A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the LKRSDH gene, which encodes the Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished LKRSDH alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Bloomington Drosophila Stock Center, Department of Biology, Indiana University"],"departments":[""],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/635497264bfa84f70789c8c28e9447f7.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an LKRSDH mutant (LKRSDHCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in LKRSDH mutants typically occurs in this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus, but clots are usually larger than the one depicted here. (D and E) Dorsal views of other LKRSDH mutants (LKRSDHCR70173-TG4.0/Df(2L)ED12527) showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profile is still more rounded than the profile of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced LKRSDH alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LKRSDHΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of LKRSDH mutants and the characterization of LKRSDH alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_02 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the LKRSDH region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LKRSDHCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional LKRSDH mutations tested | ||
692 | LKRSDH1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 LKRSDH1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LKRSDHΔ11/CyO | RRID:BDSC_607941 |
| w1118; LKRSDHΔ16/CyO | Jason Tennessen |
607942 | w1118; LKRSDHΔ17/CyO | RRID:BDSC_607942 |
| w1118; LKRSDHΔ17-1/CyO | Jason Tennessen |
| w1118; LKRSDHΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the LKRSDH gene, which encodes the Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase protein responsible for two enzymatic steps in lysine degradation (LKR activity in the N-terminal half converts lysine to saccharopine and SDH activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, LKRSDH protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with LKRSDH mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the LKRSDH gene (Mi{ET1}LKRSDHMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to LKRSDH. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LKRSDHMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LKRSDHMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LKRSDHMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LKRSDHMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was LKRSDH itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (LKRSDH, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in LKRSDH (LKRSDHCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LKRSDHMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional LKRSDH alleles. Four alleles (LKRSDHΔ11, LKRSDHΔ16 , LKRSDHΔ17 and LKRSDHΔ17-1) are CRISPR-induced, frameshift mutations early in the coding sequence generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. The fifth allele is the original LKRSDH mutation, LKRSDH1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving LKRSDH1 and hemolymph leakage was rare, suggesting that LKRSDH1 is hypomorphic. LKRSDH1 is a complex chromosomal rearrangement disrupting the 5’ end of LKRSDH (St. Clair et al. 2017), but my results and results presented by St. Clair et al. suggest there is residual gene expression, perhaps initiated from a cryptic transcription start site. The sixth allele (LKRSDHΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the LKR activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant LKRSDH protein (Figure 1F) with a disrupted LKR domain but an intact SDH domain.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that LKRSDH mutants (LKRSDHCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and LKRSDH mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in LKRSDH mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between LKRSDH protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in LKRSDH mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
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","pubmedId":"","doi":"10.1002/arch.22071"},{"reference":"Morgan TH, Bridges CB, Sturtevant AH. 1925. The genetics of Drosophila. Gravenhage: M. Nijhoff.
","pubmedId":"","doi":""},{"reference":"Morrow H, Mirth CK. 2024. Timing Drosophila development through steroid hormone action. Current Opinion in Genetics & Development 84: 102148.
","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Sheldon TJ, Miguel-Aliaga I, Gould AP, Taylor WR, Conklin D. 2007. A novel family of single VWC‐domain proteins in invertebrates. FEBS Letters 581: 5268-5274.
","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase mutants of Drosophila melanogaster
","reviews":[{"reviewer":{"displayName":"Keith A. Maggert"},"openAcknowledgement":true,"status":{"submitted":true}},{"reviewer":{"displayName":"Eric Spana"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Aoife Larkin"},"openAcknowledgement":false,"submitted":null}]},{"id":"d5330025-4d03-4026-bf44-09086ff4954e","decision":"revise","abstract":"A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the LKRSDH gene, which encodes the Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished LKRSDH alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Bloomington Drosophila Stock Center, Department of Biology, Indiana University"],"departments":[""],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/abf9f501c707c23acbfc11878e1ccf25.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an Lkrsdh mutant (LkrsdhCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in Lkrsdh mutants typically occurs in three positions (this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus), but clots are usually larger than the one depicted here. (D and E) Dorsal views of other individuals with the same genotype showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profiles are still more rounded than the profiles of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced Lkrsdh alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LkrsdhΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of LKRSDH mutants and the characterization of LKRSDH alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_03 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the LKRSDH region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LKRSDHCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional LKRSDH mutations tested | ||
692 | LKRSDH1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 LKRSDH1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LKRSDHΔ11/CyO | RRID:BDSC_607941 |
| w1118; LKRSDHΔ16/CyO | Jason Tennessen |
607942 | w1118; LKRSDHΔ17/CyO | RRID:BDSC_607942 |
| w1118; LKRSDHΔ17-1/CyO | Jason Tennessen |
| w1118; LKRSDHΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein responsible for two enzymatic steps in lysine degradation (Lkr activity in the N-terminal half converts lysine to saccharopine and Sdh activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, Lkrsdh protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with Lkrsdh mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the Lkrsdh gene (Mi{ET1}LkrsdhMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to Lkrsdh. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LkrsdhMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LkrsdhMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LkrsdhMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LkrsdhMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was Lkrsdh itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (Lkrsdh, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in Lkrsdh (LkrsdhCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LkrsdhMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional Lkrsdh alleles. Four alleles (LkrsdhΔ11, LkrsdhΔ16, LkrsdhΔ17 and LkrsdhΔ17-1) are CRISPR-induced, frameshift mutations generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. These four mutations are positioned to truncate the Lkrsdh protein and eliminate domains necessary for both Lkr and Sdh activities as well as the protein region that binds to the ecdysone receptor EcR in regulating histone methylation (Cakouros et al. 2008; Letunic and Bork 2026; Paysan-Lafosse et al. 2025). Lkr activity requires two domains: a catalytic domain and a domain that binds NAD+. The Lkr catalytic domain is likely intact in proteins produced by all four alleles (assuming the truncated proteins are stable), but its activity depends on the domain for binding NAD+, which would be absent—strongly suggesting these four alleles are amorphic. The fifth allele is the original Lkrsdh mutation, Lkrsdh1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving Lkrsdh1 and hemolymph leakage was rare, suggesting that Lkrsdh1 is hypomorphic. Lkrsdh1 is a complex chromosomal rearrangement deleting the normal translation start site, inserting repetitive sequences before the seventh codon and introducing an indel (St. Clair et al. 2017), but my phenotypic results and results presented by St. Clair et al. showing an 80% reduction in transcription suggest there is residual gene expression—likely initiated from a transcription start site within the insertion. The presence of a methionine codon within the inserted sequences near the Lkrsdh coding sequences suggests that a variant protein is produced with a shorter, novel N-terminus. The sixth allele (LkrsdhΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the Lkr activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant Lkrsdh protein (Figure 1F) with a disrupted Lkr domain but intact Sdh and EcR-binding domains.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that Lkrsdh mutants (LkrsdhCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and Lkrsdh mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in Lkrsdh mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between Lkrsdh protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in Lkrsdh mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
","pubmedId":"41939508","doi":""},{"reference":"Grell EH. 1961. The genetics and biochemistry of red fat cells in Drosophila melanogaster. Genetics 46: 925-933.
","pubmedId":"","doi":"10.1093/genetics/46.8.925"},{"reference":"Labropoulou V, Wang L, Magkrioti C, Smagghe G, Swevers L. 2024. Single domain von Willebrand factor type C “cytokines” and the regulation of the stress/immune response in insects. Archives of Insect Biochemistry and Physiology 115: 10.1002/arch.22071.
","pubmedId":"","doi":"10.1002/arch.22071"},{"reference":"Letunic I, Bork P. 2025. SMART v10: three decades of the protein domain annotation resource. Nucleic Acids Research 54: D499-D503.
","pubmedId":"","doi":"10.1093/nar/gkaf1023"},{"reference":"Morgan TH, Bridges CB, Sturtevant AH. 1925. The genetics of Drosophila. Gravenhage: M. Nijhoff.
","pubmedId":"","doi":""},{"reference":"Morrow H, Mirth CK. 2024. Timing Drosophila development through steroid hormone action. Current Opinion in Genetics & Development 84: 102148.
","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Paysan-Lafosse T, Andreeva A, Blum M, Chuguransky SR, Grego T, Pinto BL, et al., Bateman. 2024. The Pfam protein families database: embracing AI/ML. Nucleic Acids Research 53: D523-D534.
","pubmedId":"","doi":"10.1093/nar/gkae997"},{"reference":"Sheldon TJ, Miguel-Aliaga I, Gould AP, Taylor WR, Conklin D. 2007. A novel family of single VWC‐domain proteins in invertebrates. FEBS Letters 581: 5268-5274.
","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase mutants of Drosophila melanogaster
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Aoife Larkin"},"openAcknowledgement":false,"submitted":"1791890022085"}]},{"id":"db6f0e13-d10b-43d5-8ce0-32ea08db49c4","decision":"accept","abstract":"A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished Lkrsdh alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Bloomington Drosophila Stock Center, Department of Biology, Indiana University"],"departments":[""],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/abf9f501c707c23acbfc11878e1ccf25.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an Lkrsdh mutant (LkrsdhCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in Lkrsdh mutants typically occurs in three positions (this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus), but clots are usually larger than the one depicted here. (D and E) Dorsal views of other individuals with the same genotype showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profiles are still more rounded than the profiles of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced Lkrsdh alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LkrsdhΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of Lkrsdh mutants and the characterization of Lkrsdh alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_03 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the Lkrsdh region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LkrsdhCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional Lkrsdh mutations tested | ||
692 | Lkrsdh1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 Lkrsdh1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LkrsdhΔ11/CyO | RRID:BDSC_607941 |
| w1118; LkrsdhΔ16/CyO | Jason Tennessen |
607942 | w1118; LkrsdhΔ17/CyO | RRID:BDSC_607942 |
| w1118; LkrsdhΔ17-1/CyO | Jason Tennessen |
| w1118; LkrsdhΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein responsible for two enzymatic steps in lysine degradation (Lkr activity in the N-terminal half converts lysine to saccharopine and Sdh activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, Lkrsdh protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with Lkrsdh mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the Lkrsdh gene (Mi{ET1}LkrsdhMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to Lkrsdh. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LkrsdhMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LkrsdhMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LkrsdhMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LkrsdhMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was Lkrsdh itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (Lkrsdh, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in Lkrsdh (LkrsdhCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LkrsdhMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional Lkrsdh alleles. Four alleles (LkrsdhΔ11, LkrsdhΔ16, LkrsdhΔ17 and LkrsdhΔ17-1) are CRISPR-induced, frameshift mutations generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. These four mutations are positioned to truncate the Lkrsdh protein and eliminate domains necessary for both Lkr and Sdh activities as well as the protein region that binds to the ecdysone receptor EcR in regulating histone methylation (Cakouros et al. 2008; Letunic and Bork 2026; Paysan-Lafosse et al. 2025). Lkr activity requires two domains: a catalytic domain and a domain that binds NAD+. The Lkr catalytic domain is likely intact in proteins produced by all four alleles (assuming the truncated proteins are stable), but its activity depends on the domain for binding NAD+, which would be absent—strongly suggesting these four alleles are amorphic. The fifth allele is the original Lkrsdh mutation, Lkrsdh1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving Lkrsdh1 and hemolymph leakage was rare, suggesting that Lkrsdh1 is hypomorphic. Lkrsdh1 is a complex chromosomal rearrangement deleting the normal translation start site, inserting repetitive sequences before the seventh codon and introducing an indel (St. Clair et al. 2017), but my phenotypic results and results presented by St. Clair et al. showing an 80% reduction in transcription suggest there is residual gene expression—likely initiated from a transcription start site within the insertion. The presence of a methionine codon within the inserted sequences near the Lkrsdh coding sequences suggests that a variant protein is produced with a shorter, novel N-terminus. The sixth allele (LkrsdhΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the Lkr activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant Lkrsdh protein (Figure 1F) with a disrupted Lkr domain but intact Sdh and EcR-binding domains.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that Lkrsdh mutants (LkrsdhCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and Lkrsdh mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in Lkrsdh mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between Lkrsdh protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in Lkrsdh mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
","pubmedId":"41939508","doi":""},{"reference":"Grell EH. 1961. The genetics and biochemistry of red fat cells in Drosophila melanogaster. Genetics 46: 925-933.
","pubmedId":"","doi":"10.1093/genetics/46.8.925"},{"reference":"Labropoulou V, Wang L, Magkrioti C, Smagghe G, Swevers L. 2024. Single domain von Willebrand factor type C “cytokines” and the regulation of the stress/immune response in insects. Archives of Insect Biochemistry and Physiology 115: 10.1002/arch.22071.
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","pubmedId":"","doi":"10.1093/nar/gkaf1023"},{"reference":"Morgan TH, Bridges CB, Sturtevant AH. 1925. The genetics of Drosophila. Gravenhage: M. Nijhoff.
","pubmedId":"","doi":""},{"reference":"Morrow H, Mirth CK. 2024. Timing Drosophila development through steroid hormone action. Current Opinion in Genetics & Development 84: 102148.
","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Paysan-Lafosse T, Andreeva A, Blum M, Chuguransky SR, Grego T, Pinto BL, et al., Bateman. 2024. The Pfam protein families database: embracing AI/ML. Nucleic Acids Research 53: D523-D534.
","pubmedId":"","doi":"10.1093/nar/gkae997"},{"reference":"Sheldon TJ, Miguel-Aliaga I, Gould AP, Taylor WR, Conklin D. 2007. A novel family of single VWC‐domain proteins in invertebrates. FEBS Letters 581: 5268-5274.
","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine ketoglutarate reductase/saccharopine dehydrogenase mutants of Drosophila melanogaster
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Aoife Larkin"},"openAcknowledgement":false,"submitted":null}]},{"id":"f1fe17b5-66a3-41d2-a119-fce575ee5009","decision":"edit","abstract":"A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished Lkrsdh alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Bloomington Drosophila Stock Center, Department of Biology, Indiana University"],"departments":[""],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/abf9f501c707c23acbfc11878e1ccf25.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an Lkrsdh mutant (LkrsdhCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in Lkrsdh mutants typically occurs in three positions (this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus), but clots are usually larger than the one depicted here. (D and E) Dorsal views of other individuals with the same genotype showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profiles are still more rounded than the profiles of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced Lkrsdh alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LkrsdhΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of Lkrsdh mutants and the characterization of Lkrsdh alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_03 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the Lkrsdh region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LkrsdhCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional Lkrsdh mutations tested | ||
692 | Lkrsdh1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 Lkrsdh1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LkrsdhΔ11/CyO | RRID:BDSC_607941 |
| w1118; LkrsdhΔ16/CyO | Jason Tennessen |
607942 | w1118; LkrsdhΔ17/CyO | RRID:BDSC_607942 |
| w1118; LkrsdhΔ17-1/CyO | Jason Tennessen |
| w1118; LkrsdhΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein responsible for two enzymatic steps in lysine degradation (Lkr activity in the N-terminal half converts lysine to saccharopine and Sdh activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, Lkrsdh protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with Lkrsdh mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the Lkrsdh gene (Mi{ET1}LkrsdhMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to Lkrsdh. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LkrsdhMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LkrsdhMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LkrsdhMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LkrsdhMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was Lkrsdh itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (Lkrsdh, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in Lkrsdh (LkrsdhCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LkrsdhMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional Lkrsdh alleles. Four alleles (LkrsdhΔ11, LkrsdhΔ16, LkrsdhΔ17 and LkrsdhΔ17-1) are CRISPR-induced, frameshift mutations generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. These four mutations are positioned to truncate the Lkrsdh protein and eliminate domains necessary for both Lkr and Sdh activities as well as the protein region that binds to the ecdysone receptor EcR in regulating histone methylation (Cakouros et al. 2008; Letunic and Bork 2026; Paysan-Lafosse et al. 2025). Lkr activity requires two domains: a catalytic domain and a domain that binds NAD+. The Lkr catalytic domain is likely intact in proteins produced by all four alleles (assuming the truncated proteins are stable), but its activity depends on the domain for binding NAD+, which would be absent—strongly suggesting these four alleles are amorphic. The fifth allele is the original Lkrsdh mutation, Lkrsdh1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving Lkrsdh1 and hemolymph leakage was rare, suggesting that Lkrsdh1 is hypomorphic. Lkrsdh1 is a complex chromosomal rearrangement deleting the normal translation start site, inserting repetitive sequences before the seventh codon and introducing an indel (St. Clair et al. 2017), but my phenotypic results and results presented by St. Clair et al. showing an 80% reduction in transcription suggest there is residual gene expression—likely initiated from a transcription start site within the insertion. The presence of a methionine codon within the inserted sequences near the Lkrsdh coding sequences suggests that a variant protein is produced with a shorter, novel N-terminus. The sixth allele (LkrsdhΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the Lkr activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant Lkrsdh protein (Figure 1F) with a disrupted Lkr domain but intact Sdh and EcR-binding domains.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that Lkrsdh mutants (LkrsdhCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and Lkrsdh mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in Lkrsdh mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between Lkrsdh protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in Lkrsdh mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
","pubmedId":"41939508","doi":""},{"reference":"Grell EH. 1961. The genetics and biochemistry of red fat cells in Drosophila melanogaster. Genetics 46: 925-933.
","pubmedId":"","doi":"10.1093/genetics/46.8.925"},{"reference":"Labropoulou V, Wang L, Magkrioti C, Smagghe G, Swevers L. 2024. Single domain von Willebrand factor type C “cytokines” and the regulation of the stress/immune response in insects. Archives of Insect Biochemistry and Physiology 115: 10.1002/arch.22071.
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","pubmedId":"","doi":"10.1093/nar/gkaf1023"},{"reference":"Morgan TH, Bridges CB, Sturtevant AH. 1925. The genetics of Drosophila. Gravenhage: M. Nijhoff.
","pubmedId":"","doi":""},{"reference":"Morrow H, Mirth CK. 2024. Timing Drosophila development through steroid hormone action. Current Opinion in Genetics & Development 84: 102148.
","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Paysan-Lafosse T, Andreeva A, Blum M, Chuguransky SR, Grego T, Pinto BL, et al., Bateman. 2024. The Pfam protein families database: embracing AI/ML. Nucleic Acids Research 53: D523-D534.
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","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine ketoglutarate reductase/saccharopine dehydrogenase mutants of Drosophila melanogaster
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Aoife Larkin"},"openAcknowledgement":false,"submitted":null}]},{"id":"e66a97fb-d5b9-4da2-9a55-8b09eed4e6f1","decision":"edit","abstract":"A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished Lkrsdh alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Indiana University"],"departments":["Bloomington Drosophila Stock Center, Department of Biology"],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/abf9f501c707c23acbfc11878e1ccf25.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an Lkrsdh mutant (LkrsdhCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in Lkrsdh mutants typically occurs in three positions (this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus), but clots are usually larger than the one depicted here. (D and E) Dorsal views of other individuals with the same genotype showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profiles are still more rounded than the profiles of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced Lkrsdh alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LkrsdhΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of Lkrsdh mutants and the characterization of Lkrsdh alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_03 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the Lkrsdh region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LkrsdhCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional Lkrsdh mutations tested | ||
692 | Lkrsdh1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 Lkrsdh1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LkrsdhΔ11/CyO | RRID:BDSC_607941 |
| w1118; LkrsdhΔ16/CyO | Jason Tennessen |
607942 | w1118; LkrsdhΔ17/CyO | RRID:BDSC_607942 |
| w1118; LkrsdhΔ17-1/CyO | Jason Tennessen |
| w1118; LkrsdhΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein responsible for two enzymatic steps in lysine degradation (Lkr activity in the N-terminal half converts lysine to saccharopine and Sdh activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, Lkrsdh protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with Lkrsdh mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the Lkrsdh gene (Mi{ET1}LkrsdhMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to Lkrsdh. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LkrsdhMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LkrsdhMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LkrsdhMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LkrsdhMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was Lkrsdh itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (Lkrsdh, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in Lkrsdh (LkrsdhCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LkrsdhMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional Lkrsdh alleles. Four alleles (LkrsdhΔ11, LkrsdhΔ16, LkrsdhΔ17 and LkrsdhΔ17-1) are CRISPR-induced, frameshift mutations generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. These four mutations are positioned to truncate the Lkrsdh protein and eliminate domains necessary for both Lkr and Sdh activities as well as the protein region that binds to the ecdysone receptor EcR in regulating histone methylation (Cakouros et al. 2008; Letunic and Bork 2026; Paysan-Lafosse et al. 2025). Lkr activity requires two domains: a catalytic domain and a domain that binds NAD+. The Lkr catalytic domain is likely intact in proteins produced by all four alleles (assuming the truncated proteins are stable), but its activity depends on the domain for binding NAD+, which would be absent—strongly suggesting these four alleles are amorphic. The fifth allele is the original Lkrsdh mutation, Lkrsdh1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving Lkrsdh1 and hemolymph leakage was rare, suggesting that Lkrsdh1 is hypomorphic. Lkrsdh1 is a complex chromosomal rearrangement deleting the normal translation start site, inserting repetitive sequences before the seventh codon and introducing an indel (St. Clair et al. 2017), but my phenotypic results and results presented by St. Clair et al. showing an 80% reduction in transcription suggest there is residual gene expression—likely initiated from a transcription start site within the insertion. The presence of a methionine codon within the inserted sequences near the Lkrsdh coding sequences suggests that a variant protein is produced with a shorter, novel N-terminus. The sixth allele (LkrsdhΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the Lkr activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant Lkrsdh protein (Figure 1F) with a disrupted Lkr domain but intact Sdh and EcR-binding domains.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that Lkrsdh mutants (LkrsdhCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and Lkrsdh mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in Lkrsdh mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between Lkrsdh protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in Lkrsdh mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
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","pubmedId":"","doi":"10.1093/nar/gkaf1023"},{"reference":"Morgan TH, Bridges CB, Sturtevant AH. 1925. The genetics of Drosophila. Gravenhage: M. Nijhoff.
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","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Paysan-Lafosse T, Andreeva A, Blum M, Chuguransky SR, Grego T, Pinto BL, et al., Bateman. 2024. The Pfam protein families database: embracing AI/ML. Nucleic Acids Research 53: D523-D534.
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","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine ketoglutarate reductase/saccharopine dehydrogenase mutants of Drosophila melanogaster
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Aoife Larkin"},"openAcknowledgement":false,"submitted":null}]},{"id":"8e8e8af9-97b8-4249-ba95-6f138fecb4a1","decision":"publish","abstract":"A remarkable series of coincidences led the author to show that mutations in a Drosophila melanogaster gene encoding a protein responsible for degrading lysine and regulating ecdysone-induced histone methylation are associated with abnormal morphogenesis of the dorsal thorax. Specifically, loss-of-function mutations in the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein, result in loss of the scutoscutellar sulcus, the hingelike cuticular groove between the two major divisions of the fly thorax.
","acknowledgements":"I thank Jason Tennessen, Sophie Fleck and Arthur Luhur of Indiana University for stocks with unpublished Lkrsdh alleles and for helpful discussions and my colleagues at the Bloomington Drosophila Stock Center for their support. Stocks were obtained from the Department of Drosophila Genomics and Genetic Resources of the Kyoto Institute of Technology (Kyoto Stock Center) and the Bloomington Drosophila Stock Center. Genetic information was accessed via FlyBase, supported by grants from the National Human Genome Research Institute (U24 HG013300) and the Wellcome Trust.
","authors":[{"affiliations":["Indiana University, Bloomington, IN, United States"],"departments":["Bloomington Drosophila Stock Center, Department of Biology"],"credit":["conceptualization","fundingAcquisition","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kercook@iu.edu","firstName":"Kevin R.","lastName":"Cook","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9260-364X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grant P40 OD018537 and the Kathy Matthews Endowment.
","image":{"url":"https://portal.micropublication.org/uploads/abf9f501c707c23acbfc11878e1ccf25.png"},"imageCaption":"(A) Dorsal view of a wild type (Canton-S) fly showing the indentation of the dorsal thorax between the scutum and scutellum forming the scutoscutellar sulcus. The normal position of the sulcus is marked by arrowheads in all panels. (B) Dorsal view of an Lkrsdh mutant (LkrsdhCR70173-TG4.0/Df(2L)ED12527) showing near absence of the sulcus. There is a smooth transition from the scutum to the scutellum rather than a deep groove. (C) Lateral view of the same fly to demonstrate the rounded, “grooveless” profile. The arrow points to a small clot from hemolymph leakage. As in grooveless mutants, leakage in Lkrsdh mutants typically occurs in three positions (this position near the humeral bristles, slightly dorsal and posterior to the sternopleural bristles and/or at the lateral ends of the scutoscutellar sulcus), but clots are usually larger than the one depicted here. (D and E) Dorsal views of other individuals with the same genotype showing that the cuticular area where the sulcus would normally form can sometimes appear crumpled and distorted. In flies with this phenotype, the thoracic profiles are still more rounded than the profiles of normal flies. This phenotype also appears in grooveless (gvl) mutants, though it was not mentioned by Cook and Mauthner (2026). F. Newly sequenced Lkrsdh alleles. The top lines show the positions of deletions in terms of Release 6 genomic coordinates. In two alleles, extra nucleotides were introduced at the breakpoint junctions by DNA repair processes. For the first four alleles, the bottom lines show N-terminal amino acids remaining from the normal sequences followed by novel residues introduced by frame-shifted translation. For LkrsdhΔ163, the bottom line shows replacement of 61 amino acids of the normal sequence with FFFFFHF. (Note that panel A was recycled from Cook and Mauthner (2026).)
","imageTitle":"The thoracic phenotypes of Lkrsdh mutants and the characterization of Lkrsdh alleles
","methods":"The stocks used in complementation tests are listed in the Reagents Table. More information about these stocks may be obtained via the website for the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Genetic nomenclature and gene models reflect FlyBase release 2026_03 (Öztürk-Çolak et al. 2024). DNA was extracted from frozen samples of adult flies using standard protocols and sequenced as single-end reads on the Ultima Genomics platform by the University of Minnesota Model Organism Sequencing Service (https://genomics.umn.edu/service/model-organism-sequencing).
","reagents":"Stock number | Genotype | RRID or source |
Deficiency Kit stocks | ||
90 | Df(2L)C144, dppd-ho ed1/In(2LR)Gla, wgGla-1 PPO1Bc EgfrE1 | RRID:BDSC_90 |
744 | Df(2L)M24F-B/SM1 | RRID:BDSC_744 |
3084 | Df(2L)ast2/SM1 | RRID:BDSC_3084 |
3548 | Df(2L)al, dsal/In(2L)Cy, DuoxCy | RRID:BDSC_3548 |
5330 | Df(2L)ed1/CyO; P{ry+t7.2=ftz-lacC}1 | RRID:BDSC_5330 |
6338 | Df(2L)BSC6, dpyov1 cn1/SM6a | RRID:BDSC_6338 |
6507 | y1 w*; Df(2L)drm-P2, P{w+mC=lacW}ND-PDSWk10101/SM6b | RRID:BDSC_6507 |
6648 | Df(2L)dppd14/In(2LR)Gla, wgGla-1 | RRID:BDSC_6648 |
7144 | Df(2L)BSC37, dppEP2232/CyO | RRID:BDSC_7144 |
7492 | w1118; Df(2L)Exel6005, P{w+mC=XP-U}Exel6005/CyO | RRID:BDSC_7492 |
7495 | w1118; Df(2L)Exel6009, P{w+mC=XP-U}Exel6009/CyO | RRID:BDSC_7495 |
7497 | w1118; Df(2L)Exel6011, P{w+mC=XP-U}Exel6011/CyO | RRID:BDSC_7497 |
7744 | w1118; Df(2L)Exel6277, P{w+mC=XP-U}Exel6277/CyO | RRID:BDSC_7744 |
7783 | w1118; Df(2L)Exel7011/CyO | RRID:BDSC_7783 |
7807 | w1118; Df(2L)Exel7034/CyO | RRID:BDSC_7807 |
8673 | w1118; Df(2L)BSC107/SM6a | RRID:BDSC_8673 |
8674 | w1118; Df(2L)BSC109/CyO | RRID:BDSC_8674 |
8835 | w1118; Df(2L)BSC110/CyO | RRID:BDSC_8835 |
8836 | w1118; Df(2L)BSC111/CyO | RRID:BDSC_8836 |
8901 | w1118; Df(2L)ED19, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED19/SM6a | RRID:BDSC_8901 |
8906 | w1118; Df(2L)ED678, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED678/SM6a | RRID:BDSC_8906 |
8908 | w1118; Df(2L)ED94, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED94/SM6a | RRID:BDSC_8908 |
9176 | w1118; Df(2L)ED136, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED136/SM6a | RRID:BDSC_9176 |
9270 | w1118; Df(2L)ED250, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED250/In(2L)CyLtR In(2R)Cy, DuoxCy amosRoi-1 | RRID:BDSC_9270 |
9341 | w1118; Df(2L)ED385, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED385/SM6a | RRID:BDSC_9341 |
9343 | w1118; Df(2L)ED334, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED334/SM6a | RRID:BDSC_9343 |
9353 | w1118; Df(2L)ED5878, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED5878/SM6a | RRID:BDSC_9353 |
9502 | w1118; Df(2L)BSC142/CyO | RRID:BDSC_9502 |
9560 | w1118; Df(2L)BSC169/CyO | RRID:BDSC_9560 |
9600 | w1118; Df(2L)BSC165/CyO | RRID:BDSC_9600 |
9605 | w1118; Df(2L)BSC172/CyO | RRID:BDSC_9605 |
9610 | w1118; Df(2L)BSC180/CyO | RRID:BDSC_9610 |
9615 | w1118; Df(2L)BSC188/CyO | RRID:BDSC_9615 |
9631 | w1118; Df(2L)BSC204/CyO | RRID:BDSC_9631 |
9704 | w1118; Df(2L)BSC227/CyO | RRID:BDSC_9704 |
9708 | w1118; Df(2L)BSC233/CyO | RRID:BDSC_9708 |
23676 | w1118; Df(2L)BSC291/CyO | RRID:BDSC_23676 |
23677 | w1118; Df(2L)BSC292/CyO | RRID:BDSC_23677 |
23680 | w1118; Df(2L)BSC295/CyO | RRID:BDSC_23680 |
24118 | w1118; Df(2L)ED105, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED105/SM6a | RRID:BDSC_24118 |
24123 | w1118; Df(2L)ED247, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED247/SM6a | RRID:BDSC_24123 |
24124 | w1118; Df(2L)ED7853, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED7853/SM6a | RRID:BDSC_24124 |
24127 | w1118; Df(2L)ED489, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED489/SM6a | RRID:BDSC_24127 |
24132 | w1118; Df(2L)ED629, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED629/SM6a | RRID:BDSC_24132 |
24378 | w1118; Df(2L)BSC354/CyO | RRID:BDSC_24378 |
24626 | w1118; Df(2L)ED50001, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED50001/SM6a | RRID:BDSC_24626 |
24652 | w1118; Df(2L)ED441, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED441/SM6a | RRID:BDSC_24652 |
24958 | w1118; Df(2L)BSC454/SM6a | RRID:BDSC_24958 |
24959 | w1118; Df(2L)BSC455/SM6a | RRID:BDSC_24959 |
25743 | w1118; Df(2L)BSC653/SM6a | RRID:BDSC_25743 |
26540 | w1118; Df(2L)BSC688, P+PBac{w+mC=XP3.WH3}BSC688/SM6a | RRID:BDSC_26540 |
26544 | w1118; Df(2L)BSC692, P+PBac{w+mC=XP3.RB5}BSC692/SM6a | RRID:BDSC_26544 |
27381 | w1118; Df(2L)BSC810/SM6a | RRID:BDSC_27381 |
94697 | w1118; Df(2L)ED4651/SM6a | RRID:BDSC_94697 |
Additional deficiency stocks | ||
9274 | w1118; Df(2L)ED499, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED499/SM6a | RRID:BDSC_9274 |
9616 | w1118; Df(2L)BSC189/CyO | RRID:BDSC_9616 |
9617 | w1118; Df(2L)BSC190/CyO | RRID:BDSC_9617 |
9618 | w1118; Df(2L)BSC191/CyO | RRID:BDSC_9618 |
9619 | w1118; Df(2L)BSC192/CyO | RRID:BDSC_9619 |
24128 | w1118; Df(2L)ED501, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED501/SM6a | RRID:BDSC_24128 |
24129 | w1118; Df(2L)ED12527, P{w+mW.Scer\\FRT.hs3=3'.RS5+3.3'}ED12527/SM6a | RRID:BDSC_24129 |
Mutations tested in the Lkrsdh region | ||
14288 | y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}CG7149KG06170 | RRID:BDSC_14288 |
14997 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}r2d2EY00028 | RRID:BDSC_14997 |
16450 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}Cdc14EY10303 | RRID:BDSC_16450 |
17058 | w1118; P{w+mC=EP}HerpEP2450 | RRID:BDSC_17058 |
20640 | y1 w67c23; P{y+mDint2 w+mC=EPgy2}CG7149EY10883 | RRID:BDSC_20640 |
29231 | w1118; Mi{GFPE.3xP3=ET1}r2d2MB11618/SM6a | RRID:BDSC_29231 |
32068 | P{GMR-w.RNAi}13D; P{ry+t7.2=neoFRT}40A r2d2S165fsX | RRID:BDSC_32068 |
37074 | y1 w*; Mi{y+mDint2=MIC}HerpMI03683 | RRID:BDSC_37074 |
54575 | y1 w*; r2d21 P{ry+t7.2=neoFRT}40A/CyO, amosRoi-1 | RRID:BDSC_54575 |
60537 | y1 w67c23; Mi{PT-GFSTF.2}HerpMI03683-GFSTF.2 | RRID:BDSC_60537 |
79333 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.2}Cdc14CR00871-TG4.2 | RRID:BDSC_79333 |
92724 | y1 w*; TI{GFP3xP3.cLa=CRIMIC.TG4.0}LkrsdhCR70173-TG4.0/SM6a | RRID:BDSC_92724 |
600152 | w*; Df(2L)LanB1/CyO, P{w+mC=GAL4-Kr.C}DC3, P{w+mC=UAS-GFP.S65T}DC7 | RRID:BDSC_600152 |
Additional Lkrsdh mutations tested | ||
692 | Lkrsdh1 rc1; ss1 | RRID:BDSC_692 |
101560 | wgSp-1 Lkrsdh1 rc1 J1/SM1 | Kyoto Stock Center |
607941 | w1118; LkrsdhΔ11/CyO | RRID:BDSC_607941 |
| w1118; LkrsdhΔ16/CyO | Jason Tennessen |
607942 | w1118; LkrsdhΔ17/CyO | RRID:BDSC_607942 |
| w1118; LkrsdhΔ17-1/CyO | Jason Tennessen |
| w1118; LkrsdhΔ163/CyO | Jason Tennessen |
Stock used for the photograph of a normal scutoscutellar sulcus | ||
64349 | Canton-S | RRID:BDSC_64349 |
My colleague Stephanie Mauthner and I recently reported (Cook and Mauthner 2026) that homozygous mutations in the fourth chromosome gene grooveless (gvl, formerly CG31997) reduce or eliminate the scutoscutellar sulcus—the transverse cuticular crease separating the larger subdivision of the notum, the scutum, from the smaller subdivision, the scutellum (Figure 1A). These mutations also cause hemolymph leakage from the thorax at lower penetrance. The gvl gene encodes a protein with a single von Willibrand factor type C (SVWC) domain, likely involved in binding other proteins, and a cell export signal, but no other known protein motifs (Sheldon et al. 2007). SVWC proteins appear to be restricted to arthropods, but the biological functions of few of these proteins have been studied in detail (Labropoulou et al. 2024).
At the same time we were characterizing gvl mutations, I was working on an unrelated project involving the Lkrsdh gene, which encodes the Lysine ketoglutarate reductase/saccharopine dehydrogenase protein responsible for two enzymatic steps in lysine degradation (Lkr activity in the N-terminal half converts lysine to saccharopine and Sdh activity in the C-terminal half converts saccharopine to alpha-aminoadipic semialdehyde; this bifunctional protein is also called aminoadipate semialdehyde synthase (St. Clair et al. 2017; Tan et al. 2023)). At least in Drosophila, Lkrsdh protein also inhibits ecdysone-induced methylation of histone H3 (Cakouros et al. 2008). In an effort to map a mutation that interacts with Lkrsdh mutations, I had generated more than a hundred chromosomes carrying a transposon disrupting the Lkrsdh gene (Mi{ET1}LkrsdhMB01942) linked to a variety of chromosomal deletions, transposon insertions and point mutations located proximal to Lkrsdh. When I combined 45 of these “double-mutation” chromosomes in various pairwise combinations in flies via crosses, I was surprised to see the same phenotypes I was seeing in gvl mutants: shallow scutoscutellar sulci in crosses with all 45 chromosomes and hemolymph leakage in crosses with 32 of them. To my knowledge, these phenotypes (Figure 1) had never been associated with any gene besides gvl and two other genes (ascute and ascutex) identified by Calvin Bridges (Bridges 1935; Bridges and Morgan 1923; Morgan et al. 1925), so it was an amazing coincidence to see them in a different context at the same time I was working with gvl mutants!
The causative mutation must have been present on the Mi{ET1}LkrsdhMB01942 chromosome used to make the double-mutation chromosomes. For it to have been homozygosed in all the crosses described above, it must lie distal to the y+-marked Mi{MIC}MI04503 transposon insertion in polytene subdivision 30B, because I screened for single crossovers between the cis-linked insertions Mi{ET1}LkrsdhMB01942 and Mi{MIC}MI04503 to eliminate Mi{MIC}MI04503 and place the deletions, insertions and mutations on the same chromosome as Mi{ET1}LkrsdhMB01942. To identify this gvl-like mutation, I combined one of the double-mutation chromosomes (Mi{ET1}LkrsdhMB01942 Df(2L)Exel8022) with the 54 tiling chromosomal deficiencies from the Bloomington Deficiency Kit that remove 30B to the 2L tip (see Reagents Table). In aggregate, these deficiencies removed 2,983 of the 3,010 genes distal to Mi{MIC}MI04503. I found two overlapping deficiencies, Df(2L)BSC142 and Df(2L)BSC233, that failed to complement the mutation. With crosses of the same double-mutation chromosome to an additional seven deficiencies (see Reagents Table), I further narrowed the region with the mutation to the overlap of Df(2L)ED12527 and Df(2L)ED499. Much to my surprise, one of the four genes within this overlap was Lkrsdh itself! These deficiencies also failed to complement a sampling of six additional double-mutation chromosomes.
To identify the mutated gene in this small, four-gene region, I complementation tested seven double-mutation chromosomes against mutations in three of the four genes (Lkrsdh, lncRNA:CR44825, Herp and r2d2) as well as mutations in the genes immediately flanking this region (CG7149 and Cdc14) (see Reagents Table). Remarkably, I saw that the only noncomplementing mutation was one in Lkrsdh (LkrsdhCR70173-TG4.0; Figure 1)! Obviously, the Mi{ET1}LkrsdhMB01942 insertion on the double-mutation chromosomes was responsible for the grooveless phenotypes rather than a mutation linked to it.
I have now seen the grooveless phenotypes in complementation crosses with six additional Lkrsdh alleles. Four alleles (LkrsdhΔ11, LkrsdhΔ16, LkrsdhΔ17 and LkrsdhΔ17-1) are CRISPR-induced, frameshift mutations generated in screens by Arthur Luhur and Jason Tennessen (Figure 1F). These crosses exhibited phenotypic penetrance and expressivity similar to the crosses described above. These four mutations are positioned to truncate the Lkrsdh protein and eliminate domains necessary for both Lkr and Sdh activities as well as the protein region that binds to the ecdysone receptor EcR in regulating histone methylation (Cakouros et al. 2008; Letunic and Bork 2026; Paysan-Lafosse et al. 2025). Lkr activity requires two domains: a catalytic domain and a domain that binds NAD+. The Lkr catalytic domain is likely intact in proteins produced by all four alleles (assuming the truncated proteins are stable), but its activity depends on the domain for binding NAD+, which would be absent—strongly suggesting these four alleles are amorphic. The fifth allele is the original Lkrsdh mutation, Lkrsdh1, which was previously called lysine1 (Grell 1961; St. Clair et al. 2017). Interestingly, the scutoscutellar phenotype shows weak penetrance and expressivity in genotypes involving Lkrsdh1 and hemolymph leakage was rare, suggesting that Lkrsdh1 is hypomorphic. Lkrsdh1 is a complex chromosomal rearrangement deleting the normal translation start site, inserting repetitive sequences before the seventh codon and introducing an indel (St. Clair et al. 2017), but my phenotypic results and results presented by St. Clair et al. showing an 80% reduction in transcription suggest there is residual gene expression—likely initiated from a transcription start site within the insertion. The presence of a methionine codon within the inserted sequences near the Lkrsdh coding sequences suggests that a variant protein is produced with a shorter, novel N-terminus. The sixth allele (LkrsdhΔ163) also appears to be hypomorphic. It came from one of the CRISPR screens mentioned above and consists of 183 bp in the region encoding the Lkr activity replaced by a thymidine-rich sequence (likely TTTTTTTTTTTTTTCCATTTT) that has proved difficult to sequence consistently. The hypomorphic phenotypes suggest the substituted sequence has the appropriate number of thymidines to allow in-frame translation of a variant Lkrsdh protein (Figure 1F) with a disrupted Lkr domain but intact Sdh and EcR-binding domains.
Flexion between the scutum and scutellum at the scutoscutellar sulcus is part of normal flight (Boettinger and Furshpan 1952; Miyan and Ewing 1985), but, similar to gvl mutants (Cook and Mauthner, 2026), I saw that Lkrsdh mutants (LkrsdhCR70173-TG4.0/Df(2L)ED12527) with strong grooveless phenotypes are capable of flight. It seems likely that the thoracic malformations in gvl and Lkrsdh mutants affect flight efficiency, but in neither case has the details of flight mechanics been examined.
I do not know the developmental basis of “groovelessness”. Shallow scutoscutellar sulci and hemolymph leakage could be related to weak or improperly timed flight muscle contractions during pupal development, altered cuticle formation during metamorphosis, or both. Since many biological processes underlie muscle and cuticle development, groovelessness may be a relatively common mutant phenotype. The shallow sulcus phenotype may simply have escaped notice most of the time because it is fairly subtle. (I doubt I would have noticed it in Lkrsdh mutants if I had not been working with gvl mutants.) Indeed, there may be no close functional relationship between Lkrsdh protein and the secreted SVWC protein encoded by gvl. Currently, it is impossible to say if the grooveless phenotypes in Lkrsdh mutants are attributable to the widespread effects of increased lysine on metabolic processes (St. Clair et al. 2017), to the widespread effects of misregulating ecdysone-dependent processes on development (Morrow and Mirth 2024), or a mixture of both.
","references":[{"reference":"Bridges CB. 1935. The mutants and linkage data of chromosome four of Drosophila melanogaster. Biologicheskii Zhurnal. 4:401-420.
","pubmedId":"","doi":""},{"reference":"Bridges CB, Morgan TH. 1923. The third-chromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst Publ, Washington. 327:pp. 1-251.
","pubmedId":"","doi":""},{"reference":"Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S. 2008. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes. J Cell Biol 182(3): 481-95.
","pubmedId":"18695041","doi":""},{"reference":"Cook KR, Mauthner SE. 2026. The grooveless gene encodes a single von Willebrand factor type C domain protein necessary for proper formation of the scutoscutellar sulcus in Drosophila melanogaster. MicroPubl Biol 2026: 10.17912/micropub.biology.002111.
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","pubmedId":"","doi":"10.1016/j.gde.2023.102148"},{"reference":"Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, et al., Lovato. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227: 10.1093/genetics/iyad211.
","pubmedId":"","doi":"10.1093/genetics/iyad211"},{"reference":"Paysan-Lafosse T, Andreeva A, Blum M, Chuguransky SR, Grego T, Pinto BL, et al., Bateman. 2024. The Pfam protein families database: embracing AI/ML. Nucleic Acids Research 53: D523-D534.
","pubmedId":"","doi":"10.1093/nar/gkae997"},{"reference":"Sheldon TJ, Miguel-Aliaga I, Gould AP, Taylor WR, Conklin D. 2007. A novel family of single VWC‐domain proteins in invertebrates. FEBS Letters 581: 5268-5274.
","pubmedId":"","doi":"10.1016/j.febslet.2007.10.016"},{"reference":"St. Clair SL, Li H, Ashraf U, Karty JA, Tennessen JM. 2017. Metabolomic analysis reveals that the Drosophila melanogaster gene lysine influences diverse aspects of metabolism. Genetics 207: 1255-1261.
","pubmedId":"","doi":"10.1534/genetics.117.300201"},{"reference":"Tan Y, Chrysopoulou M, Rinschen MM. 2023. Integrative physiology of lysine metabolites. Physiological Genomics 55: 579-586.
","pubmedId":"","doi":"10.1152/physiolgenomics.00061.2023"}],"title":"The serendipitous discovery of thoracic malformations in Lysine ketoglutarate reductase/saccharopine dehydrogenase mutants of Drosophila melanogaster
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