Tissue-specific Expression of Phospholipase C Encoded by the norpA ...

THEJOURNAL OF BIOLOGICAL CHEMISTRV

Vol. 268, No. 21, Issue of July 25, pp. 15994-16001, 1993 Printed in U.S.A.

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Tissue-specific Expression of Phospholipase C Encoded by the norpA Gene ofDrosophila melanogaster” (Received for publication, February 12,

1993, and in revised form, April 8, 1993)

Liqing ZhuS, Richard R. McKay, and RandallD. Shortridgeg From the Department of Biological Sciences, State Uniuersity of New York, Buffalo, New York 14260

Mutations in the norpA gene of Drosophila melano- family of enzymes that participate at a centralposition in one gaster severely affect the light-evoked photoreceptor of the most widely used second messenger signal-generating potentialwithstrongmutationsrenderingthe fly pathways known (Rhee et al., 1989; Meldrum et al., 1991). blind. Molecular cloning of the norpA gene revealed Activated PLC hydrolyzes phosphatidylinositol 4,5-bisphosthat it encodes phosphatidylinositol-specific phospho- phate (PIPZ) to yieldtwo intracellular second messenger lipase C, which enzymes play a pivotal role in one of molecules, inositol trisphosphate and diacylglycerol, which the largest classes of signaling pathways known. We function in mobilizing calcium and activating protein kinase have used Northern analysis, Western blots, phospho- C, respectively (Berridge, 1987). Signaling pathways that utilipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific lize PLC are activated by a diverse spectrum of stimuli and expression of the norpA gene and found that it is ex- modulate a wide variety of cellular processes such as invertepressed in a varietyof tissues besides the eye. Hybrid- brate phototransduction(Pak andShortridge, 1991),secretion ization of norpA cRNA probe toblots of poly(A+)RNA (Putney, 1988), contraction (Volpe et al., 1986; Stull et al., reveals that thegene encodes at least four transcripts: 1988), fertilization (Slack et al., 1986),cell growth and differa 7.5-kilobase (kb) transcript that is expressed in eye entiation (Michell, 1989), and long-term potentiation(Collingridge, 1987). PLC enzymes have been purified from a variety and 6.5-, 5.5-, and5.0-kb transcripts that are expressed in adult body or early stages of development. of tissue sources and many have been extensively characterAntiserum generated against the major gene product ized biochemically(reviewedby Meldrum et al., 1991).Several of norpA recognizes a 130-kDa protein that is abun- of the purified enzymes were found to differ in molecular dant in eyes but severely reduced or absent in norpA weight, calcium dependence, substrate preference, tissue dismutants, a result which is consistent with previous tribution, and immunological reactivity, leading to the proconclusions that the norpA gene encodes an essential posal that thereare at least five different types of mammalian component of the visual system. However, the norpA PLC (Rhee et al., 1989). Molecular cloning of PLC cDNAs antiserum also recognizes a 130-kDa protein in adult have identified enzymes falling into three major classes, deslegs, thorax, and male abdomen, but not female abdo- ignated PLCp, PLCr, and PLCG, whose members differ widely men. These localizations are supported by results of in structural characteristics and molecular weight, yet retain phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in small regions that are conserved among the different classes each of the tissues in which norpA protein can be (Rhee et al., 1989; Rhee and Choi, 1992). There are multiple detected. Furthermore, immunohistochemical staining subtypes of PLC within each of the three type classes and of tissue sections with the norpA antiserum demon- these aredesignated by adding numbers after the Greek letter norpA protein is abundant in the retinatype designation, such as PLCpl, PLCp2, or PLCp3 (Rhee strates that the and ocelli and is present toa lesser extent in the brain and Choi, 1992). The diversity of PLC enzymes, coupled with their disparate and thoracic nervoussystem. Since some of the above mentioned tissues that express norpA (such as thorax, tissue distributions (Ross et al., 1989), seem to point to a legs, and abdomen) have no known photoreceptor tis- model of selective coupling of individual PLC isozymes to sue, we conclude that the norpA gene product is also different receptors, signaling pathways, or cellular processes. likely to have arole in signaling pathways otherthan However, very little is known about the role that PLC diverphototransduction. sity plays in physiological processes, mostly because it has been difficult to identify the specific signaling pathway which utilizes a particular subtype of PLC. Mammalian PLCy has Phosphatidylinositol-specific phospholipase C (PLC)’ is a been tentatively identified as being coupled to growth factor receptors (Pandiella et al., 1989; Wahl et al., 1989; Rhee and * This work was supported by the National Science Foundation Choi, 1992), but it is not known if all subtypes of PLCy Grant IBN-9120866.The costs of publication of this article were participate in growth factor signaling or if a single PLC-y is defrayed in part by the payment of page charges. This article must activated by more than one type of signaling pathway. Sigtherefore be hereby marked “aduertisement” in accordance with 18 naling pathways that utilize individual subtypes of mammaU.S.C. Section 1734 solely to indicate this fact. $ Current address: Fred Hutchinson Cancer Research Center, Hu- lianPLCp or mammalian PLCG have yet to be identified man Genetics Program, 1124 Columbia St., Seattle, WA 98104-2092. (Rhee and Choi, 1992). § To whom correspondence should be addressed State University Two PLC-encoding genes have been identified in Drosophof New York at Buffalo, Dept. of Biological Sciences, Cooke Hall Rm. ila and these share a high similarity in structure to their 109,Buffalo, NY 14260-1300.Tel.: 716-645-3122; Fax: 716-645-2975. mammalian counterparts (Bloomquist et al., 1988; Shortridge ’ The abbreviations used are: PLC, phospholipase C; PIP*, phos- et al., 1991). One of these is the norpA ( n o receptor potential) phatidylinositol4,5-bisphosphate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albu- gene, which mutants have long been known to be deficient in light-induced electrical responses in the compound eye (Pak min; kb, kilobase(s).

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et al., 1970; Hotta and Benzer, 1970) and ocellus (Hu et al., 1996 in Bloomquist etal., 1988) which was clonedinto the pGEM-3Z 1978) with strong mutations rendering thefly blind. Molecu- plasmid (Promega). Generation of Antiserum against the norpA Protein-A 492-base lar cloning and analysisof the norpA gene (Bloomquist et al., pair Sac1 restriction fragment of the norpA cDNA (nucleotides 8481988) revealed that it encodes a homologue of mammalian 1340 in Bloomquist et al., 1988) was cloned in frame with the glutaPLCP, a finding which wassignificant for at least two reasons.thione S-transferase gene in the pGEX-KG expression vector (Guan and Dixon, 1991) to express part of the norpA protein as a fusion to First, since norpA mutants lack photoreceptorfunction,it demonstrates that PLC is an essential componentof photo- glutathione S-transferase in Escherichia coli JMlOl cells. Cells were a collected by centrifugation and the fusion protein isolated (in inclutransduction in Drosophila and lends critical support to bodies) essentially asdescribed by Harlow and Lane(1988). The growing body of evidence that invertebratevision is mediated sion fusion protein was purified by resolving themixture by sodium by calcium mobilization, inositol phosphate production, and dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ona PLC activity(reviewed byPak and Shortridge, 1991). Second, 12% gel, staining briefly in Coomassie Blue, destaining in distilled it identifiesa particular signaling pathway in which a specific water, and cutting out the fusion protein band. The gel slice was isozyme of PLC is utilized, which has been difficult to do in lyophilized to dryness, ground intoa fine powder using a mortar and (150 mM NaCI,10 mM sodium the cases of other isozymes of PLC identified thus far. How- pestle, andresuspendedinPBS ever, it's unknown if the PLC encoded by the norpA gene is phosphate, p H 7.2). Approximately 300 pg of the protein was emulsified withanequal volume of Freund'scompleteadjuvantand utilized exclusively in phototransduction or if it is also utilized injected intradermally and subcutaneously into a 13-week-old white in other signaling pathways. rabbit. The rabbit was boosted using the same amount of protein in A growing body of evidence suggests that alternative splic- Freund's incomplete adjuvant a t monthly intervals. Ten days after ing of mRNA contributes further to diversity of enzymes each booster injection, blood was collected and the serumharvested. Western Blot Analysis-Western blotting was performed by first within PLC subtypegroupings. Shortridge et al. (1991) cloned a Drosophila PLCP gene, designated plc21, and found that it grinding fly tissues in 50 mM Tris-C1, pH 7.5, 250 mM KC1, 0.05% encodes two subtypes of PLCP from alternative splicing of sodium deoxycholate, 0.1 mM phenylmethanesulfonyl fluoride using Teflonpestlesin 1.5-mlmicrocentrifuge tubes. The homogenates mRNA. The two subtypes of protein differ by seven consec- were centrifuged brieflyfor2 min at 12,000 X g to remove the utive amino acids, out of 1305 total, that are found in oneof particulate matter, boiled for 5 min in an equal volume of 2 X SDS the proteins and not the other. Based on Northern hybridi- sample buffer (125 mM Tris-C1, pH 6.8, 4% SDS, 20% glycerol, 4% zations, the two forms of plc21 are expressed indifferent P-mercaptoethanol, 10 pg/ml bromphenol blue), fractionated by 7% anitrocellulose membrane tissues andat different stagesof development, and thus, these SDS-PAGE,andelectroblottedonto (Towbin etal., 1979). The membrane was preincubated in a blocking 7 amino acids presumably confer a specific function to the solutioncontaining 3% BSA, 2% Tween 20, 1% goat serum,and enzyme subtype that is required in an individual tissue. A 0.04% sodium azide in PBS at room temperature for 1 h. The rabbit similar case of alternative splicing is found in a rat brain antiserum was diluted 1:3000 in freshblocking solution and incubated PLCP gene which appears to encodetwo proteins that canbe with the membrane a t room temperature for 2 h. The filter was then resolved using Western analysis (Suh et al., 1988). However, washedfour timesin PBS for 20 min each. Binding of primary the identityof the signaling pathways that utilize subtypes of antibody was detected with an alkaline phosphatase-conjugated goat by diluting the antibody either Drosophila plc21 or mammalian PLCP is unknown. anti-rabbit antibody (Boehringer Mannheim) Neither is it clear whethersplice variants from a single gene 1:6000 in blocking buffer, incubating the filter in the buffer a t room temperature for 1 h, and washing the filter four times in PBSfor 20 are involved in one type of receptor signaling pathway or if min each. Immunolabeling was visualized by reaction at room temthey are utilized in a number of diverse pathways. We have perature in 100 mM Tris-C1, pH 9.5, 100 mM NaCI, 5 mM MgCI2,0.41 found that thenorpA gene, like the two PLCP encoding genes mM 4-nitro blue tetrazolium chloride, and 0.38 mM 5-bromo-4-chlorodescribedabove, is acomplex gene that produces multiple 3-indolylphosphate. As control,theprimaryantiserum was first transcripts and possibly multiple isoforms of norpA protein. preincubatedwith excess of the fusion protein for 1 h a t room temperature prior to incubation with thenitrocellulose membrane. Inordertoassessthe role that the norpA gene playsin Phospholipase C Activity Assays-PLC activity assayswere carried phototransductionandothersignalingpathways, we have out a t room temperature for 5 min in a 0.1-ml volumeof 50 mM Trisexamined its tissue-specific expression and found that it is c1, p H 7.5, M CaCI,, 0.1 mg/ml BSA, 0.2 mM phosphatidylinoexpressed in a variety of tissues and developmental stages. sitol, 44,000 dpm [3HH]phosphatidylinositol 4,5-bisphosphate, and Since some of these tissues do not have photoreceptor struc- Drosophila tissueextractessentiallyas described by Inoueet al. tures, we conclude that norpA isozymes are utilized in both (1988). Tissue extractswere prepared by homogenization using Teflon phototransduction and other signaling pathways besides pho- pestles in 1.5-ml microcentrifuge tubes in a buffer of 50 mM Tris-C1, p H 7.5, 250 mM KC1, 0.05% sodium deoxycholate, 0.1 mM dithiothretotransduction. MATERIALS AND METHODS

Drosophila Strains-Drosophila melanoguster white ( ILP'~) mutant was used in all experiments asa control group, because it eliminates visual pigment which can interfere with some assays and its genetic backgroundismostsimilarto that of norpA mutants utilized in experiments. In the Western blots, Canton-S (wild-type) strain was used. norpAm4, norpAP1*,and norpAEE5are strong phenotypic alleles (Lindsley and Zimm, 1992) in which norpA mRNA is known to be severely reduced or absent. eyes absent (eya) mutant (Sved, 1986), which completely lacks compound eyes, was utilized for comparisons to identify norpA gene products that are expressed in theeye, RNA Isolation and Northern Blot Analysis-Flies were grown at 25 "C for specified times after a 12-h egg-laying period and used as a tissue source for preparation of RNA (Shortridge et al., 1991). Preparation of RNA from tissues as well as Northern blots were carried out as described by Shortridge et al. (1991) except that radiolabeled cRNAprobes were used. Probe was constructed by synthesizing antisense or sense norpA cRNA in the presenceof [32P]UTPusing a Promega Riboprobe Systems Kit. The cRNA probes were transcribed from a 1.1-kb BamHI fragment of norpA cDNA (nucleotides 892-

itol, and 0.1 mM phenylmethanesulfonyl fluoride. Following homogenization, tissues were centrifuged briefly at 12,000 X g to remove particulate matter. Protein concentration in the homogenates was determined using theBCA protein assay (Pierce Chemical Co.), with BSA as a standard, and an appropriate amount of extract (amount empirically determined to yield linear results with respect to time) added to the reaction mixture. The reactions were stopped by addition of 2 ml of chloroform:methanol(2:1) and 0.5 ml of 1 N HCI containing 5 mM EDTA, vortexing briefly, and centrifuging to separate phases. 0.7 ml of the upper (aqueous) layer was removed and counted by liquid scintillation. Immunohistochemistry-Drosophila tissues were imbedded in O.C.T. compound (Tissue-Tek) and frozen on dry ice. 10-pm-thick tissue sections were cut on a Reichert cryostat and transferred onto gelatin-subbed slides (Gall and Pardue, 1971). The sections were fixed for 30 min in a freshly made solution of 150 mM sodium phosphate, p H 7.0, 75 y M lysine, 10 p M sodium metaperiodate, and2% paraformaldehyde(McLeanandNakane, 1974).Following tissuefixation, sections were washed two times in PBS for 5 min each. The sections were blocked, incubated with antibodies, washed, and developed in the same way asWesternblotsmentioned aboveexcept primary antibody inccbations were carried out overnight a t 4 "C.

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present in the body of norpAm4 mutant, suggesting that they are indeed derived from the m r p A gene and are not a result of cross-hybridization to non-norpA mRNA species. Transcripts of similar size can also be detectedin 0-24-h-old embryo (Fig. 1B). Moreover, a 6.5-kb transcript is prominent is lanes of poly(A+)RNA from 3-day-old Drosophila (Fig. 1B). When the blots described above were reprobed with norpA sense cRNA, no bands were detected (data not shown), demonstrating that the transcripts seen in Fig. 1are derived from the same DNA strand as norpA mRNA. Therefore, we conclude that the norpA gene encodes multiple transcripts, at least one of which is expressed in the eye and others in body or at early stages of development. The significance of lighter hybridization signals in the size range of 0.5-2.4 kb (Fig. 1, B and C ) is unknown, although these transcripts are too small to encode proteins in the size range of any eukaryotic PLC isolated thus far. In addition, the significance of two prominent bands of 4.5 and 5.0 kb in lanes of head (Fig. 1C)arenot known, as these are not observed when identical blots are carried out using poly(A+) RNA isolated from Drosophila Canton-S head (data not A eya P24 B C shown). Generation of Antiserum against the norpA Protein-norpAE 3 5 7 9 H B H B HEB3 5 7 9 HB specific antiserum was generated in rabbits against a bacterial-norpA fusion protein. A 492-base pair Sac1 restriction fragment from a norpA cDNA was cloned into thepGEX-KG -9.5 expression vector (Guan and Dixon, 1991) so that a small -7.5 region of the norpA protein would be expressed as a fusion m protein linked to glutathione S-transferase. The basic idea I was to generate antibodies against a nonconserved region of I the norpA protein so that they would not cross-react with other PLCisozymes. The small expressed region of the norpA protein was chosen because it is relatively nonconserved among known eukaryotic PLC enzymes. -1.4 Antiserumgeneratedagainst the norpA-glutathione Stransferase fusion protein recognizes the norpA proteinin Western transfers of Drosophila tissue homogenates (Fig. 2). 0.3 The antiserum (designatedabRN) specifically stains a protein of 130-kDa molecular mass that is abundantly expressed in D adult head, but reduced or no staining is seen when an equal amount of head protein from threestrong norpA alleles, mrpAm4, norpAP1*,and norpAEESwere loaded in gel lanes. Moreover, essentially no abRN staining of the 130-kDa protein is seen in extracts from eya heads, strongly suggesting FIG.1. Northern analysis showing that the norpA gene is that theprotein is localized in the eye. This is identical to the expressed in adult head, adult body, and during early stages findings of Schneuwly et al. (1991) who identified a 130-kDa of development. Approximately 5 pg of poly(A+) RNA was loaded eye-specific protein as the major norpA gene product. per gel lane and probed with a 1.2-kb norpA cRNA fragment (antiReduced amounts of 130-kDa protein are detectable inadult sense strand). Lanes are: E , 0-24-h embryo; 3, 3-day-old; 5, 5-dayold; 7, 7-day-old; 9,9-day-old; H , adult head; B, adult body; eya H bodies (see next section), eya mutant heads, and norpAEE5 and B, eyes absent mutant adult head and body; P24 H and B , mrpAm4 mutant heads (data not shown) when Western transfers are mutant adult head and body. B shows a 5-fold overexposure of the intentionally overstained. Staining of norpA protein inbodies four leftmost lanes in A . C shows a 3-fold underexposure of the adult is in agreement with the Northern data showing that norpA head andbody lanes shown inA. Mobility of RNA size standards (in kilobases) are indicated on theright. D shows signals (0.6 kb) which mRNA is present in body (Fig. 1).Moreover, the presence of result from reprobing the blot with Drosophila RP49 cDNA (O'Con- small amounts of norpA protein in head of eya and norpAEE5 nell and Rosbash, 1984) as a control for mRNA loading. The mrpA mutants is consistent with the following information. First, cRNA probe hybridizes to a major transcript of 7.5 kb in the adult eya mutants lack compound eyes, but areknown to have ocelli head, but not body (lanes H and B ) . This transcript is missing from (Sved, 1986). Schneuwly et al. (1991) demonstrated that the head of eya mutant and n0rpAfq4mutant. Two transcripts (5.5 and 5.0 kb) can be detected in adult body (including body of eya mutant), norpA protein is expressed in ocelli, and thus, eya mutant but not in body of norpA"' mutants (lanes B, eya B, and P24 B, heads would be expected to contain small amounts of m r p A respectively). Similar sized transcripts (5.5 and 5.0 kb) can be detected protein. Second, norpA mRNA has been shown to be present in early embryos, and a 6.5-kb transcript is prominent in 3-day-old in low amounts in the head of norpAEEsmutant (Bloomquist (transcripts markedby arrows on left of A ) . The significance of small et al., 1988), which correlates exactly with our observation transcripts (0.3-2.4 kb) in various lanes or two prominent (4.4 and that there isdetectable amount of norpA protein in norpAEE5 5.0 kb) transcripts in head (visible in lane H,panel C ) are unknown head. Staining of the 130-kDa protein does not occur in wild(see text). When this blot was reprobed using norpA cRNA sense strand, no detectable signals were observed, even after very long (2 type heads if preimmune serum is used (Fig. 2B) or if the week) exposure times (data not shown). immune serum is first preincubated with an excess of the RESULTS

Detection of MultiplenorpA TranscriptsonNorthern Blots-When radiolabeled norpA cRNA probes are hybridized to blots of poly(A+) RNA isolated from 0-24-h old embryos, 3-, 5-, 7-, and 9-day-old developing organisms, and from adult heads or bodies, at least four transcripts of different sizes can be detected (Fig. 1).The 3-, 5-, 7-, and 9-day-old organisms correspond approximately to first and second instar larvae, third instar larvae, early pupae, and late pupae, respectively. As shown in Fig. 1,a major 7.5-kb transcript is easily detected in adult head, but not head of n0rpAm4 mutant. Moreover, this 7.5-kb transcript is absent in head of eya (eyes absent) mutant, a mutant thatcompletely lacks compound eyes (Sved, 1986), suggesting that this 7.5-kb transcript is present in the eyes of the fly. These data agree with results obtained by Bloomquist et al. (1988) who described a 7.5-kb transcript as the major norpA gene product. As shown in Fig. 1, transcripts of 5.5 and 5.0 kb can alsobe detected in adultbody, including body of eya mutant. However, these two transcripts are not

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FIG.2. Western blot stained with antiserum generated against thenorpA protein. Lanes were loaded with protein extracts from approximately 10 heads or two bodies from wild-type flies or mutant flies listed at the top of each lane. The antiserum (designated abRN) stainsa protein that is abundant inwild type ( w t ) heads (see arrow), but severely reduced or absent in heads of eya (eyes absent) mutant, heads of norpAm4, norpA"'*, or norpAEEs mutant, or wildtype body. The apparent molecular mass of this protein (130 kDa) matches the deduced molecular mass of the norpA protein (mobility of protein size standards are indicated on the right). Small amounts norpAER5head, eya head of the 130-kDa protein are detectable in (data not shown), or wild-type body (Fig. 3) when blots are subjected to color staining for prolonged periods.The 130-kDa protein does not stain if preimmune serum is used ( B )or if the abRN immune serum is first preincubatedwiththe fusion proteinantigen used in the immunizations ( C ) .

fusion protein used in immunizations (Fig. 2C). Therefore, we conclude that this 130-kDa protein is, indeed, the major product of the norpA gene, and the abRN antiserumspecifically reacts with the m r p A protein. Detection of norpAProteins in Adult Legs, Thorax, and Abdomen-When Western transfers were stained with the m r p A antiserum (abRN) and thenintentionally subjected to prolonged incubations in thecolor substrate solutions, it was possible to identify small amounts of the 130-kDa norpA protein in adult body. To further examine the distribution of norpA protein in body, we loaded homogenates of adult legs, male thorax, female thorax, male abdomen, and female abdomen separately ingel lanes on Westernblots. norpA tissues were also tested in these blots for comparison with wild type. As shown in Fig. 3, we found clear abRN staining of norpA protein in legs, male thorax, female thorax, and male abdomen, but not female abdomen. Again, there is no staining observed in mrpAm4 mutant thorax or abdomen (Fig. 3). Moreover, abRN staining in mrpAm4 legs was not observed, but clear staining was seen, as expected, in eya mutant legs (data not shown). Thus, the m r p A protein appears to be present in a variety of tissues in adult body and might be expressed in a sex-specific manner in adult abdomen. To examine if there was a sex-specific difference in norpA expression in head, we loaded male and female heads separately in gel lanes on Western blots and stainedusing abRN antiserum. We observed heavy staining of norpA protein in wild-type heads, reduced staining in eya mutant heads, and

FIG.3. Immunodetection of the norpA protein on Western blots of homogenates of legs, thorax, and abdomen. Lanes were loaded with protein from approximately six head, thorax, orabdomen or 100 pl of leg fragments. Thorax and abdomen are abbreviated in the lane labels shown at the top of the figure, and males or females are shown by universal symbol designations. Staining of the norpA protein with abRN wasvisualizedusing an alkaline phosphataseconjugated secondary antibody and intentionally overdeveloped by prolonged incubation in a solution of nitro blue tetrazolium chloride and bromochloroindolyl phosphate which results in visualization of a variety of background proteins. The 130-kDa norpA gene product (arrow), which is abundant in wild-type head ( f a r right lane; here loaded in a nonadjacent lane to minimize lane spillover), canalso be detected in homogenates of legs, male thorax, female thorax, and maleabdomen.Thisproteinisnotdetectablein homogenates of female abdomen (on right), or legs (not shown),male thorax, female thorax, or male abdomen of norpA mutants. No sex-specific differences in staining arevisible when comparing male and female heads, including heads of eya mutant (data not shown).

no staining in norpAm4 mutant head, but no differences between males and females (data not shown). Reduced Phospholipase C Activity in norpA Mutant Tissues-Yoshioka et al. (1985) found that phospholipase C activity, which is easily measured in wild-type Drosophila heads, is severely reduced in heads of norpA mutants. Moreover, Toyoshima et al. (1990) demonstrated that this missing activity is derived from the norpA gene and that the norpA gene encodes the predominant PLC activityin head. To further evaluate the relationship of norpA gene products to phospholipase C activityand toconfirm which tissues express the norpA gene, we examined the ability of homogenates of various Drosophila tissues to cleave PIP2 in an in vitro activity assay. In agreement with resultsdescribed by others (Yoshioka et al., 1985; Toyoshima et al. 1990), we find that normal Drosophila heads contain a high amount of measurable PLC activity that is severely reduced in heads of norpA mutants (Fig. 4A). Moreover, the majority of this PIP2-hydrolyzing activity is localized in the eye, as shown by the reduction of PLC activity in heads of eya mutant. However, the eya mutation only eliminates approximately 65% of PLC activity in wild-type heads, whereas the norpAm4, norpAPl2, and mrpAEE5 mutations eliminate more than 95%. This residual PLC activity in eya mutant heads is likely due to expression of the m r p A gene in non-eye tissues of the head, such as theocellus. We also tested a variety of homogenates of adult body tissues and found that PIPz-hydrolyzing activity could easily be measured, although the specific activity is much less than

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8 'b 8 '6 FIG. 4. Phospholipase C activity is reduced in a variety of tissues in norpA mutants. Tissue homogenates were tested for ability to cleave [3H]PIP2in an in vitro biochemical reaction and the results expressed as specific activity of the homogenate (picomoles of [3H]PIP, cleaved per 5 min/mg of protein). The results shown are an average of three determinations (error bars indicate the range of standard deviation). PLC activity in adult heads that are wild-type for norpA is reduced in heads of eya (eyes absent)mutant and severely reduced in heads of three strong norpA mutant alleles ( A ) which is consistent with previous results showing that the norpA protein is the predominant PLC activity in heads (Toyoshima et al., 1990). The norpA mutation also reduces PLC activity in legs, thorax, and male abdomen, but not in female abdomen ( B ) ,in agreement with the amounts of norpA protein that is detectable on Western blots (Fig.3).

that found in heads (Fig. 4B).The norpA mutation reduces and Western blots localizing norpA gene products in eya heads PLC activity in adult legs, male thorax, female thorax, male and wild-type body. In addition, intense abRN staining was abdomen, but not female abdomen, in complete agreement observed in theocelli (Fig. 5 A ) , in agreementwith the concluwith our resultsfrom Western analyses (previous section; Fig. sion of Schneuwly et al. (1991) that ocelli express the norpA 3). Thus, the reduction of PLC activity in norpA body ho- gene. Furthermore, localization of the norpA protein in the mogenates confirms the presence of norpA gene products in a retina, optic lobe, and cerebrum is incomplete agreement variety of body tissues and further supports the proposal thatwith spatiallocalization of norpA mRNA in tissue sectionsof PLC(Bloomquist et al., 1988; head (Bloomquistet al., 1988) and isvery similar to the spatial the norpA geneencodes distribution of PIP2,which is the relevant substratefor PLC, Schneuwly et al., 1991; Pak and Shortridge,1991). in head (Suzuki andHirosawa, 1992). ImmunohistochemicalLocalization of norpAProtein-To examine the spatial distribution of norpA protein in adult DISCUSSION tissues, we performed immunostainingof adult tissue sections. Staining of abdomens with the norpA antiserum(abRN) norpA mutants have long been known to exhibit defects in resulted in high amounts of nonspecific staining, even in the vision and, more recently, it has been proposed that thenorpA gene encodes a phospholipase C enzyme that is essential for absence of primary antibody (data not shown), making it norpA protein. phototransduction (Bloomquistet al., 1988; Schneuwly et al., difficult to identify tissuesexpressingthe However, as shown in Figs. 5 and 6 , there is a clear staining 1991; Pak and Shortridge, 1991). The primary question addressed here is whether the norpA gene is expressed excluof head and of norpA protein by abRN in tissue sections or the proteinis also thorax. abRN strongly stains the adult retina, which is con- sively in the phototransduction pathway sistent with the proposal that norpA plays a role in thevisual utilized in other signal transduction pathways. In Northern system. Retina of norpAm4 does not stain (Fig. 5 B ) showing blots, a t least four norpA transcripts were detected in head, that the staining in retina is specific for the norpA protein. body, or at early stages of development (Fig. l),indicating Furthermore, preimmune serumdoes not stain the retina nor that there is a differential transcriptionof the norpA gene in does the immune serum if it is preincubated in an excess of tissues and developmentalstages. One of the norpA transcripts (7.5 kb) is easily detected in the fusion protein used in immunizations (Fig. 5, C and D). in mutant heador at earlier abRN staining is also observed in the optic lobes and brain adult head, but not detectableeya (cerebrum) and the thoracic nervous systemof flies that are stages of development. Moreover, this transcript is not deeye is developing. The absence wild-type for norpA, but not innorpAm4 mutants (Fig. 5A and tectable in late pupae when the Fig. 6 ) . The staining in the optic lobes and cerebrum appears of this particular transcript from eya mutant head and from the early developmental stages suggests that this transcriptis to be darkest in the corticalregions. localized in the eye. Its absence from late pupae supports the The observation that abRN staining occurs in brain and of norpA encodes a product thoracic ganglia is consistent with the results of Northern view that this particular transcript

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FIG. 5. Immunostaining of the norpA protein in horizontal tissue sections of adult head. 10-pm sections were cut horizontally through adult heads of a white mutant ( A ) or norpAm4/whitedouble-mutant ( R )and stained with abRN immune serum.white mutants lack photopigment which can interfere with photographicrecordings of antibody labeling. Photographs were taken using differential interference contrast illumination. In all four panels, anterior is up. As seen in A , abRN strongly stains the retina ( r ) , and to a lesser extent, stains the optic lobe ( 0 ) and brain ( b ) . However, abRN fails to stain these tissues in head of norpA”‘ mutant ( B ) , demonstrating that the immune serum is specific for the norpA protein. Preimmune serum fails to stain retina, optic lobes, or brain in (white) head that is wild-type for nomA (C). Moreover. staining bv abRN immune serum can be blocked in white head by preincubating the immune serum with the fusion prdtein used in immunizations ( D ) .

having a role in phototransduction (Pak and Shortridge, 1991) of the other known phenotypic characteristics of norpA murather than a developmental process. norpA transcripts of tants. It has been observed that the initiation of courtship other sizes are easily detectable in adult body (including eya and beginningof mating areprolonged in norpA mutant males (Markow and Manning, 1980; Tompkins et al., 1982). The body but notbody of norpAm4mutant) and during early stages phenomena has been explained as a loss of photoreception or of development (e.g. embryos).Sincetherearenoknown photoreceptor structures in adultbody or embryo, thesegene of visual acuity which may impede mate localization during products are alsolikely to have a role in signaling pathway(s) courtship. Although an intact visual system is important for Drosophila, the sexually dimorphic expresmating behavior in other than phototransduction. Western blots and immunohistochemical stainingof adult sion of norpA in the fly abdomen may suggest another possitissue sections both confirm the above conclusions and help bility; a subtype of PLC encoded by the norpA gene perhaps to identify individual tissues expressing the norpA protein. has somerole in fly mating behavior. The validity of this idea m r p A protein is easily detected in adult retina and ocellus in will become clear as additional studies are carried out to completeagreementwiththeresults describedabove and identify the signaling pathway involved or the cell types in elsewhere (Bloomquist et al., 1988; Pak and Shortridge,1991; the abdomen that expressnorpA. The presence of the norpA protein in legs is interesting in Schneuwly et al., 1991). However, it is alsopossible to detect light of very recent observations that norpA mutants have the norpA protein in optic lobe and brain, thoracic nervous system, legs, and male abdomen, but not female abdomen. reduced feeding responses to some sugars, namely trehalose, glucose, and sucrose.* Many insects, including fruit flies, have Some of these localizations not only serve to confirm that the been long known to have taste receptors on their legs (Duerr norpA gene is expressed in adult body, as revealed by the Northern analyses, butalsoidentifythetissues involved. and Quinn et al., 1982). The legs of Drosophila have been Moreover, activity assayswhich demonstrate the reductionof shown to containhigh inositol trisphosphate bindingactivity (Yoshikawa et al., 1992), suggesting the presence of a phosPLC activity by the norpA mutation in specific tissues both pholipase C signaling pathway. The inability of norpA mucorroborate the above conclusions as well as further support tants to taste sugars would seem to suggest that a possible the proposal that thenorpA gene is a structural gene encoding role of the norpA protein in legs is as a phospholipase C that PLC. participates in signal transductionvia taste receptors. The observation that the norpA gene is expressed in male * T. Tanimura, personal communication. abdomen, but notfemale abdomen, is interesting,given some

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Expression Tissue-specific

of mrpA

FIG.6. Immunostaining of ocelli, brain,andthoracic ganglia with abRN antiserum. abRN staining of a (10 pm) cut sagittal tissue section through a white mutant head ( A ) shows staining in a dorsal ocellus (arrow) and brain (asterisk). Darkeststainingappears in the corticalregion of the brain. abRN fails to stain these tissues in a similarsection of a norpA"' mutant head (C) showing that staining is specific for the norpA protein. In A and C, dorsalisupandanteriortothe left. abRNstaining of a sagittalsection through the thorax of white adult ( B ) shows staining in the thoracic nervous system (asterisk). Again, abRN fails to stain the thoracic ganglia in a similar sagittal section cut through a norpA"' mutant thorax (D). In B and D,dorsal is to the right and anterior is up. Photographs were taken using differential interference contrast illumination.

The role that the mrpA protein has in the optic lobes, brain, and thoracic nervous system is unknown. However, the fact that the protein is present in brain and nervous system appears to suggest that it is utilized in a signaling pathway other thanone that transduces primary sensory information. Moreover, in heads from two very strong norpA mutants, norpAP'*and norpAm4,we are unable to detect norpA protein in Western blots. However, both of these mutants are homozygous viable. Thus, it appears that the norpA protein in the nervous system might not be essential for viability of the organism. The observation that the norpA gene encodes multiple transcripts that are expressed in more than one signaling pathway sheds light on PLC diversity and at the same time opens up new questions. As previously mentioned, phosphatidylinositol-specific PLC is known to be a family of proteins that have been classified into three major groups (PLCP,

PLCr, and PLCd) that can be further divided into subtype groupings (Rhee andChoi, 1992). The subtypes of PLC identified thus far are products of separate genes. Newly emerging is the concept that subtype groupings may be further subdivided by splice variant products from single genes. A question that remains unclear is whether splice-variant enzymes encoded by a single gene operate in one type of signaling pathway or participate in a diverse class of signaling pathways. Our finding that multiple tissuesexpress the norpA gene has demonstrated thatproducts from a single gene maybe utilized in a variety of signaling pathways that respond to different stimuli. At this time it is unclear if the alternatively spliced transcripts of norpA encode more than one subtypeof the protein. Only a single 130-kDa protein band was detected on Western blots (Fig. 2). However, this does not preclude the possibility that norpA subtypes differ only in small regions of the protein

Expression Tissue-specific and that all subtypes migrate equally on electrophoresis gels. The two subtypes of the Drosophila plc21 protein differ by 7 amino acids out of a total of 1305 (Shortridge et al., 1991), and thetwo subtypes would not likely be resolved using SDSPAGE. Subtypes of norpA may also have subtle amino acid changesthat may be very importantforfunction of the particular protein in an individual signaling pathway. The identification of these putative subtypesis possible by molecular cloning and sequence analysis of other subclasses of norpA cDNA. When this is carried out, it will be possible to more fully address the question of what is the role of each subtype of PLC in individual signaling pathway(s). Acknowledgment-We thank Dr. Todd Hennessey for help and advice in carrying out PLC activity assays. REFERENCES Berridge, M. (1987) Annu. Rev Biochem. 5 6 , 159-193 Bloomquist, B. T., Shortrid e, R D , Schneuwly, S., Perdew, M., Montell, C., Steller, H., Rubin, G., a n f P a k ; W . L. (1988) Cell 5 4 , 723-733 Collingridge, G. (1987) Nature 330,604-605 Duerr, J. S., and Quinn, W. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 36463650 Gall, J. G., and Pardue, M. L. (1971) Methods Enzymol. 21, 470-480 Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192,262-267 Harlow, E., and Lane, D.(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Hotta, Y., and Benzer, S. (1970) Proc. Natl. Acad. Sci. U. S . A. 67,1156-1163 Hu, K. D., Reichert, H., and Stark, W. S. (1978) J., Comp. Physiol. 1 2 6 , 15-24 Inoue, H., Yoshloka, T., and Hotta, Y . (1988) J. Blochem. (Tokyo) 103,91-94 Lindsley, D. L., and Zimm, G. G., (1992) The Genome of Drosophila melanogaster, Academic Press, San Diego, CA Markow, T. A., and Manning, M. (1980) Behuu. Neural Biol. 2 9 , 276-280

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