Date of Award


Degree Name

Doctor of Philosophy


Biological Sciences

First Advisor

Dr. Donald Kane

Second Advisor

Dr. Pamela Hoppe

Third Advisor

Dr. Wendy Beane

Fourth Advisor

Dr. Andre Venter


Zebrafish, Cyclin B1, G2 to M arrest, cell cycle, Cyclin B2


Regulation of cell division is essential for normal embryo development. The Cyclins and their Cyclin-dependent kinases are key regulators controlling this process. In this thesis, I examine the role of cyclin B1 and cyclin B2 in zebrafish development. It is thought that both Cyclins are necessary for a cell to progress past the G2/M checkpoint into mitosis. First, I show that zygotic Cyclin B1 is essential for normal cell cycle progression, but not for cells to enter mitosis. Lack of zygotic Cyclin B1 in the early arrest mutant specter, which carries a nonsense mutation in the cyclin B1 gene, did not stop cells from entering mitosis. However, for some cells, M phase was abnormal and elongated, sometimes resulting in chromosomal abnormalities, while for other cells M phase occurred normally.

Then, using CRISPR/Cas9 gene editing, I created a new mutation in cyclin B1 that failed to complement the specter null allele, confirming that the specter mutant phenotype is the result of disrupted Cyclin B1 function. The new mutation is a recessive gain-of-function mutation that has a more severe phenotype. This mutation produces an alternative splice variant of cyclin B1 mRNA, that does not undergo non-sense mediated decay and could produce an altered Cyclin B1 protein lacking several key components. Analysis of this CRISPR mutant, reveals that it has a different effect on cell cycle progression once zygotic control takes over, as all cells pause at the G2/M checkpoint and cease to divide, eventually undergoing apoptosis. I hypothesize that lack of Cyclin B1 in the null allele, allows another protein complex to form and sustain cell cycle progression, whereas the product of the gain-of-function allele somehow prevents this alternative mechanism from occurring.

To test this hypothesis, we used CRISPR/Cas9 mediated gene editing to target cyclin B2. Using the Dual FUCCI transgene expression and antibody staining, I show that one of two putative cyclin B2 mutations, has a very severe morphological phenotype where many cells are in G2/M phase, but do not die. The other putative cyclin B2 mutant produced in the specter background, has a less severe morphological phenotype. Cells progress through G2/M phase, but mitotic cells have severe chromosomal abnormalities, much like the specter CRISPR mutant, but form double nucleated cells, suggesting incomplete mitosis. My molecular and cellular analysis suggest that I have created two independent recessive mutants that completely remove cyclin B function.

In summary, using the specter and CRISPR specter mutants, I show that there might be an alternative mechanism of cell cycle progression in zebrafish when Cyclin B1 is absent. By attempting to knockout cyclin B2 and analyzing the new mutations in a wild-type and specter mutant backgrounds, I show that Cyclin B proteins might have redundant functions in progression through the G2/M checkpoint during early zebrafish development.

Access Setting

Dissertation-Open Access