Chia-Teng Chang, Toru Kawanishi, Sandy Nandagopal, Sean G Megason, Tony Y-C Tsai
{"title":"A Knock-in Zebrafish Reporter Line for Live Visualization of Endogenous Olig2 Protein Dynamics.","authors":"Chia-Teng Chang, Toru Kawanishi, Sandy Nandagopal, Sean G Megason, Tony Y-C Tsai","doi":"10.1177/15458547251376166","DOIUrl":null,"url":null,"abstract":"<p><p>The transcription factor oligodendrocyte transcription factor 2 (Olig2) plays a central role in specifying motor neurons and oligodendrocytes during vertebrate neural development. While transgenic reporter lines such as <i>TgBAC(olig2:EGFP)</i> have been instrumental in visualizing <i>olig2</i> expression, they fall short in directly reporting endogenous protein levels and may not fully recapitulate native gene regulation. To address these limitations, we generated a <i>TgKI(olig2-mNeonGreen)</i> zebrafish line using CRISPR/Cas9-mediated knock-in at the endogenous <i>olig2</i> locus. The resulting Olig2-mNeonGreen fusion protein localizes specifically to the nucleus, enabling direct live imaging and accurate quantification of Olig2-expressing cells. We confirmed that the knock-in preserves endogenous mRNA expression and protein function, and that homozygous fish develop normally. As proof of concept, modulation of Sonic Hedgehog signaling altered Olig2-mNeonGreen+ cell numbers as expected, confirming the reporter's responsiveness to known upstream inputs. This <i>TgKI(olig2-mNeonGreen)</i> line offers a robust tool for studying neural progenitor dynamics <i>in vivo</i>.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"182-188"},"PeriodicalIF":1.2000,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Zebrafish","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1177/15458547251376166","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/9/24 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
The transcription factor oligodendrocyte transcription factor 2 (Olig2) plays a central role in specifying motor neurons and oligodendrocytes during vertebrate neural development. While transgenic reporter lines such as TgBAC(olig2:EGFP) have been instrumental in visualizing olig2 expression, they fall short in directly reporting endogenous protein levels and may not fully recapitulate native gene regulation. To address these limitations, we generated a TgKI(olig2-mNeonGreen) zebrafish line using CRISPR/Cas9-mediated knock-in at the endogenous olig2 locus. The resulting Olig2-mNeonGreen fusion protein localizes specifically to the nucleus, enabling direct live imaging and accurate quantification of Olig2-expressing cells. We confirmed that the knock-in preserves endogenous mRNA expression and protein function, and that homozygous fish develop normally. As proof of concept, modulation of Sonic Hedgehog signaling altered Olig2-mNeonGreen+ cell numbers as expected, confirming the reporter's responsiveness to known upstream inputs. This TgKI(olig2-mNeonGreen) line offers a robust tool for studying neural progenitor dynamics in vivo.