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Susov Dhakal, Josilyn Sejd, Megan Batty, Deborah L Stenkamp; Temporally-specific requirement for vascular endothelial cells during retinal neurogenesis in zebrafish embryos. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1786.
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© ARVO (1962-2015); The Authors (2016-present)
We use the embryonic zebrafish model to identify novel regulatory roles for the early ocular vasculature. Zebrafish embryos are not dependent upon circulating hemoglobin for tissue oxygenation until 5 days post fertilization, permitting the study of non-metabolic roles for vascular cells. Previously, we have shown that absence of ocular vascular endothelial cells throughout retinal neurogenesis is associated with abnormal retinal development while not causing hypoxia (Dhakal et al., 2015; Dev Dyn 244(11):1439-55). In addition, selective depletion of vascular endothelial cells between 48 and 72 hours post fertilization (hpf) in zebrafish embryos results in similarly abnormal retinal development (Dhakal et al., ARVO 2015, poster #1482). Here, we tested the roles of ocular vascular endothelial cells during retinal neurogenesis between 24hpf and 48hpf in zebrafish embryos
We treated wildtype and transgenic zebrafish embryos, Tg(kdrl:mCherry), expressing mCherry in their vascular endothelial cells, with 2µM SU5416, a selective inhibitor of vascular endothelial growth factor receptor kdrl, for two hours starting at 24hpf. Disruption of kdrl signaling inhibits proliferation of vascular endothelial cells. SU5416 treated embryos were evaluated for the presence of vascular endothelial cells, eye and lens size, retinal lamination and neuronal and non-neuronal cell differentiation using confocal and histological techniques.
Treatment of SU5416 at 24hpf impaired the development of ocular vascular system from 27-48 hpf as visualized in Tg(kdrl:mCherry) embryos. Treated embryos displayed significantly smaller eyes (p value = 0.027) and smaller lens (p value = 0.022) at 72hpf but not 48hpf. Retinal lamination and ganglion cell layer (GCL) appeared normal in SU5416 treated embryos at 48hpf. Similarly, neuronal and non-neuronal cell differentiation also appeared normal in SU5416 treated embryos at 72hpf.
Our results suggest that vascular endothelial cells may have minimal regulatory roles during retinal neurogenesis in zebrafish embryos between 24hpf and 48hpf. However, based on our previous data, abnormal retinal development occurs in absence of vascular endothelial cells between 48hpf and 72hpf in zebrafish embryos. Therefore, developing retinal cells in zebrafish embryos require regulatory signals from vascular endothelial cells in a temporally-specific manner.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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