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Travis S D'Cruz, Peter F Hitchcock, ; Midkine-a negatively regulates pro-neural signaling pathways in the developing embryonic zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1488.
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© ARVO (1962-2015); The Authors (2016-present)
Midkine-a (Mdka) is a retinoic acid-induced, heparin-binding growth factor with multiple functions in neural development and repair. In the developing zebrafish retina, Mdka controls cell cycle kinetics and functions upstream of Id2a, a HLH regulatory protein known to inhibit Notch signaling. Id2a also represses the transcription factor vsx2 (chx10) in progenitors that attain neural competence. The purpose of this study was to identify Mdka-regulated signaling networks necessary for cell cycle control.
For Mdka and Id2a knockdown, embryos at the one-cell stage were injected with morpholino oligonucleotides targeted to mdka or id2a and compared to embryos that were either uninjected or injected with 5-bp mismatch morpholino. For Mdka gain of function (GOF), the Tg(HSP70/4:mdka/egfp) line was used to induce Mdka overexpression. Wild type (WT) heat-shocked embryos were used as controls. Whole embryos were fixed with 4% paraformaldehyde at 30 hours post fertilization (hpf) and processed for cryosectioning. Also for each experimental group, gene expression was quantitatively measured by qRT-PCR and expression patterns were determined by in situ hybridization. The number of mitotically active cells in the developing retina was determined using a BrdU labeling assay. The γ-secretase inhibitor, DAPT, was used to inhibit Notch signaling.
In the developing retina, Mdka was found to repress her4, a Notch pathway readout, and vsx2. Mdka knockdown resulted in increased her4 expression (30 hpf) and Brdu staining (48hpf). Treatment with DAPT abolished her4 expression and rescued the Brdu phenotype. In Mdka GOF embryos, the expression of her4 and vsx2 was significantly reduced compared to controls. Knockdown of Id2a in GOF embryos did not rescue her4 expression. However, qRT-PCR revealed that the loss of vsx2 expression in Mdka GOF retinae is partially, but significantly, reversed by Id2a knockdown.
These results indicate that Mdka negatively regulates pro-neural genes during early retinal development, particularly acting on the Notch and Vsx2 pathways. Mdka facilitates cell cycle exit in retinal progenitors by inhibiting components of Notch signaling. The action of Mdka on the Notch pathway does not require Id2a. However, Id2a is involved in Mdka mediated repression of vsx2 in the developing retina.
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