Robo2 regulates synaptic oxytocin content by affecting actin dynamics
- Weizmann Institute of Science, Israel
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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Robo2 regulates synaptic oxytocin content by affecting actin dynamics
eLife 8:e45650.
https://doi.org/10.7554/eLife.45650
Further reading
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- Chromosomes and Gene Expression
- Developmental Biology
Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.
