To further investigate oligodendrocyte differentiation, we performed quantitative real-time PCR to assay the relative expression levels of the CNS myelin genes myelin protein zero and 36K at 4 dpf. The reduction in dorsal OPCs and the previously proposed role of pes in cell proliferation raised the possibility that pes Barlerin function is necessary for the division of OPCs after they migrate out of the pMN domain. To assay the proliferation of OPCs in Napabucasin mutant larvae we exposed the larvae to the thymidine analogue BrdU, an S-phase marker, at various time-points during early development. However, this experiment revealed that mutant spinal cords had a five-fold increase in the total number of BrdU + cells, which were mostly adjacent to the central canal and medial septum. Additionally, the number of cells that were labeled by the M-phase marker phosphohistone H-3 was dramatically increased in mutants. Nevertheless, there was no significant difference in the total number of spinal cord cells between mutant and wild type. One possible explanation for an increase of cell cycle activity without an accompanying increase in cell number is that cell division is balanced by cell death. To investigate this possibility we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling assay to label dying cells. Mutant larvae had less than two-fold more TUNEL + cells than wild type, less than the five-fold increase of dividing cells. Therefore, elevated levels of cell death may not fully account for the lack of increase in total cell number despite the substantial increase in apparent dividing cells. An alternative explanation is that pes function is necessary for cell cycle progression. To test this idea, we investigated the cell cycle kinetics of pes null larvae. To determine the length of time that spinal cord cells spend in S/G2 phase, we performed a BrdU pulse-chase experiment in which larvae were labeled with BrdU and then fixed at various time-points followed by co-labeling with anti-BrdU and anti-PH3 antibodies.Our results indicate that whereas spinal cord cells of wild-type larvae progressed from labeling to M phase within roughly six hours, mutant larvae had cells that were still entering mitosis as long as 10 hours after the pulse, the longest interval we collected.