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

Area of Science:

• Neuroscience
• Developmental Biology
• Genetics

Background:

• Oligodendrocyte transcription factor 2 (Olig2) is crucial for motor neuron and oligodendrocyte development in vertebrates.
• Existing reporter lines like TgBAC(olig2:EGFP) have limitations in reflecting endogenous Olig2 protein levels and native gene regulation.

Purpose of the Study:

• To generate a more accurate reporter zebrafish line for visualizing endogenous Olig2 protein.
• To enable direct live imaging and quantification of Olig2-expressing cells in vivo.
• To provide a tool for studying neural progenitor dynamics.

Main Methods:

• CRISPR/Cas9-mediated knock-in of mNeonGreen fluorescent protein at the endogenous Olig2 locus in zebrafish.
• Generation of the TgKI(olig2-mNeonGreen) zebrafish line.
• Validation of reporter function through assessment of protein localization, mRNA expression, protein function, and response to Sonic Hedgehog signaling modulation.

Main Results:

• The TgKI(olig2-mNeonGreen) line successfully expresses a nuclear-localized Olig2-mNeonGreen fusion protein.
• Knock-in preserves endogenous Olig2 mRNA expression and protein function, with homozygous fish developing normally.
• The reporter line accurately reflects Olig2+ cell numbers in response to altered Sonic Hedgehog signaling, validating its responsiveness.

Conclusions:

• The TgKI(olig2-mNeonGreen) zebrafish line provides a robust and accurate tool for real-time monitoring of Olig2 expression.
• This reporter facilitates in vivo studies of neural progenitor cell dynamics and oligodendrocyte development.
• The line overcomes limitations of previous reporter systems, offering improved insights into vertebrate neural development.