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. 2019 Apr 4;2019:10.17912/micropub.biology.000097. doi: 10.17912/micropub.biology.000097

TRiP stocks contain a previously uncharacterized loss-of-function sevenless allele

Spencer E Escobedo 1, Jonathan Zirin 2, Vikki M Weake 1,§
Reviewed by: Anonymous
PMCID: PMC7252292  PMID: 32550423

Figure 1.

Figure 1.

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Identification of a new sevenless (sev) allele in a subset of TRiP stocks A Representative images showing adult retinas imaged using optical neutralization for male flies from the TRiP collection that show wild type (BL42511) or sev (BL50662) phenotypes. White arrow indicates expected position of missing R7 rhabdomere. B Summary table describing presence of the eye phenotype and sev mutation in tested TRiP stocks. Eye phenotypes were analyzed using optical neutralization on male flies. Note, BL35781 and BL32261 females were outcrossed to Oregon R males with y1 sc v1; +/CyO and y1 sc v1; +/TM3, Sb1 male progeny used to assess presence/absence of R7. C Chromatogram showing sequence analysis of the sev gene in BL42511 and BL50662 stocks; the position of the 1107648A>T mutation corresponding to the new sev21 allele is shown.

Description

The Transgenic RNAi Project (TRiP) has generated more than 12,000 transgenic RNAi fly stocks that have been distributed to the community via the Bloomington Drosophila Stock Center (Ni et al. 2007; Ni et al. 2011; Perkins et al. 2015). These stocks express long double-stranded RNA hairpins (dsRNAs) or short RNA hairpins (shRNAs) under GAL4/UAS control (Brand and Perrimon 1993), and provide powerful tools for targeted genetic screens. Unexpectedly, as part of a genetic screen examining retinal degeneration in flies, we identified a defect in eye development associated with many of the TRiP stocks. Drosophila have a compound eye composed of repeating units, termed ommatidia, that each contain eight photoreceptor cells (R cells 1 – 8) (Ready, Hansen and Benzer 1976). The light-sensing organelle, the rhabdomere, in seven of these photoreceptors can be directly visualized in wild-type flies using light microscopy either by optical neutralization or by examining the deep pseudopupil; R7/R8 are stacked on top of each other so only one is visible in a given vertical plane (Franceschini and Kirschfeld 1971).Whereas seven rhabdomeres could be counted per ommatidium in wild-type flies (Fig. 1A), a subset of the TRiP lines tested show characteristic loss of a single rhabdomere (Fig. 1A, Fig. 1B). This single photoreceptor loss phenotype is reminiscent of sevenless (sev) mutants; sev (FBgn0003366) encodes a receptor tyrosine kinase essential for development of R7; thus, loss of function sev mutations result in ommatidia that lack R7 (Harris et al. 1976; Simon, Bowtell and Rubin 1989). Preliminary observations suggested that the sev phenotype was X-linked and observed only in TRiP stocks containing a scute (sc) allele of unknown origin denoted sc*. Whole genome sequencing data for one of the TRiP stocks with the X chromosome containing this sc allele (y1 sc* v1) revealed the presence of an A>T mutation at position X:1107648 in sev, which would result in a premature stop codon at K665X. We tested several of the TRiP stocks that showed the sev phenotype using PCR sequencing, and found that all contained the same mutation (Fig. 1C). We have named this new allele sev21. We note that we observed both sev21 and wild-type flies in BL32421, suggesting that this stock is mixed. Since this premature stop codon would result in a severely truncated protein, it is likely that the sev21 allele would represent a loss-of-function mutation. Supporting this, our newly identified sev21 allele did not complement the known sev14 loss-of-function allele (BL67947, BL32421, BL50662). Since stocks generated by the TRiP at Harvard Medical School and their collaborators at Tsinghua University show the sev phenotype, but stocks generated by TRiP collaborators at the National Institute of Genetics in Japan do not, we suspected that the mutation was likely present in the stocks used to balance the TRiP lines (BL35781 and BL32261). PCR sequencing revealed that both of these stocks carry the sev21 allele. Together, these data show that many of the TRiP RNAi stocks balanced with BL35781 or BL32261 contain a newly identified loss-of-function sev allele, sev21. TRiP stocks containing this sev21 allele, including both RNAi and sgRNA lines (TRiP-CRISPR Overexpression and KnockOut) (Port et al. 2014; Jia et al. 2018), will be annotated on Flybase and at BDSC. The presence of the sev21 mutation will not generally affect the use of these stocks, as the X chromosome is typically segregated out or heterozygous during experiments.

Reagents

BL25709 (RRID:BDSC_25709): y1 v1 P{y+t7.7=nos-phiC31\int.NLS}X; P{y+t7.7=CaryP}attP40

BL25710 (RRID:BDSC_25710): y1 sc* v1 P{y+t7.7=nos-phiC31\int.NLS}X; P{y+t7.7=CaryP}attP2

BL35781 (RRID:BDSC_35781): y1 sc* v1; In(2LR)Gla, wgGla-1 PPO1Bc/CyO

BL32261 (RRID:BDSC_32261): y1 sc* v1; Dr1 e1/TM3, Sb1

BL67947 (RRID:BDSC_67947): y1 sc* v1; P{y+t7.7 v+t1.8=TRiP.HMS05772}attP40

BL32421 (RRID:BDSC_32421): y1 sc* v1; P{ y+t7.7 v+t1.8=TRiP.HMS00416}attP2

BL50662 (RRID:BDSC_50662): y1 sc* v1; P{ y+t7.7 v+t1.8=TRiP.HMC03063}attP2

BL42511 (RRID:BDSC_42511): y1 v1; P{ y+t7.7 v+t1.8=TRiP.HMJ02076}attP40

BL5690 (RRID:BDSC_5690): sev14; Ras85De2F/TM3, Sb1

BL5691 (RRID:BDSC_5691): sev14; drke0A/CyO

Acknowledgments

Acknowledgments

We would like to thank Dr. Andrew Zelhof for confirming the presence of the sev phenotype, Dr. Yanui Hu for compiling list of variants in sev from TRiP stock whole genome sequence, and Dr. Kevin Cook and Dr. Annette Parks for providing Drosophila stocks and for their input on the manuscript.

Funding

Research reported in this publication was supported by the National Eye Institute of the NIH under Award Number R01EY024905 to VW.

References

  1. Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim HS, Miller A, Housden A, Foos M, Randkelv S, Kelley C, Namgyal P, Villalta C, Liu LP, Jiang X, Huan-Huan Q, Wang X, Fujiyama A, Toyoda A, Ayers K, Blum A, Czech B, Neumuller R, Yan D, Cavallaro A, Hibbard K, Hall D, Cooley L, Hannon GJ, Lehmann R, Parks A, Mohr SE, Ueda R, Kondo S, Ni JQ, Perrimon N. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics. 2015 Aug 28;201(3):843–852. doi: 10.1534/genetics.115.180208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ni JQ, Zhou R, Czech B, Liu LP, Holderbaum L, Yang-Zhou D, Shim HS, Tao R, Handler D, Karpowicz P, Binari R, Booker M, Brennecke J, Perkins LA, Hannon GJ, Perrimon N. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods. 2011 Apr 01;8(5):405–407. doi: 10.1038/nmeth.1592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ni JQ, Markstein M, Binari R, Pfeiffer B, Liu LP, Villalta C, Booker M, Perkins L, Perrimon N. Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods. 2007 Dec 16;5(1):49–51. doi: 10.1038/nmeth1146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun 01;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  5. Ready DF, Hanson TE, Benzer S. Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol. 1976 Oct 15;53(2):217–240. doi: 10.1016/0012-1606(76)90225-6. [DOI] [PubMed] [Google Scholar]
  6. Franceschini N, Kirschfeld K. [Pseudopupil phenomena in the compound eye of drosophila]. Kybernetik. 1971 Nov 01;9(5):159–182. doi: 10.1007/BF02215177. [DOI] [PubMed] [Google Scholar]
  7. Harris WA, Stark WS, Walker JA. Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J Physiol. 1976 Apr 01;256(2):415–439. doi: 10.1113/jphysiol.1976.sp011331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Simon MA, Bowtell DD, Rubin GM. Structure and activity of the sevenless protein: a protein tyrosine kinase receptor required for photoreceptor development in Drosophila. Proc Natl Acad Sci U S A. 1989 Nov 01;86(21):8333–8337. doi: 10.1073/pnas.86.21.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jia Y, Xu RG, Ren X, Ewen-Campen B, Rajakumar R, Zirin J, Yang-Zhou D, Zhu R, Wang F, Mao D, Peng P, Qiao HH, Wang X, Liu LP, Xu B, Ji JY, Liu Q, Sun J, Perrimon N, Ni JQ. Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM. Proc Natl Acad Sci U S A. 2018 Apr 16;115(18):4719–4724. doi: 10.1073/pnas.1800677115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Port F, Chen HM, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 01;111(29):E2967–E2976. doi: 10.1073/pnas.1405500111. [DOI] [PMC free article] [PubMed] [Google Scholar]

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