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Pain Medicine  |   October 2003
Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 
Author Affiliations & Notes
  • Sumiko Gamo, M.D.
    *
  • Junya Tomida, M.S.
  • Katsuyuki Dodo, M.S.
  • Dai Keyakidani, M.S.
    §
  • Hitoshi Matakatsu, Ph.D.
  • Daisuke Yamamoto, Ph.D.
    #
  • Yoshiharu Tanaka, Ph.D.
    **
  • * Professor, ** Lecturer, Department of Earth and Life Sciences, Osaka Prefecture University. † Former position: Graduate Student, Department of Earth and Life Sciences, Osaka Prefecture University. Current position: Graduate Student, Kyoto University, School of Medicine, Kyoto, Japan. ‡ Former position: Graduate Student, Department of Earth and Life Sciences, Osaka Prefecture University. Current position: Staff Biopatent, Patent Department, Takara Bio, Inc., Otsu, Japan. § Former position: Student, Department of Earth and Life Sciences, Osaka Prefecture University. Current position: Graduate Student, Nara Institute of Science and Technology, Graduate School of Information Science, Nara, Japan. ∥ Former position: Graduate Student, Department of Earth and Life Sciences, Osaka Prefecture University. Current position: Associate Researcher, Department of Zoology, University of Wisconsin, Madison, Wisconsin. # Professor, Department of Human Sciences and Advanced Research, Waseda University, Tokorozawa, Japan.
  • Received from the Department of Earth and Life Sciences, Osaka Prefecture University, Sakai, Osaka, Japan.
Article Information
Pain Medicine
Pain Medicine   |   October 2003
Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 
Anesthesiology 10 2003, Vol.99, 867-875. doi:
Anesthesiology 10 2003, Vol.99, 867-875. doi:
TO understand the mechanisms of action of anesthetic agents, studies have shown two phenotypes of anesthetic sensitivity: hypersensitive and resistant to volatile anesthetics. The hypersensitive mutants require a lower (> 20%) concentration of anesthetics, whereas resistant mutants need higher (> 20%) concentrations than the wild type. A Drosophila  mutant, Eth  AR201  (old name, Eth-29  ), is resistant to diethylether, chloroform, and halothane, and different genes control each anesthetic phenotype. 1 Another Drosophila  halothane-resistant mutant, har  , induced by a chemical mutagenesis, 2 and many mutants hypersensitive and resistant to diethylether by using the mutagenesis of a transposon tagging of the P-element insertion, 3 have been isolated. A number of mutated genes, hypersensitive and resistant to volatile anesthetics, have been identified in Drosophila  , 3–5 Caenorhabditis elegans  , 6–10 and mice. 11,12 
This article is accompanied by an Editorial View. Please see: Morgan PG: A glimpse into the many possibilities that lie ahead. Anesthesiology 2003; 99:771–3.
In Drosophila  , one of the candidate genes in the hypersensitive mutants is the para  gene that encodes a voltage-gated sodium channel α subunit. 5 The Na+channel is known as the generator of the action potential and is expressed primarily in the nervous system of the fly. 13 Mutations of the para  gene have been isolated by heat-induced paralysis (29°–37°C), and some of them are temperature-sensitive to grow at lower temperatures than the wild type. 14–16 Almost all mutants show hypersensitivity to diethylether. 17 The mutant, para  hd838, is the most hypersensitive to diethylether, but it shows no paralysis at the lowest (29°C) temperature. 18 It suggests that the underlying mechanisms are not identical, e.g.  , simple reduction of channel function, but rather distinct sites of the protein for diethylether anesthesia and heat-induced paralysis. In C. elegans  , a candidate sodium ion channel subunit (UNC-8) and a close homolog of the mammalian stomatin (UNC-1) complex is considered a possible target molecule for diethylether and halothane anesthesia. 6 Stomatin is thought to regulate an as yet unknown ion channel to control sodium flux in a ball-and-chain fashion. Mutations in unc-1  and unc-8  genes alter sensitivities to volatile anesthetics, and some mutants of unc-1  suppress unc-8  mutation. 6,7 In the model animals, mutated genes encode the proteins that alter the sensitivity to volatile anesthetics, including the target molecules of volatile anesthetics. They are almost all membrane proteins such as sodium channel subunits and stomatin described above, Drosophila  A kinase anchor protein 200 (DAKAP200), 4 insulin-like growth factor type 1 receptor–like, 4 syntaxin and syntaxin-binding proteins, 9 G-protein α subunit (GOA), 10 the complex I of the nematode respiratory chain, 8 γ-aminobutyric acid type A receptor, 11 and α-2A adrenoceptor 12 (reviewed in Gamo 19).
We have isolated another hypersensitive mutant, eth  as311  , which has a transposon in the Drosophila  calreticulin gene, crc  . 3 It is the first fertile mutation of the crc  gene. Calreticulin is known to have multiple functions and diverse cellular distribution (reviewed in Michalak et al.  20 and Krause and Michalak 21). Briefly, the protein has three domains, N, P, and C, and resides mainly in the lumen of the endoplasmic reticulum in nonmuscle cells. On the basis of several in vitro  and in vivo  mammalian studies, the N-domain is unique to calreticulin and functions outside of the endoplasmic reticulum. That is, calreticulin modulates both the α-integrin adhesive function and integrin-initiated Ca2+-signaling into the cell and binds to DNA-binding domain of steroid hormone receptors to regulate the gene expression. Recently, calreticulin was found to mediate nuclear export of glucagon receptors, and glucagon receptor export is facilitated by its DNA-binding domain, which is shown to function as a nuclear export signal. 22 The P-domain has the chaperoning action of glycoproteins, coworking with calnexin and protein–protein interacting action with sarcoplasmic/endoplasmic reticulum Ca2+-adenosine triphosphatase and inositol triphosphate (InsP3) receptor in endoplasmic reticules. The C-domain has a high-capacity Ca2+-binding site. The P- and C-domains play a central role in intracellular Ca2+homeostasis.
To confirm that the mutated crc  gene of eth  as311  causes altered phenotype (hypersensitivity to diethylether), we cloned and sequenced the crc  cDNA and examined the expression of crc  at various developmental stages. We also examined the relations between the crc  gene and phenotype in the mutant, revertants, and transgenic flies. Furthermore, we determined the 50% anesthetizing concentrations (EC50s) of other anesthetics, isoflurane and halothane, and speculated on the possible relation between the phenotype and functions of crc  .
Materials and Methods
Drosophila  Stocks
The Drosophila  mutant hypersensitive to diethylether, eth  as311  , is named as an eth  er-a  nesthetic s  ensitive mutant, is found as a mutation on chromosome 3  , and is the 11  th mutant in our laboratory. The mutant that has a P-element in the crc  locus and the P-element excision–induced revertants, revertant-2  , -3  , -5  , and -44  , have been isolated previously. 3 CyO/Sp;SbP[ry  +Δ2–3 99B]/TM6 (jump starter), w;+;PrDr/TM3  (balancer), Canton-S  (wild type), and w  (white  ) were used in this study. 23 CyO  , TM6  , and TM3  are balancers that inhibit the crossing over. P[ry  +Δ2–3 99B] is a P-element construct to produce transposase and inserted in the third chromosome at 99B. The flies were raised on a standard medium (agar, yeast, glucose, corn grits) or on an egg-collecting medium (grape juice, whisky, agar) for collecting embryos at 25°C. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the participating institutions in Department of Earth and Life Sciences Osaka Prefecture University.
cDNA Cloning and DNA Sequence Analysis
The general cloning techniques described by Sambrook et al.  24 were followed. A 2.1-kb genomic DNA fragment (pr-clone in fig. 1A) was isolated by the plasmid rescue method 25 and used as a probe to isolate cDNAs from the crc  locus. Using the pr-clone as a probe, wild-type cDNA clones were isolated from a Canton-S  adult male library in the phage λ gt10. 26 We determined the complete nucleotide sequence of the cDNA clone on both strands. The cDNA insert was isolated from the phage vector, subcloned into the Bluescript KS+vector (Stratagene, Burlingame, CA), and sequenced by a dideoxynucleotide chain termination procedure using Dye Deoxy Terminator Cycle Sequencing Kit in a model 373A autosequencer (Perkin Elmer ABI, Foster City, CA). Analysis of sequence data was searched using BLAST programs provided by the NCBI server (National Institutes of Health, Bethesda, MD) and programs provided by the Berkeley Drosophila  Genome Project server (Berkeley, CA). All reagents used in this study were supplied by Nacalai tesque Inc. (Nara, Japan) unless otherwise mentioned.
Fig. 1. Structure of the crc  gene. (A  ) (Upper portion  ) Genomic DNA with a P-element insertion (triangle  ) and restriction sites (Ps  =Pst  I; K  =Kpn  I; B  =Bam  HI; Bg  =Bgl  II; E  =Eco  RI). (Center portion  ) 2.1-kb genomic DNA clone (pr-clone  ). 3 (Lower portion  ) Exon–intron organization of crc  (exon:black boxes  = translated regions; white boxes  = untranslated regions). (B  ) Drosophila  calreticulin model. Drosophila  calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40 The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C  ) Northern blot analysis. Embryos, (CE, EE  ), first and second instar larve (C12, E12  ), third instar larvae (C3, E3  ), pupae (CP, EP  ), and adults (CA, EA  ) of Canton-S  (C) and ethas311  (E), respectively. A 1.4 kb is a transcript of crc  and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41 (D  ) Calreticulin expression is normalized by rp49 in Canton-S  (black bars  ) and eth  as311  (gray bars  ).
Fig. 1. Structure of the crc 
	gene. (A 
	) (Upper portion 
	) Genomic DNA with a P-element insertion (triangle 
	) and restriction sites (Ps 
	=Pst 
	I; K 
	=Kpn 
	I; B 
	=Bam 
	HI; Bg 
	=Bgl 
	II; E 
	=Eco 
	RI). (Center portion 
	) 2.1-kb genomic DNA clone (pr-clone 
	). 3(Lower portion 
	) Exon–intron organization of crc 
	(exon:black boxes 
	= translated regions; white boxes 
	= untranslated regions). (B 
	) Drosophila 
	calreticulin model. Drosophila 
	calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C 
	) Northern blot analysis. Embryos, (CE, EE 
	), first and second instar larve (C12, E12 
	), third instar larvae (C3, E3 
	), pupae (CP, EP 
	), and adults (CA, EA 
	) of Canton-S 
	(C) and ethas311 
	(E), respectively. A 1.4 kb is a transcript of crc 
	and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41(D 
	) Calreticulin expression is normalized by rp49 in Canton-S 
	(black bars 
	) and eth  as311 
	(gray bars 
	).
Fig. 1. Structure of the crc  gene. (A  ) (Upper portion  ) Genomic DNA with a P-element insertion (triangle  ) and restriction sites (Ps  =Pst  I; K  =Kpn  I; B  =Bam  HI; Bg  =Bgl  II; E  =Eco  RI). (Center portion  ) 2.1-kb genomic DNA clone (pr-clone  ). 3 (Lower portion  ) Exon–intron organization of crc  (exon:black boxes  = translated regions; white boxes  = untranslated regions). (B  ) Drosophila  calreticulin model. Drosophila  calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40 The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C  ) Northern blot analysis. Embryos, (CE, EE  ), first and second instar larve (C12, E12  ), third instar larvae (C3, E3  ), pupae (CP, EP  ), and adults (CA, EA  ) of Canton-S  (C) and ethas311  (E), respectively. A 1.4 kb is a transcript of crc  and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41 (D  ) Calreticulin expression is normalized by rp49 in Canton-S  (black bars  ) and eth  as311  (gray bars  ).
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RNA Isolation, Northern Blot Hybridization, and 5′RACE
Total RNA was isolated from the embryos, larvae, pupae and adult flies. Poly(A)+RNA was separated on oligo(dT)-cellulose columns (Amersham Life Science, Buckinghamshire, United Kingdom). Next, 5 μg per lane of poly(A)+RNA was loaded per lane of the formaldehyde-agarose gel. After electrophoresis, the RNA was transferred to a Hybond-N membrane (Amersham). Fluorescein labeling of the probes, hybridization, and detection of mRNA were performed according to the instructions provided by the supplier (Amersham). Furthermore, to determine the 5′ ends of the mRNA transcripts, 200 ng poly(A)+was applied for 5′ RACE using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) kit (Ambion Inc., Austin, TX). Briefly, the RNA is treated with calf intestinal phosphatase in a 20-μl reaction mixture to remove free 5′-phosphates and to remain the cap structure found on intact 5′ ends of mRNA. After phenol:chloroform extract, the RNA is then treated with Tobacco acid pyrophosphatase to remove the cap structure, and a 45-base RNA adapter oligonucleotide is ligated to the treated RNA using T4 RNA ligase. A random-primed reverse-transcription reaction and nested polymerase chain reaction then amplifies the 5′ end of crc  transcript: outer 5′RLM-RACE polymerase chain reaction using 5′ RACE outer primer and an outer crc  primer (N-2, 5′-gatattctgctcgtgcttcac-3′, 391–371 nt) and inner 5′RLM-RACE polymerase chain reaction using 5′ RACE inner primer and an inner crc  primer (5′-gttggagaagccatcaaact-3′, 340–321 nt).
Expression of mRNA from the crc  Locus
To study the expression patterns of the crc  mRNA, we synthesized digoxigenin-UTP-labeled single-stranded DNA probes. Sense and antisense probes were synthesized by polymerase chain reaction using each N, P, and C domain primers of crc  cDNA subclone: sense primers, N-1 (5′-tctgaaggagaatttcgacaac-3′, 148–169 nt), P-1 (5′-aggacgactgggacttcctg-3′, 672–691 nt), and C-1 (5′-gttaagtccggcactatcttc-3′, 1043–1063 nt); and antisense primers, N-2, P-2 (5′-tttggctgccactcgccctt-3′, 915–896 nt), and C-2 (5′-caattcgtcgtgttcgctttga-3′, 1300–1279 nt). Probes were hybridized in situ  to whole mounts of embryos and developing brains according to the method of Tautz and Pfeifle. 27 The preparation mounted in glycerol was viewed using an Olympus BX60 light microscope (Olympus Optical Co., Ltd., Osaka, Japan).
Isolation of P-element Excision–induced Revertants of crc 
Recently, a lethal excision derivative, revertant-s20-1  , was isolated by dysgenic cross. 28 Briefly, flies carrying the crc  with inserted P-element of eth  as311  (w;+;p  ) were crossed to +;CyO/Sp;SbP[ry  +Δ2–3 99B]/TM6. Dysgenic progeny carrying CyO;Sb  were crossed to w;+;PrDr/TM3  flies twice. w;+;p*/TM3  (p*  represents excised P-element) progeny were selected for lines. 3 revertant-s20-1  is a line in which no homo p*/p*  progeny emerged, i.e.  , lethal.
Transgenic crc  Lines
Transgenic lines with the wild-type crc  gene were produced. A  new P-factor for driving genes behind the hsp70 promoter of the plasmid, pCaspeR-hs, was constructed with w  +as the selectable marker. 29 This vector, containing an Eco  RI fragment of the crc  cDNA, was injected into w  embryos. Chromosomal localization of the transgenes and the generation of homozygotes for the transgenes were performed by standard crosses. The crc  +transgene resides on the second chromosome (crc-T1  and crc-T2  ) and mutated crc  gene on the third chromosome in crc-T1;eth  as311  and cr  c-T2;eth  as311  , respectively.
Anesthetic Techniques and Statistical Analysis
For the estimation of anesthetic concentration, each of 20 1-day-old female and male flies were administered a given concentration of anesthetics, and their responses were scored using avoidance reflex from stimulation by a fine brush as an endpoint. 3 Diethylether, isoflurane (Forane; Abbott Laboratories Inc., Park, IL), and halothane (Fluothane; Takeda Chemical Industries, Osaka, Japan) were used. The dose–response curves from at least four to nine data points were linearized by log-probit transformation to estimate EC50with 95% confidence limits. Comparison of EC50s was conducted by analysis of covariance. 18 
Results
Drosophila  Calreticulin Locus
eth  as311  , a hypersensitive mutant to diethylether anesthesia, was screened by determining EC50of diethylether. It has a modified P-element between the eight base repeats (CTGACTGA) of 26–33-bp upstream from the initiation codon (ATG) within the first exon of the crc  locus. 3 We obtained a 1.4-kb cDNA, sequenced it, and performed a 5′ RACE analysis (EMBL sequence accession No. AB000718). We identified a 1420-bp full-length cDNA including the 5′ and 3′ untranslated regions of crc  , 82-bp and 117-bp, respectively (fig. 1A). The open reading frame and deduced amino acid sequence were similar to those predicted from the genomic sequencing reported by Smith. 30 Drosophila  calreticulin has high homology (∼ 70%) to calreticulins of other eukaryote species, indicating structural and functional similarities among them. The model of Drosophila  calreticulin shown in figure 1Bis built from a mammalian model of calreticulin. 20,21 Northern blotting analysis showed that only a 1.4-kb transcript was found at all developmental stages in the wild type and eth  as311  (fig. 1C). In the wild type, the highest expression was observed at pupa stage; however, the mRNA level was much lower in the mutant than the wild type at all developmental stages, including pupal stage (fig. 1D). These results show that eth  as311  is a low-expression mutation in the crc  gene and support that eth  as311  is a fertile mutation. In fact, the endogenous calreticulins were detected in the wild type and the mutant (Sumiko Gamo, M.D., and Dai Keyakidani, M.S., Department of Earth and Sciences, Osaka Prefecture University, Sakai, Osaka, Japan, unpublished data, April 1999). However, another Drosophila  mutant, l(3)s114307  , and the knockout mice of calreticulin gene are lethal. 31,32 
crc  mRNA Expression at Various Embryonic Stages
crc  mRNA is expressed at all developmental stages in wild-type Drosophila  . Whole-mount in situ  hybridization of the crc  transcripts was performed to identify the cells and tissues that are stained by the antisense cDNA of the probe at various embryonic stages. At the cellular blastoderm, where embryonic cells distribute on the egg surface, the perinuclear cytoplasm was stained, but no staining of the cell nucleus (fig. 2A-1) was found compared to a sense RNA probe (fig. 2A-2). The cells on the egg surface were stained, but the yolk granules were not stained at the central region of the egg. At embryonic stage 9, the crc  mRNA was expressed ubiquitously, especially at pericephalic and ventral neuroblast regions of the central nervous system (fig. 2B-1), but no expression was detected when a sense probe of crc  was used (fig. 2B-2). Strong expression was detected in the head region, ventral nervous systems, trachea, and hind gut at embryonic stage 15 in the wild type (fig. 2C-1), but disruption of the crc  expression was found in the mutant embryo (data not shown); the pattern was similar to that of the wild-type embryo when a sense RNA probe was used (fig. 2C-2).
Fig. 2. In situ  hybridization of crc  transcripts in wild-type embryos. The crc  antisense was used as a probe in A-1  , B-1  , and C-1  , and the sense probes were used in A-2  , B-2  , and C-2  . (A-1  ) Pole cells (pc  ) and yolk granules (yg  ) at embryonic stage 5. (B-1  ) Procephalic neuroblasts (pnb  ) and ventral neuroblasts (vnb  ) at embryonic stage 9. (C-1  ) Salivary gland (sg  ) in the head region, trachea (t  ), hind gut (hg  ), and midgut (mg  ) at stage 15. (Left  ) anterior egg position; (right  ) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
Fig. 2. In situ 
	hybridization of crc 
	transcripts in wild-type embryos. The crc 
	antisense was used as a probe in A-1 
	, B-1 
	, and C-1 
	, and the sense probes were used in A-2 
	, B-2 
	, and C-2 
	. (A-1 
	) Pole cells (pc 
	) and yolk granules (yg 
	) at embryonic stage 5. (B-1 
	) Procephalic neuroblasts (pnb 
	) and ventral neuroblasts (vnb 
	) at embryonic stage 9. (C-1 
	) Salivary gland (sg 
	) in the head region, trachea (t 
	), hind gut (hg 
	), and midgut (mg 
	) at stage 15. (Left 
	) anterior egg position; (right 
	) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42Bar 
	= 50 μm.
Fig. 2. In situ  hybridization of crc  transcripts in wild-type embryos. The crc  antisense was used as a probe in A-1  , B-1  , and C-1  , and the sense probes were used in A-2  , B-2  , and C-2  . (A-1  ) Pole cells (pc  ) and yolk granules (yg  ) at embryonic stage 5. (B-1  ) Procephalic neuroblasts (pnb  ) and ventral neuroblasts (vnb  ) at embryonic stage 9. (C-1  ) Salivary gland (sg  ) in the head region, trachea (t  ), hind gut (hg  ), and midgut (mg  ) at stage 15. (Left  ) anterior egg position; (right  ) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
×
crc  mRNA Expression in the Central Nervous System
We performed in situ  hybridization to find when and where the highest level of the crc  transcript appears. At the third instar larvae, the outer surface neurons in the brain of the wild type were stained most strongly (fig. 3A-1). In addition, the leg and wing disks and ring gland were stained but very weakly stained in the thoracic and abdominal ganglia (data not shown) as well as the ventral ganglion (fig. 3A-1). No strong crc  mRNA expression was observed when sense probes were used in wild-type brain (fig. 3A-2) and in the brain of eth  as311  by using antisense probes (fig. 3A-3). We focused on the crc  expression in the brains of pupae and adults. Unlike most other larval organs, the central nervous system persists into the adult stage in Drosophila  . The staining intensity of newly emerged adult type neurons (outside of brain) was stronger than the larval type neurons (inside of brain) in the 19-h-old pupae (fig. 3B-1). The strongest expression was observed at 19 h after puparization in the wild type in comparison to the control straining (fig. 3B-2), and the level of crc  mRNA was markedly low in the eth  as311  brain even in 19-h-old pupae (fig. 3B-3). Subsequently, the expression decreased gradually in wild-type brain (fig. 3C-1) and was still clearer than the control of a sense probe (fig. 3C-2). In 48-h-old pupae, more differentiation of optic lobes occurred and the staining of neuronal cell bodies was stronger than the axons in the optic lobes, brains, and the subesophageal ganglia that develop from the larval part of the ventral ganglion and esophagus (fig. 3C-1). In 90-h-old pupae (figs. 3D-1 and -2), the brain was morphologically similar to the adult brain, and the staining was markedly weak (fig. 3D-1). In 1-day-old adult brains, staining was found at the limited small regions (fig. 3E-1), no staining was found in the control brain (fig. 3E-2), and little was found in the mutant brain (fig. 3E-3). During the formation of adult brain, the maximum expression is apparent in the earlier pupal stage, but it decreases gradually to minimal expression in the adult brain. Therefore, these staining patterns are due to expression of crc  mRNA and consistent with the results of Northern blot analysis of crc  (fig. 1D).
Fig. 3. The highest crc  expression in the brains of early pupae. The crc  antisense was used as a probe in A-1  , B-1  , C-1  , D-1  , and E-1  in Canton-S  and A-3  , B-3  , and E-3  in ethas311  , and the sense probes were used in A-2  , B-2  , C-2  , D-2  , and E-2  in Canton-S  . (A  ) Larval brain hemispheres (bh  ) including the optic lobes (ol  ) and ventral ganglion (vg  ) in the third instar larvae. (B  ) The brain of a 19-h-old pupa. (C  ) Eye ganglia (arrows  ), subesophageal ganglia (sub  ), and esophagus (es  ) in the head region of a 48-h-old pupa. (D  ) Staining of the brain and optic lobes of a 90-h-old pupa. (E  ) In the adult brain, staining was limited to arrow regions (arrows  ). For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
Fig. 3. The highest crc 
	expression in the brains of early pupae. The crc 
	antisense was used as a probe in A-1 
	, B-1 
	, C-1 
	, D-1 
	, and E-1 
	in Canton-S 
	and A-3 
	, B-3 
	, and E-3 
	in ethas311 
	, and the sense probes were used in A-2 
	, B-2 
	, C-2 
	, D-2 
	, and E-2 
	in Canton-S 
	. (A 
	) Larval brain hemispheres (bh 
	) including the optic lobes (ol 
	) and ventral ganglion (vg 
	) in the third instar larvae. (B 
	) The brain of a 19-h-old pupa. (C 
	) Eye ganglia (arrows 
	), subesophageal ganglia (sub 
	), and esophagus (es 
	) in the head region of a 48-h-old pupa. (D 
	) Staining of the brain and optic lobes of a 90-h-old pupa. (E 
	) In the adult brain, staining was limited to arrow regions (arrows 
	). For names of organs and developmental stages, refer to Hartenstein. 42Bar 
	= 50 μm.
Fig. 3. The highest crc  expression in the brains of early pupae. The crc  antisense was used as a probe in A-1  , B-1  , C-1  , D-1  , and E-1  in Canton-S  and A-3  , B-3  , and E-3  in ethas311  , and the sense probes were used in A-2  , B-2  , C-2  , D-2  , and E-2  in Canton-S  . (A  ) Larval brain hemispheres (bh  ) including the optic lobes (ol  ) and ventral ganglion (vg  ) in the third instar larvae. (B  ) The brain of a 19-h-old pupa. (C  ) Eye ganglia (arrows  ), subesophageal ganglia (sub  ), and esophagus (es  ) in the head region of a 48-h-old pupa. (D  ) Staining of the brain and optic lobes of a 90-h-old pupa. (E  ) In the adult brain, staining was limited to arrow regions (arrows  ). For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
×
Low crc  Expression Is Associated with Hypersensitivity to Diethylether
We demonstrated here that in the eth  as311  , the insertion of a P-element into the crc  gene results in a decrease in mRNA levels. To confirm that the inserted P-element causes the mutant phenotype (hypersensitivity to diethylether anesthesia), we determined EC50s in the excised P-element revertants. The revertants of eth  as311  were isolated, and their genomic DNA were analyzed by Southern blot hybridization to show complete removal of the P-element from the eth  as311  mutants to revert wild type. 3 In the revertant-2  , -3  , -5  , and -44  , EC50s were of similar values to those of wild-type strains, Canton-S  and w  , 1.94% and 1.95% atm in these female flies and 1.91% and 1.98% atm in the male flies, respectively (table 1). These results strongly indicate that the hypersensitivity to diethylether of the mutant phenotype is caused by the inserted P-element in the crc  locus, which induced a lower expression of the gene.
Table 1. Concentration of Diethylether (EC50) in Drosophila Mutants
Image not available
Table 1. Concentration of Diethylether (EC50) in Drosophila Mutants
×
Furthermore, we prepared transgenic lines, crc-T1  and crc-T2  , by introducing wild-type crc  gene under a heat shock promoter, which expressed the gene by heat treatment and examined whether they could be rescued by heat shock from the mutant phenotype to wild type. Transgenic lines crossed to eth  as311  to emerge as double mutants, crc-T1;eth  as311  and crc-T2;eth  as311  . Their EC50s for diethylether were compared to the wild-type and mutant strains (table 1). Double-mutant strains without heat shock had significantly lower EC50s than those of the wild type, although their ED50s were higher than those of eth  as311  . When exposed to heat (37°C), their EC50s were not significantly different from those of the wild type. Thus, the transgenic flies were rescued from the mutation phenotypes to the wild type. The results also strongly indicate that crc  is the gene that causes the hypersensitivity to diethylether in eth  as311  . Three types of heat treatment were performed: heata, heatb, and heatcwere performed 1 h/day for the whole life and 4 days and 3 days before EC50determination, respectively. However, their EC50s were not different from one another. A double mutant with a crc  +transgene plus a lethal revertant-s20-1  (crc-T1;s20-1  ) with heat treatment recovered and was still alive at adulthood in the homozygotes. The results indicate that a transgene could be expressed by heat treatment.
crc  Mutant Phenotypes to Volatile Anesthetics
Previous genetic studies have identified several mutated genes that alter the sensitivity to various volatile anesthetics. 3–12 In some cases, the mutant phenotypes to volatile anesthetics are not parallel, and some are different among themselves. 6,7,9 Therefore, we compared the sensitivity to isoflurane and halothane between eth  as311  and the wild type. The dose–response curves for isoflurane were plotted to determine the EC50s by using loss of avoidance response as an endpoint (fig. 4). The EC50s of isoflurane were higher in eth  as311  than in the wild type in both sexes. eth  as311  was resistant to isoflurane but hypersensitive to diethylether. To confirm that the inserted P-element causes the isoflurane-resistant phenotype, we determined EC50s in the revertant and transgenic strains as well as hypersensitivity to diethylether anesthesia. In the revertant-2  , -3  , -5  , and -44  , EC50s were of similar values to those of wild-type strains, Canton-S  and w  , 0.75% and 0.77% atm in these female flies and 0.80% and 0.79% atm in the male flies, respectively (table 2). crc-T1;eth  as311  without heat shock had significantly higher EC50s than those of the wild type; their EC50s with heat shock did not differ significantly from those of the wild type. Thus, the transgenic flies were rescued from the isoflurane-resistant phenotypes to the wild type. These results also strongly indicate that crc  is the gene that causes resistance to isoflurane anesthesia in eth  as311  . On the other hand, the EC50s of halothane were not different in eth  as311  and wild type (table 2), suggesting that calreticulin is not involved in the halothane anesthesia pathway or halothane does not seem to affect the functional site of the protein.
Fig. 4. Dose–response curves of isoflurane in female (filled circles  ) and male flies (unfilled circles  ) of Canton-S  and female (filled squares  ) and male flies (unfilled squares  ) of eth  as311  . The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
Fig. 4. Dose–response curves of isoflurane in female (filled circles 
	) and male flies (unfilled circles 
	) of Canton-S 
	and female (filled squares 
	) and male flies (unfilled squares 
	) of eth  as311 
	. The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
Fig. 4. Dose–response curves of isoflurane in female (filled circles  ) and male flies (unfilled circles  ) of Canton-S  and female (filled squares  ) and male flies (unfilled squares  ) of eth  as311  . The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
×
Table 2. Concentrations of Isoflurane and Halothane (EC50) in Drosophila Mutants
Image not available
Table 2. Concentrations of Isoflurane and Halothane (EC50) in Drosophila Mutants
×
Discussion
We had collected Drosophila  mutants with altered sensitivity to diethylether anesthesia, which are transposon tagging strains to facilitate gene cloning. 3 In the current study, we demonstrated that the 1.4 kb of crc  mRNA is expressed at all developmental stages and is widely distributed in tissues, including the central nervous system. In the cellular blastoderm, the crc  mRNA was already expressed in the peripheral nuclei in the cells. Increased expression in the ectoderm was observed especially in the central nervous system and then increased in mesoderm tissues with advancement of embryonic stage. crc  gene was expressed in the cell proliferation regions. At late larval and early pupal stages, its expression markedly increased in the brain; the highest expression of the mRNA was detected in newly developed adult neurons at 19 h after puparization and then decreased until adult stage. During the formation of adult brain and optic lobes, the mRNA expression was stronger in cell bodies of neurons than in axons. Considered together, our results suggest that calreticulin is required for all development stages of Drosophila  and especially for the formation of the brain and optic lobes at the late larval and early pupal stages (figs. 3A-1 and B-1).
We also showed that eth  as311  , which is the first fertile mutation in crc  , has lower transcriptional levels of the crc  gene at all developmental stages and leads to the mutant phenotypes. The crc  mutants, revertant-s20-1  (this study) and l(3)s114307  , 31 cause death during embryonic maturation and are probably null mutation. In addition, knockout mice for the calreticulin gene die during embryonic development. 32 In this regard, calreticulin is important for the development, organization, and pathfinding of the peripheral nervous system in Drosophila  embryos 31 and for formation of the heart, brain, and ventral body wall closure in mice. 32 In fibroblasts, the proteins influence cell migration in calcium- and substrate-dependent manners. 33 The crc  may play a role in cellular adhesion during cell proliferation, differentiation, movement, and synapse formation in the brain, similar to knockout mice and Aplysia  . 32–34 
The eth  as311  shows different sensitivities to various volatile anesthetics, i.e.  , hypersensitivity to diethylether, resistance to isoflurane, and wild-type sensitivity to halothane (tables 1 and 2). These findings support the possibility that diethylether and isoflurane interact with the different sites of calreticulin to induce anesthesia, but halothane does not interact with any functional site of the protein. In crc-T1;eth  as311  , a transgene of crc  controlled by a heat shock promoter is expressed only by heat shock. Transgenic flies that were not exposed to heat shock showed hypersensitivity to diethylether and resistance to isoflurane (tables 1 and 2). The transgenic flies that were exposed to heat shock were rescued to show the wild-type anesthetic responses. crc-T1;eth  as311  exposed to heat (37°C) for 1 h a day for 3 days just before anesthesia showed wild-type sensitivities to diethylether and isoflurane. This suggests strongly that the mutant phenotypes are caused by low expression of calreticulin in the adult flies.
At clinical concentrations, halothane and isoflurane cause a leak of calcium into the cytosol, deplete InsP3-sensitive calcium stores, and prevent the increase in cytosolic calcium concentration. 35 Halothane reduces endoplasmic reticulum Ca2+content in airway smooth muscle cells via  increased Ca2+leak through both InsP3and ryanodine receptors, and it alters InsP3receptors directly and indirectly via  modulating InsP3levels. 36 These results suggest that halothane targets as InsP3receptor, but it does not interact with calreticulin because lower levels of crc  expression cause diethylether hypersensitivity and isoflurane resistance but normal response to halothane in Drosophila  . The reduced amount of calreticulin in the mutant may induce higher cellular Ca2+levels in resting cells and require a higher concentration of isoflurane to induce anesthesia, causing isoflurane resistance.
Diethylether might act on calreticulin directly; calreticulin easily loses its function in the mutant, leading to hypersensitivity to diethylether. A possible explanation of the hypersensitivity to diethylether and isoflurane resistance is that the two volatile anesthetics act on different regions of calreticulin to induce opposite effects. We speculated previously that isoflurane interacts with the P- and C-domains of calreticulin, which control intracellular Ca2+levels. Another function in the N-domain of calreticulin is as a modulator of both α-integrin adhesion and integrin-initiated Ca2+influx signaling in fibroblasts of knockout mice. 33 Furthermore, the N-domain does not only decrease tyrosine phosphorylation of β-catenin, resulting in the modulation of cell adhesiveness, 37,38 but it also is known as the receptor for thrombospondin, one of the de-adhesive matricellular proteins. 39 The low level of calreticulin in the crc  mutant may lead to hypersensitivity to diethylether via  weakness of the adhesion complexes and the signaling pathway.
Last, the highest crc  expression was noted during development in the newly formed adult brain at the pupa stage of 19 h. In the insect, steroid hormone ecdysone induces metamorphosis. The marked reduction of calreticulin in the pupa stage may affect the formation of adult tissues by nuclear export of the glucocorticoid receptor, 22 down-regulation of steroid hormone receptor functions, and chaperoning function to glycoproteins. 20,21 These effects may be disregarded as the cause of sensitivity to diethylether and isoflurane directly, but because of their indirect effects they may not be ruled out. However, we cannot ignore the interaction between the anesthetics and other proteins, particularly in the case of halothane.
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Fig. 1. Structure of the crc  gene. (A  ) (Upper portion  ) Genomic DNA with a P-element insertion (triangle  ) and restriction sites (Ps  =Pst  I; K  =Kpn  I; B  =Bam  HI; Bg  =Bgl  II; E  =Eco  RI). (Center portion  ) 2.1-kb genomic DNA clone (pr-clone  ). 3 (Lower portion  ) Exon–intron organization of crc  (exon:black boxes  = translated regions; white boxes  = untranslated regions). (B  ) Drosophila  calreticulin model. Drosophila  calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40 The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C  ) Northern blot analysis. Embryos, (CE, EE  ), first and second instar larve (C12, E12  ), third instar larvae (C3, E3  ), pupae (CP, EP  ), and adults (CA, EA  ) of Canton-S  (C) and ethas311  (E), respectively. A 1.4 kb is a transcript of crc  and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41 (D  ) Calreticulin expression is normalized by rp49 in Canton-S  (black bars  ) and eth  as311  (gray bars  ).
Fig. 1. Structure of the crc 
	gene. (A 
	) (Upper portion 
	) Genomic DNA with a P-element insertion (triangle 
	) and restriction sites (Ps 
	=Pst 
	I; K 
	=Kpn 
	I; B 
	=Bam 
	HI; Bg 
	=Bgl 
	II; E 
	=Eco 
	RI). (Center portion 
	) 2.1-kb genomic DNA clone (pr-clone 
	). 3(Lower portion 
	) Exon–intron organization of crc 
	(exon:black boxes 
	= translated regions; white boxes 
	= untranslated regions). (B 
	) Drosophila 
	calreticulin model. Drosophila 
	calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C 
	) Northern blot analysis. Embryos, (CE, EE 
	), first and second instar larve (C12, E12 
	), third instar larvae (C3, E3 
	), pupae (CP, EP 
	), and adults (CA, EA 
	) of Canton-S 
	(C) and ethas311 
	(E), respectively. A 1.4 kb is a transcript of crc 
	and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41(D 
	) Calreticulin expression is normalized by rp49 in Canton-S 
	(black bars 
	) and eth  as311 
	(gray bars 
	).
Fig. 1. Structure of the crc  gene. (A  ) (Upper portion  ) Genomic DNA with a P-element insertion (triangle  ) and restriction sites (Ps  =Pst  I; K  =Kpn  I; B  =Bam  HI; Bg  =Bgl  II; E  =Eco  RI). (Center portion  ) 2.1-kb genomic DNA clone (pr-clone  ). 3 (Lower portion  ) Exon–intron organization of crc  (exon:black boxes  = translated regions; white boxes  = untranslated regions). (B  ) Drosophila  calreticulin model. Drosophila  calreticulin has 404 amino acid residues, and a signal sequence (1–19 residues). 40 The N-domain binds to KXGFFKR of steroid hormone receptors, retinoic receptors, and α-integrin. The P-domain has two sets of three sequence repeats, and a high-affinity Ca2+-binding site. The C-domain binds more than 25 mol of Ca2+with a relatively high capacity and low affinity. A C-terminal HDEL is ER retrieval signal. (C  ) Northern blot analysis. Embryos, (CE, EE  ), first and second instar larve (C12, E12  ), third instar larvae (C3, E3  ), pupae (CP, EP  ), and adults (CA, EA  ) of Canton-S  (C) and ethas311  (E), respectively. A 1.4 kb is a transcript of crc  and the 0.5 kb of ribosomal protein 49 (rp49) as a positive control. 41 (D  ) Calreticulin expression is normalized by rp49 in Canton-S  (black bars  ) and eth  as311  (gray bars  ).
×
Fig. 2. In situ  hybridization of crc  transcripts in wild-type embryos. The crc  antisense was used as a probe in A-1  , B-1  , and C-1  , and the sense probes were used in A-2  , B-2  , and C-2  . (A-1  ) Pole cells (pc  ) and yolk granules (yg  ) at embryonic stage 5. (B-1  ) Procephalic neuroblasts (pnb  ) and ventral neuroblasts (vnb  ) at embryonic stage 9. (C-1  ) Salivary gland (sg  ) in the head region, trachea (t  ), hind gut (hg  ), and midgut (mg  ) at stage 15. (Left  ) anterior egg position; (right  ) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
Fig. 2. In situ 
	hybridization of crc 
	transcripts in wild-type embryos. The crc 
	antisense was used as a probe in A-1 
	, B-1 
	, and C-1 
	, and the sense probes were used in A-2 
	, B-2 
	, and C-2 
	. (A-1 
	) Pole cells (pc 
	) and yolk granules (yg 
	) at embryonic stage 5. (B-1 
	) Procephalic neuroblasts (pnb 
	) and ventral neuroblasts (vnb 
	) at embryonic stage 9. (C-1 
	) Salivary gland (sg 
	) in the head region, trachea (t 
	), hind gut (hg 
	), and midgut (mg 
	) at stage 15. (Left 
	) anterior egg position; (right 
	) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42Bar 
	= 50 μm.
Fig. 2. In situ  hybridization of crc  transcripts in wild-type embryos. The crc  antisense was used as a probe in A-1  , B-1  , and C-1  , and the sense probes were used in A-2  , B-2  , and C-2  . (A-1  ) Pole cells (pc  ) and yolk granules (yg  ) at embryonic stage 5. (B-1  ) Procephalic neuroblasts (pnb  ) and ventral neuroblasts (vnb  ) at embryonic stage 9. (C-1  ) Salivary gland (sg  ) in the head region, trachea (t  ), hind gut (hg  ), and midgut (mg  ) at stage 15. (Left  ) anterior egg position; (right  ) posterior egg position. For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
×
Fig. 3. The highest crc  expression in the brains of early pupae. The crc  antisense was used as a probe in A-1  , B-1  , C-1  , D-1  , and E-1  in Canton-S  and A-3  , B-3  , and E-3  in ethas311  , and the sense probes were used in A-2  , B-2  , C-2  , D-2  , and E-2  in Canton-S  . (A  ) Larval brain hemispheres (bh  ) including the optic lobes (ol  ) and ventral ganglion (vg  ) in the third instar larvae. (B  ) The brain of a 19-h-old pupa. (C  ) Eye ganglia (arrows  ), subesophageal ganglia (sub  ), and esophagus (es  ) in the head region of a 48-h-old pupa. (D  ) Staining of the brain and optic lobes of a 90-h-old pupa. (E  ) In the adult brain, staining was limited to arrow regions (arrows  ). For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
Fig. 3. The highest crc 
	expression in the brains of early pupae. The crc 
	antisense was used as a probe in A-1 
	, B-1 
	, C-1 
	, D-1 
	, and E-1 
	in Canton-S 
	and A-3 
	, B-3 
	, and E-3 
	in ethas311 
	, and the sense probes were used in A-2 
	, B-2 
	, C-2 
	, D-2 
	, and E-2 
	in Canton-S 
	. (A 
	) Larval brain hemispheres (bh 
	) including the optic lobes (ol 
	) and ventral ganglion (vg 
	) in the third instar larvae. (B 
	) The brain of a 19-h-old pupa. (C 
	) Eye ganglia (arrows 
	), subesophageal ganglia (sub 
	), and esophagus (es 
	) in the head region of a 48-h-old pupa. (D 
	) Staining of the brain and optic lobes of a 90-h-old pupa. (E 
	) In the adult brain, staining was limited to arrow regions (arrows 
	). For names of organs and developmental stages, refer to Hartenstein. 42Bar 
	= 50 μm.
Fig. 3. The highest crc  expression in the brains of early pupae. The crc  antisense was used as a probe in A-1  , B-1  , C-1  , D-1  , and E-1  in Canton-S  and A-3  , B-3  , and E-3  in ethas311  , and the sense probes were used in A-2  , B-2  , C-2  , D-2  , and E-2  in Canton-S  . (A  ) Larval brain hemispheres (bh  ) including the optic lobes (ol  ) and ventral ganglion (vg  ) in the third instar larvae. (B  ) The brain of a 19-h-old pupa. (C  ) Eye ganglia (arrows  ), subesophageal ganglia (sub  ), and esophagus (es  ) in the head region of a 48-h-old pupa. (D  ) Staining of the brain and optic lobes of a 90-h-old pupa. (E  ) In the adult brain, staining was limited to arrow regions (arrows  ). For names of organs and developmental stages, refer to Hartenstein. 42 Bar  = 50 μm.
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Fig. 4. Dose–response curves of isoflurane in female (filled circles  ) and male flies (unfilled circles  ) of Canton-S  and female (filled squares  ) and male flies (unfilled squares  ) of eth  as311  . The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
Fig. 4. Dose–response curves of isoflurane in female (filled circles 
	) and male flies (unfilled circles 
	) of Canton-S 
	and female (filled squares 
	) and male flies (unfilled squares 
	) of eth  as311 
	. The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
Fig. 4. Dose–response curves of isoflurane in female (filled circles  ) and male flies (unfilled circles  ) of Canton-S  and female (filled squares  ) and male flies (unfilled squares  ) of eth  as311  . The ordinate is the proportion of anesthetized flies in probit scale, and the abscissa represents isoflurane concentration (percent atm) in logarithmic scale.
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Table 1. Concentration of Diethylether (EC50) in Drosophila Mutants
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Table 1. Concentration of Diethylether (EC50) in Drosophila Mutants
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Table 2. Concentrations of Isoflurane and Halothane (EC50) in Drosophila Mutants
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Table 2. Concentrations of Isoflurane and Halothane (EC50) in Drosophila Mutants
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