Cell division is controlled by genes that regulate the cell cycle. Here we show that the zebrafish mutant specter (spr) is a mutation in cyclin B1, a gene necessary for the G2 to M transition of the cell cycle. The spr mutant phenotype becomes visible by the 7-somite stage as subtle differences in the head-to-tail ratio. Initial studies showed that the spr mutant arrests with a body shape at roughly midsegmentation and eventually exhibits signs of massive cell death in the nervous system. Investigation of the nervous system reveals that notch1b and deltaA are more weakly expressed in neural stem cells that give rise to fewer, but bigger neuronal precursors. Likewise blood markers reveal mutant embryos also have fewer but bigger blood cells.
The spr mutation was mapped to the zebrafish cyclin B1 gene. Sequencing showed a transition (C139T) that causes a nonsense mutation in exon 2 of the cyclin B1 gene. In situ analysis revealed that cyclin B1 mRNA is absent in the mutant embryo by the end of gastrulation suggesting that spr is a loss of cyclin B1 function. Phospho-histone-3 antibody staining showed that the cell cycle is abnormal in the mutant embryo. The expression of the Dual FUCCI transgene in live spr mutant embryos confirmed that cells are delayed in the S/G2/early M phase of the cell cycle. We confirmed that spr is a mutation in cyclin B1 using CRISPR/Cas9 mediated germline mutagenesis. This produced a mutation that failed to complement the original mutant allele confirming that the absence of CyclinB1 causes the mutant phenotype. Interestingly, the CRISPR cyclin B1 mutation expresses morphological changes that are more severe and can be detected earlier. Phospho-histone-3 antibody staining and the expression of the Dual FUCCI transgene in the live CRISPR spr mutant embryos suggested that the cell cycle abnormalities are more severe: very few cells enter mitosis, and a majority of the cells remains in the S and G2 phase of the cell cycle. In situ hybridization of cyclin B1 revealed that mRNA transcripts are still present in the CRISPR mutant and reverse transcriptase/PCR showed variable mRNA sizes. These results suggest that the new mutation is possibly a splicing variant that causes a gain-of function mutation, although we have yet to identify an obvious dominant phenotype.
We conclude that the spr mutant phenotype is caused by the mutation in the cell cycle gene cyclin B1, an essential regulator of the cell cycle progression from the G2 to M phase of the cell cycle. This leads to mitotic abnormalities, such as delayed cell cycle progression, developmental arrest, and activation of apoptotic pathways.