©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Subtypes of Phospholipase C Are Encoded by the norpA Gene of Drosophila melanogaster(*)

Sunkyu Kim , Richard R. McKay , Karen Miller , Randall D. Shortridge (§)

From the (1)Department of Biological Sciences, State University of New York, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.


INTRODUCTION

Phosphatidylinositol-specific phospholipase C (PLC)()cleaves phosphatidylinositol 4,5-bisphosphate to yield two preeminent second messenger molecules, inositol trisphosphate and diacylglycerol, in one of the largest classes of cellular signaling pathways known (Berridge, 1987; Rhee et al., 1989). Signaling pathways that are mediated by PLC are found in both plants (Drobak, 1992) and animals (Ryu et al., 1987) and have been shown to modulate a variety of cellular processes such as invertebrate phototransduction (Pak and Shortridge, 1991), contraction (Volpe et al., 1986), secretion (Putney, 1988), fertilization (Miyazaki et al., 1993), growth, differentiation (Michell, 1989), phagocytosis, and chemotaxis (Lew, 1990).

PLC is known to be a diverse family of enzymes whose members differ in structure and tissue distribution (Rhee et al., 1989; Meldrum et al., 1991). PLC enzymes have been grouped into four major classes (PLC-, PLC-, PLC-, and PLC-), based on immunological and structural differences (Rhee et al., 1989). The PLC-, PLC-, and PLC- enzymes have been further classified into subgroups (PLC-1 through PLC-4, PLC-1 through PLC-3, and PLC-1 through PLC-3) based on differences in amino acid sequence and that each subtype is encoded by a separate gene (Rhee and Choi, 1992a, 1992b). Emerging evidence indicates that an additional level of diversity exists beyond the subgroup classifications because individual genes have been shown to encode multiple PLC products by alternative splicing of RNA (Shortridge et al., 1991; Ferreira et al., 1993; Bahk et al., 1994).

The major types of PLC appear to be activated by different receptor classes (Meldrum et al., 1991; Jones and Carpenter, 1992; Rhee and Choi, 1992a, 1992b). PLC- activation appears to be modulated by canonical heptahelical receptors coupled to G-proteins, while PLC- enzymes appear to be coupled to growth factor receptors via tyrosine kinase phosphorylation. It is thought that individual PLCs within a class are coupled to a single type of receptor (eg. G-protein-coupled receptors), yet this has not been proven because so few PLC subtypes have been linked to a particular receptor or cellular process in vivo. Moreover, it is unknown whether splice-variant subtypes of PLC are utilized in the same signaling pathway, a particular type of signaling pathway, or in completely diverse and separate signaling pathways.

A PLC for which a function in vivo has been identified is encoded by the norpA gene of Drosophila melanogaster. Strong mutations in the norpA gene have long been known to abolish the photoreceptor potential, rendering the fly blind (Hotta and Benzer, 1970; Pak et al., 1970). The identification of the norpA gene product as a PLC emerged from findings that norpA mutations severely reduce PLC activity in eye (Yoshioka et al., 1985) and that the deduced protein is similar in structure and amino acid sequence to mammalian PLC (Bloomquist et al., 1988). These findings, along with a large body of evidence indicating that invertebrate phototransduction is mediated by PLC, led to the conclusion that the norpA gene encodes a PLC that is utilized in phototransduction in Drosophila (Bloomquist et al., 1988; Pak and Shortridge, 1991).

However, norpA is also known to be a complex gene whose expression is not limited to the retina nor utilized exclusively in phototransduction. Zhu et al.(1993) demonstrated that the norpA gene is expressed in embryo, adult body (thorax and abdomen), leg, and brain, as well as retina. Since body, leg, and brain are not known to contain visual machinery, it was proposed that the norpA-encoded PLC is utilized in signaling pathways in addition to phototransduction. Zhu et al.(1993) also observed different sizes of norpA transcripts on Northern analyses, raising questions whether norpA transcripts are alternatively spliced and if this leads to yet unidentified subtypes of norpA protein. If so, this would be in parallel with three other PLC- genes that are highly homologous to norpA (Shortridge et al., 1991; Ferreira et al., 1993; Bahk et al., 1994). It is unknown what role the cognate splice-variant subtypes play in signaling processes.

An advantage of using the norpA mutant of Drosophila as a model to study PLC function is that it is much easier to deduce the identity of the signaling pathways involved by examining the mutant phenotype. Here, we demonstrate that the norpA gene encodes at least two subtypes of PLC by alternative splicing of the nascent RNA and that each is likely to be utilized in completely different signaling pathways.


MATERIALS AND METHODS

Drosophila Strains

D. melanogaster white (w) mutant was used in all experiments as a control group because it lacks visual pigment, which can interfere with some assays, and its genetic background is most similar to that of norpA mutants used in experiments. Hereafter, w is referred to as wild type because it is wild type for norpA expression. The norpA/w mutant was used in all studies as a negative control and is known to exhibit total blindness (Bloomquist et al., 1988), to have severely reduced amounts of phospholipase C activity in eye, and to lack detectable amounts of norpA protein (Zhu et al., 1993). The norpA mutant is known to express small amounts of norpA RNA.()The eya (eyes absent) mutant (Bonini et al., 1993), which completely lacks compound eyes, was used for comparisons to identify norpA gene products that are expressed in eye.

Cloning and Sequence Analysis of norpA cDNAs

norpA cDNAs were obtained by reverse transcribing Drosophila poly(A) RNA followed by amplifying the norpA cDNA using the polymerase chain reaction (RT-PCR) (Fuqua et al., 1990). Poly(A) RNA used as template for RT-PCR was prepared from eya mutant body (thorax and abdomen) and leg according to methods described by Shortridge et al.(1991). eya mutant was used as a source for body RNA to obviate the problem of having the preparations contaminated with tissue from the compound eye. Priming of the poly(A) RNA in reverse transcription reactions was done using oligo-d(T) as well oligonucleotide primers that are complementary to the norpA cDNA (Bloomquist et al., 1988). Amplification of norpA cDNA by PCR was carried out using three sets of oligonucleotide primers made to overlapping segments of the norpA cDNA (nucleotides 594-1956, 1796-2551, 2417-4051; numbering according to Bloomquist et al. (1988)). During synthesis of the PCR primers, restriction endonuclease sites were added to the 5`-ends to facilitate cloning into plasmids (Kaufman and Evans, 1990). Standard methods (Sambrook et al., 1989) were used for cloning the amplified norpA cDNA fragments into plasmid vectors, transforming the recombinant plasmids into Escherichia coli, and determining the nucleotide sequence of the cloned inserts. A set of 34 oligonucleotide sequencing primers made against various regions along the norpA cDNA were used to facilitate sequencing of the cloned DNAs.

To map the location of exons of norpA (3, 4, 4A, and 5) within ge-nomic DNA, a cosmid clone (COSM1) that contains the norpA gene (Bloomquist et al., 1988) was used as template for PCR reactions. PCR was carried out on COSM1 DNA using oligonucleotide primers made to sites within exons. Location of the exons within the genomic DNA and approximate size of the intervening sequences was deduced by identifying and sizing the amplified products on agarose gels. Nucleotide sequences at intron/exon boundaries, as well as within the exons, was determined by carrying out DNA sequencing reactions on COSM1 DNA using oligonucleotide primers made against sites within the exons.

Northern Blots

Northern blots were carried out as described by Zhu et al.(1993) except that DNA probes were used. Probes were constructed by amplifying an 80-bp region of norpA cDNA corresponding to either exon 4 or exon 4A by the polymerase chain reaction (Innis et al., 1990). The amplified DNA fragments were purified by fractionation on a 2% agarose gel and extraction of the DNA from an excised plug of agarose (Sambrook et al., 1989). Radiolabeling of the DNA probes were carried out by primer extension reactions using Klenow polymerase in the presence of [P]dCTP (Sambrook et al., 1989).

RT-PCR Analyses

RNA used as template for RT-PCR was prepared by grinding approximately 100 µg of tissue in 1 volume of homogenizing buffer (0.15 M sodium acetate, 5 mM EDTA, 1% SDS, 50 mM Tris-Cl, pH 9.0), volume of phenol, and volume of a mixture of chloroform/isoamyl alcohol (100:1) (Montell et al., 1985) using Teflon pestles in 1.5-ml microcentrifuge tubes (Kontes). The homogenates were extracted once with chloroform/isoamyl alcohol, four times with phenol/chloroform (1:1), and once with chloroform/isoamyl alcohol, followed by precipitation of the RNA in ethanol (Sambrook et al., 1989). RNA was resuspended in water, and its concentration was estimated from the absorbance at 260 nm (Sambrook et al., 1989). Approximately 5 µg of RNA was used as template for reverse transcription. Next, a 1,363-bp norpA cDNA fragment (nucleotides 594-1956) that encompasses the site of alternative splicing was amplified by PCR, and the amplified products were analyzed by Southern blotting after fractionating on 1% agarose gels (Sambrook et al., 1989). The alternatively spliced norpA products were detected by hybridizing to digoxygenin-labeled 80-bp probes that comprise either norpA exon 4 or exon 4A. Digoxygenin labeling of probes, hybridization reactions, and visualization in nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate was carried out using the Genius system (Boehringer Mannheim) according to the manufacturer's instructions.

In Situ Hybridization

10-micron-thick frozen sections of adult head (0-4 days after eclosion) were prepared on glass slides as described by Zhu et al.(1993). In situ hybridization of norpA exon 4 or exon 4A probes to the tissue sections were carried out using modifications of the procedure of Tautz and Pfeifle(1989). Tissue sections were air dried for approximately 4 h after cutting followed by fixation for 20 min in a freshly made solution of 4% paraformaldehyde in 1 PBS (150 mM NaCl, 10 mM sodium phosphate, pH 7.22). After fixing, tissues were rinsed in 3 PBS and then 1 PBS for 5 min each followed by dehydration in a graded (30-100%) ethanol series (Hafen et al., 1983) and air drying for 2 h. The dried tissue sections were treated by rehydrating for 5 min in 1 PBS and then immediately incubating in 0.2 N HCl for 20 min. The treated sections were then incubated in 2 SSC (1 SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) for 30 min at 65 °C followed by digesting for 3 min in 1 PBS containing 50 µg/ml proteinase K. Following proteinase K digestion, sections were incubated for 2 min in 1 PBS containing 2 mg/ml glycine and then fixed as before in a solution of 4% paraformaldehyde in PBS. After rinsing in PBS, tissues were acetylated by incubation in 500 ml of 0.1 M triethanolamine-Cl, pH 8.0, to which 0.625 ml of acetic anhydride had freshly been added. Sections were then rinsed twice in PBS for 5 min each, 2 SSC for 5 min, and then dehydrated in a graded ethanol series as before.

Probes were generated by terminal labeling of 80-bp DNA fragments that correspond to norpA exon 4 or exon 4A using terminal transferase in the presence of digoxygenin 11-dUTP (Boehringer Mannheim) according to manufacturer's instructions. The treated tissue sections were hybridized overnight at 37 °C in hybridization solution (35% deionized formamide, 5% dextran sulfate, 1 Denhardt's solution (0.2 mg/ml Ficoll, 0.2 mg/ml bovine serum albumin, 0.2 mg/ml polyvinylpyrrolidone), 5 SSC, 100 µg/ml salmon testes DNA, and 25 µg/ml E. coli tRNA), to which the digoxygenin-labeled DNA probe was added. Following hybridization, the slides were washed by incubating at 37 °C in fresh hybridization solution for 20 min followed by incubations in 50% hybridization solution in 1 PBS and then 1 PBS for 20 min each. Visualization of the digoxygenin-labeled probe was carried out according to instructions in the Genius Kit (Boehringer Mannheim) using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as a chromogen.


RESULTS

Cloning and Sequence Analysis of Splice-variant Subtypes of norpA

Zhu et al.(1993) proposed that the norpA-encoded PLC is not exclusively utilized in phototransduction because norpA expression is not solely found in retina but is also present in tissues not known to contain visual system machinery. Moreover, Zhu et al.(1993) identified at least three major sizes of norpA transcripts (one in adult eye and two in adult body), suggesting that norpA RNA is alternatively spliced. To ascertain whether norpA RNA encodes other subtypes of norpA protein, we utilized RT-PCR (Fuqua et al., 1990) to obtain norpA cDNA from body (thorax and abdomen) and leg and then compared the nucleotide sequences of the amplified products to the head-specific cDNA sequence described by Bloomquist et al. (1988).

Amplification of norpA cDNA was carried out using PCR primer sets (see ``Materials and Methods'') that are complimentary to the previously reported norpA cDNA (Bloomquist et al., 1988). A variety of primer set combinations were tested in the RT-PCR reactions, and only single amplification products were observed (data not shown). Nucleotide sequences of the amplified norpA cDNA matched the norpA sequence reported by Bloomquist et al.(1988) except within a small region of 80 nucleotides occurring between nucleotides 1038 and 1119 (Fig. 1). This difference was found in both body and leg cDNA and corresponds to a region of the norpA cDNA that Masai and Hotta(1991) described as exon number 4. However, the exon 4 domain of the Bloomquist sequence is missing from these amplified cDNAs. The region that replaces exon 4 in these body and leg tissues was designated as exon 4A. No other sequence differences were found between the Bloomquist sequence and the norpA cDNA obtained from body or leg.


Figure 1: Nucleotide sequences of alternatively spliced regions of the norpA gene. The nucleotide sequences of exon 4 and exon 4A are shown in capitalletters with intervening sequences at the junction sites shown in lowercaseletters. Numbersbelow the sequences correspond to the location of these exons in the norpA cDNA reported by Bloomquist et al. (1988). Consensus sequences at intron/exon junctions in Drosophila (Mount et al., 1992) are shown above the proposed intron/exon boundary regions. M indicates A or C, R indicates A or G, Y indicates C or T, W indicates A or T, and N is any nucleotide. Invariant positions within the splicing consensus are overlined, and the angled line marks the splice site. Notice that the nucleotides at the 5`- and 3`-boundaries of exon 4A match splicing consensus sequences exactly, strongly supporting the proposal that exon 4A is an alternatively spliced exon.



To determine whether the putative exon 4A occurs within the norpA gene, we performed DNA sequence analysis on the norpA genomic clone, COSM1 (Bloomquist et al., 1988). Indeed, there is a region of COSM1 DNA that matches exon 4A exactly (Fig. 1). Moreover, the nucleotide sequences surrounding exon 4A match consensus sequences found at intron/exon boundaries in other Drosophila genes (Fig. 1) (Mount et al., 1992). Furthermore, exon 4A localizes approximately 300 bp upstream from exon 4 in the genomic DNA (Fig. 2, panelA). Since exon 4A is present within norpA genomic DNA as well as norpA cDNAs from body and leg, and the nucleotides surrounding exon 4A match those at splice sites in other Drosophila genes, we conclude that exon 4A is an alternatively spliced exon that is retained in some norpA transcripts when exon 4 is excluded.


Figure 2: Splice-variant subtypes of phospholipase C encoded by the norpA gene. PanelA schematically illustrates the proposed mutually exclusive splicing of norpA RNA and relative location of the exons within genomic DNA. Exon 4A is located in the genomic DNA approximately 300 nucleotides upstream of exon 4. PanelB schematically illustrates the deduced subtypes of norpA protein and the location of amino acid differences. The highly conserved box X and box Y regions (Rhee and Choi, 1992a, 1992b) that are present in major types of PLC are represented by cross-hatched rectangles. Numbersabove the schematic of subtype I protein refer to locations of significant regions within norpA protein (Bloomquist et al., 1988). PanelC shows an alignment of amino acids within the alternatively spliced region of norpA protein with corresponding regions in other PLC- isozymes (Dros plc21 (Shortridge et al., 1991), bovine PLC-1 (Suh et al., 1988), human PLC-2 (Park et al., 1992), rat PLC-3 (Jhon et al., 1993), and bovine PLC-4 (Ferreira et al., 1993, Lee et al., 1993a)). There are 12 amino acids in this region that are identical in norpA subtype I and subtype II proteins, and 4 of these are invariantly conserved among the PLC- enzymes (indicated by asterisksabove the alignment). Numbers refer to the location of the amino acids within each PLC- enzyme, and those in parentheses refer to tentative numbering assigned to rat PLC-3 because the N terminus of the mature protein is not yet identified (Jhon et al., 1993).



We have designated the putative norpA protein encoded by an exon 4-containing transcript as subtype I protein and one that is encoded by an exon 4A-containing transcript as subtype II. The differences between deduced norpA subtype I and subtype II proteins occur within a small domain of 26 amino acids(130-155), which localizes outside of the highly conserved box X and box Y domains (Rhee et al., 1989) shared among major types of PLC (Fig. 2, panelB). Within the domain of 26 amino acids, 12 are identical in both subtypes of norpA protein. Moreover, alignment of this region with similar domains in other PLC- enzymes reveals that four of the conserved residues occur at invariant positions (Fig. 2, panelC).

Northern Analysis of Adult RNA

When radiolabeled norpA cDNA probes are hybridized to blots of poly(A) RNA, three major transcripts can be identified. As shown in Fig. 3(panelA), a major norpA transcript that is 7.5 kb in length is easily detected in wild-type head but is absent from head of eya mutant. The absence of the 7.5-kb transcript from eya head suggests that it is expressed in the compound eye. Two other transcripts, one that is 5.5 kb and one that is 5.0 kb in length, are visible in body. None of these transcripts are detectable in head or body of norpA mutant (Zhu et al., 1993), suggesting that they are encoded by the norpA gene.


Figure 3: Northern blot analysis of norpA transcripts in adult Drosophila tissues. Approximately 5 µg of poly(A) RNA was loaded in each lane and probed with a 3.4-kb norpA cDNA fragment (nucleotides 1-3453) (A), an 80-bp exon 4 cDNA fragment (B), or an 80-bp exon 4A cDNA fragment (C). Lane designations indicate RNA isolated from adult head or body (thorax and abdomen) of wild-type (WT) Drosophila, eyes absent (eya) mutant, or norpA mutant. Mobility of RNA size standards (in kilobases) are indicated on the right. PanelsD-F show the result of reprobing the blots with Drosophila RP49 cDNA (O'Connell and Rosbash, 1984) as a control to test for RNA loading. The 3.4-kb norpA cDNA probe detects three major transcripts in adults (arrows in panelA). The largest is approximately 7.5-kb in length and is visible in the wild-type head RNA lane but not in head of eya mutant (panelA). Two other transcripts of 5.5 and 5.0 kb (arrows) appear in wild-type body as well as body of eya mutant. None of these bands are visible in RNA obtained from head or body of norpA mutant (data not shown). Notice that norpA subtype I (exon 4) probe detects the 7.5-kb transcript in head but not the 5.5- or 5.0-kb transcripts in body (panelB). Conversely, the subtype II (exon 4A) probe detects the 5.5- and 5.0-kb transcripts in body but not the 7.5-kb transcript in head (panelC). None of these transcripts are detectable in norpA mutant tissues. The significance of two major bands (5.0 and 4.4 kb) in wild-type head (panelA) or smearing in the head RNA lanes is unknown (Zhu et al., 1993). However, these other bands and smearing are not visible in head RNA prepared from Canton-S strain (data not shown). Differences in signal intensity between body lanes (panelsA and C) should not be taken to reflect relative amounts of RNA but is likely due to experimental fluctuations augmented by weak signals and extensive autoradiographic exposure times.



To differentiate between which of the bands appearing on the Northern analysis are norpA subtype I or subtype II transcripts, we radiolabeled 80-mer DNA probes that correspond to norpA exon 4 or exon 4A and used them individually as probe in the hybridization reactions. As shown in Fig. 3(panelB), exon 4 hybridizes to a 7.5-kb transcript that is present in wild-type head but is absent from body as well as eya mutant head and norpA mutant head. This 7.5-kb transcript appears to be the same 7.5-kb transcript identified by hybridization to the much longer norpA DNA probes (Fig. 3, panelA). Conversely, exon 4A probe specifically hybridizes to 5.5- and 5.0-kb transcripts in wild-type body and eya mutant body but does not detect transcripts in wild-type head or tissues of norpA mutant (Fig. 3, panelC). Again, these appear to be the same transcripts detected by the longer norpA DNA probes. These data suggest that the distributions of the two subtypes of norpA RNA are mutually exclusive and that norpA subtype I (exon 4) transcripts are expressed in eye and that subtype II (exon 4A) transcripts are expressed in body. In similar Northern analyses, norpA transcripts could not be detected at earlier times of development when the 80-mer exon-specific DNA probes were used (data not shown).

RT-PCR Analysis of Tissue and Developmental Distribution of norpA RNA

To corroborate the results from the Northern analyses and to identify tissues expressing norpA transcripts in too low abundance to detect on Northern blots, we utilized RT-PCR (Fuqua et al., 1990) to amplify the norpA products from tissue- and developmentally specific RNA pools. RNA used in the RT-PCR analyses was prepared from 0-12-h-old embryo, 12-24-h embryo, 3-, 5-, 7-, and 9-day-old developing organisms, and head, leg, thorax, and abdomen of adult. The 3-, 5-, 7-, and 9-day-old Drosophila correspond approximately to first and second instar larvae, third instar larvae, early pupae, and late pupae, respectively (Zhu et al., 1993). A single primer set was used in the PCR amplification reactions so that both splice-variant products would be amplified as 1,363-bp DNA fragments (see ``Materials and Methods''). Differentiation between subtype I and subtype II amplification products was carried out by hybridization to individual 80-bp exon 4 or exon 4A probes.

As shown in Fig. 4, norpA subtype I (exon 4) transcripts can easily be detected by RT-PCR in wild-type head and to a lesser extent in eya head and norpA mutant head, but not in body or at earlier developmental times. Conversely, norpA subtype II transcripts are detectable in all lanes tested but most easily detected in adult body (thorax and abdomen) (Fig. 4). These data are in agreement with the Northern blot results that show norpA subtype I RNA is most abundantly found in head, more specifically in compound eye, and subtype II RNA to be most abundant in body. However, the ability of RT-PCR assays to detect lower abundance transcripts refine and expand the Northern blots by identifying other tissues and developing times that express norpA.


Figure 4: RT-PCR analyses showing tissue and developmental distribution of norpA subtype I and II RNA. Total RNA was extracted from tissues, reverse transcribed into cDNA, and used as template for PCR to generate 1,363-bp norpA cDNA products. The amplified DNAs were separated on 1% agarose gels, blotted to nylon filters, and probed with a digoxygenin-labeled norpA exon 4 or exon 4A probe (see ``Materials and Methods''). Lanes are norpAHead, norpA, mutant head; eyaHead, eyes absent mutant head; WT Head, wild-type head; Thorax, wild-type thorax; Abdomen, wild-type abdomen; Leg, wild-type legs; 9Day, 7Day, 5Day, and 3Day, developing Drosophila (wild type) collected on the days indicated after egg laying; 12-24 Hour, 12-24-h-old (wild-type) embryos; 0-12 Hour, 0-12-h-old (wild-type) embryos. Notice that the norpA subtype I (exon 4) probe easily detects amplification products (arrow) from wild-type head and to a lesser extent from head of norpA mutant and eya mutant (upperpanel). Conversely, subtype II (exon 4A) probe detects amplification products from all of the tissues tested, but signals are most intense in thorax and abdomen of adults (arrow, lowerpanel).



Spatial Localization of norpA Transcripts in Head

To determine the spatial localization of norpA transcripts in adult head, the 80-bp exon-specific DNAs were used as probe in tissue in situ hybridization reactions. As shown in Fig. 5, norpA subtype I transcripts (exon 4) appear to localize specifically in retina. norpA subtype II transcripts (exon 4A) are not detectable in retina, but weak signals are visible over the cortex of brain (Fig. 5). This localization of norpA expression in brain and retina agrees with findings of Zhu et al.(1993) that norpA protein is localized in brain as well as retina. Moreover, these results agree with those from the Northern analyses that show subtype I transcripts are localized in the compound eye.


Figure 5: Spatial localization of subtypes of norpA RNA in adult head. Digoxygenin-labeled norpA DNA probes were used for in situ hybridization analysis on 10-µm tissue sections cut horizontally through head. Signals were developed with an anti-digoxygenin antibody conjugated to alkaline phosphatase using 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate as chromogen. Anterior is up in both panels. The 80-bp norpA exon 4 probe (specific for subtype I RNA) hybridizes to transcripts in retina (r), but no signals are seen in brain (b) (panelA). Conversely, exon 4A probe (norpA subtype II) does not detect transcripts in retina, but weak signals do appear over the cortex of brain (arrows, panelB).




DISCUSSION

Mutations in the norpA gene have long been known to drastically affect the light-evoked responses of photoreceptor cells, and it is well accepted that the norpA gene encodes phospholipase C that is essential for phototransduction (Bloomquist et al., 1988; Pak and Shortridge, 1991; Ranaganathan et al., 1991). However, norpA gene products have also been proposed to operate in other signaling pathways in addition to phototransduction (Zhu et al., 1993). Here, we have identified a second subtype of norpA (designated subtype II) and find that it is not detectable in retina but is easily detected in other tissues not known to contain phototransduction machinery. Conversely, norpA subtype I transcripts are easily detectable in retina but not easily detected in other tissues. Since norpA subtype I transcripts are found in retina, but subtype II transcripts are not, we conclude that norpA subtype I protein is utilized in phototransduction. Moreover, norpA subtype II transcripts are found in tissues not known to contain phototransduction machinery, indicating that subtype II protein is utilized in signaling pathways other than phototransduction.

The results of the RT-PCR analyses showing that norpA subtype I transcripts are present in head of eya mutant (Fig. 4) do not contradict results from the Northern blot analyses, which fail to detect subtype I expression in eya head (Fig. 3). RT-PCR is known to be much more sensitive than Northern blots in detecting very low amounts of RNA products (Fuqua et al., 1990). Even though eya mutants lack compound eyes, the ocellar visual organs on the top of the head are present (Bonini et al., 1993). Moreover, the light-evoked responses in ocelli are known to be abolished by strong norpA mutations (Hu et al., 1978). Inasmuch as norpA subtype I protein is utilized in the phototransduction pathway in compound eyes, it is logical to expect that the ocellar phototransduction pathway utilizes subtype I protein as well. Thus, the norpA subtype I transcripts detected in eya mutant head by RT-PCR may be derived from the ocellar visual organs and are too low in abundance in eya head RNA pools to be detected by Northern blot analysis.

Moreover, the capacity of RT-PCR analyses to detect low abundance transcripts is likely the reason that RT-PCR detects norpA subtype II transcripts in wild-type head (Fig. 4), but these are not observed in the Northern analysis (Fig. 3). Zhu et al. (1993) reported that norpA protein is expressed in brain as well as compound eyes and ocelli. This localization in brain is in complete agreement with the tissue in situ hybridization analyses shown in the present work that localizes norpA subtype II RNA in brain (Fig. 5). However, these signals are only weakly visible in brain whenever the tissue in situ hybridization reactions are carried out on head (data not shown). Thus, it is likely that norpA subtype II transcripts are not expressed in sufficient amounts in head to be detected by Northern blot analysis. Furthermore, the ability of RT-PCR to detect low abundance transcripts is why norpA RNA is detected in norpA mutant (Fig. 4).

Currently, it is unclear how many signaling pathways utilize the different subtypes of norpA or if there are more splice-variant subtypes yet to be identified. norpA mutants were originally identified as a result of their visual system defect (Hotta and Benzer, 1970; Pak et al., 1970). norpA mutants have also been shown to exhibit altered mating behavior (Markow and Manning, 1980; Tompkins et al., 1982) as well as defective olfactory responses in maxillary palps (Riesgo-Escovar et al., 1995). There are likely additional mutant characteristics that remain unidentified, especially since the presence of norpA expression in brain and leg point to involvement in signaling pathways other than those affecting the above mentioned mutant defects. While these data suggest that upwards of three completely different signaling processes utilize the norpA-encoded PLC, results of the present work indicate that the two identified subtypes of norpA comprise the major splice-variant forms. In the Northern blot analyses, the two exon-specific probes detect all three of the major transcripts of norpA that are identified when much longer cDNA probes are used (Fig. 3). However, this does not preclude the possibility that there are yet unidentified splice-variant forms of norpA that are expressed only in a small subset of cells or in a very low abundance in tissues.

The significance of the present work lies in its contribution to defining the role of subtypes of PLC in cellular signaling. As previously mentioned, phosphatidylinositol-specific PLC is known to be a family of proteins that have been classified into major groups (PLC-, PLC-, PLC-, and PLC-) that can be further divided into subtype groupings (Rhee and Choi, 1992a, 1992b). Recent findings indicate that subtype groupings may be further divided into splice-variant products that are encoded by single genes. Including the present work, four different PLC- encoding genes that produce splice-variant subtypes have been identified (Shortridge et al., 1991; Ferreira et al., 1993; Bahk et al., 1994). Comparison of the splicing differences in these PLCs reveals that all occur outside of the box X and box Y conserved regions shared among the major PLC types (Fig. 6). A growing body of evidence suggests that the regions outside of the box X and box Y conserved domains comprise regulatory sites responsible for interacting with signaling components such as G-proteins (Lee et al., 1993b; Wu et al., 1993a, 1993b). It is thus likely that the differences among subtypes caused by alternative splicing are a reflection of the need for each subtype to interact with individual signaling components of different signal-generating pathways. This idea is supported by the results of the present work showing that the two subtypes of norpA likely operate in completely different types of signaling pathways, one being phototransduction and the others, although yet unidentified, are not phototransduction pathways.


Figure 6: Differences in splice-variant subtypes of PLC-. Schematic representations of four different PLC- enzymes are shown: norpA, Drosophila norpA-encoded PLC (Bloomquist et al., 1988); plc21, Drosophila plc21-encoded PLC (Shortridge et al., 1991); PLC-1, rat PLC-1 (Suh et al., 1988; Bahk et al., 1994); and PLC-4, bovine PLC-4 (Ferreira et al., 1993; Lee et al., 1993a). The cross-hatched rectangles represent the box X and box Y regions that are highly conserved among major types of PLC (Rhee and Choi, 1992a, 1992b). Splice-variant differences between individual subtypes are illustrated by stippledboxesabove or below each schematic. Numbersabove each schematic represent amino acid positions at boundaries of the splice-variant domains, and those in parenthesesbelow the names indicate total length (in amino acids) or the range of length of the PLC subtypes. Numbering of PLC-4 is according to Lee et al. (1993a). Note that all of the splicing differences occur outside of the box X and box Y conserved domains.



Transformation of a norpA subtype I mini-gene into the germ line of norpA mutant has recently been shown to rescue the phototransduction defect concomitant with rescuing PLC activity in retina (McKay et al., 1995). A future experiment may include the expression of norpA subtype II protein in mutant retina to see if it can substitute for subtype I and rescue the visual defect. If it turns out that norpA subtype II protein is able to provide PLC activity in retina without rescuing the light-evoked responses of photoreceptors, it would demonstrate that the 14 amino acids found in subtype I protein but absent from subtype II are critical for interaction with components of the phototransduction machinery.


FOOTNOTES

*
This work was supported by the National Science Foundation Grant IBN-9120866. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: State University of New York at Buffalo, Dept. of Biological Sciences, Cooke Hall, Rm. 109, Buffalo, NY 14260-1300. Tel.: 716-645-3122; Fax: 716-645-2975; E-mail: rds@ubvms.cc.buffalo.edu.

The abbreviations used are: PLC, phospholipase C; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair(s); kb, kilobase(s).

S. Kim, R. R. McKay, K. Miller, and R. D. Shortridge, unpublished results.


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