Loss of function in the Drosophila melanogaster gene julius seizure (jus) causes the fly to have a seizure-like condition known as bang-sensitivity. Here, we characterize the expression patterns of two different jus reporters, a jus-GAL4 construct and a jus protein trap allele, in the central nervous system during the developmental stages that jus expression is required. The majority of cells that expressed the jus-GAL4 construct co-expressed a GAL80 enhancer trap in the homeotic gene teashirt (tsh); expression of jus in this subpopulation was necessary to prevent bang-sensitivity.
","acknowledgements":"Acknowledgements: The authors are very grateful to Nancy Piatczyc and Clara Ives for their technical assistance with the confocal microscope. Pei-Wen Chen of Williams College kindly helped us develop a protocol for counting mCherry-labeled nuclei. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Bioinformatic information was obtained from FlyBase (Jenkins et al., 2022).
","authors":[{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["conceptualization","dataCuration","writing_originalDraft","writing_reviewEditing","fundingAcquisition","methodology","investigation","formalAnalysis","project","supervision","validation","visualization"],"email":"ddean@williams.edu","firstName":"Derek M.","lastName":"Dean","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0000-4135-1518"},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"lec4@williams.edu","firstName":"Lillian E.","lastName":"Codd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"mye1@williams.edu","firstName":"Mira","lastName":"Eras","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","writing_reviewEditing","validation"],"email":"kol2@williams.edu","firstName":"Katherine","lastName":"Lopez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"huynpham200@gmail.com","firstName":"Huy","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"dmataylor9@gmail.com","firstName":"Dylan","lastName":"Taylor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","investigation","validation","writing_reviewEditing","methodology"],"email":"esa3@williams.edu","firstName":"Aisha","lastName":"Abakura","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"zg2@williams.edu","firstName":"Zoe","lastName":"Gabriel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"alyg@stanford.edu","firstName":"Alyana J.","lastName":"Granados","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["investigation","formalAnalysis","methodology","validation","writing_reviewEditing"],"email":"bml3@williams.edu","firstName":"Ben","lastName":"Litvak-Hinenzon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"jjw4@williams.edu","firstName":"Jeremy","lastName":"Wen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, MA, US"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"cyturralde24@gmail.com","firstName":"Catalin","lastName":"Yturralde","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Cornell University, Ithaca, NY, US"],"departments":["Neurobiology and Behavior"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","project","resources","supervision","validation","writing_reviewEditing"],"email":"dld14@cornell.edu","firstName":"David L","lastName":"Deitcher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This project was funded internally by Williams College.
","image":{"url":"https://portal.micropublication.org/uploads/55df65647ce89bdc0daa2f40e2d08ccd.jpg"},"imageCaption":"Top left: color code for the confocal colors seen in (A-D): magenta nuclei are from jus-GAL4 driving nls-mCherry expression (“jus > nls-mCh”), green cell bodies express the JusGFSTF fusion protein, and colocalization between these two expression patterns either appears as a magenta dot within a green cell, or as yellow if the colors overlap (high magnification examples shown at bottom left of panel A). A legend lists the symbols and abbreviations used in (A-D). White scale bar in each confocal image indicates 100 µm. During pupal stages P2 (A) and P4 (B), jus > nls-mCh and JusGFSTF were both expressed widely in the ventral nerve cord (VNC), with some expression across the central brain, and virtually no expression in the optic lobes. Some colocalization was seen in the VNC, particularly in cells along the midline and lateral margins of the posterior segments. (C) By stage P5ii, and through to the end of the critical period (D) jus > nls-mCh expression was strongest in the most posterior and anterior VNC segments, while JusGFSTF expression was most prominent in the mid-thoracic region of the VNC; colocalization of the two signals was seen in a narrow, lateral band of cells that wrapped around the mid-thoracic ganglia. At the transition between P6 and P7, additional colocalization was seen in a band of cells across the protocerebrum, a region of the brain that receives and processes information from the optic lobe. (E) jus > nls-mCh expression in the CNS of a P4 pupa that did not carry jusGFSTF. As in panel B, mCherry signal was seen throughout the VNC. Even in the absence of JusGFSTF, the head cuticle and fat body strongly fluoresced green, confirming that this was a non-specific, autofluorescent signal. (F) The number of jus > nls-mCh-expressing nuclei was strongly reduced in pupae that also carried the tsh-GAL80 construct. This reduction was particularly noticeable throughout the VNC. (G) tsh > nls-mCh expression was ubiquitous in the VNC, posterior central brain, and compound eyes. (H) When jus-GAL4 was used to express UAS-jusRNAi (“jus > jusRNAi”), 98% of flies were bang-sensitive. tsh-GAL80 strongly suppressed jus > jusRNAi bang-sensitivity. UAS-jusRNAi misexpression using a tsh-GAL4 driver (“tsh> jusRNAi”) caused 100% bang-sensitivity. n = 150 or more for each sample group. Two-tailed Fisher’s exact tests: p < 1.1e-110 (jus > jusRNAi vs. RNAi-only and GAL4-only controls) p = 1.1e-43 (jus > jusRNAi vs. jus > jusRNAi + tsh-GAL80), and p < 2.1e-89 (tsh > jusRNAi vs. RNAi-only and GAL4-only controls).
","imageTitle":"(A-D) Confocal microscope imaging of the expression of two different julius seizure (jus) reporters in the central nervous system during the critical period for jus expression, (E-G) comparison of jus-GAL4 expression to that of teashirt (tsh)-GAL80 and -GAL4 constructs, and (H) bang sensitivity of jus > jusRNAi, jus > jusRNAi + tsh-GAL80, and tsh > jusRNAi flies
","methods":"Fly stocks, diet, and experimental crosses: The fly strains that were used in our experiments are listed below. Stocks received from the Bloomington Drosophila Stock Center are denoted by “BDSC [stock number]”.
● jus-GAL4: w1118; P{y+t7.7 w+mC=GMR90B09-GAL4}attP2 (BDSC 607421; Bermudez et al., 2023)
● Control strain for jus-GAL4 background: w1118 (BDSC 6326)
● UAS-jusRNAi: y1 v1; P{y+t7.7 v+t1.8=TRiP.JF03192}attP2 (BDSC 28764; Perkins et al., 2015)
● Control strain for jusRNAi background: y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303; Groth et al., 2004)
● jusGFSTF: w; Kr/CyO; jusGFSTF/TM6C Sb Tb (BDSC 607422; Dean et al., 2018). For our experiments, the Kr and CyO second chromosomes had been crossed out of the background, leaving a strain with the genotype w; +/+;jusGFSTF/TM6C Sb Tb
● UAS-nls-mCh (for crosses involving jus-GAL4): w; P{w+mC=UAS-mCherry.NLS}3 (BDSC 38424; Caussinus et al., 2008)
● A jus > nls-mCh stock was made by recombining the jus-GAL4 and UAS-nls-mCh constructs described above onto the same chromosome and a w1118; jus-GAL4 UAS-nls-mCh strain was made.
● jus-GAL4 jusGFSTF: This stock was generated by recombining jus-GAL4 and jusGFSTF onto the same chromosome and balancing over TM6C. Full genotype was w1118; jus-GAL4/TM6C Sb Tb.
● tsh-GAL80: w; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; TM2/TM6B (BDSC 605556; Clyne and Miesenbock, 2008). For our experiments, the tsh-GAL80 construct was rebalanced over the CyO, P{Wee-P.ph0}Bacc[Wee-P20] chromosome from BDSC 52267; this balancer ubiquitously expresses GFP, enabling us to identify pupae that carry the tsh-GAL80 chromosome (Clyne et al., 2003).
● tsh-GAL80; jus-GAL4: A strain carrying both the jus-GAL4 and tsh-GAL80 constructs was made with standard crosses (details available by request). Full genotype: w1118; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; P{y+t7.7w+mC=GMR90B09-GAL4}attP2
● tsh-GAL4: y1 w1118; P{w+mW.hs=GawB}tsh[md621]/CyO; P{w+mC=UAS-y.C}MC1/TM2 (BDSC 3040; Soller et al., 2006)
● UAS-nls-mCh (for crosses involving tsh-GAL4): w; P{w+mC=UAS-mCherry.NLS}2 (BDSC 38425; Caussinus et al., 2008). This strain was crossed to tsh-GAL4 to generate the image in Figure 1, while crosses involving jus-GAL4 and mCherry used the related UAS-nls-mCh stock described above (BDSC 38424).
Larvae and flies were fed a modified yeast/dextrose/cornmeal diet (Dean et al., 2016; Dean et al., 2020). Parental stocks were maintained at room temperature, while experimental crosses were incubated at 25°C as the progeny developed. Mating schemes for experimental crosses were as follows (see stock list immediately above for full parental genotypes):
● Figure 1A-D (Comparing jus > nls-mCh and jusGFSTF expression in CNS)
o jus > nls-mCh females X jusGFSTF males
● Figure 1E-G (Expression of tsh-GAL80 and tsh-GAL4 constructs in CNS)
o jus > nls-mCh females X w1118 males
o jus > nls-mCh females X tsh-GAL80/CyO, P{Wee-P.ph0}Bacc[Wee-P20] males. Progeny carrying tsh-GAL80 were identified by a lack of GFP expression from the CyO, P{Wee-P.ph0}Bacc[Wee-P20] balancer (Clyne et al., 2003).
o tsh-GAL4 females X nls-mCh males
● Figure 1H (Bang-sensitive assays)
o jus-RNAi-only control
w1118 females X UAS-jusRNAi males
o jus-GAL4-only control
jus-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
o jus > jusRNAi
jus-GAL4 females X UAS-jusRNAi males
o jus > jusRNAi with tsh-GAL80
tsh-GAL80; jus-GAL4 females X UAS-jusRNAi males
o tsh-GAL4-only control
tsh-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
o tsh > jusRNAi
tsh-GAL4 females X UAS-jusRNAi males
CNS preparation for confocal microscopy: Wandering larvae and pupal stages P1-P7 were identified by morphological criteria (Bainbridge and Bownes, 1981; Chyb and Gompel, 2013). Each CNS was dissected in cold PBS, fixed in PBS + 4% formaldehyde for 30 minutes at room temperature with gentle rocking, then rinsed twice in PBS + 0.3% Triton X-100 (all chemicals obtained from Sigma-Aldrich).
CNS tissue was transferred to microscope slides and overlaid with Vectashield Antifade Mounting Media (Vector Labs), then with a #1.5 18 x 18 mm coverslip (Fisher Scientific). Coverslip edges were sealed with nail polish. Slides were stored in the dark at 4ºC until they were imaged at the Williams College confocal microscope facility.
Confocal microscopy and post-production image processing: Within a week after slide preparation, pupal CNS tissue was imaged using a Nikon Ti2 inverted microscope and Nikon A1R HD software.
For Figures 1A-G, Z stack series were collected with the following settings: 20X objective lens, 2 µm steps, 32X averaging resonant scan, 0.9 pinhole, 1024 x 1024 pixel resolution, and power/off-set/gain settings were FITC 25/0/43 and TRITC 0.8/0/65. To reduce background from cuticle and fat body at the later pupal stages, FITC power was decreased to as low as 4.5 while holding other settings constant. Z-series were saved as ND2 files. Each Figure 1 image was made by merging a Z-series into a focused EDF document and saving as an uncompressed TIFF.
For quantification of jus > nls-mCh-labeled nuclei at the P4 stage (genotypes shown in Figure 1E,F), Z stack series were captured with similar settings to those described above, but a 10X objective lens was used, and power/off-set/gain settings were FITC 4.5/0/43 and TRITC 0.4/0/61. Sections were collectively exported into a multipage TIFF file, imported into Fiji, color channels were split, the TRITC channel was selected, and 3D objects were counted using a threshold of 800 and a minimum size of 35, parameters which appeared to reliably identify each visible nucleus.
Bang-sensitivity assays: 3-7-day-old adult flies were vortex assayed for bang-sensitive paralysis using a protocol based on Bermudez et al. (2023). Adult progeny of experimental crosses were allowed to emerge over 4 days, anesthetized with CO2, transferred to glass vials containing fresh diet, and vials were capped with plastic foam plugs (Fisher Scientific)—we specify the container materials because, in our experience, bang-sensitivity was enhanced if flies were vortexed in glass vials with foam plugs compared to standard plastic vials and cotton plugs, perhaps because the latter materials would have softer impact on the flies during vortexing. Vials were stored at room temperature for 3 additional days; during this period, to avoid making the flies refractory to bang-sensitive paralysis, vials were not jostled or otherwise moved (Pavlidis and Tanouye, 1995). After recovery, each vial was gently picked up then pressed upside down against a Vortex Genie (Sigma-Aldrich) on maximum setting for 10 seconds to agitate the flies against the cap and vial wall. Immediately afterwards, the number of paralyzed flies were counted and compared to the total number of living flies in the vial.
Image editing, data analysis, and figure generation: The focused EDF TIFF files from confocal imaging were imported into Adobe Photoshop 2021. Exposure was increased +1, and to increase accessibility for red/green colorblind readers, the red mCh channel was converted to magenta by adding a Hue/Saturation Adjustment Layer and altering the hue of the reds -35 (Summerbell, 2019). Otherwise, no image adjustments were made. Vortex data were graphed and analyzed using JMP Student Version 18. The Figure 1 graph was saved as an editable JPG. Photos and the graph were assembled into a figure and annotated using Adobe Illustrator 2021.
AI statement: No AI was used in this project for data collection, data analysis, writing, figure generation, or editing.
","reagents":"","patternDescription":"The fruit fly Drosophila melanogaster has been an informative model system for studying seizure disorders. Mutations in certain genes cause Drosophila adults to have seizure-like neural activity and muscle spasms, followed by a period of paralysis before recovery. This behavioral sequence can be induced by external stimuli, including extreme temperature changes; electrical shock; strobe lighting; and mechanical shock, for example from shaking the culture vial upon a lab vortex machine. Given the ease and effectiveness of mechanical shock, fly mutants with this seizure-like phenotype are considered “bang-sensitive” (Baraban, 2007; Parker et al., 2011a; Mituzaite et al., 2021; Fischer et al., 2023).
Most loci that affect bang-sensitivity are associated with basic neurophysiological function. For examples, paralytic(para) encodes a subunit of a voltage gated Na+ channel, and ATPα encodes a subunit of Na+/K+-ATPase (Schubiger et al., 1994; Parker et al., 2011b). In contrast, julius seizure (jus, formerly known as slamdance and sda) has a more cryptic role in regulating neural activity because, other than two predicted transmembrane spans that bracket a cysteine-rich, extracellular loop, the Jus protein has no conserved domains that imply a specific function (Horne et al., 2017; Dean et al., 2018). In preprint, we have reported that Jus physically associates with 23 other proteins, including ATPα and Nrv3, which are α and β subunits of the Na+/K+-ATPase, so it is possible that Jus affects the assembly, positioning, stability, and/or activity of a channel that is essential for maintaining resting membrane potential (Bermudez et al., 2023). In two separate experiments, we had used RNAi to knock down jus transcript at specific developmental stages and found evidence that jus expression may be required as early as the wandering larval phase and as late as pupal stage P7, with a peak requirement at about P4-5, when the adult head epidermis everts and surrounds the brain (Horne et al., 2017; experiment replication/refinement described in preprint Bermudez et al., 2023). We also found that jus expression is required in neurons—most significantly in cholinergic neurons—and not in glia, to protect against bang-sensitivity (Horne et al., 2017; Dean et al., 2018). With this as a starting point, our goals were (1) to determine the expression pattern of jusduring its critical period for expression, and (2) to begin to characterize the spatial distribution of neurons that must express jus to protect the adult against bang-sensitivity.
We used two reporters to assess jus expression in the pupal CNS: (1) jusGFSTF is a protein trap allele that inserts an EGFP-FlAsH-StrepII-TEV-3xFlag multi-tag cassette in the putative extracellular domain of Jus; this fusion protein mislocalizes to the cytoplasm of neuronal cell bodies, and its expression can be detected by GFP fluorescence (Diao et al., 2015; Dean et al., 2018). (2) GMR90B09-GAL4, hereafter referred to as jus-GAL4, is a construct that uses the juspromoter region to drive GAL4 expression (Jennet et al., 2012; Bermudez et al., 2023). A GAL4 construct that reflects endogenous jus expression would be expected to cause severe bang-sensitivity when used to express UAS-jusRNAi. Retesting jus-GAL4 for this study, we found that, while GAL4-only and RNAi-only controls were not bang-sensitive, 98% of jus > jusRNAi flies were, confirming that this jus-GAL4 construct is expressed in neurons that express jus and that affect bang-sensitivity (Figure 1H).
To compare the expression patterns of jus-GAL4 and Jus protein, jus > nls-mCh and jusGFSTF stocks were mated, and progeny were examined during metamorphosis. Throughout the critical period for jus expression, both nls-mCh and GFP were expressed widely in the ventral nerve cord (VNC) and somewhat in the central brain, with little expression in the optic lobes (Figure 1A-D). Despite the similar spatial distribution of nls-mCh and GFP signals across the nervous system, few cells strongly expressed both nls-mCh and GFP, with most examples located in the VNC, and later in the protocerebrum, a region of the central brain that directly associates with the optic lobe. There are several possible explanations for the apparent mismatch between the jus > nls-mCh and JusGFSTF expression patterns: 1) jus > nls-mCh may not fully reflect endogenous jus expression, because although the jus-GAL4 construct includes the entire juspromoter region, it may be missing enhancer sequences that refine the expression pattern. 2) JusGFSTF may not fully reflect wild type Jus+ protein expression, because the mutant protein mislocalizes from the axon to the cell body (Dean et al., 2018), suggesting it is structurally defective. Therefore, JusGFSTF stability/turnover rate may differ from that of Jus+protein. 3) jus > nls-mCh and JusGFSTF expression may overlap more significantly than suggested by confocal microscopy, but much of this overlap may have been undetected due to differing expression levels or protein turnover kinetics of nls-mCh vs. JusGFSTF in any given cell. Regardless of which of these hypotheses is correct, jus > jusRNAi caused very high rates of bang-sensitivity, so jus-GAL4 could help identify and characterize neuronal subpopulations that strongly affect bang-sensitivity.
Given the extensive expression of jus-GAL4 and JusGFSTF in the VNC, we considered whether a GAL80 construct associated with teashirt (tsh), a homeotic gene that directs development of the VNC, might be expressed in jus > nls-mCh-labeled cells (Röder et al., 1992; Rubio-Ferrera et al., 2023). Throughout the critical period, jus > nls-mCh pupae carrying a tsh-GAL80 enhancer trap construct had noticeably fewer brightly labeled nls-mCh-labeled nuclei across the VNC (Figure 1E vs. F). This effect was quantified in pupae at P4, the peak of the critical period: relative to jus > nls-mCh controls without GAL80, jus > nls-mCh pupae carrying tsh-GAL80 had only 29.9% the average number of mCherry-labeled nuclei fluorescing above a set brightness threshold in their CNS (averages of 113.4 ± 7.7 SEM vs. 379.6 ± 26.5 SEM, n=9 for each group; p = 3.6e-6, Student’s t-test). tsh-GAL4, a construct swapped into the same enhancer trap sequence as tsh-GAL80, was used to misexpress UAS-nls-mCh. Throughout the critical period, tsh > nls-mCh was ubiquitously expressed across the VNC, with some labeling within the posterior section of the central brain, and high expression in the compound eyes (Figure 1G).
Finally, we tested if jus expression is required in cells that express teashirt-associated constructs to prevent bang-sensitivity. In a previous study, tsh-GAL80-expressing neurons were shown to protect against seizure-like behavior in Drosophila larvae, using an optogenetic model and electrical shock (Giachello and Baines, 2015). In our experiments, tsh-GAL80 strongly suppressed the bang-sensitivity of jus > jusRNAi flies (36.6% with tsh-GAL80 vs. 98.0% without; Figure 1H), and when tsh-GAL4, a construct swapped into the same enhancer trap sequence as tsh-GAL80, was used to misexpress jusRNAi, 100% of flies were bang-sensitive (Figure 1H; the end of the figure caption discusses statistics).
Considering the involvement of tsh-GAL80- and tsh-GAL4-expressing neurons in regulating jus-associated bang-sensitivity, along with the persistent expression of these markers in the pupal VNC, an intriguing possibility is that bang-sensitivity can be caused by loss of jus expression in neural tissue outside of the brain. However, given that tsh-GAL4 seems to be expressed in the central brain, at least to some extent (Figure 1G), it is certainly possible that jus expression in the central brain is what protects the fly against bang-sensitivity, and that jus expression in the VNC has a supporting or distinct function. In future experiments, jusGFSTF, jus-GAL4, tsh-GAL80, and tsh-GAL4 could be useful tools to functionally characterize neurons that affect bang-sensitivity.
","references":[{"reference":"Bainbridge SP, Bownes M. 1981. Staging the metamorphosis of Drosophila melanogaster. J Embryol Exp Morphol 66: 57-80.
","pubmedId":"6802923","doi":""},{"reference":"Baraban SC. 2007. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Opin Neurol 20(2): 164-8.
","pubmedId":"17351486","doi":""},{"reference":"Bermudez S, Dean DM, Alkin V, Wang Z, Pham H, Kondo E, Amini A, Deitcher DL. 2023. A Complex Containing the Na\n +\n /K\n +\n ATPase Regulates Epileptogenesis in\n Drosophila melanogaster. : 10.1101/2023.09.22.559031.
","pubmedId":"","doi":"10.1101/2023.09.22.559031"},{"reference":"Caussinus E, Colombelli J, Affolter M. 2008. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol 18(22): 1727-34.
","pubmedId":"19026547","doi":""},{"reference":"Chyb, S., and N. Gompel, 2013 Atlas of Drosophila morphology: wild-type and classical mutants. Academic Press, London; Waltham, MA.
","pubmedId":"","doi":""},{"reference":"Clyne PJ, Brotman JS, Sweeney ST, Davis G. 2003. Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements. Genetics 165(3): 1433-41.
","pubmedId":"14668392","doi":""},{"reference":"Clyne JD, Miesenböck G. 2008. Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133(2): 354-63.
","pubmedId":"18423205","doi":""},{"reference":"Cole SH, Carney GE, McClung CA, Willard SS, Taylor BJ, Hirsh J. 2005. Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280(15): 14948-55.
","pubmedId":"15691831","doi":""},{"reference":"Dean DM, Maroja LS, Cottrill S, Bomkamp BE, Westervelt KA, Deitcher DL. 2015. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development. G3 (Bethesda) 6(2): 299-310.
","pubmedId":"26613949","doi":""},{"reference":"Dean D, Weinstein H, Amin S, Karno B, McAvoy E, Hoy R, et al., Deitcher D. 2018. Extending julius seizure, a bang-sensitive gene, as a model for studying epileptogenesis: Cold shock, and a new insertional mutation. Fly (Austin) 12(1): 55-61.
","pubmedId":"29125376","doi":""},{"reference":"Dean DM, Deitcher DL, Loehlin DW, Banta LM. 2020. Mapping a Mutation to its Gene: The \"Fly Lab\" as a Modern Research Experience. CourseSource 7: 10.24918/cs.2020.51.
","pubmedId":"","doi":"10.24918/cs.2020.51"},{"reference":"Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, et al., White BH. 2015. Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes. Cell Rep 10(8): 1410-21.
","pubmedId":"25732830","doi":""},{"reference":"Fischer FP, Karge RA, Weber YG, Koch H, Wolking S, Voigt A. 2023. Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview. Front Mol Neurosci 16: 1116000.
","pubmedId":"36873106","doi":""},{"reference":"Groth AC, Fish M, Nusse R, Calos MP. 2004. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166(4): 1775-82.
","pubmedId":"15126397","doi":""},{"reference":"Giachello CN, Baines RA. 2015. Inappropriate Neural Activity during a Sensitive Period in Embryogenesis Results in Persistent Seizure-like Behavior. Curr Biol 25(22): 2964-8.
","pubmedId":"26549258","doi":""},{"reference":"Horne M, Krebushevski K, Wells A, Tunio N, Jarvis C, Francisco G, et al., Deitcher DL. 2017. julius seizure, a Drosophila Mutant, Defines a Neuronal Population Underlying Epileptogenesis. Genetics 205(3): 1261-1269.
","pubmedId":"28082408","doi":""},{"reference":"Mituzaite J, Petersen R, Claridge-Chang A, Baines RA. 2021. Characterization of Seizure Induction Methods in Drosophila. eNeuro 8(4): pii: ENEURO.0079-21.2021. 10.1523/ENEURO.0079-21.2021.
","pubmedId":"34330816","doi":""},{"reference":"Jenett A, Rubin GM, Ngo TT, Shepherd D, Murphy C, Dionne H, et al., Zugates CT. 2012. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2(4): 991-1001.
","pubmedId":"23063364","doi":""},{"reference":"Jenkins VK, Larkin A, Thurmond J, FlyBase Consortium. 2022. Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol 2540: 1-34.
","pubmedId":"35980571","doi":""},{"reference":"Parker L, Howlett IC, Rusan ZM, Tanouye MA. 2011. Seizure and epilepsy: studies of seizure disorders in Drosophila. Int Rev Neurobiol 99: 1-21.
","pubmedId":"21906534","doi":""},{"reference":"Parker L, Padilla M, Du Y, Dong K, Tanouye MA. 2011. Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics 187(2): 523-34.
","pubmedId":"21115970","doi":""},{"reference":"Pavlidis P, Tanouye MA. 1995. Seizures and failures in the giant fiber pathway of Drosophila bang-sensitive paralytic mutants. J Neurosci 15(8): 5810-9.
","pubmedId":"7643221","doi":""},{"reference":"Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, et al., Perrimon N. 2015. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201(3): 843-52.
","pubmedId":"26320097","doi":""},{"reference":"Röder L, Vola C, Kerridge S. 1992. The role of the teashirt gene in trunk segmental identity in Drosophila. Development 115(4): 1017-33.
","pubmedId":"1360402","doi":""},{"reference":"Röder L, Vola C, Kerridge S. 1992. The role of the teashirt gene in trunk segmental identity in Drosophila. Development 115(4): 1017-33.
","pubmedId":"1360402","doi":""},{"reference":"Schubiger M, Feng Y, Fambrough DM, Palka J. 1994. A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis. Neuron 12(2): 373-81.
","pubmedId":"8110464","doi":""},{"reference":"Soller M, Haussmann IU, Hollmann M, Choffat Y, White K, Kubli E, Schäfer MA. 2006. Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons. Curr Biol 16(18): 1771-82.
","pubmedId":"16979554","doi":""},{"reference":"Summerbell E. (2019) How to make scientific figures accessible to readers with color-blindness. American Society for Cell Biology.
","pubmedId":"","doi":""}],"title":"julius seizure acts within teashirt-expressing neurons to protect Drosophila melanogaster from bang-sensitivity
","reviews":[{"reviewer":{"displayName":"Toshihiro Kitamoto"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":"1775542876134"}]},{"id":"62b12daa-64f1-40c5-9e31-2e954b01d2b4","decision":"accept","abstract":"Loss of function in the Drosophila melanogaster gene julius seizure (jus) causes the fly to have a seizure-like condition known as bang-sensitivity. Here, we characterize the expression patterns of two different jus reporters, a jus-GAL4 construct and a jus protein trap allele, in the central nervous system during the developmental stages that jus expression is required. The majority of cells that expressed the jus-GAL4 construct co-expressed a GAL80 enhancer trap in the homeotic gene teashirt (tsh); expression of jus in this subpopulation was necessary to prevent bang-sensitivity.
","acknowledgements":"The authors are very grateful to Nancy Piatczyc and Clara Ives for their technical assistance with the confocal microscope. Pei-Wen Chen of Williams College kindly helped us develop a protocol for counting mCherry-labeled nuclei. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Bioinformatic information was obtained from FlyBase (Jenkins et al., 2022).
","authors":[{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["conceptualization","dataCuration","writing_originalDraft","writing_reviewEditing","fundingAcquisition","methodology","investigation","formalAnalysis","project","supervision","validation","visualization"],"email":"ddean@williams.edu","firstName":"Derek M.","lastName":"Dean","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0000-4135-1518"},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"lec4@williams.edu","firstName":"Lillian E.","lastName":"Codd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"mye1@williams.edu","firstName":"Mira","lastName":"Eras","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","writing_reviewEditing","validation"],"email":"kol2@williams.edu","firstName":"Katherine","lastName":"Lopez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"huynpham200@gmail.com","firstName":"Huy","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"dmataylor9@gmail.com","firstName":"Dylan","lastName":"Taylor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation","validation","writing_reviewEditing","methodology"],"email":"esa3@williams.edu","firstName":"Aisha","lastName":"Abakura","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"zg2@williams.edu","firstName":"Zoe","lastName":"Gabriel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"alyg@stanford.edu","firstName":"Alyana J.","lastName":"Granados","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["investigation","formalAnalysis","methodology","validation","writing_reviewEditing"],"email":"bml3@williams.edu","firstName":"Ben","lastName":"Litvak-Hinenzon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"jjw4@williams.edu","firstName":"Jeremy","lastName":"Wen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"cyturralde24@gmail.com","firstName":"Catalin","lastName":"Yturralde","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Cornell University, Ithaca, New York, United States"],"departments":["Neurobiology and Behavior"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","project","resources","supervision","validation","writing_reviewEditing"],"email":"dld14@cornell.edu","firstName":"David L","lastName":"Deitcher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded internally by Williams College.
","image":{"url":"https://portal.micropublication.org/uploads/1e8471ffe50be68cafab9d9ed05fb4b5.jpg"},"imageCaption":"Top left, schematic of a neuronal cell body, indicating the colors emitted by the two jus expression reporters that were used in this study. Magenta nuclei are from jus-GAL4 driving UAS-nls-mCherry expression (“jus-GAL4 > nls-mCh”), green cell bodies express the JusGFSTF fusion protein, and colocalization between these two expression patterns either appears as a magenta dot within a green cell, or as yellow if the colors overlap. A legend lists the symbols and abbreviations used in (A-K). White scale bar in each confocal image indicates 100 µm. During pupal stages P2 (A) and P4 (B), jus-GAL4 > nls-mCh and JusGFSTF were both expressed widely in the ventral nerve cord (VNC), with some expression across the central brain (CB), and virtually no expression in the optic lobes (OL). Some colocalization was seen in the VNC, particularly in cells along the midline and lateral margins of the posterior segments. The head cuticle (c) tended to autofluoresce green. (C) By stage P5ii, and through to the end of the critical period (D), jus-GAL4 > nls-mCh expression was strongest in the most posterior and anterior VNC segments, while JusGFSTF expression was most prominent in the mid-thoracic region of the VNC; colocalization of the two signals was seen in a narrow, lateral band of cells that wrapped around the mid-thoracic ganglia. Fat body (f) often autofluoresced green. At the transition between P6 and P7 (D), additional colocalization was seen in a band of cells across the protocerebrum (PR), a region of the brain that receives and processes information from the optic lobe. (E) and (F) show higher magnification examples of nls-mCh/GFP colocalization in the VNC. (F) is a transverse section of an abdominal segment to show colocalization on the dorsal (top) as well ventral (bottom) margins of the VNC. (G) shows a higher magnification example of colocalization in a lateral band across the mid-thorax. (H) is a higher magnification view of colocalization in the protocerebrum (PR) as well as a small number of cells in the central brain (CB). (I) jus-GAL4 > nls-mCh expression in the CNS of a P4 pupa that did not carry jusGFSTF. As in panel B, mCherry signal was seen throughout the VNC. Even in the absence of JusGFSTF, the head cuticle (c) and fat body (f) strongly fluoresced green, confirming that these were non-specific, autofluorescent signals. (J) The number of jus-GAL4 > nls-mCh-expressing nuclei was significantly reduced in pupae that also carried the tsh-GAL80 construct. Loss of nls-mCh signal was particularly noticeable throughout the VNC. This particular specimen also happens to have reduced green autofluorescence, but this was not specific to the jus-GAL4 > nls-mCh + tsh-GAL80 genotype, i.e., cuticle and fat body autofluorescence varied widely in all sample groups [compare panels (B), (I), and (J)]. (K) tsh-GAL4 > nls-mCh expression was ubiquitous in the VNC, posterior central brain, and compound eyes (CE). (L) When jus-GAL4 was used to express UAS-jusRNAi (“jus-GAL4 > jusRNAi”), 98.0% of flies were bang-sensitive. tsh-GAL80 suppressed jus-GAL4 > jusRNAi bang-sensitivity down to 36.6%. UAS-jusRNAi expression using a tsh-GAL4 driver (“tsh-GAL4 > jusRNAi”) caused 100% bang-sensitivity. n = 150 or more for each sample group. Two-tailed Fisher’s exact tests: p < 1.1e-110 (jus-GAL4 > jusRNAi vs. RNAi-only and GAL4-only controls) p = 1.1e-43 (jus-GAL4 > jusRNAi vs. jus-GAL4 > jusRNAi + tsh-GAL80), and p < 2.1e-89 (tsh-GAL4 > jusRNAi vs. RNAi-only and GAL4-only controls).
","imageTitle":"(A-D) Confocal microscope imaging of the expression of two different julius seizure (jus) reporters in the central nervous system (CNS) during the critical period for jus expression, (E-H) higher magnification examples of jus reporter colocalization, (I-K) comparison of jus-GAL4 expression to that of teashirt (tsh)-GAL80 and tsh-GAL4 constructs, and (L) bang sensitivity of jus-GAL4 > jusRNAi, jus-GAL4 > jusRNAi + tsh-GAL80, and tsh-GAL4 > jusRNAi flies
","methods":"Fly stocks, diet, and experimental crosses: The fly strains that were used in our experiments are listed below. Stocks received from the Bloomington Drosophila Stock Center are denoted by “BDSC [stock number]”.
● jus-GAL4: w1118; P{y+t7.7 w+mC=GMR90B09-GAL4}attP2 (BDSC 607421; Bermudez et al., 2023)
● Control strain for jus-GAL4 background: w1118 (BDSC 6326)
● UAS-jusRNAi: y1 v1; P{y+t7.7 v+t1.8=TRiP.JF03192}attP2 (BDSC 28764; Perkins et al., 2015)
● Control strain for jusRNAi background: y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303; Groth et al., 2004)
● jusGFSTF: w; Kr/CyO; jusGFSTF/TM6C Sb Tb (BDSC 607422; Dean et al., 2018). For our experiments, the Kr and CyO second chromosomes had been crossed out of the background, leaving a strain with the genotype w; +/+; jusGFSTF/TM6C Sb Tb
● UAS-nls-mCh (for crosses involving jus-GAL4): w; P{w+mC=UAS-mCherry.NLS}3 (BDSC 38424; Caussinus et al., 2008)
● A jus-GAL4 > nls-mCh stock was made by recombining the jus-GAL4 and UAS-nls-mCh constructs described above onto the same chromosome and a w1118; jus-GAL4 UAS-nls-mCh strain was made.
● jus-GAL4 jusGFSTF: This stock was generated by recombining jus-GAL4 and jusGFSTF onto the same chromosome and balancing over TM6C. Full genotype was w1118; jus-GAL4 jusGFSTF/TM6C Sb Tb.
● tsh-GAL80: w; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; TM2/TM6B (BDSC 605556; Clyne and Miesenbock, 2008). For our experiments, the tsh-GAL80 construct was rebalanced over the CyO, P{Wee-P.ph0}Bacc[Wee-P20] chromosome from BDSC 52267; this CyO balancer ubiquitously expresses GFP, enabling us to identify pupae that carried the tsh-GAL80 chromosome (Clyne et al., 2003).
● tsh-GAL80; jus-GAL4: A strain carrying both the jus-GAL4 and tsh-GAL80 constructs was made. Full genotype: w1118; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; P{y+t7.7w+mC=GMR90B09-GAL4}attP2
● tsh-GAL4: y1 w1118; P{w+mW.hs=GawB}tsh[md621]/CyO; P{w+mC=UAS-y.C}MC1/TM2 (BDSC 3040; Soller et al., 2006)
● UAS-nls-mCh (for crosses involving tsh-GAL4): w; P{w+mC=UAS-mCherry.NLS}2 (BDSC 38425; Caussinus et al., 2008). This strain was crossed to tsh-GAL4 to generate the image in Figure 1K, while crosses involving jus-GAL4 and mCherry used the related UAS-nls-mCh stock described above (BDSC 38424).
Larvae and flies were fed a modified yeast/dextrose/cornmeal diet (Dean et al., 2016; Dean et al., 2020). Parental stocks were maintained at room temperature, while experimental crosses were incubated at 25°C as the progeny developed. Mating schemes for experimental crosses were as follows (see stock list immediately above for full parental genotypes):
● Figure 1A-H (Comparing jus-GAL4 > nls-mCh and jusGFSTF expression in CNS)
o jus-GAL4 > nls-mCh females X jusGFSTF males
● Figure 1I-K (Expression of tsh-GAL80 and tsh-GAL4 constructs in CNS)
o Figure 1I: jus-GAL4 > nls-mCh females X w1118 males
o Figure 1J: jus-GAL4 > nls-mCh females X tsh-GAL80/CyO, P{Wee-P.ph0}Bacc[Wee-P20] males. Progeny carrying tsh-GAL80 were identified by a lack of GFP expression from the CyO, P{Wee-P.ph0}Bacc[Wee-P20] balancer (Clyne et al., 2003).
o Figure 1K: tsh-GAL4 females X nls-mCh (BDSC 38425) males
● Figure 1L (Bang-sensitive assays)
o jus-RNAi-only control
w1118 females X UAS-jusRNAi males
o jus-GAL4-only control
jus-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
o jus-GAL4 > jusRNAi
jus-GAL4 females X UAS-jusRNAi males
o jus-GAL4 > jusRNAi with tsh-GAL80
tsh-GAL80; jus-GAL4 females X UAS-jusRNAi males
o tsh-GAL4-only control
tsh-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
o tsh-GAL4 > jusRNAi
tsh-GAL4 females X UAS-jusRNAi males
CNS preparation for confocal microscopy: Wandering larvae and pupal stages P1-P7 were identified by morphological criteria (Bainbridge and Bownes, 1981; Chyb and Gompel, 2013). Each CNS was dissected in cold PBS, fixed in PBS + 4% formaldehyde for 30 minutes at room temperature with gentle rocking, then rinsed twice in PBS + 0.3% Triton X-100 (all chemicals obtained from Sigma-Aldrich).
CNS tissue was transferred to microscope slides and overlaid with Vectashield Antifade Mounting Media (Vector Labs), then with a #1.5 18 x 18 mm coverslip (Fisher Scientific). Coverslip edges were sealed with nail polish. Slides were stored in the dark at 4ºC until they were imaged at the Williams College confocal microscope facility.
Confocal microscopy and post-production image processing: Within a week after slide preparation, pupal CNS tissue was imaged using a Nikon Ti2 inverted microscope and Nikon A1R HD software.
Z stack series were collected with the following settings: 20X objective lens, 2 µm steps, 32X averaging resonant scan, 0.9 pinhole, 1024 x 1024 pixel resolution, and power/off-set/gain settings were FITC 25/0/43 and TRITC 0.8/0/65. To reduce background from cuticle and fat body at the later pupal stages, FITC power was decreased to as low as 4.5 while holding other settings constant. Z-series were saved as ND2 files. Each Figure 1 image was made by merging a Z-series into a focused EDF document and saving as an uncompressed TIFF.
For quantification of jus-GAL4 > nls-mCh-labeled nuclei at the P4 stage (genotypes shown in Figure 1I-J), Z stack series were captured with similar settings to those described above, but a 10X objective lens was used, and power/off-set/gain settings were FITC 4.5/0/43 and TRITC 0.4/0/61. Sections were collectively exported into a multipage TIFF file, imported into Fiji, color channels were split, the TRITC channel was selected, and 3D objects were counted using a threshold of 800 and a minimum size of 35, parameters which appeared to reliably identify each visible nucleus.
Bang-sensitivity assays: 3-7-day-old adult flies were vortex assayed for bang-sensitive paralysis using a protocol based on Bermudez et al. (2023). Adult progeny of experimental crosses were allowed to emerge over 4 days, anesthetized with CO2, transferred to glass vials containing fresh diet, and vials were capped with plastic foam plugs (Fisher Scientific)—we specify the container materials because, in our experience, bang-sensitivity was enhanced if flies were vortexed in glass vials with foam plugs compared to standard plastic vials and cotton plugs, perhaps because the latter materials would have softer impact on the flies during vortexing. Vials were stored at room temperature for 3 additional days; during this period, to avoid making the flies refractory to bang-sensitive paralysis, vials were not jostled or otherwise moved (Pavlidis and Tanouye, 1995). After recovery, each vial was gently picked up then pressed upside down against a Vortex Genie (Sigma-Aldrich) on maximum setting for 10 seconds to agitate the flies against the cap and vial wall. Immediately afterwards, the number of paralyzed flies were counted and compared to the total number of living flies in the vial.
Image editing, data analysis, and figure generation: The focused EDF TIFF files from confocal imaging were imported into Adobe Photoshop 2021. Exposure was increased +1, and to increase accessibility for red/green colorblind readers, the red mCh channel was converted to magenta by adding a Hue/Saturation Adjustment Layer and altering the hue of the reds -35 (Summerbell, 2019). Otherwise, no image adjustments were made. Vortex data were graphed and analyzed using JMP Student Version 18. The Figure 1L graph was saved as an editable JPG. Photos and the graph were assembled into a figure and annotated using Adobe Illustrator 2021.
AI statement: No AI was used in this project for data collection, data analysis, writing, figure generation, or editing.
","reagents":"","patternDescription":"The fruit fly Drosophila melanogaster has been an informative model system for studying seizure disorders. Mutations in certain genes cause Drosophila adults to have seizure-like neural activity and muscle spasms, followed by a period of paralysis before recovery. This behavioral sequence can be induced by external stimuli, including extreme temperature changes; electrical shock; strobe lighting; and mechanical shock, for example from shaking the culture vial upon a lab vortex machine. Given the ease and effectiveness of mechanical shock, fly mutants with this seizure-like phenotype are considered “bang-sensitive” (Baraban, 2007; Parker et al., 2011a; Dean et al., 2018; Mituzaite et al., 2021; Fischer et al., 2023).
Most loci that affect bang-sensitivity are associated with basic neurophysiological function. For examples, paralytic (para) encodes a subunit of a voltage gated Na+ channel, and ATPα encodes a subunit of Na+/K+-ATPase (Schubiger et al., 1994; Parker et al., 2011b). In contrast, julius seizure (jus, formerly known as slamdance and sda) has a more cryptic role in regulating neural activity because, other than two predicted transmembrane spans that bracket a cysteine-rich, extracellular loop, the Jus protein has no conserved domains that imply a specific function (Horne et al., 2017; Dean et al., 2018). In preprint, we have reported that Jus physically associates with 23 other proteins, including ATPα and Nrv3, which are α and β subunits of the Na+/K+-ATPase, so it is possible that Jus affects the assembly, positioning, stability, and/or activity of a channel that is essential for maintaining resting membrane potential (Bermudez et al., 2023). In two separate experiments, we had used RNAi to knock down jus transcript at specific developmental stages and found evidence that jus expression may be required as early as the wandering larval phase and as late as pupal stage P7, with a peak requirement at about P4-5, when the adult head epidermis everts and surrounds the brain (Horne et al., 2017; experiment replication/refinement described in preprint Bermudez et al., 2023). We also found that jus expression is required in neurons—most significantly in cholinergic neurons—and not in glia, to protect against bang-sensitivity (Horne et al., 2017; Dean et al., 2018). With this as a starting point, our goals were (1) to determine the expression pattern of jus during its critical period for expression, and (2) to begin to characterize the spatial distribution of neurons that must express jus to protect the adult against bang-sensitivity.
We used two reporters to assess jus expression in the pupal CNS: (1) jusGFSTF is a protein trap allele that inserts an EGFP-FlAsH-StrepII-TEV-3xFlag multi-tag cassette in the putative extracellular domain of Jus; this fusion protein mislocalizes to the cytoplasm of neuronal cell bodies, and its expression can be detected by GFP fluorescence (Diao et al., 2015; Dean et al., 2018). (2) GMR90B09-GAL4, hereafter referred to as jus-GAL4, is a construct that uses the jus promoter region to drive GAL4 expression (Jennet et al., 2012; Bermudez et al., 2023). A GAL4 construct that reflects endogenous jus expression would be expected to cause severe bang-sensitivity when used to express UAS-jusRNAi. Retesting jus-GAL4 for this study, we found that, while GAL4-only and RNAi-only controls were not bang-sensitive, 98% of jus-GAL4 > jusRNAi flies were, confirming that this jus-GAL4 construct is expressed in neurons that express jus and that affect bang-sensitivity (Figure 1L).
To compare the expression patterns of jus-GAL4 and Jus protein, jus-GAL4 > nls-mCh and jusGFSTF stocks were mated, and progeny were examined during metamorphosis. Throughout the critical period for jus expression, both nls-mCh and GFP were expressed widely in the ventral nerve cord (VNC) and somewhat in the central brain, with little expression in the optic lobes (Figure 1A-D). Despite the similar spatial distribution of nls-mCh and GFP signals across the nervous system, few cells strongly expressed both nls-mCh and GFP, with most examples located in the VNC, and later in the protocerebrum, a region of the central brain that directly associates with the optic lobe (Figure 1E-H). There are several possible explanations for the apparent mismatch between the jus-GAL4 > nls-mCh and JusGFSTF expression patterns: 1) jus-GAL4 > nls-mCh may not fully reflect endogenous jus expression, because although the jus-GAL4 construct includes the entire jus promoter region, it may be missing enhancer sequences that refine the expression pattern. 2) JusGFSTF may not fully reflect wild type Jus+ protein expression, because the mutant protein mislocalizes from the axon to the cell body (Dean et al., 2018), suggesting it is structurally defective. Therefore, JusGFSTF stability/turnover rate may differ from that of Jus+ protein. 3) jus-GAL4 > nls-mCh and JusGFSTF expression may overlap more significantly than suggested by confocal microscopy, but much of this overlap may have been undetected due to differing expression levels or protein turnover kinetics of nls-mCh vs. JusGFSTF in any given cell. Regardless of which of these hypotheses is correct, jus-GAL4 > jusRNAi caused very high rates of bang-sensitive paralysis, so jus-GAL4 must be expressed in neuronal subpopulations that strongly affect bang-sensitivity.
Given the broad expression of jus-GAL4 and JusGFSTF across the VNC, we considered whether a GAL80 construct associated with teashirt (tsh), a homeotic gene that directs development of the VNC, might be expressed in jus-GAL4 > nls-mCh-labeled cells (Röder et al., 1992; Rubio-Ferrera et al., 2023). If tsh-GAL80 and jus-GAL4 > nls-mCh expression patterns overlap, tsh-GAL80 would be expected to block GAL4 function, reducing the number of mCh-labelled nuclei. Consistent with this prediction, jus-GAL4 > nls-mCh + tsh-GAL80 pupae, throughout the jus critical period, had a low number of mCh-labelled nuclei in the VNC (Figure 1I vs. J). Quantifying at P4, the peak of the critical period, it was found that jus-GAL4 > nls-mCh + tsh-GAL80 central nervous systems had 29.9% the average number of labeled nuclei seen in jus-GAL4 > nls-mCh controls (averages of 113.4 ± 7.7 SEM vs. 379.6 ± 26.5 SEM, n=9 for each group; p = 3.6e-6, Student’s t-test). tsh-GAL4, a construct swapped into the same enhancer trap sequence as tsh-GAL80, was used to express UAS-nls-mCh. Throughout the critical period, tsh-GAL4 > nls-mCh was ubiquitously expressed across the VNC, with some labeling within the posterior section of the central brain, and high expression in the compound eyes (Figure 1K).
Finally, we tested if jus expression is required in tsh-GAL80- and tsh-GAL4-expressing cells to prevent bang-sensitivity. In a previous study, tsh-GAL80-expressing neurons were shown to protect against seizure-like behavior in Drosophila larvae, using an optogenetic model and electrical shock (Giachello and Baines, 2015). In our experiments, tsh-GAL80 strongly suppressed the bang-sensitivity of jus-GAL4 > jusRNAi flies (36.6% with tsh-GAL80 vs. 98.0% without; Figure 1L), and when tsh-GAL4 was used to express jusRNAi, 100% of flies were bang-sensitive (Figure 1L; the end of the figure caption discusses statistics).
Considering the involvement of tsh-GAL80- and tsh-GAL4-expressing neurons in regulating jus-associated bang-sensitivity, along with the persistent expression of these markers in the pupal VNC, an intriguing possibility is that bang-sensitivity can be caused by loss of jus expression in neural tissue outside of the brain. However, given that tsh-GAL4 seems to be expressed in the central brain, at least to some extent (Figure 1K), it is certainly possible that jus expression in the central brain is what protects the fly against bang-sensitivity, and that jus expression in the VNC has a supporting or distinct function. Future experiments could test these two hypotheses, and otherwise spatially and functionally characterize the jus-expressing neurons that affect bang-sensitivity.
","references":[{"reference":"Bainbridge SP, Bownes M. 1981. Staging the metamorphosis of Drosophila melanogaster. J Embryol Exp Morphol 66: 57-80.
","pubmedId":"6802923","doi":""},{"reference":"Baraban SC. 2007. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Opin Neurol 20(2): 164-8.
","pubmedId":"17351486","doi":""},{"reference":"Bermudez S, Dean DM, Alkin V, Wang Z, Pham H, Kondo E, Amini A, Deitcher DL. 2023. A Complex Containing the Na\n +\n /K\n +\n ATPase Regulates Epileptogenesis in\n Drosophila melanogaster. : 10.1101/2023.09.22.559031.
","pubmedId":"","doi":"10.1101/2023.09.22.559031"},{"reference":"Caussinus E, Colombelli J, Affolter M. 2008. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol 18(22): 1727-34.
","pubmedId":"19026547","doi":""},{"reference":"Chyb, S., and N. Gompel, 2013 Atlas of Drosophila morphology: wild-type and classical mutants. Academic Press, London; Waltham, MA.
","pubmedId":"","doi":""},{"reference":"Clyne PJ, Brotman JS, Sweeney ST, Davis G. 2003. Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements. Genetics 165(3): 1433-41.
","pubmedId":"14668392","doi":""},{"reference":"Clyne JD, Miesenböck G. 2008. Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133(2): 354-63.
","pubmedId":"18423205","doi":""},{"reference":"Cole SH, Carney GE, McClung CA, Willard SS, Taylor BJ, Hirsh J. 2005. Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280(15): 14948-55.
","pubmedId":"15691831","doi":""},{"reference":"Dean DM, Maroja LS, Cottrill S, Bomkamp BE, Westervelt KA, Deitcher DL. 2015. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development. G3 (Bethesda) 6(2): 299-310.
","pubmedId":"26613949","doi":""},{"reference":"Dean D, Weinstein H, Amin S, Karno B, McAvoy E, Hoy R, et al., Deitcher D. 2018. Extending julius seizure, a bang-sensitive gene, as a model for studying epileptogenesis: Cold shock, and a new insertional mutation. Fly (Austin) 12(1): 55-61.
","pubmedId":"29125376","doi":""},{"reference":"Dean DM, Deitcher DL, Loehlin DW, Banta LM. 2020. Mapping a Mutation to its Gene: The \"Fly Lab\" as a Modern Research Experience. CourseSource 7: 10.24918/cs.2020.51.
","pubmedId":"","doi":"10.24918/cs.2020.51"},{"reference":"Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, et al., White BH. 2015. Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes. Cell Rep 10(8): 1410-21.
","pubmedId":"25732830","doi":""},{"reference":"Fischer FP, Karge RA, Weber YG, Koch H, Wolking S, Voigt A. 2023. Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview. Front Mol Neurosci 16: 1116000.
","pubmedId":"36873106","doi":""},{"reference":"Groth AC, Fish M, Nusse R, Calos MP. 2004. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166(4): 1775-82.
","pubmedId":"15126397","doi":""},{"reference":"Giachello CN, Baines RA. 2015. Inappropriate Neural Activity during a Sensitive Period in Embryogenesis Results in Persistent Seizure-like Behavior. Curr Biol 25(22): 2964-8.
","pubmedId":"26549258","doi":""},{"reference":"Horne M, Krebushevski K, Wells A, Tunio N, Jarvis C, Francisco G, et al., Deitcher DL. 2017. julius seizure, a Drosophila Mutant, Defines a Neuronal Population Underlying Epileptogenesis. Genetics 205(3): 1261-1269.
","pubmedId":"28082408","doi":""},{"reference":"Mituzaite J, Petersen R, Claridge-Chang A, Baines RA. 2021. Characterization of Seizure Induction Methods in Drosophila. eNeuro 8(4): pii: ENEURO.0079-21.2021. 10.1523/ENEURO.0079-21.2021.
","pubmedId":"34330816","doi":""},{"reference":"Jenett A, Rubin GM, Ngo TT, Shepherd D, Murphy C, Dionne H, et al., Zugates CT. 2012. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2(4): 991-1001.
","pubmedId":"23063364","doi":""},{"reference":"Jenkins VK, Larkin A, Thurmond J, FlyBase Consortium. 2022. Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol 2540: 1-34.
","pubmedId":"35980571","doi":""},{"reference":"Parker L, Howlett IC, Rusan ZM, Tanouye MA. 2011. Seizure and epilepsy: studies of seizure disorders in Drosophila. Int Rev Neurobiol 99: 1-21.
","pubmedId":"21906534","doi":""},{"reference":"Parker L, Padilla M, Du Y, Dong K, Tanouye MA. 2011. Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics 187(2): 523-34.
","pubmedId":"21115970","doi":""},{"reference":"Pavlidis P, Tanouye MA. 1995. Seizures and failures in the giant fiber pathway of Drosophila bang-sensitive paralytic mutants. J Neurosci 15(8): 5810-9.
","pubmedId":"7643221","doi":""},{"reference":"Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, et al., Perrimon N. 2015. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201(3): 843-52.
","pubmedId":"26320097","doi":""},{"reference":"Röder L, Vola C, Kerridge S. 1992. The role of the teashirt gene in trunk segmental identity in Drosophila. Development 115(4): 1017-33.
","pubmedId":"1360402","doi":""},{"reference":"Röder L, Vola C, Kerridge S. 1992. The role of the teashirt gene in trunk segmental identity in Drosophila. Development 115(4): 1017-33.
","pubmedId":"1360402","doi":""},{"reference":"Schubiger M, Feng Y, Fambrough DM, Palka J. 1994. A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis. Neuron 12(2): 373-81.
","pubmedId":"8110464","doi":""},{"reference":"Soller M, Haussmann IU, Hollmann M, Choffat Y, White K, Kubli E, Schäfer MA. 2006. Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons. Curr Biol 16(18): 1771-82.
","pubmedId":"16979554","doi":""},{"reference":"Summerbell E. (2019) How to make scientific figures accessible to readers with color-blindness. American Society for Cell Biology.
","pubmedId":"","doi":""}],"title":"julius seizure acts within teashirt-expressing neurons to protect Drosophila melanogaster from bang-sensitivity
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"6a2e88e3-6fca-4e56-a1ca-9add90fb435a","decision":"publish","abstract":"Loss of function in the Drosophila melanogaster gene julius seizure (jus) causes the fly to have a seizure-like condition known as bang-sensitivity. Here, we characterize the expression patterns of two different jus reporters, a jus-GAL4 construct and a jus protein trap allele, in the central nervous system during the developmental stages that jus expression is required. The majority of cells that expressed the jus-GAL4 construct co-expressed a GAL80 enhancer trap in the homeotic gene teashirt (tsh); expression of jus in this subpopulation was necessary to prevent bang-sensitivity.
","acknowledgements":"The authors are very grateful to Nancy Piatczyc and Clara Ives for their technical assistance with the confocal microscope. Pei-Wen Chen of Williams College kindly helped us develop a protocol for counting mCherry-labeled nuclei. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Bioinformatic information was obtained from FlyBase (Jenkins et al., 2022).
","authors":[{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["conceptualization","dataCuration","writing_originalDraft","writing_reviewEditing","fundingAcquisition","methodology","investigation","formalAnalysis","project","supervision","validation","visualization"],"email":"ddean@williams.edu","firstName":"Derek M.","lastName":"Dean","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0000-4135-1518"},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"lec4@williams.edu","firstName":"Lillian E.","lastName":"Codd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"mye1@williams.edu","firstName":"Mira","lastName":"Eras","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","writing_reviewEditing","validation"],"email":"kol2@williams.edu","firstName":"Katherine","lastName":"Lopez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"huynpham200@gmail.com","firstName":"Huy","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"dmataylor9@gmail.com","firstName":"Dylan","lastName":"Taylor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation","validation","writing_reviewEditing","methodology"],"email":"esa3@williams.edu","firstName":"Aisha","lastName":"Abakura","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"zg2@williams.edu","firstName":"Zoe","lastName":"Gabriel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","investigation","validation","writing_reviewEditing"],"email":"alyg@stanford.edu","firstName":"Alyana J.","lastName":"Granados","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["investigation","formalAnalysis","methodology","validation","writing_reviewEditing"],"email":"bml3@williams.edu","firstName":"Ben","lastName":"Litvak-Hinenzon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"jjw4@williams.edu","firstName":"Jeremy","lastName":"Wen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Williams College, Williamstown, Massachusetts, United States"],"departments":["Biology"],"credit":["formalAnalysis","methodology","validation","writing_reviewEditing","investigation"],"email":"cyturralde24@gmail.com","firstName":"Catalin","lastName":"Yturralde","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Cornell University, Ithaca, New York, United States"],"departments":["Neurobiology and Behavior"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","project","resources","supervision","validation","writing_reviewEditing"],"email":"dld14@cornell.edu","firstName":"David L","lastName":"Deitcher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded internally by Williams College.
","image":{"url":"https://portal.micropublication.org/uploads/1e8471ffe50be68cafab9d9ed05fb4b5.jpg"},"imageCaption":"Top left, schematic of a neuronal cell body, indicating the colors emitted by the two jus expression reporters that were used in this study. Magenta nuclei are from jus-GAL4 driving UAS-nls-mCherry expression (“jus-GAL4 > nls-mCh”), green cell bodies express the JusGFSTF fusion protein, and colocalization between these two expression patterns either appears as a magenta dot within a green cell, or as yellow if the colors overlap. A legend lists the symbols and abbreviations used in (A-K). White scale bar in each confocal image indicates 100 µm. During pupal stages P2 (A) and P4 (B), jus-GAL4 > nls-mCh and JusGFSTF were both expressed widely in the ventral nerve cord (VNC), with some expression across the central brain (CB), and virtually no expression in the optic lobes (OL). Some colocalization was seen in the VNC, particularly in cells along the midline and lateral margins of the posterior segments. The head cuticle (c) tended to autofluoresce green. (C) By stage P5ii, and through to the end of the critical period (D), jus-GAL4 > nls-mCh expression was strongest in the most posterior and anterior VNC segments, while JusGFSTF expression was most prominent in the mid-thoracic region of the VNC; colocalization of the two signals was seen in a narrow, lateral band of cells that wrapped around the mid-thoracic ganglia. Fat body (f) often autofluoresced green. At the transition between P6 and P7 (D), additional colocalization was seen in a band of cells across the protocerebrum (PR), a region of the brain that receives and processes information from the optic lobe. (E) and (F) show higher magnification examples of nls-mCh/GFP colocalization in the VNC. (F) is a transverse section of an abdominal segment to show colocalization on the dorsal (top) as well ventral (bottom) margins of the VNC. (G) shows a higher magnification example of colocalization in a lateral band across the mid-thorax. (H) is a higher magnification view of colocalization in the protocerebrum (PR) as well as a small number of cells in the central brain (CB). (I) jus-GAL4 > nls-mCh expression in the CNS of a P4 pupa that did not carry jusGFSTF. As in panel B, mCherry signal was seen throughout the VNC. Even in the absence of JusGFSTF, the head cuticle (c) and fat body (f) strongly fluoresced green, confirming that these were non-specific, autofluorescent signals. (J) The number of jus-GAL4 > nls-mCh-expressing nuclei was significantly reduced in pupae that also carried the tsh-GAL80 construct. Loss of nls-mCh signal was particularly noticeable throughout the VNC. This particular specimen also happens to have reduced green autofluorescence, but this was not specific to the jus-GAL4 > nls-mCh + tsh-GAL80 genotype, i.e., cuticle and fat body autofluorescence varied widely in all sample groups [compare panels (B), (I), and (J)]. (K) tsh-GAL4 > nls-mCh expression was ubiquitous in the VNC, posterior central brain, and compound eyes (CE). (L) When jus-GAL4 was used to express UAS-jusRNAi (“jus-GAL4 > jusRNAi”), 98.0% of flies were bang-sensitive. tsh-GAL80 suppressed jus-GAL4 > jusRNAi bang-sensitivity down to 36.6%. UAS-jusRNAi expression using a tsh-GAL4 driver (“tsh-GAL4 > jusRNAi”) caused 100% bang-sensitivity. n = 150 or more for each sample group. Two-tailed Fisher’s exact tests: p < 1.1e-110 (jus-GAL4 > jusRNAi vs. RNAi-only and GAL4-only controls) p = 1.1e-43 (jus-GAL4 > jusRNAi vs. jus-GAL4 > jusRNAi + tsh-GAL80), and p < 2.1e-89 (tsh-GAL4 > jusRNAi vs. RNAi-only and GAL4-only controls).
","imageTitle":"(A-D) Confocal microscope imaging of the expression of two different julius seizure (jus) reporters in the central nervous system (CNS) during the critical period for jus expression, (E-H) higher magnification examples of jus reporter colocalization, (I-K) comparison of jus-GAL4 expression to that of teashirt (tsh)-GAL80 and tsh-GAL4 constructs, and (L) bang sensitivity of jus-GAL4 > jusRNAi, jus-GAL4 > jusRNAi + tsh-GAL80, and tsh-GAL4 > jusRNAi flies
","methods":"Fly stocks, diet, and experimental crosses: The fly strains that were used in our experiments are listed below. Stocks received from the Bloomington Drosophila Stock Center are denoted by “BDSC [stock number]”.
●jus-GAL4: w1118; P{y+t7.7 w+mC=GMR90B09-GAL4}attP2 (BDSC 607421; Bermudez et al., 2023)
●Control strain for jus-GAL4 background: w1118 (BDSC 6326)
●UAS-jusRNAi: y1 v1; P{y+t7.7 v+t1.8=TRiP.JF03192}attP2 (BDSC 28764; Perkins et al., 2015)
●Control strain for jusRNAi background: y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303; Groth et al., 2004)
●jusGFSTF: w; Kr/CyO; jusGFSTF/TM6C Sb Tb (BDSC 607422; Dean et al., 2018). For our experiments, the Kr and CyO second chromosomes had been crossed out of the background, leaving a strain with the genotype w; +/+; jusGFSTF/TM6C Sb Tb
●UAS-nls-mCh (for crosses involving jus-GAL4): w; P{w+mC=UAS-mCherry.NLS}3 (BDSC 38424; Caussinus et al., 2008)
●A jus-GAL4 > nls-mCh stock was made by recombining the jus-GAL4 and UAS-nls-mCh constructs described above onto the same chromosome and a w1118; jus-GAL4 UAS-nls-mCh strain was made.
●jus-GAL4 jusGFSTF: This stock was generated by recombining jus-GAL4 and jusGFSTF onto the same chromosome and balancing over TM6C. Full genotype was w1118; jus-GAL4 jusGFSTF/TM6C Sb Tb.
●tsh-GAL80: w; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; TM2/TM6B (BDSC 605556; Clyne and Miesenbock, 2008). For our experiments, the tsh-GAL80 construct was rebalanced over the CyO, P{Wee-P.ph0}Bacc[Wee-P20] chromosome from BDSC 52267; this CyO balancer ubiquitously expresses GFP, enabling us to identify pupae that carried the tsh-GAL80 chromosome (Clyne et al., 2003).
●tsh-GAL80; jus-GAL4: A strain carrying both the jus-GAL4 and tsh-GAL80 constructs was made. Full genotype: w1118; P{y+t*=GAL80}tsh[md621-GAL80]/CyO; P{y+t7.7w+mC=GMR90B09-GAL4}attP2
●tsh-GAL4: y1 w1118; P{w+mW.hs=GawB}tsh[md621]/CyO; P{w+mC=UAS-y.C}MC1/TM2 (BDSC 3040; Soller et al., 2006)
●UAS-nls-mCh (for crosses involving tsh-GAL4): w; P{w+mC=UAS-mCherry.NLS}2 (BDSC 38425; Caussinus et al., 2008). This strain was crossed to tsh-GAL4 to generate the image in Figure 1K, while crosses involving jus-GAL4 and mCherry used the related UAS-nls-mCh stock described above (BDSC 38424).
Larvae and flies were fed a modified yeast/dextrose/cornmeal diet (Dean et al., 2016; Dean et al., 2020). Parental stocks were maintained at room temperature, while experimental crosses were incubated at 25°C as the progeny developed. Mating schemes for experimental crosses were as follows (see stock list immediately above for full parental genotypes):
●Figure 1A-H (Comparing jus-GAL4 > nls-mCh and jusGFSTF expression in CNS)
jus-GAL4 > nls-mCh females X jusGFSTF males
●Figure 1I-K (Expression of tsh-GAL80 and tsh-GAL4 constructs in CNS)
Figure 1I: jus-GAL4 > nls-mCh females X w1118 males
Figure 1J: jus-GAL4 > nls-mCh females X tsh-GAL80/CyO, P{Wee-P.ph0}Bacc[Wee-P20] males. Progeny carrying tsh-GAL80 were identified by a lack of GFP expression from the CyO, P{Wee-P.ph0}Bacc[Wee-P20] balancer (Clyne et al., 2003).
Figure 1K: tsh-GAL4 females X nls-mCh (BDSC 38425) males
●Figure 1L (Bang-sensitive assays)
jus-RNAi-only control: w1118 females X UAS-jusRNAi males
jus-GAL4-only control: jus-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
jus-GAL4 > jusRNAi: jus-GAL4 females X UAS-jusRNAi males
jus-GAL4 > jusRNAi with tsh-GAL80: tsh-GAL80; jus-GAL4 females X UAS-jusRNAi males
tsh-GAL4-only control: tsh-GAL4 females X y1 v1; P{y+t7.7=CaryP}attP2 (BDSC 36303) males
tsh-GAL4 > jusRNAi: tsh-GAL4 females X UAS-jusRNAi males
CNS preparation for confocal microscopy: Wandering larvae and pupal stages P1-P7 were identified by morphological criteria (Bainbridge and Bownes, 1981; Chyb and Gompel, 2013). Each CNS was dissected in cold PBS, fixed in PBS + 4% formaldehyde for 30 minutes at room temperature with gentle rocking, then rinsed twice in PBS + 0.3% Triton X-100 (all chemicals obtained from Sigma-Aldrich).
CNS tissue was transferred to microscope slides and overlaid with Vectashield Antifade Mounting Media (Vector Labs), then with a #1.5 18 x 18 mm coverslip (Fisher Scientific). Coverslip edges were sealed with nail polish. Slides were stored in the dark at 4ºC until they were imaged at the Williams College confocal microscope facility.
Confocal microscopy and post-production image processing: Within a week after slide preparation, pupal CNS tissue was imaged using a Nikon Ti2 inverted microscope and Nikon A1R HD software.
Z stack series were collected with the following settings: 20X objective lens, 2 µm steps, 32X averaging resonant scan, 0.9 pinhole, 1024 x 1024 pixel resolution, and power/off-set/gain settings were FITC 25/0/43 and TRITC 0.8/0/65. To reduce background from cuticle and fat body at the later pupal stages, FITC power was decreased to as low as 4.5 while holding other settings constant. Z-series were saved as ND2 files. Each Figure 1 image was made by merging a Z-series into a focused EDF document and saving as an uncompressed TIFF.
For quantification of jus-GAL4 > nls-mCh-labeled nuclei at the P4 stage (genotypes shown in Figure 1I-J), Z stack series were captured with similar settings to those described above, but a 10X objective lens was used, and power/off-set/gain settings were FITC 4.5/0/43 and TRITC 0.4/0/61. Sections were collectively exported into a multipage TIFF file, imported into Fiji, color channels were split, the TRITC channel was selected, and 3D objects were counted using a threshold of 800 and a minimum size of 35, parameters which appeared to reliably identify each visible nucleus.
Bang-sensitivity assays: 3-7-day-old adult flies were vortex assayed for bang-sensitive paralysis based on a previously established protocol (Bermudez et al., 2023). Adult progeny of experimental crosses were allowed to emerge over 4 days, anesthetized with CO2, transferred to glass vials containing fresh diet, and vials were capped with plastic foam plugs (Fisher Scientific)—we specify the container materials because, in our experience, bang-sensitivity was enhanced if flies were vortexed in glass vials with foam plugs compared to standard plastic vials and cotton plugs, perhaps because the latter materials would have softer impact on the flies during vortexing. Vials were stored at room temperature for 3 additional days; during this period, to avoid making the flies refractory to bang-sensitive paralysis, vials were not jostled or otherwise moved (Pavlidis and Tanouye, 1995). After recovery, each vial was gently picked up then pressed upside down against a Vortex Genie (Sigma-Aldrich) on maximum setting for 10 seconds to agitate the flies against the cap and vial wall. Immediately afterwards, the number of paralyzed flies were counted and compared to the total number of living flies in the vial.
Image editing, data analysis, and figure generation: The focused EDF TIFF files from confocal imaging were imported into Adobe Photoshop 2021. Exposure was increased +1, and to increase accessibility for red/green colorblind readers, the red mCh channel was converted to magenta by adding a Hue/Saturation Adjustment Layer and altering the hue of the reds -35 (Summerbell, 2019). Otherwise, no image adjustments were made. Vortex data were graphed and analyzed using JMP Student Version 18. The Figure 1L graph was saved as an editable JPG. Photos and the graph were assembled into a figure and annotated using Adobe Illustrator 2021.
AI statement: No AI was used in this project for data collection, data analysis, writing, figure generation, or editing.
","reagents":"","patternDescription":"The fruit fly Drosophila melanogaster has been an informative model system for studying seizure disorders. Mutations in certain genes cause Drosophila adults to have seizure-like neural activity and muscle spasms, followed by a period of paralysis before recovery. This behavioral sequence can be induced by external stimuli, including extreme temperature changes; electrical shock; strobe lighting; and mechanical shock, for example from shaking the culture vial upon a lab vortex machine. Given the ease and effectiveness of mechanical shock, fly mutants with this seizure-like phenotype are considered “bang-sensitive” (Baraban, 2007; Parker et al., 2011a; Dean et al., 2018; Mituzaite et al., 2021; Fischer et al., 2023).
Most loci that affect bang-sensitivity are associated with basic neurophysiological function. For examples, paralytic (para) encodes a subunit of a voltage gated Na+ channel, and ATPα encodes a subunit of Na+/K+-ATPase (Schubiger et al., 1994; Parker et al., 2011b). In contrast, julius seizure (jus, formerly known as slamdance and sda) has a more cryptic role in regulating neural activity because, other than two predicted transmembrane spans that bracket a cysteine-rich, extracellular loop, the Jus protein has no conserved domains that imply a specific function (Horne et al., 2017; Dean et al., 2018). In preprint, we have reported that Jus physically associates with 23 other proteins, including ATPα and Nrv3, which are α and β subunits of the Na+/K+-ATPase, so it is possible that Jus affects the assembly, positioning, stability, and/or activity of a channel that is essential for maintaining resting membrane potential (Bermudez et al., 2023). In two separate experiments, we had used RNAi to knock down jus transcript at specific developmental stages and found evidence that jus expression may be required as early as the wandering larval phase and as late as pupal stage P7, with a peak requirement at about P4-5, when the adult head epidermis everts and surrounds the brain (Horne et al., 2017; experiment replication/refinement described in preprint Bermudez et al., 2023). We also found that jus expression is required in neurons—most significantly in cholinergic neurons—and not in glia, to protect against bang-sensitivity (Horne et al., 2017; Dean et al., 2018). With this as a starting point, our goals were (1) to determine the expression pattern of jus during its critical period for expression, and (2) to begin to characterize the spatial distribution of neurons that must express jus to protect the adult against bang-sensitivity.
We used two reporters to assess jus expression in the pupal CNS: (1) jusGFSTF is a protein trap allele that inserts an EGFP-FlAsH-StrepII-TEV-3xFlag multi-tag cassette in the putative extracellular domain of Jus; this fusion protein mislocalizes to the cytoplasm of neuronal cell bodies, and its expression can be detected by GFP fluorescence (Diao et al., 2015; Dean et al., 2018). (2) GMR90B09-GAL4, hereafter referred to as jus-GAL4, is a construct that uses the jus promoter region to drive GAL4 expression (Jennet et al., 2012; Bermudez et al., 2023). A GAL4 construct that reflects endogenous jus expression would be expected to cause severe bang-sensitivity when used to express UAS-jusRNAi. Retesting jus-GAL4 for this study, we found that, while GAL4-only and RNAi-only controls were not bang-sensitive, 98% of jus-GAL4 > jusRNAi flies were, confirming that this jus-GAL4 construct is expressed in neurons that express jus and that affect bang-sensitivity (Figure 1L).
To compare the expression patterns of jus-GAL4 and Jus protein, jus-GAL4 > nls-mCh and jusGFSTF stocks were mated, and progeny were examined during metamorphosis. Throughout the critical period for jus expression, both nls-mCh and GFP were expressed widely in the ventral nerve cord (VNC) and somewhat in the central brain, with little expression in the optic lobes (Figure 1A-D). Despite the similar spatial distribution of nls-mCh and GFP signals across the nervous system, few cells strongly expressed both nls-mCh and GFP, with most examples located in the VNC, and later in the protocerebrum, a region of the central brain that directly associates with the optic lobe (Figure 1E-H). There are several possible explanations for the apparent mismatch between the jus-GAL4 > nls-mCh and JusGFSTF expression patterns: 1) jus-GAL4 > nls-mCh may not fully reflect endogenous jus expression, because although the jus-GAL4 construct includes the entire jus promoter region, it may be missing enhancer sequences that refine the expression pattern. 2) JusGFSTF may not fully reflect wild type Jus+ protein expression, because the mutant protein mislocalizes from the axon to the cell body (Dean et al., 2018), suggesting it is structurally defective. Therefore, JusGFSTF stability/turnover rate may differ from that of Jus+ protein. 3) jus-GAL4 > nls-mCh and JusGFSTF expression may overlap more significantly than suggested by confocal microscopy, but much of this overlap may have been undetected due to differing expression levels or protein turnover kinetics of nls-mCh vs. JusGFSTF in any given cell. Regardless of which of these hypotheses is correct, jus-GAL4 > jusRNAi caused very high rates of bang-sensitive paralysis, so jus-GAL4 must be expressed in neuronal subpopulations that strongly affect bang-sensitivity.
Given the broad expression of jus-GAL4 and JusGFSTF across the VNC, we considered whether a GAL80 construct associated with teashirt (tsh), a homeotic gene that directs development of the VNC, might be expressed in jus-GAL4 > nls-mCh-labeled cells (Röder et al., 1992; Rubio-Ferrera et al., 2023). If tsh-GAL80 and jus-GAL4 > nls-mCh expression patterns overlap, tsh-GAL80 would be expected to block GAL4 function, reducing the number of mCh-labelled nuclei. Consistent with this prediction, jus-GAL4 > nls-mCh + tsh-GAL80 pupae, throughout the jus critical period, had a low number of mCh-labelled nuclei in the VNC (Figure 1I vs. J). Quantifying at P4, the peak of the critical period, it was found that jus-GAL4 > nls-mCh + tsh-GAL80 central nervous systems had 29.9% the average number of labeled nuclei seen in jus-GAL4 > nls-mCh controls (averages of 113.4 ± 7.7 SEM vs. 379.6 ± 26.5 SEM, n=9 for each group; p = 3.6e-6, Student’s t-test). tsh-GAL4, a construct swapped into the same enhancer trap sequence as tsh-GAL80, was used to express UAS-nls-mCh. Throughout the critical period, tsh-GAL4 > nls-mCh was ubiquitously expressed across the VNC, with some labeling within the posterior section of the central brain, and high expression in the compound eyes (Figure 1K).
Finally, we tested if jus expression is required in tsh-GAL80- and tsh-GAL4-expressing cells to prevent bang-sensitivity. In a previous study, tsh-GAL80-expressing neurons were shown to protect against seizure-like behavior in Drosophila larvae, using an optogenetic model and electrical shock (Giachello and Baines, 2015). In our experiments, tsh-GAL80 strongly suppressed the bang-sensitivity of jus-GAL4 > jusRNAi flies (36.6% with tsh-GAL80 vs. 98.0% without; Figure 1L), and when tsh-GAL4 was used to express jusRNAi, 100% of flies were bang-sensitive (Figure 1L; the end of the figure caption discusses statistics).
Considering the involvement of tsh-GAL80- and tsh-GAL4-expressing neurons in regulating jus-associated bang-sensitivity, along with the persistent expression of these markers in the pupal VNC, an intriguing possibility is that bang-sensitivity can be caused by loss of jus expression in neural tissue outside of the brain. However, given that tsh-GAL4 seems to be expressed in the central brain, at least to some extent (Figure 1K), it is certainly possible that jus expression in the central brain is what protects the fly against bang-sensitivity, and that jus expression in the VNC has a supporting or distinct function. Future experiments could test these two hypotheses, and otherwise spatially and functionally characterize the jus-expressing neurons that affect bang-sensitivity.
","references":[{"reference":"Bainbridge SP, Bownes M. 1981. Staging the metamorphosis of Drosophila melanogaster. J Embryol Exp Morphol 66: 57-80.
","pubmedId":"6802923","doi":""},{"reference":"Baraban SC. 2007. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Opin Neurol 20(2): 164-8.
","pubmedId":"17351486","doi":""},{"reference":"Bermudez S, Dean DM, Alkin V, Wang Z, Pham H, Kondo E, Amini A, Deitcher DL. 2023. A Complex Containing the Na\n +\n /K\n +\n ATPase Regulates Epileptogenesis in\n Drosophila melanogaster. : 10.1101/2023.09.22.559031.
","pubmedId":"","doi":"10.1101/2023.09.22.559031"},{"reference":"Caussinus E, Colombelli J, Affolter M. 2008. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol 18(22): 1727-34.
","pubmedId":"19026547","doi":""},{"reference":"Chyb, S., and N. Gompel, 2013 Atlas of Drosophila morphology: wild-type and classical mutants. Academic Press, London; Waltham, MA.
","pubmedId":"","doi":""},{"reference":"Clyne PJ, Brotman JS, Sweeney ST, Davis G. 2003. Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements. Genetics 165(3): 1433-41.
","pubmedId":"14668392","doi":""},{"reference":"Clyne JD, Miesenböck G. 2008. Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133(2): 354-63.
","pubmedId":"18423205","doi":""},{"reference":"Cole SH, Carney GE, McClung CA, Willard SS, Taylor BJ, Hirsh J. 2005. Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280(15): 14948-55.
","pubmedId":"15691831","doi":""},{"reference":"Dean DM, Maroja LS, Cottrill S, Bomkamp BE, Westervelt KA, Deitcher DL. 2015. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development. G3 (Bethesda) 6(2): 299-310.
","pubmedId":"26613949","doi":""},{"reference":"Dean D, Weinstein H, Amin S, Karno B, McAvoy E, Hoy R, et al., Deitcher D. 2018. Extending julius seizure, a bang-sensitive gene, as a model for studying epileptogenesis: Cold shock, and a new insertional mutation. Fly (Austin) 12(1): 55-61.
","pubmedId":"29125376","doi":""},{"reference":"Dean DM, Deitcher DL, Loehlin DW, Banta LM. 2020. Mapping a Mutation to its Gene: The \"Fly Lab\" as a Modern Research Experience. CourseSource 7: 10.24918/cs.2020.51.
","pubmedId":"","doi":"10.24918/cs.2020.51"},{"reference":"Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, et al., White BH. 2015. Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes. Cell Rep 10(8): 1410-21.
","pubmedId":"25732830","doi":""},{"reference":"Fischer FP, Karge RA, Weber YG, Koch H, Wolking S, Voigt A. 2023. Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview. Front Mol Neurosci 16: 1116000.
","pubmedId":"36873106","doi":""},{"reference":"Groth AC, Fish M, Nusse R, Calos MP. 2004. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166(4): 1775-82.
","pubmedId":"15126397","doi":""},{"reference":"Giachello CN, Baines RA. 2015. Inappropriate Neural Activity during a Sensitive Period in Embryogenesis Results in Persistent Seizure-like Behavior. Curr Biol 25(22): 2964-8.
","pubmedId":"26549258","doi":""},{"reference":"Horne M, Krebushevski K, Wells A, Tunio N, Jarvis C, Francisco G, et al., Deitcher DL. 2017. julius seizure, a Drosophila Mutant, Defines a Neuronal Population Underlying Epileptogenesis. Genetics 205(3): 1261-1269.
","pubmedId":"28082408","doi":""},{"reference":"Mituzaite J, Petersen R, Claridge-Chang A, Baines RA. 2021. Characterization of Seizure Induction Methods in Drosophila. eNeuro 8(4): pii: ENEURO.0079-21.2021. 10.1523/ENEURO.0079-21.2021.
","pubmedId":"34330816","doi":""},{"reference":"Jenett A, Rubin GM, Ngo TT, Shepherd D, Murphy C, Dionne H, et al., Zugates CT. 2012. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2(4): 991-1001.
","pubmedId":"23063364","doi":""},{"reference":"Jenkins VK, Larkin A, Thurmond J, FlyBase Consortium. 2022. Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol 2540: 1-34.
","pubmedId":"35980571","doi":""},{"reference":"Parker L, Howlett IC, Rusan ZM, Tanouye MA. 2011. Seizure and epilepsy: studies of seizure disorders in Drosophila. Int Rev Neurobiol 99: 1-21.
","pubmedId":"21906534","doi":""},{"reference":"Parker L, Padilla M, Du Y, Dong K, Tanouye MA. 2011. Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics 187(2): 523-34.
","pubmedId":"21115970","doi":""},{"reference":"Pavlidis P, Tanouye MA. 1995. Seizures and failures in the giant fiber pathway of Drosophila bang-sensitive paralytic mutants. J Neurosci 15(8): 5810-9.
","pubmedId":"7643221","doi":""},{"reference":"Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, et al., Perrimon N. 2015. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201(3): 843-52.
","pubmedId":"26320097","doi":""},{"reference":"Röder L, Vola C, Kerridge S. 1992. The role of the teashirt gene in trunk segmental identity in Drosophila. Development 115(4): 1017-33.
","pubmedId":"1360402","doi":""},{"reference":"Schubiger M, Feng Y, Fambrough DM, Palka J. 1994. A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis. Neuron 12(2): 373-81.
","pubmedId":"8110464","doi":""},{"reference":"Soller M, Haussmann IU, Hollmann M, Choffat Y, White K, Kubli E, Schäfer MA. 2006. Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons. Curr Biol 16(18): 1771-82.
","pubmedId":"16979554","doi":""},{"reference":"Summerbell E. (2019) How to make scientific figures accessible to readers with color-blindness. American Society for Cell Biology.
","pubmedId":"","doi":""}],"title":"julius seizure acts within teashirt-expressing neurons to protect Drosophila melanogaster from bang-sensitivity
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