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fruitless tunes functional flexibility of courtship circuitry during development

  1. Jie Chen
  2. Sihui Jin
  3. Dandan Chen
  4. Jie Cao
  5. Xiaoxiao Ji
  6. Qionglin Peng  Is a corresponding author
  7. Yufeng Pan  Is a corresponding author
  1. The Key Laboratory of Developmental Genes and Human Disease, School of Life Science and Technology, Southeast University, China
  2. Co-innovation Center of Neuroregeneration, Nantong University, China
Short Report
Cite this article as: eLife 2021;10:e59224 doi: 10.7554/eLife.59224
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
fruM is required during pupation for female-directed courtship in adult males.

(A) Knocking down fruM using RNAi throughout development and adulthood eliminated male courtship toward virgin females. n = 24 for each. ***p<0.001, unpaired t-test. (B) A schematic of genetic strategy to knockdown fruM at different developmental stages for 2 days. Stages 1–9 refer to specific developmental stages from embryos to newly eclosed adults with interval of 2 days. (C and D) Courtship indices of males with fruM knocked down at specific developmental stages as indicated above toward virgin females. Males with fruM knocked down at stage 5 for 2 days (a period of pupation from stage 5 to 6, see above picture) rarely courted virgin females (C), and none successfully mated (D). Knocking down fruM at stages near 5 (e.g., stage 4 or 6) also partially impairs courtship and mating success. Knocking down fruM at earlier or later stages has no obvious effect on courtship and mating. n = 24 for each. Error bars indicate SEM. (E–H) Two day heat shock at 30°C effectively knocks down fruM expression during development. Anti-V5 and anti-FruM signals are dramatically decreased after heat shock at stage 5 (E and F) or 7 (G and H) in tub-GAL80ts/UAS-fruMi; fruGAL4/fruV5 males. Scale bars, 100 μm. Representative of five samples each.

Figure 1—figure supplement 1
fruM microRNA efficiency and fruM expression patterns across development.

(A) The structure of the fruitless gene and designed primers against P1 and P4 transcripts. (B and C) The fruM microRNA significantly reduced fruM mRNA (B), but not common fru mRNA from the P4 promotor (C). n = 4 for each, ***p<0.001, n.s., not significant, Mann–Whitney U test. Error bars indicate SEM. (D–K) Expression pattern of fruGAL4 in different developmental stages. Scale bars, 50 μm. Representative of three samples each.

Figure 1—figure supplement 2
Validation of anti-FruM antibody and the fruV5.

Knock-in line. (A–D) The anti-FruM antibody shows intensive male-specific signals (A), and several pairs of false-positive neurons in females (B) as well as in fruM mutants (C and D). (E and F) Anti-V5 signals indicate male-specific FruM expression in fruV5/+ males. Scale bars, 100 μm. Representative of five samples each.

Figure 1—figure supplement 3
Adult fruM expression after 2 day induction of fruM microRNA during development.

(A–J) tub-GAL80ts/UAS-fruMi; fruGAL4/fruV5 males were heat shocked at 30°C for 2 days at each developmental stage (stages 1–9) and dissected at adulthood to check FruM expression using anti-V5 (left) and anti-FruM (middle) antibodies. Note that the anti-V5 signals were relatively stronger than the anti-FruM signals. Adult fruM expression was effectively restored if heat shocked at earlier stages (stages 1–5), but only partially restored if heat shocked at later stages (stages 6–9). Scale bars, 100 μm. Representative of five samples each.

Figure 1—figure supplement 4
fruM is required during a specific developmental period for regular neuronal development.

(A and B) GFP expression driven by fruGAL4 under regular condition (A) and restricted condition using GAL80ts at 18°C (B). Note that GAL80ts suppresses the majority but not 100% of fruGAL4 expression. (C) Labeling and morphological changes of the GRNs innervating VNC in males with fruM knocked down during specific developmental periods. The fruM-positive GRNs were only labeled if tub-GAL80ts/UAS-mCD8GFP; fruGAL4/UAS-fruMi males were heat shocked after stage 4 (yellow arrows). For those males heat shocked after stage 4, white arrows indicate whether there is midline crossing in these GRNs innervating VNC. (D) Percentage of males heat shocked at different developmental stages showed defect of midline crossing of the GRNs. Knocking down fruM at stages 5 and 6, but not later stages including adulthood, results in defect of midline crossing of the GRNs. Genotypes as indicated. Scale bars, 100 μm. Representative of five samples each.

fruM functions during adulthood to inhibit male–male courtship behaviors.

(A–C) Courtship behaviors performed by males that express traF or fruMi specifically during adulthood for 4 days. For male–female courtship (A), n = 17, 26, 23, 23, 24, 27, 24, and 28, respectively (from left to right), n.s., not significant, unpaired t-test. For single-pair male–male courtship (B), n = 18 for each. n.s., not significant, *p<0.05, unpaired t-test. For male chaining among eight males as a group (C), n = 8, 8, 8, 10, 8, 18, 8, and 18, respectively (from left to right). n.s., not significant, *p<0.05, **p<0.01, ***p<0.001, Mann–Whitney U test. Error bars indicate SEM. Genotypes as indicated. (D and E) Anti-V5 and anti-FruM signals are dramatically decreased after heat shock during adulthood for 4 days in tub-GAL80ts/UAS-fruMi; fruGAL4/fruV5 males. Scale bars, 100 μm. Representative of five samples each.

Figure 3 with 4 supplements
fruM tunes functional flexibility of the fruM circuitry.

(A and B) Wild-type males courted intensively toward virgin females (A, left bar), but rarely courted males (A, right bar) or displayed chaining behavior in groups of eight males (B). n = 24, 24, 8, respectively. ***p<0.001, unpaired t-test. (C) FruLexA/fru4-40 (fruM null) males rarely courted either females or males. n = 24 for each, **p<0.01, Mann–Whitney U test. (D) FruLexA/fru4-40 males did not show chaining behavior after 3 hr group-housing, but developed intensive chaining behavior after1-3 days. n = 8. (E) A summary of courtship acquisition independent of fruM. (F) RNAi against fruM efficiently decreased but not fully eliminated fruM expression. n = 4. ***p<0.001, Mann–Whitney U test. (G) Knocking down fruM in all fruGAL4 neurons generated males that have reversed sexual orientation such that they rarely courted females but intensively courted males. n = 24 and 19, respectively. ***p<0.001, unpaired t-test. (H) Males with fruM knocked down in all fruGAL4 neurons showed intensive chaining behavior at all time points (from 3 hr to 3 days upon group-housing). n = 7. (I) Distinct roles of low fruM (RNAi) and high fruM (wild-type) in regulating male–male and male–female courtship. (J) Males with fruM knocked down in fruGAL4 neurons in the brain had a lower level of courtship toward females, but their sexual orientation was not changed. n = 24 and 23, respectively. ***p<0.001, unpaired t-test. (K) Males with fruM knocked down in fruGAL4 neurons in brain showed low male chaining behavior initially but increasing levels of chaining behavior over 1–3 days. n = 6. (L) A summary of the role of fruM in brain in promoting male–female courtship and suppressing the experience-dependent acquisition or progression of male chaining behavior. (M) Males with fruM knocked down in fruGAL4 neurons outside brain generated bisexual males that have intensive male–female and male–male courtship. n = 24 for each. n.s., not significant, unpaired t-test. (N) Males with fruM knocked down in fruGAL4 neurons outside brain showed high male chaining behavior initially, but decreased levels of chaining behavior over 1–3 days. n = 8. (O) A summary of the role of fruM outside brain in suppressing male–male courtship behavior. Error bars indicate SEM.

Figure 3—figure supplement 1
Comparison of male courtship with or without food.

Male–male courtship in fruM knocked down males is higher in the presence of food. n = 15, 17, 15, 18, 18, 18, 18, and 18 from left to right, respectively. ***p<0.001, unpaired t-test. Error bars indicate SEM.

Figure 3—figure supplement 2
Courtship behaviors in fruM null males.

(A and B) Control fru4-40/+ males courted intensively toward virgin females (A, left bar) but rarely courted males (A, right bar), and hardly displayed male chaining behavior in groups of eight males during a 3 day test (B). n = 12, 12, and 8, respectively. ***p<0.001, Mann–Whitney U test. (C–F) fru4-40/ fruSat15 (C and D) and fru4-40/ fruAJ96u3 (E and F) males rarely courted either females or males but developed intensive male chaining behavior after 1–3 days. n = 12, 12, 8, 12, 12, and 8, respectively. ***p<0.001, Mann–Whitney U test. Error bars indicate SEM.

Figure 3—figure supplement 3
Dividing fruM expression into two complementary parts.

(A) Expression pattern of the fruM circuitry revealed by fruGAL4 driving UAS-mCD8GFP (green), co-stained with nc82 (magenta). (B and C) Restricted fruGAL4 expression in brain (B) or outside brain (C) using a genetic strategy to divide fruGAL4 neurons into two parts. (D and E) Expression pattern of fruGAL4 driving UAS-mCD8GFP in forelegs (D, scale bars, 100 mm) and antennae (E, scale bars, 50 mm) in males with indicated genotypes. (F–I) FruM expression in spatially restricted knockdown of fruM revealed by anti-V5 signals. Genotypes as indicated. Scale bars, 100 μm. Representative of three samples each.

Figure 3—figure supplement 4
The role of fruM in P1 and ppk23-expressing neurons for male courtship.

(A) Knocking down fruM in P1 neurons did not affect male courtship behavior toward either females or males. n = 18 of each. n.s., not significant, Mann–Whitney U test. (B) Knocking down fruM in P1 neurons did not significantly affect male–male chaining behavior. n = 8 for each. ***p<0.001, n.s., not significant, Mann–Whitney U test. (C) Knocking down fruM in ppk23-expressing neurons did not affect male courtship toward females, but mildly increased male–male courtship. n = 18 of each. n.s., not significant, ***p<0.001, Mann–Whitney U test. (D) Knocking down fruM in ppk23-expressing neurons did not significantly affect male–male chaining behavior. n = 8 for each. *p<0.05, n.s., not significant, Mann–Whitney U test. Error bars indicate SEM.

A summary of fruM function in male courtship.

(A) FruM is required during pupation for neuronal development and possibly circuit wiring that builds the potential for innately female-directed courtship, while its function during adulthood is involved in inhibiting male–male courtship. Anti-V5 signals indicate FruM expression in larva, pupa and adult males (from left to right). Scale bars, 100 μm. (B) The sex circuitry without fruM or with different levels/patterns of fruM has different properties such that males would have experience-dependent courtship acquisition, or innate courtship but with different sexual orientation (heterosexual, homosexual, or bisexual). Such flexibility of the sex circuitry is tuned by different fruM expression. Triangles and circles represent corresponding fruM levels and courtship levels (triangles: male–female courtship; circles: male–male courtship). Gray indicates systemic low level of fruM; green and magenta indicate spatially low level of fruM.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
 AntibodyMouse monoclonal anti-Bruchpilot antibody (nc82)Developmental Studies Hybridoma BankCat# nc82, RRID:AB_2314866IHC (1:50)
 AntibodyRabbit polyclonal anti-GFPThermo Fisher ScientificCat# A-11122, RRID:AB_221569IHC (1:1000)
 AntibodyDonkey polyclonal anti-Rabbit, Alexa Fluor 488Thermo Fisher ScientificCat# A-21206, RRID:AB_2535792IHC (1:500)
 AntibodyDonkey polyclonal anti-Mouse, Alexa Fluor 555Thermo Fisher ScientificCat# A-31570, RRID:AB_2536180IHC (1:500)
 AntibodyMouse monoclonal anti-V5-Tag:DyLight550Bio-RadCat# MCA1360D550GA, RRID:AB_2687576IHC (1:500)
 AntibodyRabbit polyclonal anti-FruMThis studyN/AIHC (1:200)
 PlasmidpCFD4Addgene# 49411
 PlasmidpHD-DsRedAddgene# 51434
 PlasmidpET-28aSigma–Aldrich# 69864
 Chemical compound, drugNormal Goat Serum (NGS)Jackson ImmunoResearch LaboratoriesCode# 005-000-121
RRID:AB_2336990
Chemical compound, drugParaformaldehyde (PFA)Sigma–AldrichCAS# 30525-89-44% PFA in 1× PBS
Genetic reagent
(D. melanogaster)
fruV5This studyN/ADescribed below
Genetic reagent
(D. melanogaster)
UAS-mCD8GFP; fruGAL4Stockinger et al., 2005N/A
Genetic reagent
(D. melanogaster)
UAS-fruMiMeissner et al., 2016N/A
Genetic reagent
(D. melanogaster)
UAS-fruMiScrMeissner et al., 2016N/A
Genetic reagent
(D. melanogaster)
fruLexAMellert et al., 2010N/A
Genetic reagent
(D. melanogaster)
fru4-40Pan and Baker, 2014N/A
Genetic reagent
(D. melanogaster)
fruSat15Pan and Baker, 2014N/A
Genetic reagent
(D. melanogaster)
fruAJ96uPan and Baker, 2014N/A
Genetic reagent
(D. melanogaster)
ppk23-GAL4Thistle et al., 2012N/A
Genetic reagent
(D. melanogaster)
Otd-FlpAsahina et al., 2014N/A
Genetic reagent
(D. melanogaster)
tub-GAL80tsBloomington Drosophila Stock CenterBDSC_7019
Genetic reagent
(D. melanogaster)
tub>GAL80>Bloomington Drosophila Stock CenterBDSC_38881
Genetic reagent
(D. melanogaster)
tub>stop>GAL80Bloomington Drosophila Stock CenterBDSC_39213
Genetic reagent
(D. melanogaster)
UAS-traFBloomington Drosophila Stock CenterBDSC_4590
Genetic reagent
(D. melanogaster)
R15A01-ADBloomington Drosophila Stock CenterBDSC_68837
Genetic reagent
(D. melanogaster)
R71G01-DBDBloomington Drosophila Stock CenterBDSC_69507
Software, algorithmImageJNational Institutes of Healthhttps://imagej.nih.gov/ij/
Software, algorithmPrism 8GraphPadhttps://www.graphpad.com/

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, Figure 3-figure supplement 1, 2 and 4.

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