Robo2 regulates synaptic oxytocin content by affecting actin dynamics
Abstract
The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of robo2 or slit3 displayed decreased synaptic OXT content and robo2 mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses.Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we showed that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.
Data availability
All source data files and codes were uploaded. Source data as summary tables is provided for all graphs and plots shown (Fig. 1E; 2B-E and G-J, 3E-H and J,K,M,N, 4D-F, 5O,R, 6I, 7C,D). R codes used for data import and analysis is provided.
Article and author information
Author details
Funding
Israel Science Foundation (1511/16)
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
Israel Science Foundation (2137/16)
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
Minerva Foundation ((Minerva Stiftung))
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
United States-Israel Binational Science Foundation (2017325)
- Michael Gliksberg
- Gil Levkowitz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Experiments involving zebrafish were approved by the Weizmann Institute'sInstitutional Animal Care and Use Committee (protocol #27220516)
Copyright
© 2019, Anbalagan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Cell Biology
- Developmental Biology
Mechanical forces play a critical role in tendon development and function, influencing cell behavior through mechanotransduction signaling pathways and subsequent extracellular matrix (ECM) remodeling. Here, we investigate the molecular mechanisms by which tenocytes in developing zebrafish embryos respond to muscle contraction forces during the onset of swimming and cranial muscle activity. Using genome-wide bulk RNA sequencing of FAC-sorted tenocytes we identify novel tenocyte markers and genes involved in tendon mechanotransduction. Embryonic tendons show dramatic changes in expression of matrix remodeling associated 5b (mxra5b), matrilin 1 (matn1), and the transcription factor kruppel-like factor 2a (klf2a), as muscles start to contract. Using embryos paralyzed either by loss of muscle contractility or neuromuscular stimulation we confirm that muscle contractile forces influence the spatial and temporal expression patterns of all three genes. Quantification of these gene expression changes across tenocytes at multiple tendon entheses and myotendinous junctions reveals that their responses depend on force intensity, duration, and tissue stiffness. These force-dependent feedback mechanisms in tendons, particularly in the ECM, have important implications for improved treatments of tendon injuries and atrophy.