A Knock-in Zebrafish Reporter Line for Live Visualization of Endogenous Olig2 Protein Dynamics

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>.