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This has severely hampered the identification of pro-regenerative pathways. Here, we take advantage of the regenerative capacity of larval zebrafish to identify pathways that promote ON regeneration. From a small molecule screen, we identified modulators of serotonin (5-HT) signaling that inhibit ON regeneration. We find that several serotonin type-1 (5-HT1) receptor genes are expressed in retinal ganglion cells during regeneration and that inhibiting 5-HT1 receptors or components of the 5-HT pathway selectively impedes ON regeneration. We show that 5-HT1 receptor signaling is dispensable during ON development yet is required for regenerating axons to emerge from the injury site. Blocking 5-HT receptors once ON axons have crossed the chiasm does not inhibit regeneration, suggesting a selective role for 5-HT receptor signaling early during ON regeneration. Finally, we show that agonist-mediated activation of 5-HT1 receptors leads to enhanced and ectopic axonal regrowth. Combined, our results provide evidence for mechanisms through which serotonin-dependent neuromodulation directs ON regeneration in vivo.<\/jats:p>","DOI":"10.1242\/dev.204334","type":"journal-article","created":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T07:04:32Z","timestamp":1750057472000},"source":"Crossref","is-referenced-by-count":3,"title":["Serotonin neuromodulation directs optic nerve regeneration"],"prefix":"10.1242","volume":"152","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8922-2683","authenticated-orcid":false,"given":"Kristian","family":"Saied-Santiago","sequence":"first","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania Department of Cell and Developmental Biology , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa","family":"Baxter","sequence":"additional","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania Department of Cell and Developmental Biology , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9405-9772","authenticated-orcid":false,"given":"Jaffna","family":"Mathiaparanam","sequence":"additional","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania Department of Cell and Developmental Biology , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3878-9468","authenticated-orcid":false,"given":"Michael","family":"Granato","sequence":"additional","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania Department of Cell and Developmental Biology , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2025,7,7]]},"reference":[{"key":"2026090107125152800_DEV204334C1","doi-asserted-by":"publisher","first-page":"1083","DOI":"10.1016\/S0028-3908(99)00010-6","article-title":"A review of central 5-HT receptors and their function","volume":"38","author":"Barnes","year":"1999","journal-title":"Neuropharmacology"},{"key":"2026090107125152800_DEV204334C2","doi-asserted-by":"publisher","first-page":"924","DOI":"10.1016\/j.celrep.2015.09.050","article-title":"Serotonin promotes development and regeneration of spinal motor neurons in zebrafish","volume":"13","author":"Barreiro-Iglesias","year":"2015","journal-title":"Cell Rep."},{"key":"2026090107125152800_DEV204334C3","doi-asserted-by":"publisher","first-page":"2793","DOI":"10.1002\/jnr.21121","article-title":"Growth and pathfinding of regenerating axons in the optic projection of adult fish","volume":"85","author":"Becker","year":"2007","journal-title":"J. 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