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Sequential and independent probabilistic events regulate differential axon targeting during development in Drosophila melanogaster

Nature Neuroscience volume 28, pages 998–1011 (2025)Cite this article

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Abstract

Variation in brain wiring contributes to non-heritable behavioral individuality. How and when these individualized wiring patterns emerge and stabilize during development remains unexplored. In this study, we investigated the axon targeting dynamics of Drosophila visual projecting neurons called DCNs/LC14s, using four-dimensional live-imaging, mathematical modeling and experimental validation. We found that alternative axon targeting choices are driven by a sequence of two independent genetically encoded stochastic processes. Early Notch lateral inhibition segregates DCNs into NotchON proximally targeting axons and NotchOFF axons that adopt a bi-potential transitory state. Subsequently, probabilistic accumulation of stable microtubules in a fraction of NotchOFF axons leads to distal target innervation, whereas the rest retract to adopt a NotchON target choice. The sequential wiring decisions result in the stochastic selection of different numbers of distally targeting axons in each individual. In summary, this work provides a conceptual and mechanistic framework for the emergence of individually variable, yet robust, circuit diagrams during development.

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Fig. 1: DCN targeting decision precedes distal target innervation.
Fig. 2: Single DCN axons amplify to form multi-filopodial structures that are necessary but not sufficient for medulla targeting.
Fig. 3: Notch signaling temporally restricts axonal amplification.
Fig. 4: An auto-inhibition winner-takes-all feedback model describes DCN axon filopodia dynamics.
Fig. 5: Microtubule growth selects future M-DCN axons.
Fig. 6: Tubulin acts as a limiting resource in the selection of future M-DCNs.
Fig. 7: Microtubule stabilization selects future M-DCNs during a specific developmental critical period.
Fig. 8: Model. A developmental succession of probabilistic steps creates individualized axonal wiring patterns.

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Data availability

Reagents and resources are listed in the Methods section of this paper and in Supplementary Table 1. The data required to support the findings of this study are available in the paper and in Supplementary Table 2. The mathematical model can be found in the ‘Mathematical methods’ section of the Methods.

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Acknowledgements

The authors thank the Bloomington Stock Center (NIH P40OD018537), the Vienna Drosophila Resource Center, the Developmental Studies Hybridoma Bank, E. A. Kravitz and A. Hasan for flies and reagents; B. Kunz, T. Flego, L. Hellbruegge and R. Cook for technical assistance; and H. Wolfenberg, D. Johannes and N. Demirbas for animal care and help with fly stock management. We thank C. Desplan, N. Renier and all members of the Hassan, Hiesinger and Wernet laboratories for support, insightful discussion and valuable comments. Light microscopy was carried out in the laboratory of P.R.H. at Freie Universität Berlin and in the ICM.Quant core facility of the Paris Brain Institute (ICM). We gratefully acknowledge C. Lovo for her advice on the use of Bruker multiphoton. This work was supported by the Einstein-BIH program (to B.A.H. and P.R.H.), Deutsche Forschungsgemeinschaft (DFG) Research Unit 5289 RobustCircuit project P2 (to B.A.H. and P.R.H.) and RobustCircuit project Z1 (to M.v.K. and P.R.H.); DFG Research Unit Syntophagy RP7 (Hi 1886/8); funding from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme (101019191) (to P.R.H.); the Investissements d’Avenir program (ANR-10-IAIHU-06); Paris Brain Institute-ICM core funding; the Paul G. Allen Frontiers Group Allen Distinguished Investigator grant; the Roger De Spoelberch Prize; an NIH Brain Initiative R01 grant (1R01NS121874-01) (to B.A.H.); the Fondation de la Recherche Medical (FRM) postdoctoral fellowship (ARF202005011913 to M.A.); the Swiss National Science Foundation (310030_185247) (to E.S.); DFG grant number 450430223 (to C.B. and M.v.K.); and the DFG under Germany´s Excellence Strategy–Berlin Mathematics Research Center MATH+ (EXC-2046/1, project ID: 390685689) (to M.v.K.). The funders had no role in the design of the study or the decision to publish.

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  1. Suchetana Bias Dutta

    Present address: Center for Regenerative Therapies Dresden, TU Dresden, Dresden, Germany

Authors and Affiliations

  1. Institut du Cerveau-Paris Brain Institute (ICM), Sorbonne Université, Inserm, CNRS, Hôpital Pitié-Salpêtrière, Paris, France

    Maheva Andriatsilavo, Tian Tao Griffin, Suchetana Bias Dutta & Bassem A. Hassan

  2. Division of Neurobiology of the Institute for Biology, Free University, Berlin, Germany

    Maheva Andriatsilavo, Suchetana Bias Dutta, P. Robin Hiesinger & Bassem A. Hassan

  3. Department of Mathematics and Computer Science, Free University, Berlin, Germany

    Carolina Barata, Eric Reifenstein & Max von Kleist

  4. Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

    Alexandre Dumoulin & Esther T. Stoeckli

  5. Neuroscience Center Zurich, University of Zurich, Zurich, Switzerland

    Alexandre Dumoulin & Esther T. Stoeckli

  6. Project Group 5 ‘Systems Medicine of Infectious Disease’, Robert Koch Institute, Berlin, Germany

    Max von Kleist

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Contributions

M.A., P.R.H. and B.A.H. conceived the project. M.A. designed, performed and analyzed all Drosophila experiments. T.T.G. helped with microscope scanning and quantifications. M.A. and S.B.D. established the ex vivo culture protocol for DCN live imaging. A.D. and E.S. conceived, designed, performed and analyzed all chicken experiments. C.B., E.R. and M.v.K. conceived, designed, performed and analyzed all the mathematical computational modeling work. M.A. and B.A.H. wrote the manuscript, and all authors provided critical feedback. M.A., M.v.K., E.S., P.R.H. and B.A.H. acquired funding.

Corresponding author

Correspondence to Bassem A. Hassan.

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Nature Neuroscience thanks Chihiro Hama and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary information (download PDF )

Supplementary Figs. 1–9 and captions, captions for Supplementary Movies 1–19 and Supplementary Mathematical Model.

Reporting Summary (download PDF )

Supplementary Table 1. Reagents and resources. (download XLSX )

Supplementary Table 2. All quantifications used to generate the figures of this paper. (download XLSX )

Supplementary Movie 1 (download MP4 )

Supplementary Movie 1 - Intravital imaging of DCN developing axon. (Related to Fig. 1g). 21.5-h time-lapse recording sequence (one stack every 20 min), starting from 30 h APF. White arrowhead: stabilized axonal structure. Green: CD4.tdGFP. Scale bar, 20 µm.

Supplementary Movie 2 (download MP4 )

Supplementary Movie 2 - Intravital imaging of DCN developing axon in the Lo/LP chiasma. (Related to Fig. 2a) 22.33-h time-lapse recording sequence (one stack every 30 min), starting from 30 h APF. White arrowhead: stabilized axonal structure. Green: CD4.tdGFP. Scale bar, 10 µm.

Supplementary Movie 3 (download MP4 )

Supplementary Movie 3 - Intravital imaging—retracting DCN multi-filopodia axonal structure in the Lo/LP chiasma. (Related to Supplementary Fig. 1g). 22.33-h time-lapse recording sequence (one stack every 30 min), starting from 30 h APF. White arrowhead: stabilized axonal structure. Green: CD4.tdGFP. Scale bar, 10 µm.

Supplementary Movie 4 (download MP4 )

Supplementary Movie 4 - Ex vivo live imaging of DCN axon clone extending toward the chiasma with axonal amplification. (Related to Fig. 2h). 14.33-h time-lapse recording sequence (one stack every 20 min), starting from 29 h APF. Green: CD4.tdGFP clone. Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 5 (download MP4 )

Supplementary Movie 5 - Ex vivo live imaging of DCN axon clone exploring the lobula. (Related to Fig. 2i). 10-h time-lapse recording sequence (one stack every 6 min, 44 s), starting from 29 h APF. Green: CD4.tdGFP clone. Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 6 (download MP4 )

Supplementary Movie 6 - Intravital imaging of DCN developing axon in the chiasma upon a Notch signaling pathway overactivation (NotchAct). (Related to Fig. 3k). 19-h time-lapse recording sequence (one stack every 15 min), starting from 26 h APF. White arrowhead: single filopodium. Green: CD4.tdGFP. Scale bar, 10 µm.

Supplementary Movie 7 (download MP4 )

Supplementary Movie 7 - Ex vivo live imaging of tubulin dynamics in developing DCN axon in the chiasma. (Related to Fig. 5d). 9-h, 29-min time-lapse recording sequence (one stack every 20 min), starting from 30 h APF. Green: tubulin–GFP. Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 8 (download MP4 )

Supplementary Movie 8 - Ex vivo live imaging of tubulin dynamics in developing DCN axon in the chiasma. (Related to Supplementary Fig. 5b). 9-h, 29-min time-lapse recording sequence (one stack every 20 min), starting from 30 h APF. Green: tubulin–GFP. Magenta: CD4.tdTomato. High tubulin level (white arrow); low tubulin level (black arrow). Scale bar, 20 µm.

Supplementary Movie 9 (download MP4 )

Supplementary Movie 9 - Ex vivo live imaging of developing DCN axon in the chiasma expressing a cytoplasmic GFP. (Related to Supplementary Fig. 4a). 11-h time-lapse recording sequence (one stack every 10 min), starting from 29 h APF. Green: cytoplasmic GFP (eGFP). Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 10 (download MP4 )

Supplementary Movie 10 - Ex vivo live imaging of developing DCN axon in the chiasma expressing a cytoplasmic GFP. (Related to Supplementary Fig. 4b). 9-h, 10-min time-lapse recording sequence (one stack every 10 min), starting from 30 h APF. Green: cytoplasmic GFP (eGFP). Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 11 (download MP4 )

Supplementary Movie 11 - Ex vivo live imaging of developing DCN axon in the chiasma expressing a cytoplasmic GFP. (Related to Supplementary Fig. 4c). 4-h, 10-min time-lapse recording sequence (one stack every 10 min), starting from 30 h APF. Green: cytoplasmic GFP (eGFP). Magenta: CD4.tdTomato. Scale bar, 10 µm.

Supplementary Movie 12 (download MP4 )

Supplementary Movie 12 - Ex vivo live imaging of unstable multi-filopodial structure from the chiasma to the lobula. (Related to Supplementary Fig. 5a). 9-h, 29-min time-lapse recording sequence (one stack every 20 min), starting from 30 h APF. Green: tubulin–GFP. Magenta: CD4.tdTomato. Scale bar, 20 µm.

Supplementary Movie 13 (download MP4 )

Supplementary Movie 13 - Live imaging of dI1 axon growth cone at the FP border in chicken embryo spinal cord. 300-min time-lapse recording sequence (one stack every 5 min). Magenta arrowhead: ipsilateral turning axon terminal. Black arrows: retracting branch. Green arrowhead: axon terminal crossing the FP. Scale bar, 10 µm.

Supplementary Movie 14 (download MP4 )

Supplementary Movie 14 - Live imaging of dI1 axon growth cone transiently splitting during midline crossing in chicken embryo spinal cord. 145-min time-lapse recording sequence (one stack every 5 min). Black arrows: the consecutive transient splitting events. White arrowhead: the stabilized branch. Black arrowheads: the retracting branch. Scale bar, 10 µm.

Supplementary Movie 15 (download MP4 )

Supplementary Movie 15 - Live imaging of tubulin dynamics in dI1 axon growth cone transient splitting during midline crossing. (Related to Fig. 5q). 240-min time-lapse recording sequence (one stack every 10 min). White arrows: transient growth cone. Black arrowheads: retracting branch. White arrowheads: stabilized branch. Green: tubulin–GFP. Magenta: tdTomato-F. The lower panel shows a heat map visualization of the tubulin–GFP signal. The white line shows the edge of the growing axon. Hi, high; Lo, low. Scale bar, 10 µm.

Supplementary Movie 16 (download MP4 )

Supplementary Movie 16 - Ex vivo live imaging of EB1 dynamics in developing DCN axon in the chiasma. (Related to Fig. 6m). 8-h time-lapse recording sequence (one stack every 2 min), starting from 29 h APF. Green: EB1–GFP. Scale bar, 5 µm. Right panels exhibit kymographs of EB1–GFP dynamics with the highlighted filopodium (pink).

Supplementary Movie 17 (download MP4 )

Supplementary Movie 17 - Ex vivo live imaging of EB1 dynamics in developing DCN axon in the chiasma. 4-h time-lapse recording sequence (one stack every 2 min), starting from 33 h APF. Green: EB1–GFP. Scale bar, 5 µm. Right panels exhibit kymographs of EB1–GFP dynamics with the highlighted filopodium (pink).

Supplementary Movie 18 (download MP4 )

Supplementary Movie 18 - Ex vivo live imaging of tubulin dynamics in developing DCN axon in the chiasma—DMSO treatment from 30 h APF. (Related to Supplementary Fig. 8b,d). 8-h time-lapse recording sequence (one stack every 20 min). Green: tubulin–GFP. Magenta: CD4.tdTomato. White arrow: stabilized tubulin-rich axons. Scale bar, 30 µm.

Supplementary Movie 19 (download MP4 )

Supplementary Movie 19 - Ex vivo live imaging of tubulin dynamics in developing DCN axon in the chiasma—Nocodazole treatment from 30 h APF. (Related to Supplementary Fig. 8c,e). 8-h time-lapse recording sequence (one stack every 20 min). Green: tubulin–GFP. Magenta: CD4.tdTomato. White arrow: Stabilized tubulin-rich axons. Black arrow: retracting axon. Scale bar, 30 µm.

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Andriatsilavo, M., Barata, C., Reifenstein, E. et al. Sequential and independent probabilistic events regulate differential axon targeting during development in Drosophila melanogaster. Nat Neurosci 28, 998–1011 (2025). https://doi.org/10.1038/s41593-025-01937-y

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