a novel drosophila alkaline phosphatase speciﬁc to the ... · the nearer encodes aph-4, the...

Copyright 2000 by the Genetics Society of America

A Novel Drosophila Alkaline Phosphatase Specific to the Ellipsoid Bodyof the Adult Brain and the Lower Malpighian (Renal) Tubule

Ming Yao Yang, Zongsheng Wang, Matthew MacPherson, Julian A. T. Dow and Kim Kaiser

Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, United Kingdom

Manuscript received June 15, 1999Accepted for publication September 20, 1999

ABSTRACTTwo independent Drosophila melanogaster P{GAL4} enhancer-trap lines revealed identical GAL4-directed

expression patterns in the ellipsoid body of the brain and in the Malpighian (renal) tubules in theabdomen. Both P-element insertions mapped to the same chromosomal site (100B2). The genomic locus,as characterized by plasmid rescue of flanking DNA, restriction mapping, and DNA sequencing, revealedthe two P{GAL4} elements to be inserted in opposite orientations, only 46 bp apart. Three genes flankingthe insertions have been identified. Calcineurin A1 (previously mapped to 21E-F) lies to one side, andtwo very closely linked genes lie to the other. The nearer encodes Aph-4, the first Drosophila alkalinephosphatase gene to be identified; the more distant gene [l(3)96601] is novel, with a head-elevatedexpression, and with distant similarity to transcription regulatory elements. Both in situ hybridization withAph-4 probes and direct histochemical determination of alkaline phosphatase activity precisely matchesthe enhancer-trap pattern reported by the original lines. Although the P-element insertions are notrecessive lethals, they display tubule phenotypes in both heterozygotes and homozygotes. Rates of fluidsecretion in tubules from c507 homozygotes are reduced, both basally, and after stimulation by CAP2b,cAMP, or Drosophila leucokinin. The P-element insertions also disrupt the expression of Aph-4, causingmisexpression in the tubule main segment. This disruption extends to tubule pigmentation, with c507homozygotes displaying white-like transparent main segments. These results suggest that Aph-4, whilepossessing a very narrow range of expression, nonetheless plays an important role in epithelial function.

ALKALINE phosphatase (ALP) is a zinc and mag- strong 1972, 1973; Parkash et al. 1993), but none hasbeen previously characterized at the molecular geneticnesium-containing metalloenzyme (EC 3.1.3.1)level.that hydrolyzes phosphate esters with a high pH opti-

The enhancer-trap technique has proved valuable formum. It is found in most species from bacteria to hu-identification and isolation of genes with particular spa-mans. In Escherichia coli, ALP (encoded by the genetial or temporal patterns of expression (O’Kane andphoA) is found in the periplasmic space. In yeast, ALPGehring 1987; Bier et al. 1989; Wilson et al. 1989). We(encoded by the gene PHO8) is found in lysosome-likehave recently used the P{GAL4} enhancer-trap systemvacuoles. And in mammals, it is a glycoprotein attached(Brand and Perrimon 1993) to generate and screento the membrane by a glycosylphosphatidylinositolover 1400 independent Drosophila lines for patterns of(GPI) anchor (McComb et al. 1979; Trowsdale et al.GAL4-directed b-galactosidase expression in the adult1990).brain (Yang et al. 1995) and in the Malpighian (renal)In mammals, four different ALP isozymes are cur-tubules (Sozen et al. 1997). Two lines with identicalrently known, each encoded by at least one separateexpression characteristics (c507 and c232) were selectedgene. Three are tissue specific: the placental, placental-by both criteria and analyzed in detail. Expression inlike (germ cell), and intestinal isozymes. The fourththe brain is limited to only a small subset of neuronsform, previously known as the liver/bone/kidney iso-belonging to the ellipsoid body of the central complex,zyme, is tissue nonspecific and has physical and bio-a structure implicated in the coordination of motorchemical properties that clearly distinguish it from theactivity (Strauss and Heisenberg 1993; Ilius et al.other ALP isozymes (Harris 1989; Manes et al. 1990).1994). Expression in the Malpighian tubules is restrictedThere are also several independent ALP loci in Dro-to a zone associated with fluid readsorption (O’Don-sophila melanogaster (Yao 1950; Beckman and Johnsonnell and Maddrell 1995; Sozen et al. 1997).1964; Schneiderman et al. 1966; Harper and Arm-

Here we report the characterization of the genomicregion encompassing the P{GAL4} elements in linesc507 and c232. We find the expression patterns reported

Corresponding author: Kim Kaiser, Division of Molecular Genetics,by their LacZ enhancer detectors to correspond to thatUniversity of Glasgow, 56 Dumbarton Rd., Glasgow G11 6NU, United

Kingdom. E-mail: [email protected] of a nearby ALP gene (Aph-4), implying a defined role

Genetics 154: 285–297 ( January 2000)

286 M. Y. Yang et al.

Double-stranded sequencing reactions using the dideoxyfor this isozyme in specific aspects of neural and renalchain termination method were carried out as described infunction.the Sequenase Version 2.0 manual (United States BiochemicalCorp., Cleveland). The nucleotide sequence of both strandswas obtained for all transcribed regions. Intron sequences

MATERIALS AND METHODS were sometimes read on one strand only. DNA sequenceswere analyzed using MacVector and AssemblyLIGN (SequenceDrosophila strains: P{GAL4} lines described here were iso-Analysis Software), BLAST and Prosite (NCBI), GCG (Wiscon-lated in our laboratory (Yang et al. 1995). The secondarysin), and SeqVu (Garvan Institute).reporter for GAL4 activity was a second chromosome insertion

Total Drosophila RNA was isolated using the acidic guanidi-of UASG-lacZ (Brand and Perrimon 1993). There is no visiblenium isothiocyanate method (Chomczynski and Sacchi

b-gal expression in the absence of GAL4. P{lacW} lines were1987). Poly(A)1 RNA was selected by oligo(dT) chromatogra-kindly provided by Deak et al. (1998) and P{PZ} line l(3)07028 phy, separated in 1.0% 3-[N-morpholino]propane-sulfonicwas kindly provided by A. Spradling’s laboratory. All genetics acid (MOPS)/formaldehyde denaturing gels, transferred tomarkers used in the work were described by Lindsley and nitrocellulose, and probed with radiolabeled cDNA fragments.Grell (1968). Flies were maintained on standard cornmeal/ Hybridization was carried out at 428 in 50% formamide, 53yeast/agar medium at 258 (Ashburner 1989). SSPE, 0.04% bovine serum albumin/0.04% polyvinylpyrroli-Histochemistry: To obtain sections, recombinant flies car- done/0.04% Ficoll/0.1% sodium dodecyl sulfate (SDS).rying P{GAL4} and P{UASG-lacZ} were mounted in “fly collars” These filters were then stripped and reprobed with rp49

(modified from Heisenberg and Boehl 1979), soaked in (O’Connell and Rosbach 1984) as a loading control.OCT embedding medium (Miles, Kankakee, IL) for 10 min In situ hybridization: The procedure for in situ hybridizationand then embedded in the OCT medium. Twelve-micrometer to third larval instar polytene chromosomes was essentially asserial sections of head or body were cut in a cryostat (Anglia described by Pardue (1986). pBluescript and cDNAs wereScientific) at 2188. The sections were stained and mounted labeled with Bio-16-dUTP by nick-translation. Hybridizationas described by Yang et al. (1995). Thereafter sections were was detected using 393-diaminobenzidine tetrahydrochlorideexamined and photographed on a Nomarski optical micro- (DAB)/H2O2. After hybridization, the slides were stained withscope. Giemsa and mounted using DPX mountant.

For whole-mount preparations, brains or Malpighian tu- Nonradioactive in situ hybridization to tissues was carriedbules were dissected in PBS and fixed in 4% paraformaldehyde out essentially as described by Nighorn et al. (1991). Relevantfor 20 min. They were then washed three times for 20 min cDNA fragments were used as templates for the synthesis ofin PBS and stained with staining buffer and 2% X-gal for 1–2 DIG-labeled single-strand RNA probes according to the manu-hr at 378. They were then washed for 20 min in PBS, cleared facturer’s instructions (Boehringer Mannheim). Heads andovernight at 48 with PBS/12.5% hydrogen peroxide, washed bodies of wild-type flies were cut into 12-mm sections using afor 10 min with PBS, dehydrated through graded ethanol, cryostat (Yang et al. 1995) and hybridized with the relevantand mounted in glycerol gelatin. probe.

For histochemical detection of alkaline phosphatase activity, Hybridization signals were detected by two different meth-brains or Malpighian tubules were dissected in PBS and fixed ods. (1) Colorimetric detection with NBT and X-phosphate.in 4% paraformaldehyde for 20 min. They were then washed For immunological detection of the hybridized probes, thethree times for 20 min in PBS and stained with NBT/X-phos in sections were washed in PBT and incubated with 200 ml ofdigoxigenin (DIG) detection buffer for up to 1 hr (Boehringer 5% (v/v) sheep serum in PAT for 2–3 hr. The sections wereMannheim, Indianapolis). Finally, they were washed in PBS then incubated with 150–200 ml of anti-digoxigenin antibodyand mounted in glycerol gelatin (Sigma, St. Louis). conjugated with alkaline phosphatase (Boehringer Mann-

Assay of tubule secretion phenotype: Tubules were dissected heim) diluted at 1:500 in PAT for 2–3 hr at room temperaturefrom adult (3–7-day-old) flies and placed in 10 ml drops of or overnight at 48. The sections were washed twice in PBT.1:1 Schneider’s medium:Drosophila saline under paraffin oil. After extensive washes in 100 mm Tris-HCl, pH 9.5, 100 mmFluid secretion experiments were performed as described pre- NaCl, 50 mm MgCl2, and 2 mm levamisole, sections were placedviously (Dow et al. 1994). Drosophila leucokinin was a kind with 200 ml of diluted chromogenic substrate solutions (NBTgift of J. Veenstra (Bordeaux); CAP2b was a kind gift of N. J. and BCIP, X-phosphate) following the manufacturer’s instruc-Tublitz (Oregon). tions (Boehringer Mannheim), and incubated in the dark at

Molecular methods:Genomic sequences flanking the P{GAL4} room temperature for 2–4 hr. The reaction was stopped byelement were cloned by plasmid rescue using standard tech- washing in PBT for 20 min and preparations were mountedniques (Bier et al. 1989; Wilson et al. 1989). Briefly, genomic in glycerol gelatin (Sigma). (2) Colorimetric detection withDNA was digested with PstI to clone sequences “downstream” DAB and H2O2. Sections were washed in PBT and incubatedof the P{GAL4} element and with KpnI to clone sequences with 200 ml of 5% (v/v) sheep serum in PAT for 1–2 hr.“upstream” of the P{GAL4} element, followed by ligation to They were then incubated with 150–200 ml of anti-digoxigenin-form a plasmid, which was propagated in E. coli NM621. The horseradish peroxidase (anti-DIG-HRP) Fab fragments (Boeh-plasmids were used to screen a Drosophila genomic DNA ringer Mannheim), diluted at 1:500 in PAT for 2–3 hr at roomlibrary in the vector EMBL3 (K. Kaiser, unpublished results), temperature or overnight at 48. After extensive washes, signaland a head cDNA library in the vector NM1149 (Russell was detected with diaminobenzidine and hydrogen peroxide1989). The 59 rapid amplification of cDNA ends (RACE) pro- according to the manufacturer’s conditions (Boehringercedure was carried out using Superscript RT (Life Technolo- Mannheim). Reactions were stopped by washing in PBT forgies) according to the conditions recommended by the manu- 20 min and preparations were mounted in glycerol gelatinfacturer. (Sigma).

Southern blotting was carried out essentially as describedby Sambrook et al. (1989). Hybridization was carried out at648 in 63 SSC, 53 Denhardt’s reagent, 0.5% SDS, 100 mg/ RESULTSml denatured, fragmented salmon sperm DNA. Filters were

lacZ expression patterns in P{GAL4} lines c507, c232,washed in 13 SSC and 0.1% SDS for 15 min, and then in0.13 SSC and 0.1% SDS for 30 min. and P{PZ} line l(3)07028: Lines c232 and c507 report

287Drosophila Alkaline Phosphatase

Figure 1.—Expression patterns in en-hancer-trap lines and wild-type flies. (a andb) LacZ staining of ellipsoid body ring neu-rons in the adult brain of line c507. (c)Analogous section of a wild-type head show-ing Aph-4 mRNA expression detected by thestandard anti-DIG method. (d and e) LacZstaining of the lower Malpighian tubule andureter in line c507. (f) Analogous sectionof a wild-type body showing coexpressionof Aph-4 RNA as detected by the standardanti-DIG method in the presence of levami-sole. The lower right inset shows the hybrid-ization pattern seen using the DAB/peroxi-dase detection system. (g) LacZ reporterstaining of the lower Malpighian tubule andureter in semilethal line l(3)07028. (h) Or-egon R, showing wild-type natural pigmen-tation pattern of opaque main segmentsand lower tubules. (i) c507 homozygotes,displaying transparent main segments (remi-niscent of w2 mutants) and opaque lowertubules. (j) Wild-type tubule alkaline phos-phatase activity is concentrated exclusivelyin the ureter and lower tubule. (k) Alkalinephosphatase activity is concentrated in themain segment of the tubule in homozygousc507 flies. (l) Alkaline phosphatase activityis concentrated in the ureter, lower tubule,and main segment in heterozygous c507flies. CB, ring neuron cell bodies; LTR, lat-eral triangle; EB, ellipsoid body; LT, lowerMalpighian tubule; U, ureter; M, main seg-ment. Line c232 gives identical staining pat-terns.

identical, and extremely specific, expression patterns. Plasmid rescue of flanking genomic DNA from linesc507, c232, and l(3)96601: The enhancer-trap elementGAL4-directed b-gal expression in the adult occurs in

just the two cellular domains shown in Figure 1, a and contains a bacterial plasmid replicon flanked by uniquerestriction sites, permitting rescue of genomic DNAb. Expression in the brain corresponds to ring neurons

of the ellipsoid body, and more specifically to the R3 from either side of the insertion by appropriate choiceof enzyme (Wilson et al. 1989). Genomic DNAs fromand R4 morphological subtypes (Hanesch et al. 1989;

Renn et al. 1999). Neurites arising from lateral cell bod- P{GAL4} lines c507 and c232 were digested with PstI,ligated, and rescued to produce downstream (i.e., 39 toies (CBs) extend dendritic arbors within a region known

as the lateral triangle (LTR) and send axonal process GAL4 transcription unit) plasmids pPC507 and pPC232,respectively. Similarly, KpnI was used to obtain the up-toward the midline where in combination they contrib-

ute to the characteristic ellipsoid body structure. In addi- stream (i.e., 59 to GAL4 transcription unit) plasmidpKC507 from line c507 (Figure 3). In similar fashion,tion, both GAL4 lines mark the lower section and ureter

of the Malpighian tubules (Figure 1, d and e), regions genomic DNA from P{lacW} line l(3)96601 was digestedwith EcoRI or PstI to produce downstream plasmidassociated with reabsorption of fluid from the primary

urine (O’Donnell and Maddrell 1995; Sozen et al. pE96601 and upstream plasmid pP96601 (Figure 3).Although the P{GAL4} elements in lines c232 and1997).

In P{PZ} line l(3)07028, b-gal expression is seen in c507 map to the same polytene band and give rise toidentical expression patterns, the downstream rescuedthe nuclei of cells in the lower tubules and ureter (Fig-

ure 1g), as for the two GAL4 lines. No staining is seen fragments are of different size. To examine further therelationship between the two insertions, restriction map-in the adult brain, however. By in situ hybridization to

polytene chromosomes, both P{GAL4} insertions were ping and DNA sequencing analysis was performed, re-vealing the two P{GAL4} elements to be inserted 46 bpfound to reside at the same cytological location in the

proximal part of 100B (Figure 2), consistent with the apart and in opposite orientation (Figures 3 and 4).Rescued fragments from line c507 were used aslocalization of 100B2 documented for line l(3)07028

[Bloomington Drosophila Genome Project (BDGP) ac- probes to screen an EMBL3 genomic DNA library, re-sulting in three different classes of inserts (l1–l3).cession no. AQ073337].


Figure 2.—(Left) Polytene chromosome in situ hybridization. Arrows indicate hybridization signals in the proximal part of100B on the third chromosome. (a) A set of chromosomes from line c507 probed with pBluescript, which hybridizes to theplasmid replicon component of the P{GAL4} element. (b–d) Wild-type (Canton-S) chromosomes probed with pMY51 (Aph-4),pMY8 (l(3)96601), and pMY4 (CanA1) cDNA, respectively. (Right) Southern blots of wild-type Drosophila genomic DNA (Canton-S), cleaved and probed as indicated.

These were arranged into a contig by restriction map- to none of the above on the basis of cytological locationand thus has been designated Aph-4. Like most otherping and Southern blot analysis (Figure 3).

Isolation of cDNA clones: The rescued fragment from ALPs, Aph-4 has five potential N-linked glycosylationsignals, the positions of which vary between speciespPC507 contains a 0.9-kb BamHI fragment, which, on

the basis of “reverse Northern” analysis (data not (Manes et al. 1990; Itoh et al. 1991). The cDNA doesnot contain an obvious polyadenylation site, and noneshown), appeared to be transcribed. The 0.9-kb frag-

ment was thus used to probe a Drosophila head cDNA is seen in the genomic interval between Aph-4 and the39 end of the l(3)96601 transcription unit (see also Fig-library. Two independent (non-cross-hybridizing) classes

of cDNA were obtained, the longest versions of which ure 3).Figure 4 also shows the sequence of a genomic intervalwere designated pMY51 and pMY8 (Figure 3). They

hybridize to nonoverlapping regions of the genome as containing the three P-element insertion sites of P{GAL4}c507, c232, and P{PZ} l(3)07028. Flies homozygous forshown in Figure 3. The head cDNA library was also

probed with a 4.5-kb HindIII fragment from pKC507. the insertions in lines c507 and c232 are viable, whereashomozygotes of l(3)07028 are described as having aThe longest cDNA was designated pMY4 (Figure 3).

In situ hybridization to wild-type polytene chromo- semilethal phenotype (BDGP, accession no. AQ-073337). These closely spaced insertions presumablysomes with each of the above three cDNAs showed the

respective transcription units to be contained within the disrupt the promoter of the Aph-4 gene to varying ex-tents.100B2 region (Figure 2, left). Southern blotting further

confirms a single locus for each of the respective genes Among ALPs for which sequence data are available,Aph-4 is most closely related to vertebrate isozymes, frac-(Figure 2, right).

pMY51 encodes an alkaline phosphatase:Figure 4 shows tionally more so to tissue nonspecific varieties (Figures5 and 6). The closest match was to a chicken nonspecificthe nucleotide and deduced polypeptide sequences of

the pMY51 insert and of the 59 RACE product derived isoform precursor (Crawford et al. 1995) with 43%identity and 51% similarity (determined using GAP).from pMY51 cDNA. The equivalent region of genomic

DNA was also completely sequenced, showing the gene The only other insect ALP sequence available is fromthe silkworm Bombyx mori (Itoh et al. 1991; 38% identity,to be punctuated by three introns of 1332, 67, and 60

bp, respectively. The 1952 bp cDNA contains a long 47% similarity), presumably a paralogue. Metal and sub-strate phosphate binding sites determined for E. coliopen reading frame (ORF) of 578 amino acids. Database

searching showed the deduced polypeptide to be clearly ALP by X-ray crystallography (Sowadski et al. 1985),and which are strongly conserved across phyla, are alsoa member of the ALP family (see Figures 5 and 6)

containing the highly conserved ALP signature present in Aph-4 (asterisks in Figure 5).Aph-4 expression: Aph-4 mRNA abundance is highest“VPDSAGTAT.” Three Drosophila ALP loci have been

previously described (Aph-1,-2,-3), although none has during larval and adult stages (Figure 7, left). Such aprofile parallels that described for ALP activity in ayet been cloned. The gene described here corresponds


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Figure 4.—Nucleotide and deduced polypeptide sequences of Aph-4. Uppercase nucleotide sequences are from cDNA, lowercasefrom genomic DNA. The 39 end of the transcriptionally convergent l(3)96601 gene (pMY8 insert) is shown in italic type. Thehighly conserved ALP motif VPDSAGTAT is boxed. Sites of putative N-glycosylation are underlined. P{GAL4} insertions in linesc507 and c232 are in opposite orientation. The P{PZ} insertion in line l(3)07028 has the same orientation as the c507 insertion.The Aph-4 cDNA has been assigned the EMBL/GenBank/DDBJ accession no. X98402.


range of insects (McComb et al. 1979). A dramatic in- transcription factors that normally specifies the lowertubule domain.crease of Drosophila ALP activity in third instar larvae,

prior to the secretion of pupal cuticle, has been taken to Fluid secretion phenotypes: Tubules from c507 ho-mozygotes showed consistently lower basal rates of secre-suggest a role for the ALP in cuticle formation, possibly

regulated by the ring gland (Schneiderman et al. 1966). tion than wild-type tubules (Figure 8). They also showedreduced responses to a range of agonists: the cardioac-In situ hybridization to sectioned adult heads and

bodies revealed Aph-4 mRNA within the same cellular tive peptide 2b (CAP2b), which acts on principal cellsto stimulate the apical V-ATPase through intracellulardomains as revealed by GAL4-directed b-gal expression,

i.e., the ellipsoid body (Figure 1, b and c) and the Mal- nitric oxide/cyclic GMP (Davies et al. 1997; Figure 8A);cyclic AMP, which acts on principal cells to stimulatepighian tubules (Figure 1, e and f). Since the detection

of DIG-labeled probes usually relies on an ALP reporter the apical V-ATPase (O’Donnell et al. 1996; Figure 8B);and Drosophila leucokinin, which acts on stellate cellsenzyme coupled to anti-DIG antibodies, fresh levamisole

was used to block endogenous phosphatase expression to stimulate fluid production by activating the chlorideshunt conductance through intracellular Ca21 (O’Don-(McComb et al. 1979), even though ALP activity in any

case appeared to be lost during hybridization (data not nell et al. 1998; Figure 8C). As the lower tubule doesnot participate in the fluid secretion assay, these resultsshown). An independent detection system, employing

diaminobenzidine/peroxidase staining to detect a per- must be interpreted in terms of the misexpression ofAph-4 in c507 homozygotes to the tubule main segment.oxidase/anti-DIG reporter gave the same result (Figure

1f, lower right inset). This expression may disrupt the tight coupling of metab-olism and transport at either the basal or apical mem-The c507 insertion disrupts normal tubule pigmenta-

tion: Normal tubules in wild-type fly (Oregon-R) have branes.pMY8 encodes an essential polypeptide of unknownan opaque, yellowish color throughout the main seg-

ment and lower tubules (Figure 1h), caused by the accu- function: pMY8 corresponds to a single genetic locusin the 100B2 region (Figure 2). The pMY8 insert is 897mulation of hydroxy-kynurenine and associated eye pig-

ments, through the action of the white gene product bp long, excluding the 18-bp polyA tail remnant (Figure9). A partial cDNA sequence from the Berkeley Dro-and other organic solute transporters (Wessing and

Eichelberg 1978). In c507 homozygotes, this pigmen- sophila Genome Project (GenBank accession no.AA696267) provided six additional residues at the 59tation is strikingly suppressed in the main segment, but

not in the lower tubule (Figure 1i). A possible explana- end. Genomic DNA sequencing shows the gene to bepunctuated by two introns of 87 bp and 265 bp, thetion is an effect of the Aph-4 promoter on expression

of the mini-white gene contained within the nearby en- first of which lies 38 bp upstream of the putative transla-tion start site. The sequence TATAAA is 84 bp upstreamhancer-trap element (X chromosomes of c507 and c232

carry the disfunctional w1118 allele). A powerful lower of the beginning of the poly(A) tract. This is not themost common of poly(A) addition signals, but it is usedtubule enhancer/main segment repressor element

might influence not just the lacZ reporter, but also mini- occasionally (Manley 1988). The pMY8 cDNA containsan ORF of 223 amino acids. Database searches revealedwhite, in a lower-tubule-specific pattern.

The c507 insertion causes misexpression of Aph-4 to no obviously related genes, with the possible exceptionof three mammalian expressed sequence tags (ESTs)a genetically distinct domain: We found wild-type tubule

alkaline phosphatase activity to be concentrated exclu- revealed by a TBLASTN search (GenBank accession nos.AA097076, AA098325, and R86167). The two likeliestsively in the ureter and lower tubule (Figure 1j), identi-

cal to the three enhancer-trap patterns (Figure 1, d possibilities are a transcription factor and a neuropep-tide gene. The N-terminal part of the deduced proteinand g). The Aph-4 pattern in c507 homozygotes was

profoundly different, however (Figure 1k). Expression contains a number of repeats (KPAA and similar) associ-ated with histone-like DNA regulatory proteins, whichin the lower tubule is reduced compared to wild type,

while that in the main segment is greatly enhanced are found in a range of organisms. The protein alsocontains several dibasic repeats (at aa 33-4, 130-1, and(Figure 1, j and k). c507 heterozygotes have an interme-

diate pattern with expression concentrated in lower tu- 149-50) that could result in propeptide cleavage, with adownstream “GG” motif (at aa 93-4) normally associatedbule and ureter, but with some expression in main seg-

ment (Figure 1l). The main segment is a genetic domain with N-terminal amidation.Deak et al. (1998) have generated a large collectiondefined by Sozen et al. (1997), which is known to corre-

spond to the fluid-secreting portion of the tubule of third chromosome recessive lethal lines by insertionof a P{lacW} enhancer-trap element. Using the plasmid(O’Donnell and Maddrell 1995). This suggests that

the c507 insertion is impinging on a control region rescue method of Guo et al. (1996), we found two lines(96601 and 35313) with P{lacW} insertions at the samethat specifies tubule expression domains. The fact that

misexpression in the main segment corresponds so pre- nucleotide position and in the same orientation, just 59to the pMY8 coding region (Figure 9). Both lines carrycisely to the domains described by Sozen et al. (1997)

suggests that there is a combinatorial mechanism of only a single P element and die as homozygotes during


the pupal-to-pharate adult stage (Deak et al. 1998). Oneof the lines, l(3)96601, and its corresponding rescuedplasmid, p96601 (Figure 3), were chosen for furthercharacterization.

Excision of the P element caused reversion of thelethal phenotype, verifying insertion as the cause oflethality (rather than an unlinked event elsewhere onthe third chromosome). Southern blotting ofl(3)96601/wt DNA revealed the expected band shift dueto P{lacW} insertion (not shown). Northern blotting ofwild-type mRNA revealed an adult transcript of z1 kbthat is slightly elevated in the head (Figure 7, mid-dle). Northern blotting of l(3)96601/wt mRNA indi-cated a reduction of transcript abundance of 43% (nor-malized to rp49 loading control) compared with wildtype (Figure 7, right). In situ hybridization to sectionsof the adult head and body revealed generalized expres-sion throughout the organism.

pMY4 encodes calcineurin A1: pMY4 corresponds toa single genetic locus in the 100B2 region (Figure 2).The sequence of the pMY4 insert revealed it to be a

Figure 6.—Neighbor-joining plot showing phylogenetic re-partial (59-truncated) Drosophila calcineurin A1 (CanA1)lationships between representative ALPs. Protein accessioncDNA (Guerini et al. 1992). This was surprising, since numbers are shown in parentheses.

CanA1 had been previously assigned to the cytogeneticlocation 21E-F (Guerini et al. 1992). There is no evi-

in situ hybridization to tissue sections and staining fordence from either Southern blotting or in situ hybridiza-alkaline phosphatase activity show Aph-4 to have thetion to polytene chromosomes (Figure 2) that there aresame expression pattern as the two enhancer-trap lines.two such genes in the Drosophila genome. However,Aph-4 is expressed strongly, if not exclusively, in the21E-F and 100B are at similar positions to the telomeres

of 2L and 3R, respectively, and so we suggest that 100B2is the true location. In support of this, two recentlysequenced BDGP P1 clones (GenBank accession nos.AC007975 and AC008220) that have been mapped to100A-C contain sequence identical to the publishedCanA1 cDNA.

DISCUSSION

We have identified a number of genes flanking theP{GAL4} elements in the enhancer-trap lines c507 andc232. Three different genes located at 100B were clonedand characterized. One of them is calcineurin A1, aCa21/calmodulin-stimulated protein phosphatase (Gue-rini et al. 1992), which is located upstream of the Figure 7.—Northern blot analysis. A total of 10 mg of

poly(A)1 Canton-S RNA from the indicated stages and tissuesP{GAL4} element in line c507. Two other closely linkedwas hybridized with the indicated cDNAs. As a control forgenes are located downstream of the c507 element,loading, stripped filters were reprobed with rp49 cDNA

Aph-4 being closest to the site of transposon insertion. (O’Connell and Rosbach 1984). E, 0–24 hr embryo; L,Tightly linked to Aph-4, but in opposite orientation, is mixed larval instars; P, midpupal; H, adult head; B, adult body;

wt, wild type; lacW, line l(3)96601.the essential l(3)96601 locus of unknown function. Both

Figure 5.—Pileup of representative ALP protein sequences. Boxed residues are at least 50% conserved between the sevenALPs, shaded residues at least 80% similar. * indicates residues that participate in the active site of E. coli ALP. Drosophila Aph-4(X98402); chicken nonspecific isozyme precursor (U19108; Crawford et al. 1995); human nonspecific isozyme precursor (P05186;Weiss et al. 1986); human placental type 1 isozyme precursor (P05187; Knoll et al. 1988); human placental-like isozyme precursor(P10696; Millan and Manes 1988); human intestinal isozyme precursor (P09923; Henthorn et al. 1987); silk moth membrane-bound alkaline phosphatase precursor (P29523; Itoh et al. 1991).


ellipsoid body of the brain and in the Malpighian (re- vertants were generated and analyzed. One line has alarge deletion that removes part of the P element andnal) tubule.

The P{GAL4} insertions in lines c507 and c232 are flanking genomic DNA including the Aph-4, l(3)96601,and the nearby dbt (double-time, Kloss et al. 1998; seehomozygous viable, and there are no other obvious de-

fects. To generate loss-of-function alleles, imprecise Figure 3). Although flies homozygous for this deletionP-element excision has been carried out. Over 130 re- die at the late embryo or early larval stage, both

l(3)96601 and dbt are essential loci, so no conclusioncan be drawn concerning Aph-4. However, a recentlydocumented line, l(3)07028 (BDGP accession no.AQ073337; see Figure 4), in which P{PZ} is inserted 19bp closer to the 59 end of Aph-4 than the c232 insertion,has a semilethal phenotype. This may imply that Aph-4is an essential, or at least an important, Drosophila gene.

Reverse genetic analysis in most model organisms iscomplicated by the “phenotype gap,” a term that ac-knowledges the dearth of phenotypes for detailed func-tional analysis. This is particularly acute in Drosophila,where the small size of the organism militates againstphysiological study. However, the identification of cell-specific expression of Aph-4 in the Malpighian tubule(Skaer 1993) allows a more detailed examination ofthis enigmatic enzyme than would normally be possibleand has provided novel insights into the tubule itself.First, the expression pattern delineated by the reportergenes in c507, c232, and l(3)07028 defines a lower tu-bule domain (Sozen et al. 1997) that had not beendetected by classical analysis (Wessing and Eichelberg1978), but which we now know corresponds with thereabsorptive, rather than secretory, portion of the tu-bule (O’Donnell and Maddrell 1995). Second, re-porter gene expression appears precisely to match theexpression of Aph-4 mRNA (by in situ hybridization)and of alkaline phosphatase enzyme activity (by histo-chemistry) to a population of 25 6 0.4 cells. It is rela-tively rare for an enhancer-trap pattern to display suchclose agreement with the gene into which it inserts, andthis is particularly interesting in a region where the genedensity is so high, with two other genes within a kilobase,each with very different expression patterns. Third, thegene is probably essential because one of three inser-tions in the upstream sequence is semilethal. Fourth,the phenotypes of these insertions are unusual; ratherthan producing nulls or hypomorphs, they appear tointerrupt a control region that specifies the lower tubuledomain in some combinatorial fashion. Although ex-pression of alkaline phosphatase in lower tubule is re-

Figure 8.—Effect of homozygosity for P element c507 onfluid secretion. Secretion rates were measured in Oregon-R(empty square) or c507/c507 homozygous flies. Tubules werechallenged at 30 min with maximal concentrations of agonistsknown to act on principal or stellate cells. (A) 1025 m cardioac-tive peptide 2b (CAP2b); (B) 1023 m cyclic AMP; (C) 1025 mDrosophila leucokinin. Data are presented as mean 6 SEM,with the number of separate tubules in each experimentalgroup shown in parentheses.


Figure 9.—Nucleotide sequence of the l(3)96601 gene. Uppercase nucleotide sequences are from cDNA, lowercase fromgenomic DNA. The hypothetical 223-amino-acid translation product is indicated. The putative polyadenylation signal sequenceis underlined. The 59 end of the transcriptionally convergent dbt gene (Kloss et al. 1998; B. Kloss, personal communication)and the 39 end of the transcriptionally convergent Aph-4 gene (pMY51 insert) are shown in italic. The cDNA sequence has beendeposited in the EMBL/GenBank/DDBJ database and assigned accession no. X94917.

duced, it is greatly enhanced in the main segment. Our phosphatase, which is usually a membrane-associatedenzyme, may disrupt this order and so nonspecificallybest explanation of this at present is that the boundary

between main and lower tubule domains is sharpened reduce the performance of the tissue, both of agoniststhat usually act on the prinicpal cell (CAP2b and cAMP)by interacting positive and negative regulatory elements

and that the P-element insertions separate these control and of those that act on the stellate cell (Drosophilaleucokinin).regions so that they can no longer interact. The more

distal, lower-tubule-specifying control region is thus What insights can we gain as to the role of alkalinephosphatase in tubule, or indeed in brain? In humans,moved 8 kb away from the more proximal, main seg-

ment control region and can no longer repress it. As there are multiple isozymes of ALP (McComb et al.1979); the function of these is not well understood.there are a few kilobases between the CanA1 and Aph-4

coding regions (Figure 3), this might be a particularly However, ALP is a marker of osteoblast lineages, andhypophosphatasia, a recessive human disease with multi-rewarding area in which to perform a promoter analysis.

How can the ectopic expression of alkaline phospha- ple bone disorders, has been linked to mutations inALPL, the non-tissue-specific isozyme of ALP (Henth-tase lead to a reduction in fluid secretion? In the fluid

secretion assay, the lower tubule sits outside the bathing orn and Whyte 1992). The analogous gene in mouse,tissue-nonspecific alkaline phosphatase (TNAP), hasdrop, and so dies; it acts merely as a conduit for the

urine secreted by the main segment. The loss of alkaline been inactivated by homologous recombination (Way-mire et al. 1995). In this case, bone disorders were notphosphatase activity from the lower tubule is thus un-

likely to influence measured rates of fluid secretion, evident, but the homozygous mutant mice died fromseizures, apparently from an inability to metabolize vita-and so overexpression in the main segment is more

likely to be responsible. The main segment is a highly min B6 (Waymire et al. 1995). This probably reflects arole for ALPL/TNAP as an ectoenzyme, anchored toordered tissue, with semicrystalline arrays of V-ATPase

on the apical membrane of principal cells (reviewed the cell surface, that converts pyridoxal-59-phosphate topyridoxal. The suggestion from the vertebrate literatureby Dow et al. 1998), while the basolateral membrane

contains high concentrations of Na1, K1 ATPase is thus that alkaline phosphatase can play a role in themetabolism of calcium phosphates. Interestingly, the(Schubiger et al. 1994). Overexpression of alkaline


tubules are sites for deposits of calcium and magnesium other phosphoric monoester hydrolase genes will befound near the 100B region.phosphates, in the form of luminal concretion bodies,

or spherites (Wessing and Eichelberg 1978). These We thank Greg Stewart, Ping Li, Elaine Cleary, Ji Luo, Douglasbodies have traditionally been considered to be sites of Armstrong, Shireen Davies, and Jonathan Sheps for their help with

this work. We also thank Peter Deak for P{lacW} lines, Nicole Mozden“storage excretion” of phosphates of calcium, magne-and Allan Spradling for line l(3)07028, Brian Kloss for dbt, Claudesium, and other metals. As these spherites pack into theKlee for CanA1. This work was supported by the U.K. Biotechnology

initial segments of the anterior tubules, it is possible and Biological Sciences Research Council and the Medical Researchthat the deposits are laid down in the initial segment Council, and by the Wellcome Trust.and then pass though the tubule to be excreted in theurine. Alkaline phosphatase on the apical surface of thelower tubule might allow for the selective reclamation

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a novel drosophila alkaline phosphatase speciﬁc to the ... · the nearer encodes aph-4, the...

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