Correspondence to: Spyros Artavanis-Tsakonas, Massachusetts General Hospital Cancer Center and the Department of Cell Biology, Harvard Medical School, 13th Street, Charlestown, MA 02129-2000. Tel:(617) 726-6863 Fax:(617) 726-6857 E-mail:tsakonas{at}helix.mgh.harvard.edu.
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Abstract |
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The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homologue of rab6, Drab6. Vertebrate and yeast homologues of this protein have been shown to regulate Golgi network to TGN trafficking. To study the function of this protein in the development of a multicellular organism, we analyzed three different warthog mutants. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Our studies show, contrary to yeast, that the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. We present models for the modifying effect of Drab6 on Notch signaling.
Key Words: Drosophila, Drab6, warthog, bristle, Golgi network
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Introduction |
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RAB proteins comprise the largest class of the ras-like GTPase superfamily. Genetic and biochemical studies have shown their involvement in various steps of endocytosis, exocytosis, and transcytosis (
One of these proteins, rab6, has been shown to regulate trafficking from the Golgi to the TGN (
warthog (wg)1 mutations were first identified as modifiers of Notch signaling in the course of a genetic screen involving the modulation of a constitutively active Notch receptor (
Having found genomic mutations in the Drosophila homologue of this gene, we sought to characterize its role in development. Characterization of rab function has mostly been performed in the single cell organism yeast or in tissue culture cells derived from multicellular organisms. Whereas rab3, a protein specific to neuroendocrine cells, has been studied by knockout mutations in mice (
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Materials and Methods |
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Fly Culture and warthog Alleles
All fly strains were grown and collected according to standard conditions (
To create excision alleles of the P2352 allele, 50 P2352/CyO; D2-3 Sb/+ males were independently crossed to Adv/CyO; ry- virgins. Up to four ry-Sb+ male progeny carrying the altered P2352 chromosome were retained from each pair mating and were used for complementation testing against the starting P2352 stock as well as the original screen alleles. If differing phenotypes were found to exist for any of the four male progeny, one representative of each was kept. The progeny could be grouped into three different phenotypic classes. Two of the classes constituted new warthog alleles: 14 were homozygous lethal, lethal with the P2352 allele, and failed to complement the bristle phenotype of the wrt screen alleles (D1A, D5C, D6E, D9C, D12C, D15A, D17B, D22A, D23D, D24A, D28E, D29D, D37A, D39B, and D40B), whereas 9 alleles were homozygous viable with the wrt bristle phenotype and failed to complement the bristle phenotype of wrtAM4, wrtER1, wrtAS1, and wrtMEF, but did complement the lethality of P2352 (D2B, D7C, D9A, D9D, D10A, D14E, D23A, D35A, and D40C). 13 revertants constituted the third class of excision alleles; they were homozygous viable without any bristle aberrations and complemented the wrt screen alleles as well as the P2352 allele (D1B, D2D, D3A, D4A, D5A, D6B, D11A, D13E, D17E, D27A, D28B, D30D, and D31B). Molecular analysis showed this last class of alleles to be precise excisions of the P2352 insertion, whereas the former two classes were imprecise excisions with or without duplications and deletions.
For clonal analysis, the wrtP2352, wrtER1, wrtAS1, wrtD17B, and wrtD23D mutations were recombined onto chromosome 40-2pM (w; P[mini-w+; hs-pM]21C, 36F, P[ry+; hs-neo; FRT]40A). Methods described in
Genomic Walk and cDNA Screening
The Berkeley Drosophila Genome Project (BDGP) had sequenced 300 bp of genomic DNA neighboring the P insertion site of l(2)08323. To determine which P1 in the 33C-D region contained this sequence, primers were designed from either end of the corresponding STS 0355. These primers [C: ctt ctc gct ccg ctc cgc tct cac c and D: gat tcc cgct ctg gtc aca cac aac] were used in a PCR reaction with various BDGP P1 clones assigned to this chromosomal region. With P1 DS00299 as a template, a fragment of the correct size and restriction site pattern was produced, indicating it contained the genomic DNA neighboring the P insertion. The DNA from this PCR reaction was used as a probe to start a genomic walk along the P1 DS00299. Subclones extending 15 kb to the left and 10 kb to the right of the initial fragment were obtained, sequenced, and then analyzed with DNAStar software (DNASTAR Inc.). The BLASTX program (
For cDNAs within the region, both a 012-h embryonic library and an imaginal disc library (gift of T. Xu and G. Rubin) were screened with each of the genomic subclones obtained in the walk. Sequencing was performed by the W.M. Keck Foundation Biotechnology Resource Lab at Yale University. Primers were synthesized by the Oligoz-R-Us at Yale University.
Molecular Characterization of Mutations
Whole genomic Southerns were prepared with DNA from all warthog alleles digested with EcoRI or BamHI. Each of the P1 subclones from the genomic walk was used for hybridization. For a subset of mutants, additional Southerns were prepared using the following restriction enzymes: EcoRV, SacII, PstI, ClaI, or a combination of these. From these studies, the alleles D17B, D39C, and D40B were found to have retained the 3' end of the PZ insert, whereas the alleles D12C and D23E retained the 5' end. Alleles D6E, D24A, and D29D had small insertions of genomic or transposon DNA, whereas the D23D allele had a 1.8-kb genomic deletion.
For the original screen mutants, no disruption in the restriction pattern was found by whole genomic Southerns, therefore, the particular mutation was determined by sequencing subcloned PCR fragments. DNA from wrtAM4/wrtAM4, wrtMEF/wrtMEF, wrtAS1/Df 3344, wrtER1/Df 3344, and w1118 was used as templates for a PCR reaction with primers CS-T7-2 (ccc cat tat aaa cag tga gg) and CS-T3-7 (cgt gtc aat gag tta gca ttc gc) that encompass the open reading frame (ORF) of Drab6. The single band obtained from gel purification was subcloned into the pGEM-T Easy vector (Stratagene) and sequenced. More than four independent PCR reactions were performed for each mutant and for w1118 as a control.
To determine the exact extent of the genomic deletion of D23D, PCR was also performed using DNA from D23D/CyO flies with the primers CS-T3-8 [gga atc att gaa cac aga ctg gc] and 5'-1-s[cct gct ggt tag ccg ata tcc] or CS-T3-9 [ggg ata gtc atg cga aca gag gtg cgc] and 5'-1-s[cct gct ggt tag ccg ata tcc]. For each reaction, two bands of the expected sizes were obtained, a 3.3-kb fragment derived from the balancer chromosome and a 1.5-kb fragment derived from the mutant D23D chromosome. Sequencing was performed on the 1.5-kb fragment.
Transgenic Flies
P elementmediated germline transformation was performed as per
Transgenic flies of the different cDNA constructs were created using the phsCaSpeR vector. For wild-type warthog, the PCR product of w1118 flies that was used as a control for sequencing of the screen alleles was subcloned into the pGEM-T Easy vector (Stratagene), and then transferred to the phsCaSpeR vector. This DNA contained the complete ORF of Drab6 as well as 110-bp upstream and 310-bp downstream of the coding region. To overexpress the R62C mutant form of Drab6, subcloned DNA from a PCR reaction of the AM4 allele was inserted into the phsCaSpeR vector. To generate the Q71L mutant form of Drab6, the subcloned w1118 PCR product above was used as a template for site-directed mutagenesis (Stratagene). The following primers were used to induce the point mutation that resulted in Q71 being converted to a leucine: Q71L-S (g gat acg gcg gga CTC gag cga ttc cgc) and Q71L-AS (gcg gaa tcg ctc GAG tcc cgc cgt atc c).
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Results |
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Phenotype of the Notch Modifier warthog
Our lab had previously performed a genetic screen to isolate new genes that altered Notch signaling (
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In wild-type flies, bristles are part of mechanosensory organs and develop shortly after puparium formation as the trichogen, or shaft cell, sends a cytoplasmic extension from the epidermis into the overlying cuticulin (
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The five warthog alleles (wrtAM4, wrtER1, wrtAS1, wrtBU1, and wrtBN7) recovered in the screen had considerably shortened bristles as homozygotes or transheterozygotes (Figure 2). This defect was present only for macrochaete of the ocelli, notum, and scutellum, whereas the bristles of the eye, wing, and leg appeared normal. Scanning electron micrographs of warthog bristles showed, in addition to the aberrant length, that the morphology of wrt bristles was altered. The wrt bristles did not have finely tapered ends nor did they show the regularly spaced ridges from the membranous protrusions. Instead, the tips were mangled and the surface was either smooth or had very mild and disorganized ruffling.
Cloning and Rescue of warthog Reveals It Encodes Drab6
The wrt locus had been mapped to the 32F1-3;33F1-2 region of the second chromosome (
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To determine which potential transcript corresponded to the wrt gene, rescue constructs containing different portions of the subcloned DNA were generated (Figure 4). Only fragments that contained the complete ORF of the rab6-like gene were capable of rescuing the bristle phenotype of wrtAM4, wrtMEF, wrtER1, and wrtAS1 (data not shown).
The warthog cDNA was obtained by screening two independent cDNA libraries with a genomic subclone that straddled the P insertion site sequence. A 2.1-kb cDNA was recovered from an embryonic library and a 1.9-kb cDNA from an imaginal disc library. Sequencing showed both cDNAs to be from the same transcript with varying amounts of the 5' untranslated region. Comparison to genomic DNA showed no introns but revealed several base pair polymorphisms that did not alter the amino acid sequence. Translation of the sequence showed this transcript to have 89% identity to human rab6, 72% to the yeast rhy1 protein, and has subsequently been cloned as Drab6 (
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The Notch Modifier Alleles Harbor a Point Mutation in Drab6
The molecular lesions in the warthog locus recovered from the Notch screen were determined. As none of the alleles (eAS1, eER1, and eAM4) showed molecular aberrations on whole genomic Southern blots, PCR products of the coding region were sequenced. This data showed they each harbored the same point mutation; at base pair 313 from the starting methionine, a C was converted to a T that resulted in an amino acid change of arginine 62 to a cysteine (R62C). The location of this point mutation resides one amino acid from the second conserved GTP-binding domain (Figure 5) (
It is surprising that each of the alleles contained the same mutation as they were from independent mutagenesis crosses, and it is unlikely that the same point mutation would be created multiple times from both EMS and X-ray mutagenesis. It is more probable that the mutation existed in the genetic background of our starting stock w1118 and was recovered repeatedly given the sensitivity of its interaction with the Notch phenotype used in the screen. Consistent with this interpretation, a spontaneous allele (wrtMEF), also derived from the w1118 stock, was found to contain the same R62C point mutation.
Severe Loss-of-function warthog Alleles
The original alleles recovered were homozygous viable with short bristles, but the P2352 allele used to clone the warthog gene, produced shorter macrochaete in trans to the R62C alleles and was homozygous lethal. This variability of phenotype was also seen in excision alleles generated from the P2352 allele. Upon mobilization of the P insert, two classes of new wrt mutants were recovered. Nine were phenotypically similar to the original screen alleles; they were homozygous viable with the bristle defect and they complemented the P2352 lethality giving transheterozygotic progeny with the wrt bristle phenotype. Sixteen of the excision alleles were phenotypically similar to the parental P2352 allele; they were homozygous lethal but viable with the bristle defect in trans to the original screen alleles. The phenotypic pattern of these alleles suggests the R62C mutation may be a less severe loss-of-function allele, whereas the P2352 allele and several of its excision progeny are more severe loss-of-function alleles.
Various genomic transformants were tested for the ability to rescue the lethality of the parental stock (P2352) and representative excision alleles (D1A, D5C, and D6C). As with the bristle phenotype, only genomic constructs with the complete ORF of Drab6 rescued the lethality. Different lines of the phs Drab6 cDNA transformants were also tested and all were capable of rescuing the lethality. These studies show that both the lethality and the bristle phenotype were due to perturbations in the same gene, Drab6. The original screen alleles (AS1, ER1, and AM4) and the spontaneous allele (MEF) constitute hypomorphic warthog alleles, whereas the lethal alleles (P2352, D1A, D5C, and D6C) are more severe alleles of wrt.
Unexpectedly, a subset of the Drab6 cDNA transformant lines rescued the lethality to produce flies with bristle defects more subtle than the original wrt alleles. As these same transformant lines were capable of rescuing the bristle defect of the screen alleles with the R62C point mutation, this indicates that bristle development is more sensitive to the quantity or timing of Drab6 expression or function than is lethality.
Molecular Characterization of the Severe warthog Alleles
To determine the molecular lesions associated with the different excision alleles, whole genomic Southerns were prepared and probed with each subclone of the genomic walk. The allele wrtP2352 showed a single insertion between Drab6 and Phaedra1. Restriction digests showed the expected pattern for the PZ construct, with the 3' end of the insert lying closest to the warthog locus (Figure 5). Generated excision lines from this P allele that were homozygous viable without any bristle defect were all precise excisions, showing a return of the 2.6-kb BamHI fragment to the size of wild-type flies.
The severe loss-of-function excision alleles (homozygous lethal and failed to complement the bristle defect of the original alleles) all contained molecular aberrations centering around the P insertion site of wrtP2352. Most were imprecise excisions with deletions of only one end of the PZ insert as well as some flanking genomic DNA.
Of interest was the allele D23D that showed complete excision of the PZ insert as well as a 1.8-kb genomic deletion in the region of the warthog gene (Figure 5). To determine the extent of this deletion, DNA from these flies was used as a template in PCR reactions with four different sets of primers known to extend beyond the deletion. Sequencing of the PCR products showed the deletion extended from 72-bp upstream of Phae1 to 365-bp downstream of the STOP codon of the wrt gene. Therefore, this allele constituted a genomic null of warthog. Since the deletion extended into a potential upstream regulating region of Phae1, genetic testing with the different rescue constructs was performed. Flies that were homozygotic for the D23D chromosome were viable and phenotypically wild-type if they also carried the wrt cDNA transgene or a genomic fragment with only the wrt ORF. Therefore, the wrt null did not produce any phenotypic defects other than those of the wrt deletion.
Severe warthog Mutations Are Larval Lethal
To establish the time period of Drab6 expression critical for viability, homozygotic P2352/P2352, or D23D/D23D were monitored at different stages of development. Eggs with these homozygotic genotypes would proceed through embryogenesis to the larval stage, but would not continue to develop into pupae. Therefore, the more severe alleles of warthog were larval lethal. To determine if the lack of embryonic lethality was due to a maternal contribution of wrt, the FLP-FRT system was used to generate females with wrt-/wrt- germlines. All progeny with the genotype P2352/P2352 developed past embryogenesis, showing that a maternal contribution is not responsible for survival of wrtP2352 through embryonic development.
Mosaic Analysis of wrt Mutations
To study the effect of the more severe disruptions in Drab6 function during later stages of development, mosaic clones were induced using the FLP-FRT system (
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Surprisingly, the clonal analysis also showed that wrtP2352 was nonautonomous (Figure 6 d). Whereas portions of the mosaic clones contained mutant bristles, phenotypically wild-type bristles were also present in patches of P2352/P2352 tissue, indicating that wrt protein is not required within the cell producing the shaft of the bristle.
Overexpression of Wild-type and Mutant wrt
The function of rab proteins in mammalian systems has been elicited by studying the effects of overexpression of wild-type and mutant forms of these proteins. The best characterized forms are those modeled after ras mutations and are known to alter the ability of rabs to cycle between the GDP- and the GTP-bound states. The state of continued GTP binding has been produced by altering the Q of the second conserved GTP-binding domain to a leucine (Q72L in mammalian rab6; Figure 5). This abolishes intrinsic GTPase activity and decreases GAP-stimulated hydrolysis as well (
cDNAs of wild-type Drab6, the R62C mutation, and the Q72L mutation were placed under the control of the heat shock promoter to drive expression at different stages of development. Whereas overexpression of the wild-type form and the R62C mutation produced no visible phenotype in the background of wrt+/wrt+, the Q71L mutation altered the direction of bristle growth at any point along the bristle shaft (Figure 7). Overexpression of this GTP-bound mutant produced smoothly curving bristles or bristles with sharp changes in the orientation of growth, followed by continued growth in two opposite directions. Normal morphology appeared distal to the alteration, presumably because of the return of normal Drab6 function after the pulsed overexpression of Drab6 Q71L passed. Aberrations in the circumferential ridges was also seen, indicating that the membranous protrusions from between the actin bundles was also disrupted.
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Interestingly, basal expression of the Q71L mutant cDNA without heat shock, was capable of rescuing the bristle phenotype of the R62C alleles, indicating that even small amounts of the Q71L form of Drab6 can rescue the phenotypic effects of the loss-of-function R62C mutation.
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Discussion |
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Why Notch and rab6?
The Notch signal transduction pathway is used in many species to modulate the ability of precursor cells to respond to developmental cues. This signal is activated by the binding of the ligand Delta to its receptor Notch to activate downstream proteins. However, the selection of which cells undergo this activation is influenced by the amount of the Notch receptor at the cell surface; Notch is one of only a handful of genes to produce a visible phenotype with either an extra copy of the gene or in missing one copy.
In a search for genes that modified an activated Notch phenotype, a novel bristle mutant named warthog was found. Cloning of the gene revealed it encoded the Drosophila homologue of rab6, or Drab6 (
From these tissue culture experiments, mutations in Drab6 would be expected to delay the surface presentation of the Notch receptor. Given that the amount of Notch present on the cell surface is critical for the adoption of different cellular identities, such a delay in transportation of the Notch receptor to the plasma membrane would alter Notch signaling. The phenotypic interaction of the wrt screen alleles was consistent with a decrease in the amount of N available for signaling on the cell surface (
Another explanation for the modification of Notch signaling by wrt is suggested by a paper from
Phenotypic Analysis of warthog Mutants
Having found genomic mutations in Drab6, we were able to study its effect on the development of a multicellular organism. In contrast to the single-cell organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe (
Each of these mutants affected bristle morphology in a manner similar to other bristle mutants known to affect the structural integrity of cytoskeletal components (-actinin and this activity is blocked by rab3-GTP (
However, for warthog, no additive or synergistic interactions were seen with many mutations known to affect bristle structure (e.g., Sb, sn3, f 36a, Pr1, ss, Bsb, Bsb Pr, and Pr Dr; data not shown). More importantly, the nonautonomous phenotype seen in the severe warthog mutants implies the Drosophila homologue of rab6 modifies the surface presentation of other proteins. Nonautonomous phenotypes are typically seen with secreted or transmembrane proteins that signal to neighboring cells. This effect is consistent with results from yeast and mammalian tissue culture experiments that establish the role of rab6 in the proper secretion of other proteins (
R62C Defines a Novel Rab Mutation
Mutations that have previously been studied for rab6 are those engineered based on the GTP- and GDP-bound forms of ras-like molecules. From our screen (
This hypomorphic mutation altered rab6 function differently than the Q71L mutation, which resides next to the same GTP-binding domain. Overexpression Q71L Drab6 disrupted the orientation of bristle growth, whereas overexpression of R62C Drab6 in a wild-type background elicited no effects. Q71L Drab6 was also capable of rescuing the bristle defect of the R62C mutation. Therefore, studying the R62C mutation may reveal new information of Drab6 function.
warthog and Transport Redundancy
Perhaps the most interesting aspect of this phenotypic analysis is the limited requirement of a rab6 homologue throughout development. While an essential gene, Drab6 mutations did not affect the development of the eye, wing, and leg, nor the bristle structures within these tissues. This paucity of developmental phenotypes mirrors yeast studies that show null mutations in Ypt6 or rhy1 are not lethal, implying transport redundancy exists as proteins travel to the cell membrane (
The bristle phenotype of the warthog mutants, however, reveals there is a limitation to which an organism can compensate for mutations in Drab6, even if redundant or independent pathways exist for transport through the Golgi. This limitation may also be seen only after prolonged Drab6 dysfunction.
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Acknowledgements |
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The authors thank the SAT lab for their encouragement, knowledge, and unique sense of humor. Special thanks to the smile of cj's heart, Wart, for her constant infusion of personality. Special thanks to Bruno Goud for his comments.
This work was supported by the Howard Hughes Medical Institute, the National Institutes of Health (NS26084), and the Massachusetts General Hospital.
Submitted: May 18, 1999; Revised: July 1, 1999; Accepted: July 7, 1999.
1.used in this paper: ORF, open reading frame; wrt, warthog
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References |
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