalysed ADP-ribosylation of the Go, protein during embryonic development of D. melanogaster. Radioactive bands on SDS-PAGE gels were excised and the radioactivity was counted. The autoradiogram and the radioactivity levels for each band are shown in Fig. 3A. Only one substrate for the pertussis toxin at 39,000-40,000 molecular weight was present at all different stages of embryonic development. In 2.5-4.5h and 6-10h embryos (Fig. 3A, lanes 2 and 3, respectively), the ADP-ribosylation levels were higher than in 0-1.5 h embryos (Fig. 3A, lane 1). However, a significant increase in ADP-ribosylation of Go, occurred in
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10-16 h embryos (Fig. 3A, lane 4). No further increase was observed later in development and identical radioactivity levels were obtained in older embryos (16-22 h) (Fig. 3A, lane 5). Since the pertussis toxin-catalysed ADP-ribosylation of the Go, subunit requires the integrity of the ~fl~ heterotrimer [35], that increase could be related to an enhancement in either 0t or /3V subunits. The ~ subunit concentration of the Go-like protein was estimated at the same stages of embryonic development by immunoblot analysis using the antibody raised against the carboxy-terminal sequence of this subunit. After labelling the antigen-antibody complex with [125I]Protein A, the radioactive spots on the blot were counted as a measure of the protein amount. As shown in Fig. 3B, the anti-peptide antibody recognized only one protein at 39,000-40,000 molecular weight at all stages of embryonic development. The expression of this protein increased in the course of embryonic development and reached its maximal value after 10 h of embryogenesis (Fig. 3B, lanes 4 and 5). This augmentation paralleled the increase in ADP-ribosylation levels described above. In an attempt to reveal a possible heterogeneity of the embryonic Go~ protein in Drosophila, we performed an analysis by twodimensional electrophoresis of the ADP-ribosylated membrane proteins. The embryonic ADPribosylated Go~ protein contained only one species with an isoelectric pH (pHi) of approximately 5.4. This pattern did not change during development (data not shown). By contrast, using the same technique we were able to separate two Go~ isoforms in differentiated neuroblastoma N I E-115 cells [33]. Immunohistochemical localization of the Go, protein in developing embryos A study on the localization of the Go, protein during embryogenesis has been performed on whole mount embryos using the antipeptide antibody. Although some reproducible staining was observed around the entire gut, from stage 13 of embryogenesis the Go, protein was
mainly localized in the CNS (an early stage-14 embryo is shown in Fig. 4A). Comparison of the pattern of labelling obtained with anti-Go~ antibodies with that obtained with anti-horseradish peroxidase antibodies (anti-HRP antibodies), which are known to label all neural derivatives (Fig. 4B) [36, 37], revealed that Go~ protein expression was mainly restricted to the neuropil. Indeed, anti-HRP antibodies labelled both the axonal tracts (neu in Fig. 4B) and neuronal cell bodies on each side of the scaffold (cb in Fig. 4B) whereas anti-Go~ antibodies were labelling only the axonal scaffold (neu in Fig. 4A). This feature is also observed when comparing Fig. 4C and 4D with 4E. All the axon tracts of the CNS, longitudinal axons (la) and commissural axons (ca) in both anterior (ac) and posterior (pc) commissures, seemed to be stained with anti-Go~ antibodies (Fig. 4D). Intersegmental (isn) and segmental (sn) nerves of the PNS, escaping from the CNS, were also stained with anti-Go~ antibodies but to a lesser extent than axons in the CNS (best seen in Fig. 4C). Anti-HRP antibodies labelled nerves of the PNS (pns) more strongly than anti-Go~ antibodies (Fig. 4B and 4E). It should be noticed that, even after a careful examination, we have never found any staining associated with glial cells by using anti-Go~ antibodies. The immunolocalization of the Go~ protein was followed during embryogenesis. Before the blastoderm stage, embryos were stained uniformly, with no specific localization (data not shown). The presence of the Go~ protein at this stage probably came from the maternal expression of the gene, as already mentioned [21]. The first clear localization was visible at the blastoderm stage where the membranes of all blastoderm cells were labelled (Fig. 5A). The protein seemed to accumulate principally at the basal face of the blastoderm cells (Fig. 5A'). No staining of the pole cells (pc) was observed (Fig. 5A'). During gastrulation and germ band extension, the almost uniform labelling tended to become restricted to neurogenic regions within the germ band and also to regions that corresponded to the anterior and posterior midgut invaginations (data not shown). At
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FIG. 1. Presence of one substrate at 39,000--40,000 molecular weight in membrane preparations from adult heads and embryos ADP-ribosylated with pertussis toxin (A) and recognized by an anti-peptide antibody raised against the carboxyl-terminal sequence of Go~ (B). (A) ADP-ribosylation of embryo membranes (100/~g of protein) (lane 1) and adult head membranes 30/~g of protein) (lane 2). (B) Immunoblot analysis of membrane proteins (150/tg of protein) from embryos (lane 1) and adult heads (lane 2). Arrows indicate apparent mol. wt in multiples of 1000. Only the relevant part of both autoradiogram and blot is shown.
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FIG. 2. Immunoprecipitation of ADP-ribosylated substrates in membrane preparations (30/~g of protein) from embryos (A) and adult heads (B) using an anti-peptide antibody raised against the carboxy-terminal sequence of Go~. Lanes 1 and 5, ADP-ribosylation control; lanes 2 and 6, immunoprecipitation by use of the anti-peptide antibody; lanes 3 and 7, immunoprecipitation control by preineubation of the antibody with the cognate peptide; lanes 4 and 8, immunoprecipitation control using purified IgG fraction obtained from non-immune serum. Arrows indicate apparent mol. wt in multiples of 1000. Only the relevant portion of the autoradiogram is shown. 345
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FIG. 3. Expression of the Go~ protein in the course of embryogenesis studied by ADP-ribosylation of membrane preparations (100 pg of protein) (A) and by immunoblot analysis of membrane proteins (150/ag of protein) using an anti-peptide antibody raised against the carboxy-terminal sequence of Go~ (B). Embryos were collected at different stages of development: 0-1.5 h (1), 2.5-4.5 h (2), 6-10 h (3), 10-16 h (4) and 16-22 h (5). (A) Bars represent radioactivity values for [32p]ADP-ribosylated bands. The inset shows the relevant part of the autoradiogram. (B) Bars represent radioactivity values for [125I]Protein A. The inset shows the relevant part of the blot. The experiments were repeated twice with different membrane batches. The figure shows the results from a representative experiment carried out in duplicate, the variability being < 10%.
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FIG. 4. Expression of the Go~ protein in the embryonic CNS studied by immunohistochemistry. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Anterior is left and dorsal is up in (A) and (B). Anterior is up in (C), (D) and (E). Whole mounts of embryos were stained with either anti-peptide anti-Go~ antibodies in (A), (C) and (D) or anti-HRP antibodies in (B) and (E) (see Materials and Methods). (A) In an early stage-14 embryo (around 10.5 h), Go~ protein is mainly localized in the axons of the CNS (or neuropii, neu; sec, supraoesophageal commissures). (B) An early stage-14 embryo stained with anti-HRP antibodies for comparison with (A). Besides the neuropil, anti-HRP also labels all neural derivatives: the supraoesophageal ganglion (spg), the anal plates (an), the cell bodies of the CNS (cb) and the peripheral nervous system (pns), and also the Garland gland cells (ga). (C) CNS of the posterior region of a stage-15 embryo (12 h) showing the staining by the anti-Go~ antibody of the axons of the PNS, the intersegmentai nerve (isn) and the segmental nerve (sn) (la, longitudinal axons; ca, commissural axons of the CNS). (D) Three consecutive segmental neuromeres of a stage-14 embryo (11 h). At this level of resolution, all the axons seem to be labelled. The two peripheral nerves exiting the CNS are also clearly labelled (isn and sn) whereas the neuron cell bodies are not (ac, anterior commissures; pc, posterior commissures of the CNS). (E) CNS of three consecutive segments labelled with anti-HRP antibodies for comparison with (C). The axons of the PNS (isn and sn) are comparatively more stained than with anti-Go~ antibodies. Bars = 30/~m.
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FIG. 5. Immunolocalization of the Go~ protein during embryogenesis. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Anterior is left, and, in lateral views, dorsal is up. Whole mounts of embryos were stained with anti-peptide anti-Go~ antibodies (see Material and Methods). For (D), (E), (F) and 348
Go and embryonicdevelopmentin Drosophila stage 9, the membrane of the neuroblasts (nb) was labelled, but also, to a lesser extent, the mesodermal cells (ms). By contrast, the staining of ectodermal cells (ec) was hardly visible (Fig. 5B). At the beginning of germ band shortening (late stage 11), the region of the neuromers [38] began to be clearly labelled by anti-Go~ antibodies with patches in every future segment (Fig. 5C). From this time on, the labelling was quantitatively higher than at the previous stages and the essential of the staining was concentrated in the neuropil. Go~ protein was then detected in axons growing across the ventral midline (an early stage-13 embryo is shown in Fig. 5D). The pattern of the Go~ protein expression evolved coincident with the orthogonal array of fasciculating axons: the commissural axons were first labelled, then, during dorsal closure, longitudinal axons began to be detected (a late stage-13 embryo is shown in Fig. 5E). Note in this figure that commissurai axons (ca) were more strongly labelled than longitudinal axons (la). As the CNS condensed (stage 16), the thickness and intensity of the Go~ staining in the longitudinal fascicles were greater than in the commissural fascicles (Fig. 5F). Axons of the PNS were hardly visible in these planes of focus (Fig. 5E), but, as shown
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previously (Fig. 4C and 4D), they were definitely labelled. The specificity of the labelling was assessed by depleting the antiserum on Sepharose-linked peptide against which the anti-Go~ antibodies were raised (a stage-13 embryo is shown in Fig. 5G). The reaction was no longer observed with the depleted serum, at any developmental stage, suggesting that all the observed staining was due to the specific antibodies directed against the C-terminal sequence of the Go~ protein. Adsorption of the antiserum either on Sepharose-linked thyroglobulin or BSA did not modify its reactivity. The distribution of the Go~ protein was also studied in neurogenic mutants. In this group of mutants, the determination of epithelial cells is perturbed and the nervous system of the embryo is hypertrophied [27,39]. Three of the neurogenic mutants, Enhancer of split [E(spl)], mastermind (mare) and Delta (DO were examined for the expression of Go~. One example is given in Fig. 5H for the mam mutation, but the other mutations (data not shown) gave essentially the same results: Go~ remained localized in neurons. In neurogenic mutants, axons grow normally, but neurons are in greater quantities and the overall structure of the nervous system is disrupted. In these mutants, Go~ remained
(H), the plane of focus is at the level of the CNS. (A) Cellular blastoderm stage (stage 5, 2.75 h). The plane of focus is on the surface of the embryo. The Go~ protein is detected on the membrane of all the blastoderm cells. (A') Posterior pole of the same embryo as in (A), but at higher magnification and with a midsagittai plane of focus. The basal membrane of the blastoderm cells is heavily labelled (arrow) while little or no staining is seen in the lateral or apical membranes. The pole cells (pc) are not labelled. (B) High magnification of a stage-9 germ band extending embryo (around 4 h), showing the staining of the large neuroblasts (nb) and the mesodermal cells (ms). The labelling of ectodermal cells (ec) is hardly detectable. (C) A late stage-ll embryo (7 h). The region of the neuromers (n), which is distributed as segmentally repeated patches, begin to be clearly stained. (D) A ventral view of an early stage-13 embryo (shortened germ band; 9.5 h). Go~ protein accumulation is seen in the developing neuropil in the CNS. The newly appearing axons tracts (at) are labelled. No clear staining is observed in the cell bodies. (E) Ventral view of an early stage-14 embryo (10.5 h). The Go~ protein is primarily detected on the early commissural axons (ca). Longitudinal tracts (la) have begun to form and are also labelled. A weak staining of the axons of the PNS (pns) can also be observed. (F) Lateral view of a stage-16 embryo (around 14 h). The ventral nerve cord is beginning to condense. The longitudinal axon bundles are thicker than the commissural axons (neu: neuropil; sec: supraoesophageal commissures). A labelling around the entire gut persists. (G) Lateral view of a stage-13 embryo (10h), before dorsal closure, stained with the anti-Go~ antiserum depleted of its specific antibodies. The complete absence of reactivity indicates the specificity of the antiserum. (H) A view of a mam mutant embryo stained with anti-Go~ antibodies. The nervous system is hypertrophied. The axon tracts (at) are strongly labelled. No staining is observed in the neuron cell bodies, as it is also the case in wild type embryos. Bars = 30/~m.
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specifically localized in the growing axons of the CNS. Cell bodies were not labelled. This result suggests that the specific expression of the protein in the CNS and its preferential localization in axons are not affected by the neurogenic mutations. DISCUSSION Particulate preparations from D. melanogaster embryos contained only one substrate of 39,000-40,000 molecular weight that could be ADP-ribosylated by pertussis toxin. The same substrate was also present in membranes from adult heads and we have previously demonstrated that it corresponded to a Go~-like protein using a polyclonal antibody directed against the ct subunit of the Go protein purified from bovine brain [17]. An anti-peptide antibody raised against the carboxy-terminal sequence of the ct subunit of the mammalian G O protein was able to cross-react, as shown by Western blot analysis, with a 39,000-40,000 molecular weight membrane protein in Drosophila embryos. The peptide sequence, which is specific for the ~ subunit of the G Oprotein but not for the g subunits of other G proteins [34], is conserved in the two putative Go~ proteins of D. melanogaster [18-20]. This antibody was also able specifically to immunoprecipitate the pertussis toxin ADP-ribosylated substrate in embryos. The fact that in Drosophila there is only one G protein substrate for the pertussis toxin will facilitate further investigation on the functional role of the Go protein. Immunoblotting experiments have shown that the expression of the ~ subunit of the Go protein markedly increased after 10h of embryonic development. This increase was closely correlated to an increase in ADP-ribosylation levels. This result implies that the ~ subunit could interact with fly and was fully functional at all embryonic stages [35]. In differentiated neuroblastoma N1E-I 15 cells, we have previously described the existence of two isoforms for the Go~ protein, which could be separated by their isoelectrical mobility (pH~ = 5.55 and 5.80) [33]. In Droso-
phila embryos, by using the same two-dimensional gel analysis, only one form of the ADPribosylated Go~ protein was detected at all stages of embryonic development. The isoelectric pH of this isoform was similar to that of the only form detected in Drosophila adults [17]. It should be pointed out that the two putative Go~ proteins deduced from the sequence of the two different cDNAs found in Drosophila only differ in their N-terminus [18-20] without any apparent change in their global electric charge or their molecular weight. If these two forms are really expressed during embryogenesis they could not have been resolved by the type of analysis described herein. Note that the two splice variants of Drosophila may not be equivalent to the two splice variants of the Go~ protein found in vertebrates, in which splicing involves the C-terminal third of the molecule [40]. Studies on protein localization showed that the Go~ protein was already expressed at low levels during early embryogenesis, before the onset of axonogenesis, and could therefore play a specific role in these earlier stages. The presence of the protein before and at the blastoderm stage could result from the maternal gene expression described by Schmidt et al. [21], whereas its preferential accumulation in neurogenic regions at later stages could derive from the zygotic expression, implying a specific control of gene expression in nervous territories. However, it is not likely that Go~ participates in the decision that determines the fate of neuronal cells since the specific expression of Go~ in neurogenic regions was subsequent to this primary process. Go~ expression was preserved in the neurogenic mutants E(spl), roam and DI, with the same specific pattern as in wild type embryos. This result implies that, at least in the case of the three mutants studied, the neurogenic genes do not control the spatiotemporal expression of the Go~ protein. Immunolocalization studies in Drosophila embryos also showed that Go~ is mainly localized in the axons of the CNS. Results agree with those described by Schmidt et al. [21] and Wolfgang et al. [22] in Drosophila adults, in which immunoreactivity was observed in the
GOand embryonicdevelopmentin Drosophila neuropil, and little, if any, in neuronal cell bodies. The preferential localization of the Go~ protein in growing axons in embryos suggests that it could play a direct role in axonogenesis. The onset of Go~ protein accumulation, as observed by immunoblotting, coincided with the beginning of axonogenesis (10 h) with no further increase during CNS condensation (16-22 h) and the distribution of the protein, as observed by immunochemistry, evolved concomitantly with the building of the axonal scaffold. The presence of Go~ in all the axons would be compatible with the general role assigned to second messenger systems such as calcium and protein kinase C in neurite outgrowth and axonogenesis [41]. Indeed, as already stated, Go~ seems to be implicated in the control of calcium channels [12-14] and phospholipase C [16] activities mammalian cells. It has been suggested that not only second messenger systems but also cytoskeletal elements are critical in axonal outgrowth [41]. In this context, we have previously shown in mammalian neurons that, in addition to a membrane localization, Go~ can be associated with cytoskeleton proteins such as microtubules in centriolar structures or is localized in areas rich in actin microfilaments [42]. It is interesting to compare the fact that Go~ is expressed during the period of intense axonogenesis in Drosophila with our previous results on the expression of Go~ isoforms during differentiation of NIE-I15 neuroblastoma. In these cells, differentiation is morphologically characterized by inhibition of cell growth and extension of long neurites and is accompanied by a specific increase in global Go~ level (but not in the amount of other G~ subunits) [33]. Other evidence also permits us to hypothesize that G proteins, and particularly Go, could be implicated in axonal growth and guidance. Thus, it has recently been noticed in mammals that G o, which is a major component in the growth cone membrane, can be regulated by GAP-43 [23], an axonal growth-associated protein [24]. There is, at the moment, only one indication concerning the eventual existence of a GAP-43 protein in D. melanogaster [43]. However, the
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homology between the vertebrate GAP-43 and this protein only relies on a conserved sequence of about 20 aminoacids with no other similarities in the rest of the molecule. Furthermore, unlike the GAP-43 protein, it is not expressed in growing axons. On the other hand, the possible participation of G proteins in axonal guidance is suggested by the fact that pertussis toxin blocks in vertebrates the effect of neural cell adhesion molecules on the modulation of second messenger systems [26]. Although all these data are compatible with a role of Go~ in neurite outgrowth both in mammalian and Drosophila neurons, we have to wait for the isolation of mutants in the gene coding for the ~ subunit of the G o protein to assign a definite role to G O protein in axonogenesis. However, several P-element insertions in 47A [44], within the region where the Go, gene has been localized, have recently been described in D. melanogaster [18-20]. The expression in all neurons of the Lac Z reporter gene carried by the P-element indicated the importance of the 47A region for the expression of neuronal proteins. Six of these insertions are lethal and lead to an abnormal development of the CNS [44]. Whether or not one of them is in the Go~ gene remains to be determined. Acknowledgements--The authors wish to thank Dr D. GRATECOSand Dr M. PIOVANTfor constructive
discussions. Dr J. A. FEHRENTZand Dr B. ROUOT are gratefully acknowledged for peptide synthesizing and antiserum preparing, respectively. Miss M. ASTIER is acknowledged for her excellent technical assistance. The assistance of Mrs M. PASSAMA,Mr P. WEBERand Mr L. CHARVETin preparing the manuscript is also greatly appreciated. The Spanish Ministry of Education and Science and the European Molecular Biology Organization are acknowledged for providing postdoctoral fellowships to A. GUILLI~Nfor two different periods of this work.
1. 2. 3. 4.
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25. Elkins T., Zinn K., McAllister L., Hoffmann F . M . and Goodman C.S. (1990) Cell 60, 565-575. 26. Schuch U., M. J. Lohse and M. Schner (1989) Neuron 3, 13-20. 27. Lehmann R., Jimenez F., Dietrich U. and Campos-Ortega J.A. (1983) Wilhelm Roux's Arch. Dev. Biol. 192, 62-74. 28. Campos-Ortega J. A. and Hartenstein V. (1985) In The Embryonic Development o f Drosophila melanogaster. Springer, Berlin. 29. Goldsmith P., Gierschik P., Milligan G., Unson C. G., Vinitsky R., Malech H. L. and Spiegel A. M. (1987) J. biol. Chem. 262, 14,683-14,688. 30. Mitchison T. and Sedat J. (1983) Dev. Biol. 99, 261-264. 31. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) J. biol. Chem. 193, 265275. 32. Brabet P., Pantaloni C., Rouot B., Toutant M., Garcia-Sainz A., Bockaert J. and Homburger V. (1988) Biochem. Biophys. Res. Commun. 152, 1185-1192. 33. Brabet P., Pantaloni C., Rodriguez M., Martinez J., Bockaert J. and Homburger V. (1990) J. Neurochem. 54, 1310-1320. 34. Jones D. T. and Reed R. R. (1987) J. biol. Chem. 262, 14,241-14,249. 35. Mattera R., Codina J., Sekura R. D. and Birnbaumer L. (1987) J. biol. Chem. 262, 11,247-11,251. 36. Jan L. Y. and Jan Y. N. (1982) Proc. natn. Acad. Sci. U.S.A. 72, 2700-2704. 37. Snow P. M., Patel N. H., Harrelson A. L. and Goodman C.S. (1987) J. Neurosci. 7, 4137-4144. 38. Canal I. and Ferrus A. (1986) J. Neurogenet. 3, 293-319. 39. Lehmann R., Dietrich U., Jimenez F. and Campos-Ortega J.A. (1981) Wilhelm Roux's Arch. Dev. Biol. 190, 226-229. 40. Hsu W. H., Rudolph U., Sanford J., Bertrand P., Olate J., Nelson C., Moss L. G., Boyd A. E., Codina J. and Birnbaumer L. (1990) J. biol. Chem. 265, 11,220-11,226. 41. Mattson M. P. (1988) Brain Res. Rev. 13, 179-212. 42. Gabrion J., Brabet P., Nguyen Than Dao B., Homburger V., Dumuis A., Sebben M., Rouot B. and Bockaert J. (1989) Cell. Signal. 1, 107-123. 43. Ng S. C., Perkins L. A., Conboy, G., Perrimon N. and Fishman M. C. (1989) Development 105, 629-638. 44. Bier E., Vaessin H., Shepherd S., Lee K., McCall K., Barbel S., Ackerman L., Carretto R., Uemura T., Grell E., Jan L. Y. and Jan Y. N. (1989) Genes Dev. 3, 1273-1287.
0898-6568/91 $3.00+,00 © 1991PergamonPressplc
T H E T R A N S D U C T I O N S I G N A L L I N G P R O T E I N G OD U R I N G E M B R Y O N I C D E V E L O P M E N T OF D R O S O P H I L A M E L A N O G A S T E R ALBERTO GUILLI~N, MICHEL SI~MI~RIVA,* JOEL BOCKAERT a n d VINCENT HOMBURGERt
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier Cedex 5, France and *Laboratoire de G6n6tique et Biologic Cellulaires, CNRS, 13288 Marseille Cedex 9, France (Received 22 February 1991; and accepted 10 March 1991)
A~traet--G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the • subunit of the mammalian Go protein. The Drosophila Go~ protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h of embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go~ protein was also examined in several neurogenic mutants. The Go~ protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.
Key words: Go protein, central nervous system, axonogenesis, Drosophila melanogaster embryonic development.
INTRODUCTION
cellular signalling systems including calcium channels [12-14], potassium channels [15] and phospholipase C [16]. A Go-like protein has been characterized in the nervous system of Drosophila melanogaster adults [17]. Moreover, two c D N A clones coding for an ~ subunit of a Go protein have been isolated from the fruit fly [18-20]. ln situ hybridization to Drosophila adults combined with immunohistochemical studies have revealed the presence of transcripts and Go protein throughout the optic lobes and central brain and in the thoracic and abdominal ganglia [21,22]. The G o protein present in the CNS of Drosophila adults appear to be involved in learning and memory mechanisms [17]. In order to gain a better understanding of the physiological role of the Go protein and as a preliminary approach, we undertook the biochemical characterization of a Go protein in D. melanogaster embryos and its localization
GUANINE nucleotide-binding regulatory proteins (G proteins) are a family of heterotrimeric proteins (0e//y) that couple receptors for extracellular signals to several intracellular effector systems [1-4]. In mammals, the Go protein, a member of this family that is a substrate for the pertussis toxin, is primarily expressed in the nervous system [5-7], a n d is concentrated in plasma membranes of cultured primary neurons, especially at regions of cell-cell contact [8]. The functional role of this protein remains as yet unclear, although it can interact with a number of cell surface receptors such as GABA B [9], muscarinic [10] and ~z-adrenergic receptors [11], and may affect a variety of intratAuthor to whom correspondence should be addressed at: Centre CNRS-INSERM de Pharmacologie-Endocrinologic, Rue de la Cardonille, 34094 Montpellier Cedex 5, France. Telephone: 33-67142936. Fax: 33-67542432. CELLS 3:4-F
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during embryonic development in the wild-type and some neurogenic mutants. The intermediate level of complexity of D. melanogaster and its amenability to genetic analysis make this organism particularly interesting for studying the possible role of the Go protein in the development of the nervous system. Some recent evidence suggests that G proteins and second messenger systems could be related to axonal growth and guidance. It has been described that the Go protein in mammals can be regulated by the intracellular growth cone protein GAP-43 [23], whose expression and localization correlate with axonal growth [24]. With respect to the axonal guidance and targeting, a genetic interaction has been shown in Drosophila between the fasciclin I gene encoding a neural cell adhesion molecule and the Abelson gene which encodes a tyrosine kinase [25]. It has also been proposed that neural cell adhesion molecules in vertebrates could regulate second messenger systems via pertussis toxin-sensitive G proteins [26]. D a t a presented in this report are discussed in relation to Drosophila CNS development and the possible role of Go in axonogenesis.
MATERIALS AND METHODS Drosophila stocks D. melanogaster adults and embryos were from the wild-type Canton-S and were raised at 25°C under standard laboratory conditions. Neurogenic mutant strains used in this study were kindly provided by Dr Campos-Ortega and were as follows: marez3 cn bw/Cyo, Df(3)DI FX3/TMI and Df(3)E(spl)/TM1 (R3251) [27]. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Preparation of membrane fractions and pertussis toxin-catalysed AD P-ribosylation
Membrane preparations from adult heads or embryos, dechorionated as described below, were obtained as previously reported [17]. ADP-ribosylation of membrane preparations with 0.4 #g pertussis toxin (List Biological Labs) and 2.8/tCi [ct-32P]NAD (sp. act. 800 Ci/mmol; New England Nuclear) for 90 min and electrophoretical separation of ADP-ribosylated substrates were performed according to Guill6n et al. [17] and Brabet et al. [8], respectively.
Two-dimensional electrophoretic analysis
Two-dimensional analysis of pertussis-toxin ADPribosylated substrates in membranes from embryos (50 #g of protein) consisted of isoelectrofocussing and SDS-PAGE in the first and second dimension, respectively. Isoelectric focussing was performed in accordance with Guill6n et al. [17]. Vertical isoelectrofocussing tube gels were laid onto the top of 1.5mm-thick 10% polyacrylamide slab gels to further separate proteins in the second dimension. Production of antisera and purification of antibodies
Antibodies against the peptide ANNLRGCGLY from the carboxy-terminal sequence of the Go, protein were used. The peptide was covalently linked to thyroglobulin according to Goldsmith et al. [29] and then injected in New Zealand White rabbits (HY 278, Elevage Scientifique des Dombes, France). Antibody purification was performed in accordance with Brabet et al. [8]. IgG fractions were precipitated with (NH4)2SO4 and antibodies were purified by affinity chromatography, firstly on Affi-gel l0 (Bio-Rad) linked to thyroglobulin and then on Affi-gel l0 linked to the ANNLRGCGLY peptide. Immunoprecipitation and immunoblotting
Immunoprecipitation of pertussis toxin-labelled substrates and immunoblotting experiments were carried out as previously described [17]. In immunoprecipitation assays, 20/~g of anti-peptide antibody were added to ultracentrifugation supernatant after membrane solubilization with 1% cholate. In order to assess the specificity of the immunoprecipitation, controls were performed in the presence of the anti-peptide antibody and a 10-fold excess of peptide or in the presence of 20 #g of purified IgG fraction from non-immune serum. In immunoblotting experiments, after electrotransfer onto nitrocellulose sheets of the membrane proteins previously separated by SDS-PAGE, blots were incubated overnight with 9 #g/ml of anti-peptide antibody. The antibody reaction was detected by incubation for 90 min with 250,000cpm/ml [~25I]Protein A (sp. act. 2-10 mCi/mg; New England Nuclear). Immunohistochemistry
After collection, embryos were dechorionated in 50% chlorox and rinsed with 0.1% Triton X-100. Vitelline membranes were removed as described by Mitchison and Sedat [30]. Embryos were blocked in PBS containing 0.1% Triton X-100 (PBST) and 10% heat-inactivated lamb serum (PBSTL) for 2 to 3 h at room temperature and then incubated overnight at
Go and embryonicdevelopmentin Drosophila 4°C with antibodies in PBSTL. The afffinity-purified anti-Go, antiserum was routinely used at 9/~g/ml and the primary antibody was detected as follows using Vectastain kits (Vector Labs). Embryos were washed four times for 15 min in PBSTL, then incubated for 1 h at room temperature with biotinylated goat antirabbit antibody in PBSTL. After four washes in PBST, embryos were incubated for 30 min with preformed avidin-horseradish peroxidase (HRP) complexes in PBST. Visualization of the HRP activity was as described by the manufacturer. In immunofluorescence experiments, fluorescently labelled anti-horseradish peroxidase antibodies (Cappel) were used at a 1/100 dilution in PBSTL.
Protein determination Protein concentration was determined according to the method of Lowry et al. [31], using BSA as standard. RESULTS
Characterization of a Go~ protein in embryos Particulate preparations obtained from D. melanogaster overnight (0-18h) embryos contained only one substrate ADP-ribosylated with pertussis toxin and [~-32p]NAD as observed after protein separation by SDS-PAGE (Fig. IA, lane 1). This labelled band, which did not correspond to a major protein as deduced from gels stained with Coomassie blue (data not shown), migrated with an apparent molecular weight of 39,000-40,000 and corresponded to the only ADP-ribosylated protein in membranes from D. melanogaster adult heads (Fig. 1A, lane 2). The same electrophoretical conditions allow the resolution of three pertussis toxin substrates (39,000, 40,000 and 41,000 molecular weight) in neuronal membranes from mammals [8,32,33]. We have previously demonstrated that the substrate at 39,000-40,000 molecular weight in heads of adult flies corresponds to the ~ subunit of a Go-like protein [17]. An affinity-purified polyclonal antibody raised against the carboxyterminal sequence (ANNLRGCGLY) of vertebrate [34] and fruit fly [18-20] Go, was tested by immunoblotting on membrane proteins from overnight embryos (Fig. 1B, lane 1) and from adult heads (Fig. I B, lane 2); the latter used as
343
a control. In both preparations, the antipeptide antibody detected a single band at 39,000-40,000 molecular weight. In order to determine whether the pertussis toxin-labelled substrate in embryos corresponded to the protein detected by immunoblotting, immunoprecipitation experiments were carried out (Fig. 2). Membrane proteins from embryos or adult heads were ADP-ribosylated with pertussis toxin and [~-32p]NAD, and extracted in cholate. Embryo and adult head extracts resolved by SDS-PAGE are shown in Fig. 2A, lane 1 and Fig. 2B, lane 5, respectively. After incubation of the radiolabelled extracts with the anti-peptide antibody, a single band at 39,000-40,000 molecular weight was immunoprecipitated in both embryo and adult extracts (Fig. 2A, lane 2 and Fig. 2B, lane 6, respectively). In both cases, no band was immunoprecipitated when the antibody was preincubated with the cognate peptide (Fig. 2A, lane 3 and Fig. 2B, lane 7). In addition, a nonimmune antiserum failed to precipitate the radiolabelled protein in both extracts (Fig. 2A, lane 4 and Fig. 2B, lane 8). These results strongly suggest that the ADP-ribosylated substrate at 39,000-40,000 molecular weight in membranes from D. melanogaster embryos corresponds to a Go,-like protein.
Expression of the Go, protein at different stages of embryonic development We have studied the pertussis toxin-catalysed ADP-ribosylation of the Go, protein during embryonic development of D. melanogaster. Radioactive bands on SDS-PAGE gels were excised and the radioactivity was counted. The autoradiogram and the radioactivity levels for each band are shown in Fig. 3A. Only one substrate for the pertussis toxin at 39,000-40,000 molecular weight was present at all different stages of embryonic development. In 2.5-4.5h and 6-10h embryos (Fig. 3A, lanes 2 and 3, respectively), the ADP-ribosylation levels were higher than in 0-1.5 h embryos (Fig. 3A, lane 1). However, a significant increase in ADP-ribosylation of Go, occurred in
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10-16 h embryos (Fig. 3A, lane 4). No further increase was observed later in development and identical radioactivity levels were obtained in older embryos (16-22 h) (Fig. 3A, lane 5). Since the pertussis toxin-catalysed ADP-ribosylation of the Go, subunit requires the integrity of the ~fl~ heterotrimer [35], that increase could be related to an enhancement in either 0t or /3V subunits. The ~ subunit concentration of the Go-like protein was estimated at the same stages of embryonic development by immunoblot analysis using the antibody raised against the carboxy-terminal sequence of this subunit. After labelling the antigen-antibody complex with [125I]Protein A, the radioactive spots on the blot were counted as a measure of the protein amount. As shown in Fig. 3B, the anti-peptide antibody recognized only one protein at 39,000-40,000 molecular weight at all stages of embryonic development. The expression of this protein increased in the course of embryonic development and reached its maximal value after 10 h of embryogenesis (Fig. 3B, lanes 4 and 5). This augmentation paralleled the increase in ADP-ribosylation levels described above. In an attempt to reveal a possible heterogeneity of the embryonic Go~ protein in Drosophila, we performed an analysis by twodimensional electrophoresis of the ADP-ribosylated membrane proteins. The embryonic ADPribosylated Go~ protein contained only one species with an isoelectric pH (pHi) of approximately 5.4. This pattern did not change during development (data not shown). By contrast, using the same technique we were able to separate two Go~ isoforms in differentiated neuroblastoma N I E-115 cells [33]. Immunohistochemical localization of the Go, protein in developing embryos A study on the localization of the Go, protein during embryogenesis has been performed on whole mount embryos using the antipeptide antibody. Although some reproducible staining was observed around the entire gut, from stage 13 of embryogenesis the Go, protein was
mainly localized in the CNS (an early stage-14 embryo is shown in Fig. 4A). Comparison of the pattern of labelling obtained with anti-Go~ antibodies with that obtained with anti-horseradish peroxidase antibodies (anti-HRP antibodies), which are known to label all neural derivatives (Fig. 4B) [36, 37], revealed that Go~ protein expression was mainly restricted to the neuropil. Indeed, anti-HRP antibodies labelled both the axonal tracts (neu in Fig. 4B) and neuronal cell bodies on each side of the scaffold (cb in Fig. 4B) whereas anti-Go~ antibodies were labelling only the axonal scaffold (neu in Fig. 4A). This feature is also observed when comparing Fig. 4C and 4D with 4E. All the axon tracts of the CNS, longitudinal axons (la) and commissural axons (ca) in both anterior (ac) and posterior (pc) commissures, seemed to be stained with anti-Go~ antibodies (Fig. 4D). Intersegmental (isn) and segmental (sn) nerves of the PNS, escaping from the CNS, were also stained with anti-Go~ antibodies but to a lesser extent than axons in the CNS (best seen in Fig. 4C). Anti-HRP antibodies labelled nerves of the PNS (pns) more strongly than anti-Go~ antibodies (Fig. 4B and 4E). It should be noticed that, even after a careful examination, we have never found any staining associated with glial cells by using anti-Go~ antibodies. The immunolocalization of the Go~ protein was followed during embryogenesis. Before the blastoderm stage, embryos were stained uniformly, with no specific localization (data not shown). The presence of the Go~ protein at this stage probably came from the maternal expression of the gene, as already mentioned [21]. The first clear localization was visible at the blastoderm stage where the membranes of all blastoderm cells were labelled (Fig. 5A). The protein seemed to accumulate principally at the basal face of the blastoderm cells (Fig. 5A'). No staining of the pole cells (pc) was observed (Fig. 5A'). During gastrulation and germ band extension, the almost uniform labelling tended to become restricted to neurogenic regions within the germ band and also to regions that corresponded to the anterior and posterior midgut invaginations (data not shown). At
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FIG. 1. Presence of one substrate at 39,000--40,000 molecular weight in membrane preparations from adult heads and embryos ADP-ribosylated with pertussis toxin (A) and recognized by an anti-peptide antibody raised against the carboxyl-terminal sequence of Go~ (B). (A) ADP-ribosylation of embryo membranes (100/~g of protein) (lane 1) and adult head membranes 30/~g of protein) (lane 2). (B) Immunoblot analysis of membrane proteins (150/tg of protein) from embryos (lane 1) and adult heads (lane 2). Arrows indicate apparent mol. wt in multiples of 1000. Only the relevant part of both autoradiogram and blot is shown.
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FIG. 2. Immunoprecipitation of ADP-ribosylated substrates in membrane preparations (30/~g of protein) from embryos (A) and adult heads (B) using an anti-peptide antibody raised against the carboxy-terminal sequence of Go~. Lanes 1 and 5, ADP-ribosylation control; lanes 2 and 6, immunoprecipitation by use of the anti-peptide antibody; lanes 3 and 7, immunoprecipitation control by preineubation of the antibody with the cognate peptide; lanes 4 and 8, immunoprecipitation control using purified IgG fraction obtained from non-immune serum. Arrows indicate apparent mol. wt in multiples of 1000. Only the relevant portion of the autoradiogram is shown. 345
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FIG. 3. Expression of the Go~ protein in the course of embryogenesis studied by ADP-ribosylation of membrane preparations (100 pg of protein) (A) and by immunoblot analysis of membrane proteins (150/ag of protein) using an anti-peptide antibody raised against the carboxy-terminal sequence of Go~ (B). Embryos were collected at different stages of development: 0-1.5 h (1), 2.5-4.5 h (2), 6-10 h (3), 10-16 h (4) and 16-22 h (5). (A) Bars represent radioactivity values for [32p]ADP-ribosylated bands. The inset shows the relevant part of the autoradiogram. (B) Bars represent radioactivity values for [125I]Protein A. The inset shows the relevant part of the blot. The experiments were repeated twice with different membrane batches. The figure shows the results from a representative experiment carried out in duplicate, the variability being < 10%.
346
A
FIG. 4. Expression of the Go~ protein in the embryonic CNS studied by immunohistochemistry. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Anterior is left and dorsal is up in (A) and (B). Anterior is up in (C), (D) and (E). Whole mounts of embryos were stained with either anti-peptide anti-Go~ antibodies in (A), (C) and (D) or anti-HRP antibodies in (B) and (E) (see Materials and Methods). (A) In an early stage-14 embryo (around 10.5 h), Go~ protein is mainly localized in the axons of the CNS (or neuropii, neu; sec, supraoesophageal commissures). (B) An early stage-14 embryo stained with anti-HRP antibodies for comparison with (A). Besides the neuropil, anti-HRP also labels all neural derivatives: the supraoesophageal ganglion (spg), the anal plates (an), the cell bodies of the CNS (cb) and the peripheral nervous system (pns), and also the Garland gland cells (ga). (C) CNS of the posterior region of a stage-15 embryo (12 h) showing the staining by the anti-Go~ antibody of the axons of the PNS, the intersegmentai nerve (isn) and the segmental nerve (sn) (la, longitudinal axons; ca, commissural axons of the CNS). (D) Three consecutive segmental neuromeres of a stage-14 embryo (11 h). At this level of resolution, all the axons seem to be labelled. The two peripheral nerves exiting the CNS are also clearly labelled (isn and sn) whereas the neuron cell bodies are not (ac, anterior commissures; pc, posterior commissures of the CNS). (E) CNS of three consecutive segments labelled with anti-HRP antibodies for comparison with (C). The axons of the PNS (isn and sn) are comparatively more stained than with anti-Go~ antibodies. Bars = 30/~m.
347
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FIG. 5. Immunolocalization of the Go~ protein during embryogenesis. Embryonic stages are defined according to Campos-Ortega and Hartenstein [28]. Anterior is left, and, in lateral views, dorsal is up. Whole mounts of embryos were stained with anti-peptide anti-Go~ antibodies (see Material and Methods). For (D), (E), (F) and 348
Go and embryonicdevelopmentin Drosophila stage 9, the membrane of the neuroblasts (nb) was labelled, but also, to a lesser extent, the mesodermal cells (ms). By contrast, the staining of ectodermal cells (ec) was hardly visible (Fig. 5B). At the beginning of germ band shortening (late stage 11), the region of the neuromers [38] began to be clearly labelled by anti-Go~ antibodies with patches in every future segment (Fig. 5C). From this time on, the labelling was quantitatively higher than at the previous stages and the essential of the staining was concentrated in the neuropil. Go~ protein was then detected in axons growing across the ventral midline (an early stage-13 embryo is shown in Fig. 5D). The pattern of the Go~ protein expression evolved coincident with the orthogonal array of fasciculating axons: the commissural axons were first labelled, then, during dorsal closure, longitudinal axons began to be detected (a late stage-13 embryo is shown in Fig. 5E). Note in this figure that commissurai axons (ca) were more strongly labelled than longitudinal axons (la). As the CNS condensed (stage 16), the thickness and intensity of the Go~ staining in the longitudinal fascicles were greater than in the commissural fascicles (Fig. 5F). Axons of the PNS were hardly visible in these planes of focus (Fig. 5E), but, as shown
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previously (Fig. 4C and 4D), they were definitely labelled. The specificity of the labelling was assessed by depleting the antiserum on Sepharose-linked peptide against which the anti-Go~ antibodies were raised (a stage-13 embryo is shown in Fig. 5G). The reaction was no longer observed with the depleted serum, at any developmental stage, suggesting that all the observed staining was due to the specific antibodies directed against the C-terminal sequence of the Go~ protein. Adsorption of the antiserum either on Sepharose-linked thyroglobulin or BSA did not modify its reactivity. The distribution of the Go~ protein was also studied in neurogenic mutants. In this group of mutants, the determination of epithelial cells is perturbed and the nervous system of the embryo is hypertrophied [27,39]. Three of the neurogenic mutants, Enhancer of split [E(spl)], mastermind (mare) and Delta (DO were examined for the expression of Go~. One example is given in Fig. 5H for the mam mutation, but the other mutations (data not shown) gave essentially the same results: Go~ remained localized in neurons. In neurogenic mutants, axons grow normally, but neurons are in greater quantities and the overall structure of the nervous system is disrupted. In these mutants, Go~ remained
(H), the plane of focus is at the level of the CNS. (A) Cellular blastoderm stage (stage 5, 2.75 h). The plane of focus is on the surface of the embryo. The Go~ protein is detected on the membrane of all the blastoderm cells. (A') Posterior pole of the same embryo as in (A), but at higher magnification and with a midsagittai plane of focus. The basal membrane of the blastoderm cells is heavily labelled (arrow) while little or no staining is seen in the lateral or apical membranes. The pole cells (pc) are not labelled. (B) High magnification of a stage-9 germ band extending embryo (around 4 h), showing the staining of the large neuroblasts (nb) and the mesodermal cells (ms). The labelling of ectodermal cells (ec) is hardly detectable. (C) A late stage-ll embryo (7 h). The region of the neuromers (n), which is distributed as segmentally repeated patches, begin to be clearly stained. (D) A ventral view of an early stage-13 embryo (shortened germ band; 9.5 h). Go~ protein accumulation is seen in the developing neuropil in the CNS. The newly appearing axons tracts (at) are labelled. No clear staining is observed in the cell bodies. (E) Ventral view of an early stage-14 embryo (10.5 h). The Go~ protein is primarily detected on the early commissural axons (ca). Longitudinal tracts (la) have begun to form and are also labelled. A weak staining of the axons of the PNS (pns) can also be observed. (F) Lateral view of a stage-16 embryo (around 14 h). The ventral nerve cord is beginning to condense. The longitudinal axon bundles are thicker than the commissural axons (neu: neuropil; sec: supraoesophageal commissures). A labelling around the entire gut persists. (G) Lateral view of a stage-13 embryo (10h), before dorsal closure, stained with the anti-Go~ antiserum depleted of its specific antibodies. The complete absence of reactivity indicates the specificity of the antiserum. (H) A view of a mam mutant embryo stained with anti-Go~ antibodies. The nervous system is hypertrophied. The axon tracts (at) are strongly labelled. No staining is observed in the neuron cell bodies, as it is also the case in wild type embryos. Bars = 30/~m.
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specifically localized in the growing axons of the CNS. Cell bodies were not labelled. This result suggests that the specific expression of the protein in the CNS and its preferential localization in axons are not affected by the neurogenic mutations. DISCUSSION Particulate preparations from D. melanogaster embryos contained only one substrate of 39,000-40,000 molecular weight that could be ADP-ribosylated by pertussis toxin. The same substrate was also present in membranes from adult heads and we have previously demonstrated that it corresponded to a Go~-like protein using a polyclonal antibody directed against the ct subunit of the Go protein purified from bovine brain [17]. An anti-peptide antibody raised against the carboxy-terminal sequence of the ct subunit of the mammalian G O protein was able to cross-react, as shown by Western blot analysis, with a 39,000-40,000 molecular weight membrane protein in Drosophila embryos. The peptide sequence, which is specific for the ~ subunit of the G Oprotein but not for the g subunits of other G proteins [34], is conserved in the two putative Go~ proteins of D. melanogaster [18-20]. This antibody was also able specifically to immunoprecipitate the pertussis toxin ADP-ribosylated substrate in embryos. The fact that in Drosophila there is only one G protein substrate for the pertussis toxin will facilitate further investigation on the functional role of the Go protein. Immunoblotting experiments have shown that the expression of the ~ subunit of the Go protein markedly increased after 10h of embryonic development. This increase was closely correlated to an increase in ADP-ribosylation levels. This result implies that the ~ subunit could interact with fly and was fully functional at all embryonic stages [35]. In differentiated neuroblastoma N1E-I 15 cells, we have previously described the existence of two isoforms for the Go~ protein, which could be separated by their isoelectrical mobility (pH~ = 5.55 and 5.80) [33]. In Droso-
phila embryos, by using the same two-dimensional gel analysis, only one form of the ADPribosylated Go~ protein was detected at all stages of embryonic development. The isoelectric pH of this isoform was similar to that of the only form detected in Drosophila adults [17]. It should be pointed out that the two putative Go~ proteins deduced from the sequence of the two different cDNAs found in Drosophila only differ in their N-terminus [18-20] without any apparent change in their global electric charge or their molecular weight. If these two forms are really expressed during embryogenesis they could not have been resolved by the type of analysis described herein. Note that the two splice variants of Drosophila may not be equivalent to the two splice variants of the Go~ protein found in vertebrates, in which splicing involves the C-terminal third of the molecule [40]. Studies on protein localization showed that the Go~ protein was already expressed at low levels during early embryogenesis, before the onset of axonogenesis, and could therefore play a specific role in these earlier stages. The presence of the protein before and at the blastoderm stage could result from the maternal gene expression described by Schmidt et al. [21], whereas its preferential accumulation in neurogenic regions at later stages could derive from the zygotic expression, implying a specific control of gene expression in nervous territories. However, it is not likely that Go~ participates in the decision that determines the fate of neuronal cells since the specific expression of Go~ in neurogenic regions was subsequent to this primary process. Go~ expression was preserved in the neurogenic mutants E(spl), roam and DI, with the same specific pattern as in wild type embryos. This result implies that, at least in the case of the three mutants studied, the neurogenic genes do not control the spatiotemporal expression of the Go~ protein. Immunolocalization studies in Drosophila embryos also showed that Go~ is mainly localized in the axons of the CNS. Results agree with those described by Schmidt et al. [21] and Wolfgang et al. [22] in Drosophila adults, in which immunoreactivity was observed in the
GOand embryonicdevelopmentin Drosophila neuropil, and little, if any, in neuronal cell bodies. The preferential localization of the Go~ protein in growing axons in embryos suggests that it could play a direct role in axonogenesis. The onset of Go~ protein accumulation, as observed by immunoblotting, coincided with the beginning of axonogenesis (10 h) with no further increase during CNS condensation (16-22 h) and the distribution of the protein, as observed by immunochemistry, evolved concomitantly with the building of the axonal scaffold. The presence of Go~ in all the axons would be compatible with the general role assigned to second messenger systems such as calcium and protein kinase C in neurite outgrowth and axonogenesis [41]. Indeed, as already stated, Go~ seems to be implicated in the control of calcium channels [12-14] and phospholipase C [16] activities mammalian cells. It has been suggested that not only second messenger systems but also cytoskeletal elements are critical in axonal outgrowth [41]. In this context, we have previously shown in mammalian neurons that, in addition to a membrane localization, Go~ can be associated with cytoskeleton proteins such as microtubules in centriolar structures or is localized in areas rich in actin microfilaments [42]. It is interesting to compare the fact that Go~ is expressed during the period of intense axonogenesis in Drosophila with our previous results on the expression of Go~ isoforms during differentiation of NIE-I15 neuroblastoma. In these cells, differentiation is morphologically characterized by inhibition of cell growth and extension of long neurites and is accompanied by a specific increase in global Go~ level (but not in the amount of other G~ subunits) [33]. Other evidence also permits us to hypothesize that G proteins, and particularly Go, could be implicated in axonal growth and guidance. Thus, it has recently been noticed in mammals that G o, which is a major component in the growth cone membrane, can be regulated by GAP-43 [23], an axonal growth-associated protein [24]. There is, at the moment, only one indication concerning the eventual existence of a GAP-43 protein in D. melanogaster [43]. However, the
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homology between the vertebrate GAP-43 and this protein only relies on a conserved sequence of about 20 aminoacids with no other similarities in the rest of the molecule. Furthermore, unlike the GAP-43 protein, it is not expressed in growing axons. On the other hand, the possible participation of G proteins in axonal guidance is suggested by the fact that pertussis toxin blocks in vertebrates the effect of neural cell adhesion molecules on the modulation of second messenger systems [26]. Although all these data are compatible with a role of Go~ in neurite outgrowth both in mammalian and Drosophila neurons, we have to wait for the isolation of mutants in the gene coding for the ~ subunit of the G o protein to assign a definite role to G O protein in axonogenesis. However, several P-element insertions in 47A [44], within the region where the Go, gene has been localized, have recently been described in D. melanogaster [18-20]. The expression in all neurons of the Lac Z reporter gene carried by the P-element indicated the importance of the 47A region for the expression of neuronal proteins. Six of these insertions are lethal and lead to an abnormal development of the CNS [44]. Whether or not one of them is in the Go~ gene remains to be determined. Acknowledgements--The authors wish to thank Dr D. GRATECOSand Dr M. PIOVANTfor constructive
discussions. Dr J. A. FEHRENTZand Dr B. ROUOT are gratefully acknowledged for peptide synthesizing and antiserum preparing, respectively. Miss M. ASTIER is acknowledged for her excellent technical assistance. The assistance of Mrs M. PASSAMA,Mr P. WEBERand Mr L. CHARVETin preparing the manuscript is also greatly appreciated. The Spanish Ministry of Education and Science and the European Molecular Biology Organization are acknowledged for providing postdoctoral fellowships to A. GUILLI~Nfor two different periods of this work.
1. 2. 3. 4.
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