Abstract
Purpose :
Midkine-a (Mdka) is a heparin-binding growth factor with multiple functions in neural development and repair. Previous studies using morpholino-induced protein knockdown showed that in the embryonic zebrafish, Mdka is upstream of the HLH factor Id2a and in retinal progenitors functions to control cell cycle kinetics. The purpose of this study was to characterize a mdka mutant line generated using CRISPR/Cas genome editing technology.
Methods :
CRISPR/Cas constructs were injected into single cell embryos and targeted to sequences in the second exon of the gene encoding mdka. In the line we propagated, the resulting 19 base pair deletion at the target site was predicted to generate a premature stop codon and a truncated protein. To evaluate retinal development, embryos were fixed with 4% paraformaldehyde at 30 or 48 hours post fertilization (hpf) and processed for cryosectioning followed by immunohistochemistry. Whole embryo gene expression was quantified by qPCR. Mitotically active retinal progenitors were labeled using BrdU. S-phase and cell cycle length was assessed using a pulse-chase-pulse assay.
Results :
Immunolabeling validated the absence of the Mdka in the mutant line. At 30 hpf, the retinas of mdka mutants were visibly delayed in development. At 48 hpf, the size of the eye and head was small and the body exhibited less dense melanization. Also, the number of Brdu+ progenitors was significantly greater in mutant retinas compared to wild type (WT). The S-phase length and total length of the cell cycle in retinal progenitors was significantly longer in mutants compared to WT. The expression of id2a in Mdka mutants was decreased compared to WT. By 72 hpf, the delay observed at 48hpf was recovered; the number and distribution of BrdU+ cells, retinal lamination and neuronal differentiation resemble WT. To determine if the recovery at 72 hpf might be due to the upregulation of other Midkine family members, the expression of mkdb and pleiotrophin was quantified. mdkb and pleiotrophin expression was not increased in the mutants compared to WT embryos.
Conclusions :
These results indicate that in mdka mutant embryos the changes in retinal development, cell cycle kinetics and the expression of downstream effectors recapitulate that described for Mdka morphants. Further, the recovery of the developmental delay resulting from the absence of Mdka is not compensated for by other Midkine family members.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.