An automated high-resolution in vivo screen in zebrafish to identify chemical regulators of myelination

Abstract

Myelinating oligodendrocytes are essential for central nervous system (CNS) formation and function. Their disruption is implicated in numerous neurodevelopmental, neuropsychiatric and neurodegenerative disorders. However, recent studies have indicated that oligodendrocytes may be tractable for treatment of disease. In recent years, zebrafish have become well established for the study of myelinating oligodendrocyte biology and drug discovery in vivo. Here, by automating the delivery of zebrafish larvae to a spinning disk confocal microscope, we were able to automate high-resolution imaging of myelinating oligodendrocytes in vivo. From there, we developed an image analysis pipeline that facilitated a screen of compounds with epigenetic and post-translational targets for their effects on regulating myelinating oligodendrocyte number. This screen identified novel compounds that strongly promote myelinating oligodendrocyte formation in vivo. Our imaging platform and analysis pipeline is flexible and can be employed for high-resolution imaging-based screens of broad interest using zebrafish.

A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination

“…This early promoter activation results in a faint but persistent expression of EGFP in nonoligodendrocyte cells during our treatment timeframes, likely due to the prolonged stability of EGFP protein (Progatzky, Dallman, & Lo Celso, 2013). Early et al (2018) have also recently reported a fluorescencebased myelination screening model in zebrafish using mbp regulatory sequences, which is expressed only in CNS and PNS myelinating glia and does not appear to be transiently activated in neural progenitor cells like the mpz promoter described here. Given the distinct localization and morphology of myelinating oligodendrocytes, it is easy to identify any false positive hits due to non-specific expression in other cells.…”

“…Therefore, we believe our mpz:mEGFP reporter is useful for identifying drugs that enhance CNS myelination specifically. Early et al (2018) have also recently reported a fluorescencebased myelination screening model in zebrafish using mbp regulatory sequences, which is expressed only in CNS and PNS myelinating glia and does not appear to be transiently activated in neural progenitor cells like the mpz promoter described here. While there was robust expression of our mbp reporters following injection during mosaiclabeling experiments (unpublished observations), unfortunately, the transgenic reporter lines we generated using this mbp promoter were quite dim compared to our mpz reporter.…”

“…In the future, efficient screening of 1000's of compounds for large-scale screens will require specialized equipment for automated larvae handling and imaging scripts such as developed by the Lyons lab (Early et al, 2018). In the future, efficient screening of 1000's of compounds for large-scale screens will require specialized equipment for automated larvae handling and imaging scripts such as developed by the Lyons lab (Early et al, 2018).…”

“…The scale of our screen was also limited to a small number of compounds due to temporal limitations of manual handling of larvae and labor intensive confocal imaging. In the future, efficient screening of 1000's of compounds for large-scale screens will require specialized equipment for automated larvae handling and imaging scripts such as developed by the Lyons lab (Early et al, 2018). It is also interesting to note that one itineration of the Early et al (2018) screen used a cytoplasmic marker for labeling oligodendrocytes to identify changes specifically in oligodendrocyte cell number as a parameter for identifying candidate molecules (Early et al, 2018).…”

“…In the future, efficient screening of 1000's of compounds for large-scale screens will require specialized equipment for automated larvae handling and imaging scripts such as developed by the Lyons lab (Early et al, 2018). It is also interesting to note that one itineration of the Early et al (2018) screen used a cytoplasmic marker for labeling oligodendrocytes to identify changes specifically in oligodendrocyte cell number as a parameter for identifying candidate molecules (Early et al, 2018). In contrast, our membranetethered reporter measures not only promoter activity but also membrane production, which may be independent of changes in oligodendrocyte cell numbers, for example, such as following GC1 or rapamycin treatment where the total number of mbp + oligodendrocytes was unchanged.…”