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J. Luo, L. Kakuk-Atkins, P. Hitchcock; Midkine-A Regulates Early Neurogenesis in the Retina Of Zebrafish. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5932.
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© ARVO (1962-2015); The Authors (2016-present)
Midkine is a small secreted heparin binding growth factor that has numerous biological functions, both during development and following tissue injury. There are two midkine orthologs in zebrafish, midkine-a (mdka) and midkine-b (mdkb). The purpose of this study was to use both loss- and gain-of function approaches to investigate function of Mdka during retinal neurogenesis.
Morpholino oligonucletides were injected into early-stage embryos to knock down Mdkasynthesis. The Tol2 transposon system was used to generate conditional gain-of-function transgenic lines for Mdka:EGFP fusion protein under the 5’ regulatory elements of the zebrafish heat shock gene, hsp70/4. Proliferating cells were pulse labeled by immersion in a solution of BrdU and DMSO. Cumulative BrdU labeling and determining proportions of labeled vs. unlabeled cells over time was used to characterize cell cycle kinetics. Standard methods of immunocytochemistry and in situ hybridization in tissue sections were used to identify BrdU-labeled cells or mark specific retinal cell types. Western blot analysis from whole embryo lysates was used to evaluate the loss of Mdka and the induction of Mdka:EGFP. Retinas from experimental and control animals were evaluated between 26 to 120 hours post fertilization (hpf).
The loss of Mdka does not alter gross embryonic and larval development, but does result in a transient delay in retinal differentiation. In morphants, there are fewer differentiated retinal neurons at 48hpf. Labeling with BrdU shows that all retinal cells are proliferative, though there are significantly fewer cells in M-phase. However, all major cell types are present by 72hpf. This transient delay in differentiation does not result from a delay in initiating molecular developmental events, but appears to result from lengthened cell cycle. Following heat shock treatment, synthesis of the Mdka:EGFP fusion protein is strongly induced, and initial data show that results in thicker retinal neuroepithelium and more progenitors, suggesting accelerated proliferation.
Both loss- and gain-of function experiments suggest that Mkda regulates early neurogenesis in the vertebrate retina by regulating cell cycle kinetics. The loss-of-function data resemble that reported for the disarrayed mutant (Baye LM, Link BA. BMC Dev Biol. 2007, 27:28), suggesting a possible link between disarrayed and mdka. This study provides evidence for a novel signaling pathway during early retinal development.
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