CDKN1A Deficiency in Zebrafish Promote Follicle Maturation and Degeneration Mediated by CRISPR/Cas9 Targeted Disruption
CDKN1A plays multiple roles in distinctive biological processes, such as cellular proliferation, apoptosis, DNA repairing, and etc. In mouse, Cdkn1a deficiency led to cell arrest at G1 phase, and also reported that Cdkna1-deficient mice were susceptible to tumorigenesis. However, the functions of Cdkn1a in zebrafish were still elusive due to few studies. Our previous study indicated that Cdkn1a might be closely associated with folliculogenesis, and to further investigate its specific role during folliculogenesis, Cdkn1a was targeted disrupted by CRISPR/Cas9. Interestingly, Cdkn1a-deficient zebrafish were embryonic lethality, resulted in approximate 2% homozygous mutants in adult stage. Furthermore, homozygous mutants have accelerated follicle maturation that faster than WT, and were fertile at young age (2 month). However, the matured or large follicles in mutant soon become dysfunctional due to abnormal oocyte that led to infertile at 3 months. Gradually, the abnormal follicles were undertaken follicle degeneration, resulted in PG follicle enrichment in mutant ovary at 6 months. Similar follicle degeneration phenomenon was also observed in heterozygous mutant ovary at 8 months. The degenerating follicles in mutant were convinced by in situ apoptosis detection kit. To further explore molecular mechanism, RNA-seq and qPCR validation were adopted to identify underlying molecular pathway that involved in Cdkn1a deficient apoptosis. This study firstly reported the functions of Cdkn1a in zebrafish, particular in its critical role in maintenance of normal follicle development, and the special phenotypes in folliculogenesis perfectly interpretation of its dual roles on both proliferation and apoptosis at two different phases, which replenished its novel role in folliculogenesis that expanded our insights. Overall design: Considering Cdkn1a homozygous mutants are embryonically lethal, we use heterozygous mutants for experiments. We isolated primary folliicles (PGs) from both early 50 days post-fertilization (dpf) and 8 months post-fertilization (mpf) for both the WT (Cdkn1+/+) and Cdkn1a (+/-) zebrafish. We then established bulk-RNAseq libraries according to NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. After libraries preparation, we performed sequencing using the Genomics, Bioinformatics and Single Cell Analysis Core at the Univerisity of Macau.